CN105754999B

CN105754999B - Oligonucleotide sequence for inhibiting hsa-miR-221-3p, recombinant adenovirus and preparation method and application thereof

Info

Publication number: CN105754999B
Application number: CN201610137218.0A
Authority: CN
Inventors: 梁继超; 骆爱玲; 杜春园; 孙丽娟; 陈勇
Original assignee: Hubei University
Current assignee: Hubei University
Priority date: 2016-03-11
Filing date: 2016-03-11
Publication date: 2020-05-19
Anticipated expiration: 2036-03-11
Also published as: CN105754999A

Abstract

The invention provides an oligonucleotide sequence for inhibiting hsa-miR-221-3p, which is one of the following sequences: sequence 1: (GAAACCCAGCTGACAATGTAGCTAAGA)_n≧5Sequence 2: (GAAACCCAGCCGACAATGTAGCTAAGA)_n≧5(ii) a And (3) sequence: (GAAACCCAGCGGACAATGTAGCTAAGA)_n≧5(ii) a And (3) sequence 4: (GAAACCCAGCGACAATGTAGCTAAGA)_n≧5The AAGA part in the four sequences can be replaced by other sequences, a recombinant adenovirus (Ad-s-miR-221-3p) is prepared by performing DNA homologous recombination in cells, and the application of the recombinant adenovirus in treating the non-alcoholic fatty liver disease is further provided, wherein the application comprises the steps of reducing the triglyceride level of the liver, reducing the triglyceride level of blood, improving hyperlipidemia, increasing the insulin sensitivity of an organism and providing a new solution for improving the non-alcoholic fatty liver disease.

Description

Oligonucleotide sequence for inhibiting hsa-miR-221-3p, recombinant adenovirus and preparation method and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering and biomedical treatment, and particularly relates to an oligonucleotide sequence for inhibiting hsa-miR-221-3p prepared by an oligonucleotide tandem method, a recombinant adenovirus and application thereof in reducing liver and blood triglyceride.

Background

The liver is the central organ that regulates the metabolism of the body, such as lipid metabolism, including lipid synthesis, lipoprotein absorption and secretion, fatty acid oxidation, and the like. Non-alcoholic fatty liver disease is a clinical syndrome without history of excessive drinking, characterized by degeneration and accumulation of parenchymal hepatic cell fat, is the most common liver disease, and has incidence rate of about 25% to 40% in China. With the continuous improvement of the living standard of Chinese people and the continuous change of the dietary structure, high-calorie and high-fat diet is very popular. In addition, with the acceleration of life rhythm and the increase of working pressure, many people lack of exercise, etc., the disease is in a rapid growth trend, and if not controlled, the disease is easy to develop into hepatic fibrosis and liver cancer.

the liver lipid metabolism control network is very complex, and a large number of genes directly or indirectly related to liver lipid metabolism have been discovered, such as fatty acid synthesis-related genes SCD-1, SREBP-1C, ACC, triglyceride synthesis-related genes PPAR γ, LXR α, DGAT1, DGAT2, fatty acid oxidation-related genes PPAR α, CPT1, MCAD, LCAD, UCP2, fatty acid absorption-related genes CD36, PPAR δ, and the like.

mirnas are a class of endogenous, non-protein-coding RNAs of 18 to 25 nucleotides that inhibit translation of a target protein in mammalian and human cells by binding to the 3' untranslated region of the target gene mRNA. hsa-miR-221-3p was originally found to be overexpressed in human hepatoma cells and was closely associated with hepatocyte proliferation and liver regeneration. Research shows that miR-221 is over-expressed in fat cells of obese patients. In addition, miR-221 also plays an important role in muscle differentiation and insulin resistance.

At present, no relevant research for effectively treating nonalcoholic fatty liver, hyperlipidemia, insulin resistance and the like by inhibiting hsa-miR-221-3p is available.

Disclosure of Invention

In view of the above, the invention provides an oligonucleotide sequence for inhibiting hepatocyte hsa-miR-221-3p, a recombinant adenovirus and a preparation method thereof, and application of the recombinant adenovirus in reducing liver and blood triglyceride, improving lipid metabolism abnormality of a non-alcoholic fatty liver patient and increasing insulin sensitivity.

The invention provides an oligonucleotide sequence for inhibiting hsa-miR-221-3p in a first aspect, wherein the oligonucleotide sequence is one of the following sequences:

sequence 1: (GAAACCCAGCTGACAATGTAGCTAAGA)_n≧5

Sequence 2: (GAAACCCAGCCGACAATGTAGCTAAGA)_n≧5

And (3) sequence: (GAAACCCAGCGGACAATGTAGCTAAGA)_n≧5

And (3) sequence 4: (GAAACCCAGCGACAATGTAGCTAAGA)_n≧5

Or the AAGA portion of the above sequence is replaced with another spacer sequence.

The second aspect of the invention provides prokaryotic and eukaryotic shuttle vectors and adenovirus vectors containing the above-mentioned oligonucleotide sequences for inhibiting hsa-miR-221-3 p.

The third aspect of the invention provides a recombinant adenovirus containing the oligonucleotide sequence for inhibiting hsa-miR-221-3p and an application thereof in preparing a product for treating non-alcoholic fatty liver disease.

The fourth aspect of the invention provides a preparation method of the recombinant adenovirus containing the oligonucleotide sequence for inhibiting hsa-miR-221-3p, which comprises the following steps:

a. synthesizing the oligonucleotide sequence for inhibiting the hsa-miR-221-3 p;

b. b, obtaining shRNA according to the oligonucleotide sequence obtained in the step a, and connecting the shRNA with a shuttle vector to obtain a recombinant shuttle plasmid for inhibiting hsa-miR-221-3 p;

c. linearizing the recombinant shuttle plasmid obtained in the step b, co-transforming the linearized recombinant shuttle plasmid and an adenovirus skeleton vector into competent cells in vitro, and obtaining a recombinant adenovirus vector for inhibiting hsa-miR-221-3p through homologous recombination;

d. and c, after linearization, the vector obtained in the step c is used for transfecting 293A cells by using liposome to obtain the recombinant adenovirus for inhibiting hsa-miR-221-3 p.

Advantageous effects of the inventionThe fruit is as follows: the invention provides application of recombinant adenovirus (Ad-s-miR-221-3p) for inhibiting has-miR-221-3p in the aspect of treatment of non-alcoholic fatty liver, wherein oligonucleotides containing at least more than 5 repetitive sequences are designed aiming at a mature body sequence of the has-miR-221-3p, and the adenovirus capable of inhibiting has-miR-221-3p is prepared by adopting an intracellular DNA homologous recombination method. The titer of the recombinant adenovirus for inhibiting has-miR-221-3p prepared by the invention can reach 10¹¹pfu/ml. The application of the recombinant adenovirus for inhibiting has-miR-221-3p in treating nonalcoholic fatty liver disease comprises reducing triglyceride level of liver, reducing triglyceride level of blood, improving hyperlipidemia and increasing insulin sensitivity of organism. Through experiments of treating db/db or DIO model mice by administering Ad-s-miR-221-3p through tail vein, it can be observed that treatment of Ad-s-miR-221-3p can obviously improve hyperlipidemia, improve lipid metabolism disorder, reduce liver triglyceride level of model mice, reduce blood triglyceride level and increase insulin sensitivity of mice. The Ad-s-miR-221-3p has very obvious effects and effects on reducing liver and blood triglyceride, and the invention discovers a new potential target spot and provides a new solution idea for improving the non-alcoholic fatty liver, hyperlipidemia and insulin resistance.

Drawings

FIG. 1 shows the expression levels of the liver hsa-miR-221-3p of db/db mice before and after recombinant adenovirus treatment.

FIG. 2 shows DIO mouse liver hsa-miR-221-3p expression levels before and after recombinant adenovirus treatment.

FIG. 3 shows the liver and blood triglyceride levels of DIO mice before and after recombinant adenovirus treatment.

FIG. 4 is a graph of liver and triglyceride levels in db/db mice before and after recombinant adenovirus treatment.

FIG. 5 is a DIO mouse insulin resistance curve before and after recombinant adenovirus treatment.

Detailed Description

sequence 1: (GAAACCCAGCTGACAATGTAGCTAAGA)_n≧5

Sequence 2: (GAAACCCAGCCGACAATGTAGCTAAGA)_n≧5

And (3) sequence: (GAAACCCAGCGGACAATGTAGCTAAGA)_n≧5

And (3) sequence 4: (GAAACCCAGCGACAATGTAGCTAAGA)_n≧5

Among them, the AAGA part in the four sequences is a spacer sequence, so that the substitution of the AAGA part in the above sequences with other spacer sequences also has equivalent effects.

The oligonucleotide sequence for inhibiting the hsa-miR-221-3p is prepared by designing more than 5 mature body sequences of the hsa-miR-221-3p in series, introducing an interval sequence (AAGA), and performing DNA homologous recombination in cells to prepare the recombinant adenovirus.

The second aspect of the invention provides a prokaryotic and eukaryotic shuttle vector and a recombinant adenovirus vector containing the hsa-miR-221-3p inhibition oligonucleotide sequence.

In more detail, the product for treating the non-alcoholic fatty liver disease is a product for reducing the triglyceride level of the liver, reducing the triglyceride level of blood and increasing the insulin sensitivity of the body.

In more detail, BgL2 and HindIII enzyme cutting sites are respectively introduced into two ends of the shRNA in the step a, annealing is carried out to form a double-stranded structure, and the double-stranded structure is connected with a Track-U6 shuttle vector which is cut by BgL2 and HindIII enzyme.

And d, after obtaining the recombinant adenovirus in the step d, performing amplification culture in 293A cells, and purifying and concentrating by adopting cesium chloride density gradient centrifugation. The recombinant adenovirus obtained after purification has high purity and high titer and meets the national relevant standards.

The application of the recombinant adenovirus for inhibiting hsa-miR-221-3p in the aspect of non-alcoholic fatty liver disease treatment provided by the invention is further explained by combining a specific example.

The invention designs 4 kinds of inhibition oligonucleotide sequences aiming at hsa-miR-221-3p, which are as follows:

sequence 1: (GAAACCCAGCTGACAATGTAGCTAAGA)_n≧5

Sequence 2: (GAAACCCAGCCGACAATGTAGCTAAGA)_n≧5

And (3) sequence: (GAAACCCAGCGGACAATGTAGCTAAGA)_n≧5

And (3) sequence 4: (GAAACCCAGCGACAATGTAGCTAAGA)_n≧5

The spacer AAGA portion of the above four sequences may be replaced with other sequences.

In this example, based on the difference of the designed copy number, the following 4 types of oligonucleotides were synthesized, and 16 nucleotide sequences were synthesized in total, specifically as follows:

the first type:

sponge 1-5: as shown in SEQ NO. 1;

sponge 1-6: as shown in SEQ NO. 2;

sponge 1-7: as shown in SEQ NO. 3;

sponge 1-8: as shown in SEQ NO. 4;

the second type:

sponge 2-5: as shown in SEQ NO. 5;

sponge 2-6: as shown in SEQ NO. 6;

sponge 2-7: as shown in SEQ NO. 7;

sponge 2-8: as shown in SEQ NO. 8;

in the third category:

sponge 3-5: as shown in SEQ NO. 9;

sponge 3-6: as shown in SEQ NO. 10;

sponge 3-7: as shown in SEQ NO. 11;

sponge 3-8: as shown in SEQ NO. 12;

the fourth type:

sponge 4-5: as shown in SEQ NO. 13;

sponge 4-6: as shown in SEQ NO. 14;

sponge 4-7: as shown in SEQ NO. 15;

sponge 4-8: as shown in SEQ NO. 16.

Designing shRNA according to the 16 DNA sequences, respectively introducing BgL2 enzyme cutting sites and HindIII enzyme cutting sites at two ends of the shRNA, annealing to form a double-chain structure, and directly connecting the double-chain structure with a Track-U6 shuttle vector cut by BgL2 enzyme cutting sites and HindIII enzyme cutting sites to obtain the recombinant shuttle plasmid for inhibiting hsa-miR-221-3 p.

On the basis, recombinant adenovirus is constructed, expanded and cultured, concentrated and purified. The technical scheme is as follows: and (3) linearizing the constructed shuttle vector by using PmeI, co-transforming the linearized shuttle vector and the adenovirus skeleton vector into competent cells in vitro through electroporation, and obtaining the recombinant adenovirus vector for inhibiting the hsa-miR-221-3p through homologous recombination. The vector is linearized by PacI and then transfected into 293A cells by liposome to obtain the recombinant adenovirus for inhibiting hsa-miR-221-3 p. The recombinant adenovirus is amplified in 293A cells in a large quantity, and purified and concentrated by cesium chloride density gradient centrifugation, so that the obtained recombinant adenovirus has high purity and high titer and meets the national relevant standards.

The specific operation of this embodiment is as follows:

firstly, design and subcloning of oligonucleotide fragment for inhibiting hsa-miR-221-3p

1. According to the 4 types of the 16 miRNA sponges designed above, BgL2 and HindIII enzyme cutting sites are introduced at two ends to synthesize oligonucleotide fragments, the oligonucleotide fragments are added into boiled annealing buffer solution, and the mixture is placed in a 4-degree refrigerator for natural cooling overnight.

2. 2 micrograms of Track-U6 vector was taken, 10 units of BgL2 and HindIII endonuclease were added simultaneously, and an appropriate amount of buffer was added, and the mixture was digested at 37 ℃ for 5 hours. And cutting and recovering after gel electrophoresis.

3. Take 1 microliter of the recovered product, add 7 microliter of the annealed product, 1 microliter of T4DNA ligase and buffer, and ligate overnight at 16 ℃.

10 microliters of the ligation product was taken, transformed into competent cells, cultured overnight, and then single clones were picked and sent to the company for sequencing. The sequencing result is completely consistent with the designed sequence.

Secondly, inhibiting hsa-miR-221-3p oligonucleotide fragment to construct recombinant adenovirus

1. Converting and amplifying a shuttle vector containing an hsa-miR-221-3p inhibition oligonucleotide fragment, extracting a large amount of plasmid DNA, taking 5 micrograms of high-purity shuttle plasmid, and adding 25 units of PmeI to carry out enzyme digestion for 5 hours. And cutting and recovering gel after gel electrophoresis.

2. And 6 microliters of the recovered product is taken, added with 100 nanograms of adenovirus backbone vector, uniformly mixed and added into 20 microliters of BJ5183 competent cells, and electroporation is carried out under the conditions of 2500V, 200Ohms and 25 muF. The cells were then resuspended in sterile EP tubes using 500. mu.l of LB medium preheated to 37 degrees, and incubated at 37 degrees for 3 to 5 minutes. 100. mu.l of LB-resistant plates coated with kanamycin were incubated at 37 ℃ for 16 hours.

3. Extracting recombinant plasmid, converting, preparing plasmid in large quantity, digesting and linearizing by PacI enzyme, and transfecting the linearized product into 293A cell by using liposome to obtain the recombinant adenovirus.

4. After the recombinant adenovirus is amplified in 293A cells, cesium chloride density gradient is adopted for centrifugal purification and concentration, and finally the virus titer is determined to reach 10¹¹pfu/ml。

Application of recombinant adenovirus in inhibiting hsa-miR-221-3p to reduce db/db and DIO model mouse liver and blood triglyceride

1. Preparation of experimental animal model

db/db mice, 4-6 weeks old, female, fed on normal diet, water ad libitum, and their body weights measured every other day, starting tail vein injection of recombinant adenovirus at 8-9 weeks old, and about 28 g body weight.

DIO (diet-induced obese mouse) mouse model construction: c57BL/6 female mice at 3 weeks of age were fed on a D12492 high fat diet and at 8 to 9 weeks of age, individuals with a weight 20% higher than that of the normal control group were selected for the treatment experiment.

2. Lipid lowering treatment in animal models

Female db/db or DIO mice were divided into two groups of 7 to 8 mice each, and injected with Ad-GFP (1X 10) in a single metered tail vein¹⁰pfu/0.2ml, physiological saline) and recombinant adenovirus (1X 10)¹⁰pfu/0.2ml, physiological saline).

7 days after injection, insulin resistance experiments were performed, blood triglyceride levels were measured by orbital bleeding on day eight, mice were sacrificed and liver triglyceride levels were measured.

Wherein, the method of taking blood from the orbit adopts the method of picking the eyeball, the blood serum is separated by centrifuging at 3000rpm for 10 minutes, and the sample is sent to the clinical laboratory of the hospital to detect the triglyceride level.

Weighing liver, homogenizing with lysis solution, centrifuging at 3000rpm for 10 min, and measuring triglyceride level in hospital with 200 μ l.

Results of the experiment

The liver hsa-miR-221-3p expression level of DIO and db/db mice is obviously higher than that of C57BL6 control mice.

2. The expression levels of the liver hsa-miR-221-3p of db/db mice before and after recombinant adenovirus treatment are shown in figure 1, and the expression levels of the liver hsa-miR-221-3p of DIO mice before and after recombinant adenovirus treatment are shown in figure 2. In the two mouse models, compared with the group injected with Ad-GFP, the group injected with the recombinant adenovirus inhibiting hsa-miR-221-3p has the advantage that the expression level of the liver hsa-miR-221-3p is reduced by about 70% on average.

3. Liver and blood triglyceride levels of DIO mice before and after recombinant adenovirus treatment are shown in figure 3, and liver and blood triglyceride levels of db/db mice before and after recombinant adenovirus treatment are shown in figure 4. In the two mouse models, compared with the group injected with Ad-GFP, the group injected with the recombinant adenovirus inhibiting hsa-miR-221-3p has the advantage that the average of the liver triglyceride and the blood triglyceride is obviously reduced.

4. Insulin resistance curves of DIO mice before and after recombinant adenovirus treatment are shown in figure 5, and insulin resistance experimental results show that compared with a group injected with Ad-GFP, the group injected with the recombinant adenovirus inhibiting hsa-miR-221-3p has obviously improved insulin resistance.

The application of the recombinant adenovirus provided by the embodiment in the aspect of improving the lipid metabolism disorder can be observed by injecting a db/db or DIO mouse model through tail vein, and the recombinant adenovirus designed by the invention can obviously reduce the liver and blood triglyceride levels of the two model mice and increase the insulin sensitivity. Therefore, the recombinant adenovirus for inhibiting hsa-miR-221-3p has positive, effective and reliable effects on reducing liver and blood triglyceride and improving insulin sensitivity, and provides a new solution for treating non-alcoholic fatty liver, hyperlipidemia and insulin resistance.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims

1. The application of the recombinant adenovirus containing the oligonucleotide sequence for inhibiting the hsa-miR-221-3p is characterized in that the recombinant adenovirus is applied to the preparation of a product for treating the non-alcoholic fatty liver, and the oligonucleotide sequence for inhibiting the hsa-miR-221-3p is one of the following sequences:

sequence 1: (GAAACCCAGCTGACAATGTAGCTAAGA) n ≧ 5;

sequence 2: (GAAACCCAGCCGACAATGTAGCTAAGA) n ≧ 5;

and (3) sequence: (GAAACCCAGCGGACAATGTAGCTAAGA) n ≧ 5;

and (3) sequence 4: (GAAACCCAGCGACAATGTAGCTAAGA) n ≧ 5;

2. The use of a recombinant adenovirus according to claim 1, wherein: the product for treating the non-alcoholic fatty liver disease is a product for reducing the triglyceride level of the liver, reducing the triglyceride level of blood and increasing the insulin sensitivity of the body.

3. The use of the recombinant adenovirus according to claim 1, wherein the recombinant adenovirus is prepared by a method comprising the steps of:

a. synthesizing an oligonucleotide sequence that inhibits hsa-miR-221-3p of claim 1;

4. The use of a recombinant adenovirus according to claim 3, wherein: and (b) introducing BgL2 and HindIII enzyme cutting sites at two ends of the shRNA in the step a respectively, annealing to form a double-chain structure, and connecting the double-chain structure with a Track-U6 shuttle vector cut by BgL2 and HindIII enzyme.

5. The use of a recombinant adenovirus according to claim 4, wherein: and d, after obtaining the recombinant adenovirus in the step d, performing amplification culture in 293A cells, and purifying and concentrating by adopting cesium chloride density gradient centrifugation.