CN105950619A - shRNA molecule capable of inhibiting expression of human AEBP1 gene - Google Patents
shRNA molecule capable of inhibiting expression of human AEBP1 gene Download PDFInfo
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- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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Abstract
The invention discloses a shRNA molecule capable of inhibiting expression of the human AEBP1 gene. According to the human AEBP1 gene (with a serial number of NM_001129.4) in Genbank, four shRNA molecules are designed and are synthesized targeted at the sequence of shRNA; two synthesized complementary single-stranded shRNA molecules are subjected to annealing to form a double-stranded shRNA molecule; and the double-stranded shRNA molecule is linked to a lentivirus vector to construct four lentivirus interference RNA plasmids. The shRNA plasmids are used for transfection of a Huh7 cell line containing AEBP1, and relative quantitative analysis is carried out on the AEBP1 gene through real-time quantitative PCR so as to evaluate inhibition effect on the expression of the AEBP1 gene. Results of relative quantitative analysis show that AEBP1-shRNA1 can inhibit transcription of 82.3% of AEBP1 and AEBP1-shRNA2 can inhibit transcription of 75.7% of AEBP1.
Description
Technical Field
The invention relates to a method for designing and synthesizing shRNA molecules capable of remarkably inhibiting an Adipocyte enhanced binding Protein 1 (AEBP 1) gene and evaluating the inhibition effect on liver cells.
Background
Adipocyte enhancer-binding protein 1 (AEBP 1) is a transcription inhibitor, has a molecular weight of 82kDa, is highly expressed in adipose tissue, and is involved in adipogenesis in preadipocytes. In addition, the expression level of AEBP1 in liver, lung, spleen, brain and primary macrophage is also higher.
It has been proved that AEBP1 is involved in cholesterol balance of mouse macrophage, foam cell formation and macrophage and liver cell inflammation reaction. High expression of AEBP1 promoted the release of inflammatory factors in mice. The specific mechanism is that AEBP1 promotes the release of inflammatory factors IL-6, TNF-alpha, MCP-1, iNOS and the like by activating NF-kB channel. This process relies primarily on protein-protein interactions of AEBP1 with I κ B α, binding of AEBP1 to I κ B α promotes Ser32/Ser36 phosphorylation of I κ B α, resulting in degradation of phosphorylation of I κ B α, phosphorylation of NF- κ B and transformation into the nucleus, and initiation of transcription of target genes (e.g., inflammatory factors).
In addition, it was found that AEBP1 mediated differentiation of LPS-induced macrophages into foam cells, and mice over-expressing AEBP1 were more prone to obesity. Further studies have found that overexpression of AEBP1 can inhibit the expression of PPAR γ 1(peroxisome proliferator-activator γ 1) and LXR α (liverX receptor α) as well as ABCA 1(ATP-binding cassette a1), ABCG1(ATP-binding cassette G1), apoe (apolipoprotein e) and CD36, which are important participants in the transfer of cholesterol to high-density lipoproteins. Knocking out the expression of AEBP1 in mice helps to reduce body weight. LPS stimulates macrophages, inducing upregulation of intracellular AEBP1 expression. Inhibiting the expression of AEBP1 can resist infectious shock caused by LPS and atherosclerosis caused by gram-negative bacteria infection.
Thus, it can be seen that AEBP1 is involved in inflammation and pathological reactions of diseases related to lipid metabolism, and interference with expression of AEBP1 is helpful for prevention and treatment of diseases. However, there are no shRNA molecules that inhibit the expression of human AEBP 1.
Disclosure of Invention
The shRNA molecule for inhibiting the expression of the human AEBP1 gene provided by the invention has a significant inhibiting effect on the expression of the human AEBP1 gene, and provides an application basis for the treatment of diseases related to the overexpression of the AEBP 1.
The invention is realized by adopting the following technical scheme:
an shRNA molecule for inhibiting the expression of a human AEBP1 gene, wherein the sequence number of the AEBP1 gene in GenBank is NM-001129.4. The shRNA molecule comprises shRNA1 and shRNA 2; wherein,
the sense strand template sequence of the shRNA1 molecule is:
5’-GATCCGCTATGAGGAAATGACCTTTCTTCAAGAGAGAAAGGTCATTTCCTCATAGCTTTTTTG-3’，
the antisense strand template sequence is:
5’-AATTCAAAAAAGCTATGAGGAAATGACCTTTCTCTCTTGAAGAAAGGTCATTTCCTCATAGCG-3’；
the sense strand template sequence of the shRNA2 molecule is:
5’-GATCCGGTGGTGATTACTGGCGAATCTTCAAGAGAGATTCGCCAGTAATCACCACCTTTTTTG-3’，
the antisense strand template sequence is:
AATTCAAAAAAGGTGGTGATTACTGGCGAATCTCTCTTGAAGATTCGCCAGTAATCACCACCG。
according to the invention, a group of shRNA molecules are synthesized aiming at the AEBP1 gene sequence of a human, two shRNA1 and shRNA2 are screened out, and the qRT-PCR method detects that the expression of the AEBP1 gene can be effectively reduced, so that the expression of the AEBP1 protein level is remarkably reduced, and the verification is carried out on a human liver cancer cell strain Huh 7. Quantitative analysis shows that AEBP1-shRNA1 can effectively inhibit transcription of 82.3% of AEBP1, and AEBP1-shRNA2 can inhibit transcription of 75.7% of AEBP 1.
The invention has the following application significance: some infectious diseases such as gram negative bacteria infection, virus infection (hepatitis B virus, hepatitis C virus) and the like cause the expression of AEBP1 in cells to be increased, cause continuous inflammatory reaction and lipid metabolism disorder, and promote the development process of diseases such as viral hepatitis, infectious shock, atherosclerosis and the like. Inhibition of AEBP1 expression can attenuate the progression of the disease. The shRNA molecules screened by the invention can obviously reduce the expression of AEBP 1.
Drawings
FIG. 1 is a graph showing the results of detecting the inhibition effect of shRNA transfection on AEBP1 expression by a real-time quantitative PCR method in the examples of the present invention.
Detailed Description
The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments.
The method for synthesizing shRNA molecules for inhibiting the human AEBP1 gene comprises the following steps:
according to the human AEBP1 gene in Genbank, the gene sequence number: NM-001129.4; designing 4 shRNA molecules, synthesizing aiming at shRNA sequences to obtain two complementary single-chain shRNA molecules, namely shRNA1 and shRNA2, annealing the two shRNA molecules to form double chains, and connecting the double chains to a lentiviral vector to construct 4 lentiviral interference RNA plasmids.
To test the inhibitory effect of the synthesized shRNA molecules on human AEBP1 gene expression, shRNA plasmids were transfected into a Huh7 cell line containing AEBP1 by inoculating Huh7 cells expressing AEBP1 into 24-well plates, 1.5 × 105Cell/well density, cells were cultured in 500ul of DMEM medium containing 10% fetal bovine serum, 24h later, cells reached about 75% confluence, 100ul opti-MEM was taken, 2ul liposome lipofectamin2000 was premixed with 0.8ug of plasmid, left to stand at room temperature for 20min, and added to the supernatant of cultured Huh7 cells. After 6 hours, the cells were replaced with DMEM complete medium containing 10% fetal bovine serum, and after 48 hours of culture, total RNA was extracted by Trizol.
Carrying out relative quantitative analysis on AEBP1 gene by real-time quantitative PCR on the total RNA of the transfected cells, and evaluating the inhibition effect on the expression of the AEBP1 gene; the quantitative analysis method is as follows: the one-step Real time PCR detection kit is purchased from TAKARA company, and the Real time PCR reaction system is as follows: 2 Xone Step RT-PCR Buffer III 12.5ul, Takara Ex Taq HS 0.5ul, PrimerScript RT enzyme Mix II0.5ul, upstream and downstream primers (10uM) each 0.5ul, TaqMan Probe 1ul, total RNA 2ul, RNasefrede dH2O 7.5.5 ul. The PCR reaction conditions are as follows: hold 42 ℃ for 5min, hold 95 ℃ for 10s, cycle 95 ℃ for 5s, 60 ℃ for 20s, 40 cycles. By relative quantitative analysis it follows: AEBP1-shRNA1 can effectively inhibit transcription of 82.3% of AEBP1, and AEBP1-shRNA2 can inhibit transcription of 75.7% of AEBP1, as shown in figure 1.
The Opti-MEM culture solution and DMEM culture solution selected in the above examples were purchased from Gibco, Lipofectamin2000 from Invitrogen, and fetal bovine serum from Hangzhou Sijiqing bioengineering materials, Inc; the RNA extraction reagent Trizol and the quantitative PCR kit are purchased from TAKARA company; the shRNA was synthesized by Shanghai Jima pharmaceutical technology, Inc.
The above embodiments are merely preferred embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. Meanwhile, the above description of the embodiments is only for assisting understanding of the method and the core idea of the present invention, and a person skilled in the art may change the embodiments and the application scope according to the idea of the present invention, and in summary, the content of the present description should not be understood as limiting the present invention.
Claims (1)
1. An shRNA molecule for inhibiting the expression of a human AEBP1 gene, wherein the sequence number of the AEBP1 gene in GenBank is NM-001129.4, and the shRNA molecule comprises shRNA1 and shRNA 2; wherein,
the sense strand template sequence of the shRNA1 molecule is
5’-GATCCGCTATGAGGAAATGACCTTTCTTCAAGAGAGAAAGGTCATTTCCTCATAGCTTTTTTG-3’，
The antisense strand template sequence is
5’-AATTCAAAAAAGCTATGAGGAAATGACCTTTCTCTCTTGAAGAAAGGTCATTTCCTCATAGCG-3’；
The sense strand template sequence of the shRNA2 molecule is
5’-GATCCGGTGGTGATTACTGGCGAATCTTCAAGAGAGATTCGCCAGTAATCACCACCTTTTTTG-3’，
The antisense strand template sequence is
AATTCAAAAAAGGTGGTGATTACTGGCGAATCTCTCTTGAAGATTCGCCAGTAATCACCACCG。
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| CN201610246220.1A CN105950619A (en) | 2016-04-20 | 2016-04-20 | shRNA molecule capable of inhibiting expression of human AEBP1 gene |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108686209A (en) * | 2017-04-07 | 2018-10-23 | 邢杰 | A kind of plasmid construction and application method for antiatherosclerosis |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002024741A2 (en) * | 2000-09-21 | 2002-03-28 | Ryan James W | Isolated genomic polynucleotide fragments from chromosome 7 |
| CN105238790A (en) * | 2015-11-26 | 2016-01-13 | 河南省农业科学院畜牧兽医研究所 | Promoters for regulating pig AEBP1 and construction method, and transfection carriers and construction method and application |
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- 2016-04-20 CN CN201610246220.1A patent/CN105950619A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002024741A2 (en) * | 2000-09-21 | 2002-03-28 | Ryan James W | Isolated genomic polynucleotide fragments from chromosome 7 |
| CN105238790A (en) * | 2015-11-26 | 2016-01-13 | 河南省农业科学院畜牧兽医研究所 | Promoters for regulating pig AEBP1 and construction method, and transfection carriers and construction method and application |
Non-Patent Citations (5)
| Title |
|---|
| HYO‐SUNG RO ET AL.: "Adipocyte Enhancer‐Binding Protein 1 Modulates Adiposity and Energy Homeostasis", 《OBESITY》 * |
| MAJDALAWIEH, AMIN ET AL.: "PPARγ1 and LXRα face a new regulator of", 《NUCLEAR RECEPTOR SIGNALING》 * |
| ZHANG, L ET AL.: "The Role of AEBP1 in Sex-Specific Diet-Induced Obesity", 《MOLECULAR MEDICINE》 * |
| 孙坚 等: "AEBP1在结直肠癌中的表达及其意义", 《现代生物医学进展》 * |
| 武会娟 等: "《临床科研常用分子生物学技术》", 31 July 2015 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108686209A (en) * | 2017-04-07 | 2018-10-23 | 邢杰 | A kind of plasmid construction and application method for antiatherosclerosis |
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