CN118512592A - Application of RNF125 inhibitor in preparation of medicines for preventing or treating obesity and related complications - Google Patents
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Abstract
The invention belongs to the technical field of biological medicines, and particularly relates to application of an RNF125 inhibitor in preparation of a medicament for preventing or treating obesity and related complications. The invention constructs an obesity model by knocking out the mouse RNF125 and then feeding the mouse with high-fat feed, and the result indicates that the RNF125 deficiency can improve diseases or symptoms such as obesity phenotype, hyperglycemia, hyperlipidemia, fat accumulation, nonalcoholic fatty liver and the like induced by high-fat diet. The siRNA specifically targeting RNF125 is injected into the obesity mouse model, so that the symptoms of weight increase, impaired glucose tolerance and insulin sensitivity, hyperglycemia, hyperlipidemia, fat accumulation, liver steatosis and the like caused by high-fat diet can be effectively improved. The invention develops a series of human-source targeted siRNA inhibiting RNF125 by using the target spot, and further improves the stability of the RNF125 in vivo and the silencing efficiency of the target gene by a modification means, thereby being beneficial to patent medicine.
Description
Technical Field
The invention belongs to the field of biological medicine, and particularly relates to application of an RNF125 inhibitor in preparation of a medicament for preventing or treating obesity and related complications.
Background
Obesity refers to abnormal distribution or excessive accumulation of body fat, ultimately affecting health. Obesity is mainly characterized by excessive fat accumulation. The World Health Organization (WHO) defines overweight in adults as having a Body Mass Index (BMI) of 25.0-29.9kg/m 2 and obesity as having a BMI of 30.0kg/m 2 or more.
Obesity as a healthy killer affects the entire metabolic system of the human body, leading to various complications such as type two diabetes (T2 DM), liver steatosis, cardiovascular diseases, stroke, lipid metabolism disorders, hypertension, gallbladder problems, arthritis, sleep apnea and other respiratory problems, cancers (such as breast cancer, liver cancer, ovarian cancer, prostate cancer, gallbladder cancer and colon cancer), which all increase the risk of death. The complications directly related to obesity that are most studied at present are type two diabetes, non-alcoholic fatty liver disease and cardiovascular disease. Obesity is the leading factor in the induction of type two diabetes mellitus, and the main cause of T2DM formation is insulin resistance in the liver, white Adipose Tissue (WAT) and skeletal muscle due to obesity, combined with insufficient secretion of insulin by pancreatic beta cells, which is difficult to overcome. In addition, obesity can promote cell aging in adipose tissue, liver, brain, pancreas, etc., shorten life span, and increase risk of onset of various age-related diseases and complications, including diabetes, hypertension, cancer, cognitive dysfunction, atherosclerosis, and vascular dysfunction. Therefore, the need for treatment/prevention of obesity and improvement of human quality of life is urgent, but the development of treatment schemes for obesity is difficult and serious, for example, the weight-losing therapies approved by the current regulatory authorities mostly have strong side effects, cannot be used for a long time, and have high surgical treatment cost, and do not need to be subjected to diet control for a long time once and all; long-term diet control is prone to malnutrition or recurrent episodes of obesity. If a target for treating obesity can be found out and a safe and effective new medicine is developed, good news is brought to people suffering from obesity.
The RNF125 (RING FINGER Protein 125) gene encodes a novel E3 ubiquitin ligase, comprising a ring finger domain at the N-terminus and three zinc binding motifs and a ubiquitin interaction motif at the C-terminus. The encoded protein may play a positive regulatory role in the T cell receptor signaling pathway. RNF125 is ubiquitously expressed in fat, bone marrow and 25 other tissues. However, no studies have yet demonstrated what kind of correlation exists between RNF125 and metabolic levels, obesity, insulin resistance, fatty liver, and the like.
Disclosure of Invention
First, the technical problem to be solved
In view of the above-mentioned shortcomings and drawbacks of the prior art, the present invention provides for the use of RNF125 inhibitors in the manufacture of a medicament for the prevention or treatment of obesity and related complications, providing a reliable solution for the development of safe and effective obesity therapeutic medicaments.
(II) technical scheme
The invention provides application of an RNF125 inhibitor in preparing a medicament for preventing or treating obesity and related complications.
Animal experiments prove that the siRNA targeting RNF125 can be injected into a mouse obesity model constructed by knocking out RNF125 or high-fat feed, so that the diseases such as weight increase, fat accumulation, impaired glucose tolerance, insulin resistance, hyperglycemia, hyperlipidemia and liver steatosis caused by high-fat diet can be effectively improved.
The RNF125 inhibitor is capable of producing a change in the level of any one of the following: (1) inhibiting obesity caused by high-fat diet of mice; (2) inhibiting fat accumulation due to a high fat diet; (3) Inhibiting glucose tolerance and insulin resistance caused by high-fat diet, and further preventing hyperglycemia and hyperlipidemia; (4) inhibiting hepatic steatosis caused by a high fat diet.
Wherein, the RNF125 inhibitor can inhibit the activity or expression level of the RNF125 protein, thereby achieving the effect of preventing or treating obesity and related complications.
According to a preferred embodiment of the invention, the RNF125 inhibitor is any protein molecule, nucleic acid molecule (siRNA or shRNA or miRNA, etc.), compound small molecule, viral particle or any combination of two or more of the foregoing capable of reducing the activity or expression level of RNF125 protein; or the RNF125 inhibitor is any protein molecule, nucleic acid molecule, small compound molecule, viral particle, gene editing kit, or any combination of two or more of the foregoing, capable of knocking out or knocking down or silencing an RNF125 gene or protein.
According to a preferred embodiment of the invention, the RNF125 inhibitor is any one of the following siRNA:
siRNA formed by annealing two single strands shown in SEQ ID Nos. 9-10;
siRNA formed by annealing two single strands shown in SEQ ID NO. 41-42;
siRNA formed by annealing two single strands shown in SEQ ID NO. 43-44;
siRNA formed by annealing two single strands shown in SEQ ID NO. 111-112;
siRNA formed by annealing two single strands shown in SEQ ID NO. 1-2;
siRNA formed by annealing two single strands shown in SEQ ID NO. 37-38;
siRNA formed by annealing two single strands shown in SEQ ID NO. 85-86;
siRNA formed by annealing two single strands as shown in SEQ ID nos. 205-206;
siRNA formed by annealing two single strands shown in SEQ ID NO. 3-4;
siRNA formed by annealing two single strands shown in SEQ ID NOS.87-88;
siRNA formed by annealing two single strands shown in SEQ ID NO. 45-46;
siRNA formed by annealing two single strands shown in SEQ ID NO. 5-6;
siRNA formed by annealing two single strands shown in SEQ ID NO. 55-56;
siRNA formed by annealing two single strands shown in SEQ ID NOS.159-160;
siRNA formed by annealing two single strands shown in SEQ ID NO. 23-24;
siRNA formed by annealing two single strands shown in SEQ ID NO. 29-30;
siRNA formed by annealing two single strands as shown in SEQ ID NOS.175-176;
siRNA formed by annealing two single strands shown in SEQ ID NO. 11-12;
siRNA formed by annealing two single strands shown in SEQ ID NO. 33-34;
siRNA formed by annealing two single strands shown in SEQ ID NO. 17-18;
siRNA formed by annealing two single strands shown in SEQ ID NO. 93-94;
siRNA formed by annealing two single strands shown in SEQ ID NO. 49-50;
siRNA formed by annealing two single strands shown in SEQ ID NO. 35-36;
siRNA formed by annealing two single strands shown in SEQ ID NO. 75-76;
siRNA formed by annealing two single strands shown in SEQ ID NO. 79-80; siRNA formed by annealing two single strands shown in SEQ ID NO. 109-110; siRNA formed by annealing two single strands shown in SEQ ID NO. 153-154; siRNA formed by annealing two single strands shown in SEQ ID NO. 15-16; siRNA formed by annealing two single strands shown in SEQ ID NO. 131-132; siRNA formed by annealing two single strands shown in SEQ ID NO. 59-60; siRNA formed by annealing two single strands shown in SEQ ID NO. 51-52; siRNA formed by annealing two single strands shown in SEQ ID nos. 167-168; siRNA formed by annealing two single strands shown in SEQ ID NO. 133-134; siRNA formed by annealing two single strands shown in SEQ ID NO. 77-78; siRNA formed by annealing two single strands as shown in SEQ ID nos. 219-220; siRNA formed by annealing two single strands shown in SEQ ID NO. 19-20; siRNA formed by annealing two single strands shown in SEQ ID NO. 7-8; siRNA formed by annealing two single strands shown in SEQ ID NO. 13-14; siRNA formed by annealing two single strands shown in SEQ ID NO. 27-28; siRNA formed by annealing two single strands shown in SEQ ID NO. 165-166; siRNA formed by annealing two single strands shown in SEQ ID nos. 201-202; siRNA formed by annealing two single strands shown in SEQ ID NO. 145-146; siRNA formed by annealing two single strands shown in SEQ ID NO. 123-124; siRNA formed by annealing two single strands shown in SEQ ID NO. 195-196; siRNA formed by annealing two single strands shown in SEQ ID NO. 61-62; siRNA formed by annealing two single strands shown in SEQ ID NO. 207-208; siRNA formed by annealing two single strands shown in SEQ ID NO. 39-40; siRNA formed by annealing two single strands shown in SEQ ID NO. 83-84; siRNA formed by annealing two single strands shown in SEQ ID NO. 69-70; siRNA formed by annealing two single strands shown in SEQ ID NO. 107-108; siRNA formed by annealing two single strands shown in SEQ ID NO. 47-48; siRNA formed by annealing two single strands shown in SEQ ID NO. 125-126; siRNA formed by annealing two single strands shown in SEQ ID NO. 25-26; siRNA formed by annealing two single strands shown in SEQ ID NO. 199-200; siRNA formed by annealing two single strands shown in SEQ ID NO. 31-32; siRNA formed by annealing two single strands shown in SEQ ID NO. 171-172; siRNA formed by annealing two single strands shown in SEQ ID NO. 135-136; siRNA formed by annealing two single strands shown in SEQ ID nos. 143-144; siRNA formed by annealing two single strands shown in SEQ ID NO. 89-90; siRNA formed by annealing two single strands shown in SEQ ID NO. 21-22; siRNA formed by annealing two single strands shown in SEQ ID NO. 97-98; siRNA formed by annealing two single strands shown in SEQ ID NOS.151-152; siRNA formed by annealing two single strands shown in SEQ ID NO. 221-222; siRNA formed by annealing two single strands shown in SEQ ID NO. 105-106; siRNA formed by annealing two single strands shown in SEQ ID NO. 57-58; siRNA formed by annealing two single strands shown in SEQ ID NOS.129-130; siRNA formed by annealing two single strands shown in SEQ ID NO. 229-230; siRNA formed by annealing two single strands shown in SEQ ID NO. 141-142; siRNA formed by annealing two single strands shown in SEQ ID NOS.155-156; siRNA formed by annealing two single strands shown in SEQ ID NO. 147-148; siRNA formed by annealing two single strands shown in SEQ ID NO. 65-66; siRNA formed by annealing two single strands shown in SEQ ID NO. 67-68; siRNA formed by annealing two single strands as shown in SEQ ID NO. 189-190; siRNA formed by annealing two single strands shown in SEQ ID NO. 193-194; siRNA formed by annealing two single strands shown in SEQ ID NO. 53-54; siRNA formed by annealing two single strands shown in SEQ ID NO. 73-74; siRNA formed by annealing two single strands shown in SEQ ID NO. 213-214; siRNA formed by annealing two single strands shown in SEQ ID NO. 181-182; siRNA formed by annealing two single strands shown in SEQ ID NO. 101-102; siRNA formed by annealing two single strands shown in SEQ ID NO. 139-140; siRNA formed by annealing two single strands shown in SEQ ID NO. 91-92; siRNA formed by annealing two single strands shown in SEQ ID NO. 197-198; siRNA formed by annealing two single strands shown in SEQ ID NO. 99-100; siRNA formed by annealing two single strands shown in SEQ ID NO. 137-138; siRNA formed by annealing two single strands shown in SEQ ID NO. 71-72; siRNA formed by annealing two single strands shown in SEQ ID nos. 127-128; siRNA formed by annealing two single strands shown in SEQ ID nos. 121-122; siRNA formed by annealing two single strands shown in SEQ ID NOS.115-116; siRNA formed by annealing two single strands shown in SEQ ID NOS.215-216; siRNA formed by annealing two single strands shown in SEQ ID NO. 173-174; siRNA formed by annealing two single strands shown in SEQ ID NO. 103-104; siRNA formed by annealing two single strands as shown in SEQ ID No. 177-178; siRNA formed by annealing two single strands shown in SEQ ID NO. 95-96; siRNA formed by annealing two single strands shown in SEQ ID NO. 231-232; siRNA formed by annealing two single strands shown in SEQ ID NOS 209-210; siRNA formed by annealing two single strands shown in SEQ ID NO. 119-120; siRNA formed by annealing two single strands shown in SEQ ID NO. 149-150; siRNA formed by annealing two single strands shown in SEQ ID NO. 81-82; siRNA formed by annealing two single strands shown in SEQ ID nos. 203-204; siRNA formed by annealing two single strands shown in SEQ ID NOS.157-158; siRNA formed by annealing two single strands shown in SEQ ID NO. 163-164; siRNA formed by annealing two single strands shown in SEQ ID NO. 217-218; siRNA formed by annealing two single strands shown in SEQ ID NO. 191-192; siRNA formed by annealing two single strands as shown in SEQ ID No. 187-188; siRNA formed by annealing two single strands shown in SEQ ID NO. 169-170; siRNA formed by annealing two single strands shown in SEQ ID NO. 113-114; siRNA formed by annealing two single strands shown in SEQ ID nos. 117-118; siRNA formed by annealing two single strands shown in SEQ ID NOS.63-64; siRNA formed by annealing two single strands shown in SEQ ID NO. 161-162;
siRNA formed by annealing two single strands shown in SEQ ID NO. 223-224;
siRNA formed by annealing two single strands shown in SEQ ID NO. 185-186;
siRNA formed by annealing two single strands shown in SEQ ID NOS.211-212;
siRNA formed by annealing two single strands shown in SEQ ID NOS.215-216;
siRNA formed by annealing two single strands shown in SEQ ID NO. 227-228;
siRNA formed by annealing two single strands shown in SEQ ID NO. 183-184;
siRNA formed by annealing two single strands as shown in SEQ ID No. 179-180.
According to a preferred embodiment of the present invention, the RNF125 inhibitor is a modified product obtained by subjecting the antisense strand and the sense strand of any one of the above siRNA to nucleotide modification; the nucleotide modification comprises one or more of methoxy modification, fluoro modification, galNAc coupling modification, thio modification and glycol, is used for improving the stability of siRNA in vivo, reducing degradation rate and being beneficial to improving the silencing efficiency of target genes or forming drug delivery. GalNAc is covalently coupled to the 3' -end of a nucleic acid in a trivalent state. A thio modification is a phosphorothioate linkage between two adjacent nucleotides.
According to a preferred embodiment of the present invention, the modification method is any of the following methods:
Mode one: in 21 nucleotides of the sense strand, 2 thio-modified 5 'ends, fluoro-modified 9, 10 and 11 positions respectively, methoxy-modified rest positions and coupled 3' ends with GalNAc; 2 thio modifications at the 5 'end, fluoro modifications at positions 2,5, 14 and 16, 2 thio modifications at the 3' end and methoxy modifications at the rest positions in 23 nucleotides of the antisense strand;
mode two: in 21 nucleotides of the sense strand, 2 thio modifications at the 5' end, fluoro modifications at positions 9, 10 and 11 respectively, and the nucleotides at the rest positions are methoxy and fluoro alternately modified in sequence, 2 thio modifications at the 3' end and GalNAc coupled at the 3' end; sequentially carrying out methoxy and fluoro alternate modification on 23 nucleotides of the antisense strand, 2 thio modifications at the 5 'end and 2 thio modifications at the 3' end;
Mode three: in 21 nucleotides of the sense strand, 2 thio modifications at the 5' end, fluoro modifications at 7, 9, 10 and 11 positions and methoxy modifications at the rest positions are obtained, 2 thio modifications at the 3' end and GalNAc coupling at the 3' end; 2 nd, 6 th, 8 th, 9 th, 15 th and 17 th positions of 23 th nucleotides of the antisense strand are fluoro modified, methoxy modified at the rest positions, 2 thio modified at the 5 'end and 2 thio modified at the 3' end;
Mode four: in 21 nucleotides of the sense strand, 2 thio modifications at the 5' end, fluoro modifications at 7, 9, 10 and 11 positions and methoxy modifications at the rest positions are obtained, 2 thio modifications at the 3' end and GalNAc coupling at the 3' end; 23 nucleotides of antisense strand are 2 nd, 14 th and 16 th fluoro modified, 7 th glycol nucleic acid, methoxy modified, 2 thio modified at 5 'end and 2 thio modified at 3' end.
Obesity, as used herein, refers to a chronic metabolic disorder caused by excessive fat accumulation or abnormal distribution in the body, resulting in weight gain, including genetic and environmental factors, and the interaction of various factors. Obesity-related complications include type II diabetes, hypertension, hyperlipidemia, atherosclerosis, vascular dysfunction, nonalcoholic fatty liver, stroke, lipid metabolism disorders, and sleep apnea.
In a fourth aspect, the present invention provides a medicament for the prophylaxis or treatment of obesity and related complications, wherein the medicament comprises an RNF125 inhibitor as the active ingredient; the medicine comprises an active ingredient RNF125 inhibitor and pharmaceutically acceptable auxiliary materials. The percentage of the RNF125 inhibitor in the medicine is 1-99%.
In a fifth aspect, the invention provides the use of an RNF125 gene or protein as a target in the construction of a model for the treatment of obesity.
Preferably, it is constructed by increasing the expression level of the RNF125 gene or protein in the target animal.
RNF125 is a known gene. RNF125, also known as TRAC-1, is an E3 ubiquitin ligase consisting of an N-terminal RING domain, 3 zinc finger structures in the middle (C2 HC, C2H 2) and a C-terminal ubiquitin acting motif (ubiquitin interaction motif, UIM). RNF125 was originally found to be a regulator of T cell activation, which is highly expressed in lymphoid tissues within bone marrow, spleen and thymus. Subsequent studies have also found that RNF125 is associated with a variety of diseases such as inflammatory responses, the development and progression of cancer, and innate anti-viral immune responses. However, no report has been made to reveal what kind of connection between it and obesity.
The invention firstly discloses that the RNF125 gene or protein expression is inhibited, and the diseases such as weight increase, fat accumulation, impaired glucose tolerance, insulin resistance, liver steatosis and the like caused by high-fat diet can be effectively improved; animal experiments prove that the CRISPER-Cas9 technology is utilized to knock out RNF125 in mice, and then high-fat feed is fed to construct an obesity model, and experimental results indicate that the loss of the RNF125 can improve obesity phenotypes, hyperglycemia, hyperlipidemia, fat accumulation, nonalcoholic fatty liver and the like induced by high-fat diet. The siRNA targeting RNF125 is injected into a mouse obesity model constructed by high-fat feed for treatment, and experimental results show that the siRNA can effectively improve the symptoms of weight increase, impaired glucose tolerance and insulin sensitivity, hyperglycemia, hyperlipidemia, fat accumulation, liver steatosis and the like caused by high-fat diet. From this, it was determined that RNF125 gene or protein is a new target for the treatment or therapy of obesity and related complications.
It will be appreciated that based on the foregoing findings, the innovation of the present invention is to provide a new target RNF125 for treating or preventing obesity and its complications, and that the known RNF125 inhibitors (including knockdown or knockdown agents) can be used as active ingredients of drugs for preventing or treating obesity and related complications.
For convenience and brevity, the meaning of "RNF125 inhibition" in the present application includes targeting of knockdown or silencing of RNF125, etc., while the meaning of "RNF125 inhibitor" includes any protein molecule, nucleic acid molecule, small compound molecule, gene editing kit, viral particle, or any combination of two or more of the foregoing, including, for example, but not limited to, neutralizing antibodies, siRNA, shRNA, miRNA, knockdown reagents, gene editing crispr _cas9 kit, lentiviruses, etc., capable of knocking down or knockdown or silencing RNF 125.
(III) beneficial effects
1. The present invention provides a novel target RNF125 associated with obesity, RNF125 being demonstrated to have at least one of the following activities: (1) can promote obesity caused by high-fat diet; (2) can promote fat accumulation caused by high-fat diet; (3) Can promote glucose tolerance and insulin resistance caused by high-fat diet, and cause hyperglycemia and hyperlipidemia; (4) can promote liver steatosis caused by high fat diet. Animal experiments prove that the siRNA targeting RNF125 can be injected into a mouse obesity model constructed by knocking out RNF125 or high-fat feed, so that the diseases such as weight increase, fat accumulation, impaired glucose tolerance, insulin resistance, hyperglycemia, hyperlipidemia, liver steatosis and the like caused by high-fat diet can be effectively improved. Based on the above experimental results, it was inferred that obesity and related complications can be prevented or treated by inhibiting the expression of RNF125 gene or protein.
In summary, the present invention provides applications of inhibition of RNF125 as a therapeutic target in preventing or treating obesity and related complications, which provides new directions and theoretical guidance for developing safe and effective treatment schemes and drugs for obesity and its complications.
2. The invention provides a series of siRNA sequences targeting RNF125, which are used for knocking down or silencing the expression of the gene, and the siRNA sequences provided by the invention are proved to be transfected into experimental cells at a very low concentration (1 nM) at a cell level, so that the expression of the target gene RNF125 in the cells can be effectively silenced.
3. The siRNA sequence of the target RNF125 provided by the invention is further subjected to nucleotide modification, is coupled with a GalNAc delivery system, is injected into a human transgenic mouse model body, and is subjected to blood sampling and serum separation to detect the silencing effect of the modified siRNA on target gene mRNA, and experiments show that the modified siRNA can improve the silencing efficiency of the siRNA on the target gene RNF125 in an animal model body, and different nucleotide modification modes can effectively silence the target gene. The nucleotide modification is mainly used for reducing degradation of nucleic acid molecules in animals and improving drug resistance.
Drawings
FIG. 1 is a graph showing experimental statistics of the effect of RNF125 deficiency on the feed intake and the weight-gain rate of mice in example 1.
FIG. 2 shows experimental statistics of the effect of RNF125 deficiency on hyperlipidemia-induced hyperglycemia in example 2.
FIG. 3 is experimental statistics of the effect of RNF125 deficiency on high-fat diet-induced fat accumulation in example 3.
FIG. 4 is the experimental statistics of the effect of RNF125 deficiency on high-fat diet-induced non-alcoholic fatty liver disease in example 4.
FIG. 5 shows the therapeutic effect of siRNA sequence of RNF125 on obesity caused by high-fat diet in example 5.
FIG. 6 shows the change in glucose tolerance, insulin sensitivity and blood lipid of high fat diet mice after RNF 125-specific siRNA treatment in example 6.
FIG. 7 shows the change in adipose tissue of high-fat diet mice after RNF 125-specific siRNA treatment in example 7.
FIG. 8 shows the therapeutic effect of specific siRNA sequences of RNF125 on hepatic steatosis caused by high-fat diet in example 8.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The invention firstly utilizes CRISPER-Cas9 technology to construct RNF125 knockout mice, and the mice are respectively fed with Normal diet (NCD) and high fat diet (HIGH FAT DIET, HFD) to analyze the effect of RNF125 deficiency in obesity, hyperglycemia, hyperlipidemia, fat accumulation and nonalcoholic fatty liver induced by high fat diet. Then, establishing an obesity model of the mice by using a high-fat feed, dividing the mice into two groups, and continuously feeding the mice with a high-fat diet, and simultaneously, injecting a specific siRNA sequence targeting RNF125 into a group of tail veins to form a treatment group (siRNF 125,125); the other group of tail intravenous injection sequences are control sequences which are not homologous to transcriptomes of human, rat, mouse and the like, and are control groups (siCtrl), and meanwhile, the siRNA sequences of RNF125 are used as controls to study the treatment effect of obesity and related diseases by taking common diet mice of the same age as the controls. The components of the common feed and the high-fat feed are as follows:
the experimental methods and experimental conclusions of the embodiments of the present invention are described in detail below.
1. RNF125 specific siRNA sequence screening and synthesis
Firstly, synthesizing a plurality of RNAi oligos aiming at different sites of RNF125 in a mouse body by using Jiangsu Ji Ma company, transfecting H22 mouse liver cancer cells, analyzing the RNF125 knocking down condition by using an RT-qPCR method, and picking RNAi sequences with better knocking down efficiency; the selected RNF125 RNAi oligos (RNF 125 siRNA) sequence was synthesized and its sense strand was modified in the following manner: cholesterol modification is carried out at the 3' -end, two thio skeletons at the 5' -end are modified, four thio skeletons at the 3' -end are modified, and full-chain methoxy modification is carried out. Finally, three RNF125 specific murine siRNAs were obtained as shown in Table 1:
Table 1:
2. Obese mouse model construction and treatment scheme
Male C57BL/6 mice purchased from the company of St Bei Fu Biotechnology Co at a temperature of 22.+ -. 2 ℃ were bred in SPF-class animal houses. Mice were divided into two groups after one week of acclimation, one group was fed with normal diet and one group was fed with high fat diet, and the mice were monitored for body weight, random blood glucose, fasting blood glucose, and TG and TC levels in the blood during feeding. When the weight, blood sugar, TG and TC levels of the high fat diet mice were all significantly higher than those of the normal diet group, the high fat diet mice were randomly divided into two groups, siRNA specifically targeting RNF125 and a control sequence (siCtrl) were designed and synthesized, injected into the mice by tail vein injection at a dose of 0.5OD (1.25 nM)/dose, injected once every 4 days, and the weight change of the mice was monitored, and the diet change of the mice was monitored by Columbus Instruments.
3. In the experimental process, the blood sugar of the mice, the TG and TC levels in the blood of the mice and the TG and TC levels in the liver can be measured according to the conventional method, and the operations such as Glucose Tolerance Test (GTT) and tissue H & E staining can be performed, and the following methods can be referred to:
① Blood glucose determination in mice: under normal diet conditions, random blood glucose of mice was measured using a rogowski glucometer; mice were assayed for fasting blood glucose using a rogowski glucometer under overnight starvation for 12 hours.
② TG and TC detection in mouse blood: after the mice were starved overnight for 12 hours, 100. Mu.L of blood was taken from the tail ends of the mice, and after 2 hours at room temperature, the mice were centrifuged at 3000rpm for 10 minutes, and serum was taken and assayed for TG, TC, LDL-C and FFA levels in the serum using a biochemical analyzer.
③ TG detection in mouse liver: 50mg of liver tissue was taken according to the tissue mass (g): n-propane/isopropyl alcohol volume (mL) was 1: 5-10, adding n-propane/isopropanol, homogenizing in ice bath, centrifuging at 8000g centrifugal force and 4deg.C for 10min, and measuring TG content in liver tissue with Soxhibao Triglyceride (TG) content detection kit (product number, BC 0625). The method for detecting TC in the liver is as follows: according to the tissue mass (g): isopropyl alcohol volume (mL) was 1: isopropanol is added in the proportion of 5 to 10, and the mixture is homogenized in an ice bath. Centrifuging at 10000g centrifugal force and 4 deg.C for 10min, collecting supernatant, and measuring TC content in liver tissue by using Soxhaust Total Cholesterol (TC) content detection kit (product number, BC 1985).
④ The procedure for Glucose Tolerance Test (GTT) was as follows: after 6 hours of fasted mice, glucose was intraperitoneally injected at a dose of 2g/kg, and the blood glucose of the mice was measured with a rogowski blood glucose meter at 0, 15, 30, 45, 60, 90, 120min after injection, a glucose tolerance curve was drawn, and the area under the glucose curve was calculated by the AOC method. The method of insulin resistance test (ITT) is as follows: after 6 hours of fasted mice, insulin (norand nod, insulin aspart injection) was injected intraperitoneally at a dose of 0.75U/kg, and blood glucose in tail veins of the mice was measured by a rogowski glucometer (while 20% glucose was prepared so that 100 μl of 20% glucose was injected intraperitoneally when the mice were hypoglycemic) at 0, 15, 30, 45, 60, 90, 120min after the injection, respectively, an insulin resistance curve of the mice was drawn, and the area under the blood glucose curve was calculated by an AOC method.
⑤ The method of tissue HE staining is as follows: tissue samples were fixed in 4% paraformaldehyde (seville, G1101), dehydrated, transparent, waxed, embedded, sectioned, dewaxed, and the like after 24 hours, and the dewaxed sections were stained with hematoxylin, stained with eosin, dehydrated, and sealed for viewing.
Example 1
The experiments of this example demonstrate that RNF125 deficiency can improve the high fat diet induced obesity phenotype. The experiment comprises an experimental method and an experimental result:
The experimental method comprises the following steps: RNF125 whole-body knockout mice were constructed using CRISPR-Cas9 technology. Wild type mice (WT) and RNF125 knockout mice (KO) were selected from 6-8 weeks, and each was divided into two groups, and fed with normal diet and high fat diet, respectively, and the body type and weight of the mice were monitored during feeding, and the diet changes of the mice were monitored by Columbus Instruments.
Experimental results: as shown in fig. 1. Panel A shows that RNF125 knockout resulted in mice taking more feed daily under normal diet conditions; however, the weight gain after 6 months of feeding was significantly lower than that of the wild-type mice, and the body type was significantly smaller than that of the wild-type mice (B). The daily intake of feed by RNF125 knockout mice was also significantly higher than that of wild-type mice (C) under high-fat diet conditions; however, the weight gain after 6 months of feeding was significantly lower than that of the wild-type mice, and the body type was significantly smaller than that of the wild-type mice (D)
As can be seen, RNF125 knockout mice consume significantly more food per day than wild-type mice (a) on a normal diet; however, after 6 months of feeding, the RNF125 knockout mice were slightly smaller in size than the wild-type mice, and the weight gain was significantly lower than that of the wild-type mice (B). On a high-fat diet, RNF125 knockout mice still ingest significantly more food per day than wild-type mice (C); the weight gain of wild-type mice was doubled after 6 months of high fat diet, but the weight gain of knockout mice was only about 75% greater, the weight gain was significantly lower than that of wild-type mice, and the body type was significantly smaller than that of wild-type mice (D). This indicates that after RNF125 deletion, the obese phenotype of mice was significantly improved, and mice ingested more feed, but had significantly lower body weight than wild-type mice. In the figure: * P <0.05; * P <0.01; * P <0.001; NCD, normal diet; HFD, high fat diet; WT, wild-type mice; KO, RNF125 deleted mice.
Taken together, RNF125 knockout significantly improved the high fat diet-induced obese phenotype, while RNF125 affected the appetite of mice, with RNF125 deficiency resulting in more food intake by mice, but less weight gain.
Example 2
Experiments in this example demonstrate that RNF125 deficiency is effective in ameliorating high lipid diet-induced hyperglycemia, hyperlipidemia. The experiment comprises an experimental method and an experimental result:
The experimental method comprises the following steps: the mice of example 1 were tested for blood glucose during high fat diet using a rogowski glucometer and analyzed for blood lipid changes using a biochemical analyzer, and the mice were analyzed for glucose tolerance by a Glucose Tolerance Test (GTT).
Experimental results: as shown in fig. 2. Panel A shows that random blood glucose was significantly lower in mice after RNF125 knockout than in wild-type mice; panel B shows that after overnight fasting in mice, the fasting blood glucose of RNF125 knockout mice was significantly lower than that of wild-type mice; panel C shows that the GTT profile of RNF125 knockout mice is significantly lower than that of wild-type mice on a normal diet; panel D shows that on a high fat diet, the GTT profile of RNF125 knockout mice is significantly lower than that of wild-type mice; panel E shows that the area under the curve of panels C and D, the glucose tolerance of RNF125 knockout mice is significantly higher than that of wild-type mice; panel F shows that the blood Triglyceride (TG) levels of RNF125 knockout mice were significantly lower than those of wild-type mice under normal and high-fat diet conditions; panel G shows that RNF125 knockout mice have significantly lower levels of cholesterol (TC) in the blood than wild-type mice under normal and high-fat diet conditions; panel H shows that the level of LDL-C in blood of RNF125 knockout mice is significantly lower than that of wild-type mice under normal and high-fat diet conditions; panel I shows that RNF125 knockout mice have significantly lower levels of Free Fatty Acids (FFA) in the blood than wild-type mice under normal and high fat diet conditions.
It can be seen that both random blood glucose (a) and fasting blood glucose (B) were significantly reduced in mice following RNF125 knockout. For wild-type mice, high-fat diet resulted in a significant increase in random blood glucose in wild-type mice, but RNF125 knockout restored blood glucose to normal dietary levels or slightly below normal levels, indicating that RNF125 depletion is beneficial in glycemic control. To further verify the regulatory capacity of RNF125 on blood glucose, this example analyzed the change in blood glucose in mice before and after RNF125 knockout by a Glucose Tolerance Test (GTT), and the experimental results are shown in fig. 2, panels C to E. Blood glucose levels were significantly lower in mice after RNF125 knockout than in wild-type mice, with area under the curve (AOC) also significantly reduced in group KO mice (E), whether on plain diet (C) or on high fat diet (D), indicating that the glucose tolerance of mice after RNF125 knockout was significantly enhanced. Continuing to analyze the change condition of the blood fat, wherein the analysis results are shown in F to I in fig. 2: the levels of triglyceride (F), cholesterol (G), LDL-C (H), serum free fatty acid (I) in the serum of mice after RNF125 knockout were significantly reduced, both in normal diet and in high-fat diet. The results show that the RNF125 knockout can effectively improve the problems of hyperglycemia, hyperlipidemia, impaired glucose tolerance and the like induced by animal high-fat diet.
The results show that the RNF125 deficiency can effectively improve the problems of hyperglycemia, hyperlipidemia, impaired glucose tolerance and the like induced by high-fat diet. In the figure: * P <0.05; * P <0.01; * P <0.001.NCD, normal diet; HFD, high fat diet.
Example 3
The experiments of this example demonstrate that RNF125 deficiency is effective in improving high fat diet-induced fat accumulation. The experiment comprises an experimental method and an experimental result:
The experimental method comprises the following steps: the mice on the high fat diet of example 1 were dissected for 6 months, fat accumulation in the abdominal cavity of the mice was observed, and the inguinal subcutaneous white adipose tissue (iWAT) and epididymal white adipose tissue (eWAT) of the mice were weighed and fixed with 4% paraformaldehyde and then subjected to H & E staining.
Experimental results: as shown in fig. 3. Panel A shows that after 6 months of normal or high fat diet, the mice have significantly less fat in the abdominal cavity after RNF125 knockout than wild-type mice; panel B shows that after 6 months of normal or high fat diet, the weight ratio of subcutaneous fat in RNF125 knockout mice was significantly lower than in wild-type mice; panel C shows that after 6 months of normal or high fat diet, the weight ratio of visceral fat in epididymal of RNF125 knockout mice was significantly lower than that of wild-type mice; the H & E staining results of panel D showed that RNF125 knockout mice showed significantly less adipocytes than wild type mice following a normal or high fat diet for 6 months; the H & E staining results of panel E showed that RNF125 knockout mice showed significantly less adipocytes of epididymal visceral fat than wild type mice after 6 months of normal or high fat diet.
From this, after 6 months of normal diet, fat accumulation was present around organs of wild-type mice in normal diet, but in contrast, the fat around organs of mice in which RNF125 was absent was hardly seen (on panel a); also, the high fat diet for 6 months was found to result in the wild-type mice being fat-filled in their abdomen, almost all organs being surrounded by fat, while RNF125 loss of fat in the abdomen of the mice was significantly less (panel a, below). The RNF125 knockout mice were tested for a significant reduction in subcutaneous fat after 6 months of normal diet; long-term (6 months) high-fat diet resulted in a multiple increase in subcutaneous fat in wild-type mice, with RNF125 deficiency significantly reducing the content of subcutaneous fat (panel B); furthermore, RNF125 deficiency also significantly reduced epididymal visceral fat in mice after 6 months of normal or high-fat diet (panel C). The H & E staining results showed that RNF125 knockout mice showed significantly less adipocytes of subcutaneous fat than wild type mice (D) after 6 months of normal or high fat diet, whether normal or high fat diet; likewise, after 6 months of normal or high fat diet, the mice with RNF125 knockdown of visceral fat showed significantly less adipocytes than wild-type mice (E). Overall, RNF125 deficiency significantly reduced adipose tissue accumulation and fat droplet size in fat.
The above results indicate that RNF125 deficiency is effective in improving high fat diet-induced fat accumulation. In the figure: * P <0.05; * P <0.001.NCD, normal diet; HFD, high fat diet.
Example 4
The experiments of this example demonstrate that RNF125 deficiency is effective in ameliorating high fat diet-induced non-alcoholic fatty liver disease. The experiment comprises an experimental method and an experimental result:
The experimental method comprises the following steps: mice on a high fat diet of example 1 were dissected for 6 months, their livers were weighed and fixed with 4% paraformaldehyde and then stained with H & E or oil red O to analyze the status of RNF125 deficiency on the livers.
Experimental results: as shown in fig. 4. Panel A shows the effect of RNF125 deficiency on liver morphology, size and color in mice after 6 months of normal or high-fat diet; panel B shows that RNF125 deficiency significantly reduced the liver-to-weight ratio in mice after 6 months of normal or high-fat diet; the H & E staining results of panel C show that RNF125 deficiency significantly reduced mice liver bubbling caused by high fat diet after 6 months of normal or high fat diet; panel D shows that RNF125 deficiency significantly reduced Triglyceride (TG) levels in the liver of mice after 6 months of normal or high-fat diet; panel E shows that RNF125 deficiency significantly reduced lipid drop levels in the liver after 6 months of normal or high fat diet; panel F oil red O staining results showed that RNF125 deficiency significantly reduced cholesterol (TC) levels in the liver of mice after 6 months of normal or high fat diet; panel G shows that RNF125 deficiency significantly reduced ALT levels in the blood of mice after 6 months of normal or high fat diet; panel H shows that RNF125 deficiency significantly reduced AST levels in the blood of mice after 6 months of normal or high fat diet. The results show that RNF125 deficiency can effectively improve non-alcoholic fatty liver induced by high-fat diet. In the figure: * P <0.05; * P <0.01; * P <0.001.NCD, normal diet; HFD, high fat diet.
Thus, compared with the common diet, the long-term high-fat diet causes hepatomegaly of the mice, and the mice change from dark red to beige in color, have greasy feeling and serious steatosis, but the hepatomegaly and steatosis of the mice are obviously relieved after the RNF125 is deleted (A picture); regardless of diet, the liver weight ratio of RNF125 knockout mice was significantly lower than wild-type mice (panel B). HE staining of the liver showed that long-term high-fat diet resulted in a bubbling fatty lesion in the liver of mice, with significant reduction in bubbling of the liver following RNF125 knockout (panel C). Triglyceride (panel D) and cholesterol (panel E) analyses of mouse livers and results of oil red O staining (panel F) indicate that RNF125 knockout significantly reduced liver lipid accumulation caused by high fat diets. In addition, the results of the detection of ALT (G panel) and AST (H panel) in blood show that the levels of ALT (glutamic pyruvic transaminase) and AST (glutamic pyruvic transaminase) in mice after RNF125 knockout are significantly reduced. If the liver is damaged or destroyed, transaminases from hepatocytes enter the blood and ALT and AST levels in the blood rise, suggesting a liver disease signature. The results of the foregoing experiments indicate that RNF125 deficiency alleviates liver injury caused by high fat diets.
In summary, RNF125 knockout is effective in alleviating obesity-induced nonalcoholic fatty liver disease, and RNF125 may be an important target for nonalcoholic fatty liver disease treatment.
Example 5
The experiments of this example demonstrate that RNF125 specific siRNA can treat obesity induced by high fat diet. The experiment comprises an experimental method and an experimental result:
The experimental method comprises the following steps: to investigate the effect of inhibiting RNF125 on the obese phenotype during high fat diet, a murine siRNA sequence specific for RNF125 was first designed and synthesized: siRNF125-117 (siRNA formed by annealing the two single strands shown in SEQ ID No.234 and SEQ ID No. 233), siRNF-125 (siRNA formed by annealing the two single strands shown in SEQ ID No.236 and SEQ ID No. 235), siRNF-119 (siRNA formed by annealing the two single strands shown in SEQ ID No.238 and SEQ ID No. 237), the knockdown efficiency of these sequences was analyzed. Thereafter, C57BL/6 mice were fed with a high fat diet, and when their blood glucose, body weight TG, TC levels were significantly higher than those of the normal diet group, the high fat diet mice were randomly divided into two groups, siRNF sequences and control siCtrl sequences (siRNA formed by annealing the two single strands shown in SEQ ID No.240 and SEQ ID No. 239) were injected into the mice by tail vein injection at a dose of 0.5OD (1.25 nM) alone, body weight changes of the mice were monitored every 4 days, and diet changes of the mice were monitored by Columbus Instruments.
Experimental results: as shown in fig. 5. Panel A shows the knockdown efficiency of three RNA sequences (siRNF-117, siRNF-118, siRNF-125-119) specifically targeting RNF125 in H22 cells by RT-qPCR, and it can be seen from the figure that three siRNAs can reduce the RNF125 expression level below 50%. Panel B shows the general procedure of the experiment, i.e. mice were first divided into a normal diet group and a high fat diet group, and when the blood glucose, body weight, TG, TC levels of the high fat diet group were significantly higher than those of the normal diet group, the high fat diet mice were randomly divided into two groups, and the siRNF sequence and the control sequence siCtrl were injected respectively. Panel C shows that the daily intake of high-fat diet by each mouse of the control group is 1.80+ -0.22 g, and the daily intake of high-fat diet by each mouse of the three RNF 125-targeted specific siRNA sequence treatment groups is 2.5+ -0.16 g, 2.8+ -0.39 g and 3.1+ -0.39 g, respectively, and the daily diet of the mice of the treatment group is significantly higher than that of the control group. Panel D shows that after two months of treatment with three RNF 125-targeted siRNAs, the mice body weight was significantly lower than that of the control group, more closely approximating that of the normal diet mice; the weight of the normal diet mice is 31.7+/-1.0 g, the weight of the high-fat diet control group is 47.3+/-6.1 g, the weight of the high-fat diet treatment group is 38.9+/-1.0 g, 36.2+/-1.0 g and 35.6+/-1.5 g respectively, and the weight of the mice treated by the RNF125 specific siRNA is more similar to that of the normal diet mice. Panel E shows that, after two months of treatment with three RNF 125-targeting RNAs, the mouse body shape is significantly smaller than the control group, more similar to the normal diet mouse body shape; that is, mice were lean in body mass after two months of treatment with RNF 125-specific siRNA sequences. All siRNA injection groups in the figure are high-fat diet groups; NCD, normal diet group. * P <0.05; * P <0.01; * P <0.001.
The results show that after the specific siRNA sequence is utilized to target and inhibit the RNF125 of the mice, the obesity phenotype of the mice is obviously improved, the mice can ingest more high-fat feeds, and the weight of the mice is not different from that of the ordinary diet mice. * P <0.05; * P <0.01; * P <0.001.
Example 6
The experiments of this example demonstrate that three RNF 125-specific siRNAs (siRNF-117, siRNF-125-118, siRNF-125-119) improve high-fat diet-induced glucose tolerance, insulin resistance, and hyperlipidemia. The experiment comprises an experimental method and an experimental result:
The experimental method comprises the following steps:
(1) Glucose Tolerance Test (GTT), after mice fasted for 6 hours, glucose was injected intraperitoneally at a dose of 2g/kg, the tail venous blood glucose of the mice was measured by a Rogowski glucometer at 0, 15, 30, 45, 60, 90, 120min after injection, a mouse glucose tolerance curve was drawn, and the area under the blood glucose curve was calculated by an AOC method;
(2) Insulin resistance test (ITT), mice were fasted for 6 hours, were intraperitoneally injected with insulin at a dose of 0.75U/kg, and blood glucose in the mice was measured with a rogowski glucometer (while 20% glucose was prepared, and 100 μl of 20% glucose was intraperitoneally injected for rescue) at 0, 15, 30, 45, 60, 90, 120min after injection, respectively, and a mouse insulin resistance curve was drawn and the area under the blood glucose curve was calculated by AOC;
(3) After overnight fast, the mice were taken out of serum and analyzed for blood lipid by biochemical analysis.
Experimental results: as shown in fig. 6. Experiments glucose tolerance experiments were performed on plain diet mice, high fat diet control mice, and mice treated with the three RNF125 specific siRNA. A. Panel B shows the results of a Glucose Tolerance Test (GTT); the experimental results show that when the mice after RNF125 is inhibited by specific siRNA respond to high-sugar stimulation, the blood sugar level is obviously lower than that of a control group, the AOC area of the control group is 2410.2 +/-500.3, the AOC areas treated by the three siRNAs are 1168.4 +/-531.5, 729.7+/-206.3 and 462.8 +/-160.3 respectively, and the AOC area of a common diet group is 580.9 +/-120.4. It is shown that RNF125 knockdown enhances glucose tolerance in mice during high-fat diet, and can alleviate the influence of high sugar on blood glucose in mice. C. Panel D shows the results of glucose tolerance test (ITT); experimental results show that three RNF125 specific siRNA treatment groups are more sensitive to insulin, the AOC area of a control group is 293.1 +/-172.9, the AOC areas treated by the three siRNAs are 345.0+/-24.4, 382.0 +/-66.8 and 479.1+/-64.7 respectively, and the AOC area of a common diet group is 388.1 +/-44.9. From the figure, it can be seen that the siRNA treatment group is more sensitive to insulin and can effectively improve insulin resistance caused by high-fat diet. Panel E shows that high-fat diet resulted in elevated blood TG levels, and that after treatment with three siRNA targeting RNF 125-specific, the blood TG levels were significantly reduced, approaching normal diet levels, indicating the therapeutic effect of three RNF 125-specific siRNAs on high-fat diet resulted in high-triglyceride. Panel F shows that high-fat diet resulted in elevated levels of TC in blood, and that TC levels in blood were significantly reduced following treatment with three RNF 125-specific siRNAs, approaching normal diet levels, demonstrating the therapeutic effects of three RNF 125-specific siRNAs on high cholesterol caused by high-fat diet. In the figure: siRNF125, siRNF, 125-118, siRNF, 125-119 were compared to control siCtrl, respectively, and were both high fat diets. In the figure: * P <0.05; * P <0.01; * P <0.001.NCD, normal chow diet (normal diet).
In conclusion, specific inhibition of RNF125 can improve the problems of impaired glucose tolerance and hyperlipidemia caused by a high-fat diet.
Example 7
The experiments of this example demonstrate that three RNF 125-specific siRNAs (siRNF-117, siRNF-125-118, siRNF-125-119) reduce fat accumulation from high-fat diet. The experiment comprises an experimental method and an experimental result:
the experimental method comprises the following steps: the mice were dissected after the end of the experiment of example 6, epididymal visceral fat (heft) and subcutaneous fat (iWAT) of the mice were taken and weighed with a thousandth analytical balance to calculate the weight to volume ratio; after weighing, a portion of adipose tissue was fixed in 4% paraformaldehyde, and changes in adipose tissue were observed by H & E staining.
Experimental results: as shown in fig. 7. As shown in panel a of fig. 7, visceral fat (eWAT) body weight ratio was reduced from 9.0±1.2% in the high-fat diet control group to 6.8±0.8%, 4.4±1.2%, 4.1±1.0% in the high-fat diet control group after treatment with three RNF125 specific sirnas (panel a); the weight ratio of subcutaneous fat (iWAT) was reduced from 7.9±1.6% in the high-fat diet control group to 4.1±0.9%, 1.6±0.3%, 1.9±0.7% in the high-fat diet treatment group (panel B). The H & E staining results indicated that the cell volumes of visceral and subcutaneous fat decreased and inflammatory cell infiltration decreased after three siRNA treatments (panel C). In the figure: siRNF125, siRNF, 125-118, siRNF, 125-119 are each shown as a high fat diet compared to control siCtrl (RNAi oligos with scrambled sequences). * P <0.01; * P <0.001.NCD, normal chow diet (normal diet).
These experimental results demonstrate that inhibition of RNF125 with specific siRNA during high fat diet significantly reduces visceral fat and subcutaneous fat accumulation in mice, as well as inflammatory infiltration.
Example 8
The experiments of this example demonstrate that three RNF 125-specific siRNAs (siRNF-117, siRNF-125-118, siRNF-125-119) improve high-fat diet-induced liver steatosis. The experiment comprises an experimental method and an experimental result:
The experimental method comprises the following steps: dissecting the mice after the experiment of example 6 is finished, taking the livers of the mice, photographing and weighing, taking part of the livers, fixing the liver samples in 4% paraformaldehyde, and observing the change of the liver tissues by H & E staining and oil red O staining; taking 50mg of liver tissue, grinding and crushing the liver tissue by a tissue homogenizer, and analyzing the content of Triglyceride (TG) in the liver by using a content detection kit of Solaba Triglyceride (TG); 50mg of liver tissue is taken, ground and crushed by a tissue homogenizer, and the TC content in the liver is analyzed by using a Soxhaust cholesterol (TC) content detection kit.
Experimental results: as shown in fig. 8. As shown in panel a of fig. 8, long-term high-fat diet resulted in fatty lesions, enlarged liver, softer, yellowish or blond, greasy (siCtrl control), while the liver of the three RNF 125-specific siRNA-treated groups was similar in size to that of normal diet mice, and reddish brown. The liver weights of the different treatment groups are shown in panel B, where a high fat diet resulted in an increase in liver weight in mice (siCtrl control), and the liver weight was significantly reduced after treatment with the three RNF125 specific siRNA, approaching normal diet levels. The results of liver H & E staining and oil red O staining are shown in panel C, wherein a long-term high-fat diet causes a large number of lipid droplets to accumulate in the liver, and the liver becomes vacuolated (siCtrl control), and after treatment with the three RNF 125-specific siRNAs (siRNF-117, siRNF-125-118, siRNF-119), the liver tissue morphology of the mice is substantially restored to normal with only a small number of lipid droplets. As shown in panel D, the results of the analysis of Triglyceride (TG) content in liver were that TG content in liver of normal diet mice was 30.6±6.0mg/g tissue (NCD), TG content in liver of high fat diet mice was 70.1±16.5mg/g tissue (siCtrl control group), TG content in liver of high fat diet mice after treatment with three RNF125 siRNA was 37.4±6.3mg/g, 29.8±5.5mg/g, 19.2±3.8mg/g tissue, TG level in liver of treated mice was significantly reduced, and significantly lower than that of control group siCtrl. As shown in the E graph, the results of analysis of cholesterol (TC) content in liver of the normal diet mice showed 34.2.+ -. 4.4. Mu. Mol/g tissue (NCD), TC content in liver of the high fat diet mice was 56.0.+ -. 15.3. Mu. Mol/g tissue (siCtrl control group), TC content in liver of the high fat diet mice treated with RNF 125-specific siRNA was 38.9.+ -. 3.7. Mu. Mol/g, 33.4.+ -. 5.0. Mu. Mol/g, 32.3.+ -. 4.3. Mu. Mol/g tissue, and the TC level in liver of the normal diet mice was significantly reduced after treatment, which was similar to that of the normal diet mice. Taken together, the high-fat diet resulted in significant increases in TG and TC levels in the liver of mice, and RNF 125-specific siRNA could significantly reduce TG and TC levels in the liver. Thus, inhibition of RNF125 helps to ameliorate hepatic steatosis due to obesity. In the figure: siRNF125, siRNF, 125-118, siRNF, 125-119 were compared to control siCtrl, respectively, and were both high fat diets. * P <0.05; * P <0.01; * P <0.001.NCD, normal chow diet.
In conclusion, RNF125 deficiency or targeted inhibition of RNF125 can effectively solve the problems of obesity, hyperglycemia, hyperlipidemia, fat accumulation, non-alcoholic fatty liver disease, etc. caused by high fat diet. RNF125 is an important target for the treatment of metabolic diseases such as obesity, diabetes, and nonalcoholic fatty liver disease.
Example 9
To develop siRNA targeting RNF125, this example was directed to designing an siRNA sequence targeting any position of gene RNF125 (gene bank ID: 54941) for knocking down or silencing the expression of the gene. Each siRNA sequence comprises at least one sense strand (21 bases) and one antisense strand (23 bases), the 3' end of the antisense strand of each siRNA being 2 bases more than the sense strand. The siRNA sequences designed by the invention are shown in Table 2:
Table 2:
Next, the silencing effect of the above-listed sirnas on the target gene RNF125 was verified by a cell experiment, which was as follows:
(1) Because RNF125 has low background expression on Huh7 cells, and is suitable for the cells for screening double reporter genes of siRNA, huh7 cells are selected for the experiment.
(2) Constructing a dual-report fluorescence report system plasmid: mRNA sequence of coding human RNF125 gene is inserted into psiCHECK TM -2 carrier skeleton to construct human RNF125-psiCHECK TM -2 over expression plasmid. The psiCHECK TM -2 vector is a dual luciferase reporter gene system capable of monitoring changes in expression of a target gene fused to a reporter gene. Wherein the Renilla luciferase reporter gene Renilla luciferase gene (hRluc) expression cassette promoter is a T7 RNA polymerase promoter and is used as a main reporter gene, a target gene is cloned into a polyclonal region downstream of a hRluc translation termination codon and is fused with the hRluc for expression, and fluorescence intensity is used for monitoring the change of mRNA expression quantity after siRNA induction. The firefly luciferase reporter gene firefly luciferase gene (hluc +) expression cassette promoter is an HSV-TK promoter, is independent of the expression of hRluc, is irrelevant to the expression quantity of a target gene, and can monitor the transfection efficiency of cells and normalize the fluorescent signal of the expression of the hRluc.
(3) The RNF125-psiCHECK TM -2 plasmid was co-transfected with siRNA at different concentrations (1 nM, 10 nM) into Huh7 cells, after 24h, the cells were washed with PBS and the fluorescence signal intensity values of hRluc and hluc + were detected on a multifunctional microplate reader, hRluc detection wavelength 480nM, and hluc+ detection wavelength 560 nM.
The relative expression fold calculation formula is as follows:
Normalized value = hRluc fluorescence value (main reporter gene)/hluc + (internal reference gene)
Relative expression fold = experimental/control normalized.
The relative expression levels of target mRNAs were calculated according to the above calculation method, and the target mRNAs were ranked from low to high after transfection with 1nM siRNA as shown in Table 3. The lower the relative expression of the target mRNA, the higher the silencing efficiency of siRNA on the target gene.
Table 3:
Wherein, the siRNA with sequence numbers 1-48 in Table 3 can make the mRNA expression amount in Huh7 cells less than or equal to 40%, which indicates that they have silencing efficiency of more than 60% on target gene RNF 125. The siRNA sequence numbers 49-84 resulted in mRNA expression levels in Huh7 cells ranging from 41-60%, indicating that they had 40-59% silencing efficacy against the target gene RNF 125. The siRNA sequences of sequence numbers 85-105 can enable the mRNA expression level in Huh7 cells to be between 62-80%, which indicates that the siRNA sequences have 20-38% silencing efficiency on target gene RNF 125. The silencing efficiency of siRNA sequence numbers 106-116 on target gene RNF125 in Huh7 cells is only 7-19%. Although the silencing efficiency of siRNA with sequence numbers 106-116 on target gene RNF125 is lower, the knockdown effect of the siRNA on target gene in animal models can be improved after nucleotide modification or delivery system coupling modification, and the siRNA has the possibility of patent medicine.
Example 10
15 SiRNAs were selected from the siRNAs with sequence numbers 1-48 in Table 3 of example 9, human colon cancer cells HCT116 were transfected at final concentrations of 50nM, 10nM, 2nM, 0.4nM, 0.08nM, 0.016nM, and the relative expression levels of the target gene mRNAs were examined by qPCR to test the IC50 values of the respective siRNAs. The experimental results are shown in Table 4, and the IC50 of the selected siRNA to the target gene is less than 10nM, which accords with the clinical application range.
TABLE 4 Table 4
| SiRNA name | Sequence numbering | Antisense strand | IC50(nM) |
| siRNF125-8 | SEQ ID NO.15 | UUAAAUGGUGCAAUUCAGUGGUC | 0.35 |
| siRNF125-44 | SEQ ID NO.87 | UUAUAUUAAAAUCUAUGAAAUCA | 0.40 |
| siRNF125-73 | SEQ ID NO.145 | AUCUUAAAUGGUGCAAUUCAGUG | 0.44 |
| siRNF125-6 | SEQ ID NO.11 | UUAAAUGCACUAUAACAAUAAAC | 0.52 |
| siRNF125-2 | SEQ ID NO.3 | UAUGAAAUCAUCAUAAAACAAAG | 0.56 |
| siRNF125-19 | SEQ ID NO.37 | AUACUUAUCUAUGUACUUCUGAC | 0.71 |
| siRNF125-40 | SEQ ID NO.79 | UUCAAGCAGCAUCUUAAAUGGUG | 0.75 |
| siRNF125-1 | SEQ ID NO.1 | UUAUCUAUGUACUUCUGACAAGU | 1.19 |
| siRNF125-14 | SEQ ID NO.27 | UUUUUAAAUGCACUAUAACAAUA | 1.32 |
| siRNF125-18 | SEQ ID NO.35 | UCAUCAUAAAACAAAGUGUGACU | 1.44 |
| siRNF125-10 | SEQ ID NO.19 | UUGUUGUUCUUUAGACUGGUAGC | 2.11 |
| siRNF125-23 | SEQ ID NO.45 | UCAUAAAACAAAGUGUGACUAAC | 2.83 |
| siRNF125-84 | SEQ ID NO.127 | AUAACAAUAAACAUGGACAAACA | 4.34 |
| siRNF125-21 | SEQ ID NO.41 | UUAAAAUCUAUGAAAUCAUCAUA | 7.90 |
| siRNF125-38 | SEQ ID NO.75 | UAAAUGCACUAUAACAAUAAACA | 8.87 |
Example 11
In this example, the partially targeted RNF125 human siRNA provided by the invention was modified and coupled to GalNAc structure to give a deliverable complex, which was injected into a human transgenic mouse model for silencing experiments on the target gene. The experimental content comprises the following parts:
1. Construction of human RNF125 overexpression mouse model
C57BL/6J male mice (St Bei Fu (Beijing) Biotechnology Co., ltd.) weighing around 25g at 6-8 weeks old were used for modeling of human transgenic mice according to weight distribution at random after being acclimatized for 1 week.
Human RNF125 mRNA overexpression plasmid was constructed using PiggyBac mammalian expression vector. The PiggyBac vector system comprises two vectors, one of which is called a helper plasmid, responsible for encoding the transposase; another vector, called a transposon plasmid, contains two Terminal Repeats (TRs) and a region therebetween, in which the gene expression cassette of interest is cloned, and is called a transposon plasmid. The PiggyBac transposon vector and helper plasmid are transfected into mammalian cells, and the DNA sequence on the transposon is stably integrated into the chromosome of the host cell due to the action of the transposon under the action of the transposase. mRNA sequence of coding human RNF125 gene is inserted into the region of multiple cloning site of said carrier skeleton, SEAP luciferase (secretory human placenta alkaline phosphatase) is fused and expressed, the mice are injected by hydrodynamic high-pressure method (HDI) to construct human transgenic mice model, transposon plasmid and auxiliary plasmid are mixed to form proper concentration, tail vein injection is carried out according to 8-10% volume of the weight of mice, 6-8 seconds of injection is completed, each model mouse is injected into transposon plasmid and auxiliary plasmid at 50 mug respectively, mouse tail vein blood sampling is carried out on 3, 7, 14 and 21 days after injection, and the expression level of RNF125 in human transgenic mice is detected by detecting the fluorescence expression level of SEAP in serum.
2. Silencing effect of human siRNA on target gene in human RNF125 over-expression mouse model
The modification mode of the human siRNA is as follows: in the sense strand 21 nucleotides, 2 thio modifications at the 5 'end, fluoro modifications at positions 9, 10 and 11 respectively, methoxy modifications at the rest of the nucleotides, and coupling GalNAc ([ Gal-6] s [ Gal-6 ]) at the 3' end; 2 thio modifications at the 5 'end, fluoro modifications at positions 2, 5, 14 and 16, 2 thio modifications at the 3' end and methoxy modifications at the rest positions in 23 nucleotides of the antisense strand; see table 5 for details:
Note that: m represents the methoxy modification of the left nucleotide, f represents the fluoro modification of the left nucleotide, s represents phosphorothioate linkage between the left nucleotide and the right nucleotide; ms is the simultaneous modification of m and s, and mf represents the simultaneous modification of f and s.
Selecting model mice with consistent SEAP expression quantity to perform knockdown efficiency detection of different siRNAs, subcutaneously administering 3mpk to each siRNA with GalNAc modification and siCtrl mice after neck, taking blood from each sequence on 14 th day after administration, separating serum, detecting SEAP fluorescent signal intensity, judging silencing effect of different siRNAs on target gene mRNA, and sequencing according to the expression level of the target gene mRNA from low to high: see table 6.
Table 6:
Example 12
In this example, three naked sequences, siRNF-84, siRNF-125-2, siRNF-73, etc., were selected from table 6 for four different ways of nucleotide modification (see table 7), and the silencing effect of the different modifications on the target gene in the human transgenic mouse model was compared.
Table 7:
In the above table, the lower-case letter g in modification 4 indicates that ethylene glycol nucleic acid is on the left side.
Wherein modification 1 is described in example 11; modes 2-4 are as follows:
Mode 2: in 21 nucleotides of the sense strand, 2 thio modifications at the 5' end, fluoro modifications at positions 9, 10 and 11 respectively, and the nucleotides at the rest positions are methoxy and fluoro alternately modified in sequence, 2 thio modifications at the 3' end and GalNAc coupled at the 3' end; sequentially carrying out methoxy and fluoro alternate modification on 23 nucleotides of the antisense strand, 2 thio modifications at the 5 'end and 2 thio modifications at the 3' end;
Mode 3: in 21 nucleotides of the sense strand, 2 thio modifications at the 5' end, fluoro modifications at 7, 9, 10 and 11 positions and methoxy modifications at the rest positions are obtained, 2 thio modifications at the 3' end and GalNAc coupling at the 3' end; 2 nd, 6 th, 8 th, 9 th, 15 th and 17 th positions of 23 th nucleotides of the antisense strand are fluoro modified, methoxy modified at the rest positions, 2 thio modified at the 5 'end and 2 thio modified at the 3' end;
Mode 4: in 21 nucleotides of the sense strand, 2 thio modifications at the 5' end, fluoro modifications at 7, 9, 10 and 11 positions and methoxy modifications at the rest positions are obtained, 2 thio modifications at the 3' end and GalNAc coupling at the 3' end; 23 nucleotides of antisense strand are 2 nd, 14 th and 16 th fluoro modified, 7 th glycol nucleic acid, methoxy modified, 2 thio modified at 5 'end and 2 thio modified at 3' end.
Using the human RNF125 over-expression mouse model constructed in example 11, each of the modified siRNAs and siCtrl mice were subjected to subcutaneous administration of 3mpk via the posterior cervical region, and 5 mice were each subjected to the sequence, and blood was collected on the 14 th day after the administration, serum was separated, and SEAP fluorescence signal intensity was detected, thereby judging the silencing effect of different siRNAs on target gene mRNA, and the silencing effect of the siRNA modification on human RNF125 was shown to be higher as the target gene mRNA expression level was ranked from low to high (see Table 8).
Table 8:
as can be seen from the table, after the nucleotide modification of the siRNA bare sequence is performed in different modification modes, the silencing efficiency of the modification on the human RNF125 gene in the mouse model is changed, and especially the modification mode 4 can obviously improve the silencing effect of the modification on the target gene RNF 125. This also shows that even with different ways of nucleic acid modification, the target gene can be effectively silenced after the siRNA sequence targeting RNF125 is determined, whereas modification of the naked siRNA sequence can improve the delivery effect, facilitate in vivo administration, and reduce degradation of nucleic acid drugs in vivo.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (10)
- Use of an rnf125 inhibitor in the preparation of a medicament for the prevention or treatment of obesity and related complications.
- 2. The use of claim 1, wherein the RNF125 inhibitor is capable of producing a change in the level of any one of: (1) inhibiting obesity caused by high-fat diet of mice; (2) inhibiting fat accumulation due to a high fat diet; (3) Inhibiting glucose tolerance and insulin resistance caused by high-fat diet, and further preventing hyperglycemia and hyperlipidemia; (4) inhibiting hepatic steatosis caused by a high fat diet.
- 3. The use of claim 1, wherein the RNF125 inhibitor is any protein molecule, nucleic acid molecule, small compound molecule, viral particle, or any combination of two or more thereof capable of reducing the activity or expression level of an RNF125 protein; or the RNF125 inhibitor is any protein molecule, nucleic acid molecule, small compound molecule, viral particle, gene editing kit, or any combination of two or more of the foregoing, capable of knocking out or knocking down or silencing an RNF125 gene or protein.
- 4. An siRNA targeted to inhibit RNF125, wherein any one of the following sirnas is selected from the group consisting of: siRNA formed by annealing two single strands shown in SEQ ID Nos. 9-10;siRNA formed by annealing two single strands shown in SEQ ID NO. 41-42;siRNA formed by annealing two single strands shown in SEQ ID NO. 43-44;siRNA formed by annealing two single strands shown in SEQ ID NO. 111-112;siRNA formed by annealing two single strands shown in SEQ ID NO. 1-2;siRNA formed by annealing two single strands shown in SEQ ID NO. 37-38;siRNA formed by annealing two single strands shown in SEQ ID NO. 85-86;siRNA formed by annealing two single strands as shown in SEQ ID nos. 205-206;siRNA formed by annealing two single strands shown in SEQ ID NO. 3-4;siRNA formed by annealing two single strands shown in SEQ ID NOS.87-88;siRNA formed by annealing two single strands shown in SEQ ID NO. 45-46;siRNA formed by annealing two single strands shown in SEQ ID NO. 5-6;siRNA formed by annealing two single strands shown in SEQ ID NO. 55-56;siRNA formed by annealing two single strands shown in SEQ ID NOS.159-160;siRNA formed by annealing two single strands shown in SEQ ID NO. 23-24;siRNA formed by annealing two single strands shown in SEQ ID NO. 29-30;siRNA formed by annealing two single strands as shown in SEQ ID NOS.175-176;siRNA formed by annealing two single strands shown in SEQ ID NO. 11-12;siRNA formed by annealing two single strands shown in SEQ ID NO. 33-34;siRNA formed by annealing two single strands shown in SEQ ID NO. 17-18;siRNA formed by annealing two single strands shown in SEQ ID NO. 93-94;siRNA formed by annealing two single strands shown in SEQ ID NO. 49-50;siRNA formed by annealing two single strands shown in SEQ ID NO. 35-36;siRNA formed by annealing two single strands shown in SEQ ID NO. 75-76;siRNA formed by annealing two single strands shown in SEQ ID NO. 79-80;siRNA formed by annealing two single strands shown in SEQ ID NO. 109-110;siRNA formed by annealing two single strands shown in SEQ ID NO. 153-154;siRNA formed by annealing two single strands shown in SEQ ID NO. 15-16;siRNA formed by annealing two single strands shown in SEQ ID NO. 131-132;siRNA formed by annealing two single strands shown in SEQ ID NO. 59-60;siRNA formed by annealing two single strands shown in SEQ ID NO. 51-52;siRNA formed by annealing two single strands shown in SEQ ID nos. 167-168;siRNA formed by annealing two single strands shown in SEQ ID NO. 133-134;siRNA formed by annealing two single strands shown in SEQ ID NO. 77-78;siRNA formed by annealing two single strands as shown in SEQ ID nos. 219-220;siRNA formed by annealing two single strands shown in SEQ ID NO. 19-20;siRNA formed by annealing two single strands shown in SEQ ID NO. 7-8;siRNA formed by annealing two single strands shown in SEQ ID NO. 13-14;siRNA formed by annealing two single strands shown in SEQ ID NO. 27-28;siRNA formed by annealing two single strands shown in SEQ ID NO. 165-166;siRNA formed by annealing two single strands shown in SEQ ID nos. 201-202;siRNA formed by annealing two single strands shown in SEQ ID NO. 145-146;siRNA formed by annealing two single strands shown in SEQ ID NO. 123-124;siRNA formed by annealing two single strands shown in SEQ ID NO. 195-196;siRNA formed by annealing two single strands shown in SEQ ID NO. 61-62;siRNA formed by annealing two single strands shown in SEQ ID NO. 207-208;siRNA formed by annealing two single strands shown in SEQ ID NO. 39-40;siRNA formed by annealing two single strands shown in SEQ ID NO. 83-84;siRNA formed by annealing two single strands shown in SEQ ID NO. 69-70;siRNA formed by annealing two single strands shown in SEQ ID NO. 107-108;siRNA formed by annealing two single strands shown in SEQ ID NO. 47-48;siRNA formed by annealing two single strands shown in SEQ ID NO. 125-126;siRNA formed by annealing two single strands shown in SEQ ID NO. 25-26;siRNA formed by annealing two single strands shown in SEQ ID NO. 199-200;siRNA formed by annealing two single strands shown in SEQ ID NO. 31-32;siRNA formed by annealing two single strands shown in SEQ ID NO. 171-172;siRNA formed by annealing two single strands shown in SEQ ID NO. 135-136;siRNA formed by annealing two single strands shown in SEQ ID nos. 143-144;siRNA formed by annealing two single strands shown in SEQ ID NO. 89-90;siRNA formed by annealing two single strands shown in SEQ ID NO. 21-22;siRNA formed by annealing two single strands shown in SEQ ID NO. 97-98;siRNA formed by annealing two single strands shown in SEQ ID NOS.151-152;siRNA formed by annealing two single strands shown in SEQ ID NO. 221-222;siRNA formed by annealing two single strands shown in SEQ ID NO. 105-106;siRNA formed by annealing two single strands shown in SEQ ID NO. 57-58;siRNA formed by annealing two single strands shown in SEQ ID NOS.129-130;siRNA formed by annealing two single strands shown in SEQ ID NO. 229-230;siRNA formed by annealing two single strands shown in SEQ ID NO. 141-142;siRNA formed by annealing two single strands shown in SEQ ID NOS.155-156;siRNA formed by annealing two single strands shown in SEQ ID NO. 147-148;siRNA formed by annealing two single strands shown in SEQ ID NO. 65-66;siRNA formed by annealing two single strands shown in SEQ ID NO. 67-68;siRNA formed by annealing two single strands as shown in SEQ ID NO. 189-190;siRNA formed by annealing two single strands shown in SEQ ID NO. 193-194;siRNA formed by annealing two single strands shown in SEQ ID NO. 53-54;siRNA formed by annealing two single strands shown in SEQ ID NO. 73-74;siRNA formed by annealing two single strands shown in SEQ ID NO. 213-214;siRNA formed by annealing two single strands shown in SEQ ID NO. 181-182;siRNA formed by annealing two single strands shown in SEQ ID NO. 101-102;siRNA formed by annealing two single strands shown in SEQ ID NO. 139-140;siRNA formed by annealing two single strands shown in SEQ ID NO. 91-92;siRNA formed by annealing two single strands shown in SEQ ID NO. 197-198;siRNA formed by annealing two single strands shown in SEQ ID NO. 99-100;siRNA formed by annealing two single strands shown in SEQ ID NO. 137-138;siRNA formed by annealing two single strands shown in SEQ ID NO. 71-72;siRNA formed by annealing two single strands shown in SEQ ID nos. 127-128;siRNA formed by annealing two single strands shown in SEQ ID nos. 121-122;siRNA formed by annealing two single strands shown in SEQ ID NOS.115-116;siRNA formed by annealing two single strands shown in SEQ ID NOS.215-216;siRNA formed by annealing two single strands shown in SEQ ID NO. 173-174;siRNA formed by annealing two single strands shown in SEQ ID NO. 103-104;siRNA formed by annealing two single strands as shown in SEQ ID No. 177-178;siRNA formed by annealing two single strands shown in SEQ ID NO. 95-96;siRNA formed by annealing two single strands shown in SEQ ID NO. 231-232;siRNA formed by annealing two single strands shown in SEQ ID NOS 209-210;siRNA formed by annealing two single strands shown in SEQ ID NO. 119-120;siRNA formed by annealing two single strands shown in SEQ ID NO. 149-150;siRNA formed by annealing two single strands shown in SEQ ID NO. 81-82;siRNA formed by annealing two single strands shown in SEQ ID nos. 203-204;siRNA formed by annealing two single strands shown in SEQ ID NOS.157-158;siRNA formed by annealing two single strands shown in SEQ ID NO. 163-164;siRNA formed by annealing two single strands shown in SEQ ID NO. 217-218;siRNA formed by annealing two single strands shown in SEQ ID NO. 191-192;siRNA formed by annealing two single strands as shown in SEQ ID No. 187-188;siRNA formed by annealing two single strands shown in SEQ ID NO. 169-170;siRNA formed by annealing two single strands shown in SEQ ID NO. 113-114;siRNA formed by annealing two single strands shown in SEQ ID nos. 117-118;siRNA formed by annealing two single strands shown in SEQ ID NOS.63-64;siRNA formed by annealing two single strands shown in SEQ ID NO. 161-162;siRNA formed by annealing two single strands shown in SEQ ID NO. 223-224;siRNA formed by annealing two single strands shown in SEQ ID NO. 185-186;siRNA formed by annealing two single strands shown in SEQ ID NOS.211-212;siRNA formed by annealing two single strands shown in SEQ ID NOS.215-216;siRNA formed by annealing two single strands shown in SEQ ID NO. 227-228;siRNA formed by annealing two single strands shown in SEQ ID NO. 183-184;siRNA formed by annealing two single strands as shown in SEQ ID No. 179-180.
- 5. A medicament for preventing or treating obesity and related complications, the active ingredient of the medicament being an RNF125 inhibitor, characterized in that the RNF125 inhibitor is any protein molecule, nucleic acid molecule, small molecule compound, viral particle or any combination of two or more of the foregoing capable of reducing the activity or expression level of an RNF125 protein; or the RNF125 inhibitor is any protein molecule, nucleic acid molecule, small compound molecule, viral particle, gene editing kit, or any combination of two or more of the foregoing, capable of knocking out or knocking down or silencing an RNF125 gene or protein.
- 6. The medicament according to claim 5, wherein the percentage of RNF125 inhibitor in the medicament is 1-99%.
- 7. The medicament of claim 5 or 6, wherein the RNF125 inhibitor is any one of the following siRNA:siRNA formed by annealing two single strands shown in SEQ ID Nos. 9-10;siRNA formed by annealing two single strands shown in SEQ ID NO. 41-42;siRNA formed by annealing two single strands shown in SEQ ID NO. 43-44;siRNA formed by annealing two single strands shown in SEQ ID NO. 111-112;siRNA formed by annealing two single strands shown in SEQ ID NO. 1-2;siRNA formed by annealing two single strands shown in SEQ ID NO. 37-38;siRNA formed by annealing two single strands shown in SEQ ID NO. 85-86;siRNA formed by annealing two single strands as shown in SEQ ID nos. 205-206;siRNA formed by annealing two single strands shown in SEQ ID NO. 3-4;siRNA formed by annealing two single strands shown in SEQ ID NOS.87-88;siRNA formed by annealing two single strands shown in SEQ ID NO. 45-46;siRNA formed by annealing two single strands shown in SEQ ID NO. 5-6;siRNA formed by annealing two single strands shown in SEQ ID NO. 55-56;siRNA formed by annealing two single strands shown in SEQ ID NOS.159-160;siRNA formed by annealing two single strands shown in SEQ ID NO. 23-24;siRNA formed by annealing two single strands shown in SEQ ID NO. 29-30;siRNA formed by annealing two single strands as shown in SEQ ID NOS.175-176;siRNA formed by annealing two single strands shown in SEQ ID NO. 11-12;siRNA formed by annealing two single strands shown in SEQ ID NO. 33-34;siRNA formed by annealing two single strands shown in SEQ ID NO. 17-18;siRNA formed by annealing two single strands shown in SEQ ID NO. 93-94;siRNA formed by annealing two single strands shown in SEQ ID NO. 49-50;siRNA formed by annealing two single strands shown in SEQ ID NO. 35-36;siRNA formed by annealing two single strands shown in SEQ ID NO. 75-76;siRNA formed by annealing two single strands shown in SEQ ID NO. 79-80;siRNA formed by annealing two single strands shown in SEQ ID NO. 109-110;siRNA formed by annealing two single strands shown in SEQ ID NO. 153-154;siRNA formed by annealing two single strands shown in SEQ ID NO. 15-16;siRNA formed by annealing two single strands shown in SEQ ID NO. 131-132;siRNA formed by annealing two single strands shown in SEQ ID NO. 59-60;siRNA formed by annealing two single strands shown in SEQ ID NO. 51-52;siRNA formed by annealing two single strands shown in SEQ ID nos. 167-168;siRNA formed by annealing two single strands shown in SEQ ID NO. 133-134;siRNA formed by annealing two single strands shown in SEQ ID NO. 77-78;siRNA formed by annealing two single strands as shown in SEQ ID nos. 219-220;siRNA formed by annealing two single strands shown in SEQ ID NO. 19-20;siRNA formed by annealing two single strands shown in SEQ ID NO. 7-8;siRNA formed by annealing two single strands shown in SEQ ID NO. 13-14;siRNA formed by annealing two single strands shown in SEQ ID NO. 27-28;siRNA formed by annealing two single strands shown in SEQ ID NO. 165-166;siRNA formed by annealing two single strands shown in SEQ ID nos. 201-202;siRNA formed by annealing two single strands shown in SEQ ID NO. 145-146;siRNA formed by annealing two single strands shown in SEQ ID NO. 123-124;siRNA formed by annealing two single strands shown in SEQ ID NO. 195-196;siRNA formed by annealing two single strands shown in SEQ ID NO. 61-62;siRNA formed by annealing two single strands shown in SEQ ID NO. 207-208;siRNA formed by annealing two single strands shown in SEQ ID NO. 39-40;siRNA formed by annealing two single strands shown in SEQ ID NO. 83-84;siRNA formed by annealing two single strands shown in SEQ ID NO. 69-70;siRNA formed by annealing two single strands shown in SEQ ID NO. 107-108;siRNA formed by annealing two single strands shown in SEQ ID NO. 47-48;siRNA formed by annealing two single strands shown in SEQ ID NO. 125-126;siRNA formed by annealing two single strands shown in SEQ ID NO. 25-26;siRNA formed by annealing two single strands shown in SEQ ID NO. 199-200;siRNA formed by annealing two single strands shown in SEQ ID NO. 31-32;siRNA formed by annealing two single strands shown in SEQ ID NO. 171-172;siRNA formed by annealing two single strands shown in SEQ ID NO. 135-136;siRNA formed by annealing two single strands shown in SEQ ID nos. 143-144;siRNA formed by annealing two single strands shown in SEQ ID NO. 89-90;siRNA formed by annealing two single strands shown in SEQ ID NO. 21-22;siRNA formed by annealing two single strands shown in SEQ ID NO. 97-98;siRNA formed by annealing two single strands shown in SEQ ID NOS.151-152;siRNA formed by annealing two single strands shown in SEQ ID NO. 221-222;siRNA formed by annealing two single strands shown in SEQ ID NO. 105-106;siRNA formed by annealing two single strands shown in SEQ ID NO. 57-58;siRNA formed by annealing two single strands shown in SEQ ID NOS.129-130;siRNA formed by annealing two single strands shown in SEQ ID NO. 229-230;siRNA formed by annealing two single strands shown in SEQ ID NO. 141-142;siRNA formed by annealing two single strands shown in SEQ ID NOS.155-156;siRNA formed by annealing two single strands shown in SEQ ID NO. 147-148;siRNA formed by annealing two single strands shown in SEQ ID NO. 65-66;siRNA formed by annealing two single strands shown in SEQ ID NO. 67-68;siRNA formed by annealing two single strands as shown in SEQ ID NO. 189-190;siRNA formed by annealing two single strands shown in SEQ ID NO. 193-194;siRNA formed by annealing two single strands shown in SEQ ID NO. 53-54;siRNA formed by annealing two single strands shown in SEQ ID NO. 73-74;siRNA formed by annealing two single strands shown in SEQ ID NO. 213-214;siRNA formed by annealing two single strands shown in SEQ ID NO. 181-182;siRNA formed by annealing two single strands shown in SEQ ID NO. 101-102;siRNA formed by annealing two single strands shown in SEQ ID NO. 139-140;siRNA formed by annealing two single strands shown in SEQ ID NO. 91-92;siRNA formed by annealing two single strands shown in SEQ ID NO. 197-198;siRNA formed by annealing two single strands shown in SEQ ID NO. 99-100;siRNA formed by annealing two single strands shown in SEQ ID NO. 137-138;siRNA formed by annealing two single strands shown in SEQ ID NO. 71-72;siRNA formed by annealing two single strands shown in SEQ ID nos. 127-128;siRNA formed by annealing two single strands shown in SEQ ID nos. 121-122;siRNA formed by annealing two single strands shown in SEQ ID NOS.115-116;siRNA formed by annealing two single strands shown in SEQ ID NOS.215-216;siRNA formed by annealing two single strands shown in SEQ ID NO. 173-174;siRNA formed by annealing two single strands shown in SEQ ID NO. 103-104;siRNA formed by annealing two single strands as shown in SEQ ID No. 177-178;siRNA formed by annealing two single strands shown in SEQ ID NO. 95-96;siRNA formed by annealing two single strands shown in SEQ ID NO. 231-232;siRNA formed by annealing two single strands shown in SEQ ID NOS 209-210;siRNA formed by annealing two single strands shown in SEQ ID NO. 119-120;siRNA formed by annealing two single strands shown in SEQ ID NO. 149-150;siRNA formed by annealing two single strands shown in SEQ ID NO. 81-82;siRNA formed by annealing two single strands shown in SEQ ID nos. 203-204;siRNA formed by annealing two single strands shown in SEQ ID NOS.157-158;siRNA formed by annealing two single strands shown in SEQ ID NO. 163-164;siRNA formed by annealing two single strands shown in SEQ ID NO. 217-218;siRNA formed by annealing two single strands shown in SEQ ID NO. 191-192;siRNA formed by annealing two single strands as shown in SEQ ID No. 187-188;siRNA formed by annealing two single strands shown in SEQ ID NO. 169-170;siRNA formed by annealing two single strands shown in SEQ ID NO. 113-114;siRNA formed by annealing two single strands shown in SEQ ID nos. 117-118;siRNA formed by annealing two single strands shown in SEQ ID NOS.63-64;siRNA formed by annealing two single strands shown in SEQ ID NO. 161-162;siRNA formed by annealing two single strands shown in SEQ ID NO. 223-224;siRNA formed by annealing two single strands shown in SEQ ID NO. 185-186;siRNA formed by annealing two single strands shown in SEQ ID NOS.211-212;siRNA formed by annealing two single strands shown in SEQ ID NOS.215-216;siRNA formed by annealing two single strands shown in SEQ ID NO. 227-228;siRNA formed by annealing two single strands shown in SEQ ID NO. 183-184;siRNA formed by annealing two single strands as shown in SEQ ID No. 179-180.
- 8. The drug according to claim 7, wherein the RNF125 inhibitor is a modified product obtained by nucleotide modification of the antisense strand and the sense strand of any one of the sirnas; the nucleotide modification comprises one or a combination of a plurality of methoxy modification, fluoro modification, galNAc coupling modification, thio modification and glycol.
- 9. The medicine according to claim 8, wherein the modification method is any one of the following modes:Mode one: in 21 nucleotides of the sense strand, 2 thio-modified 5 'ends, fluoro-modified 9, 10 and 11 positions respectively, methoxy-modified rest positions and coupled 3' ends with GalNAc; 2 thio modifications at the 5 'end, fluoro modifications at positions 2,5, 14 and 16, 2 thio modifications at the 3' end and methoxy modifications at the rest positions in 23 nucleotides of the antisense strand;mode two: in 21 nucleotides of the sense strand, 2 thio modifications at the 5' end, fluoro modifications at positions 9, 10 and 11 respectively, and the nucleotides at the rest positions are methoxy and fluoro alternately modified in sequence, 2 thio modifications at the 3' end and GalNAc coupled at the 3' end; sequentially carrying out methoxy and fluoro alternate modification on 23 nucleotides of the antisense strand, 2 thio modifications at the 5 'end and 2 thio modifications at the 3' end;Mode three: in 21 nucleotides of the sense strand, 2 thio modifications at the 5' end, fluoro modifications at 7, 9, 10 and 11 positions and methoxy modifications at the rest positions are obtained, 2 thio modifications at the 3' end and GalNAc coupling at the 3' end; 2 nd, 6 th, 8 th, 9 th, 15 th and 17 th positions of 23 th nucleotides of the antisense strand are fluoro modified, methoxy modified at the rest positions, 2 thio modified at the 5 'end and 2 thio modified at the 3' end;Mode four: in 21 nucleotides of the sense strand, 2 thio modifications at the 5' end, fluoro modifications at 7, 9, 10 and 11 positions and methoxy modifications at the rest positions are obtained, 2 thio modifications at the 3' end and GalNAc coupling at the 3' end; 23 nucleotides of antisense strand are 2 nd, 14 th and 16 th fluoro modified, 7 th glycol nucleic acid, methoxy modified, 2 thio modified at 5 'end and 2 thio modified at 3' end.
- Application of RNF125 gene or protein as target in constructing obesity treating model.
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