CN111110868B - NR2F6 gene and application of coded protein thereof in preparation of medicines - Google Patents
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Abstract
The invention belongs to the field of biological medicine, and discloses an application of NR2F6 gene and a coded protein thereof in preparation of medicines, in particular to an application in preparation and screening of medicines for treating non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH) and type 2 diabetes; wherein, the mRNA sequence of the mouse NR2F6 gene is shown as SEQ ID NO. 1, and the amino acid sequence of the encoded protein NR2F6 is shown as SEQ ID NO. 2; the mRNA sequence of the human NR2F6 gene is shown as SEQ ID NO. 3, and the amino acid sequence of the encoded protein NR2F6 is shown as SEQ ID NO. 4. The invention proves the important function of NR2F6 gene in regulating liver, whole body fat metabolism and sugar metabolism through in vivo and in vitro experiments for the first time, and can provide important novel targets for treating NAFLD, NASH or type 2 diabetes.
Description
Technical Field
The invention belongs to the field of biological medicine, and in particular relates to an application of NR2F6 gene and encoded protein thereof in preparing and screening medicines for treating non-alcoholic fatty liver disease, non-alcoholic steatohepatitis and type 2 diabetes.
Background
With the rapid development of economy and the change of life style of people (including intake of high calorie food, intake of high fructose beverage, lack of exercise and physical activity, etc.), non-alcoholic fatty liver disease (Non-alcoholic fatty liver disease, NAFLD) has become an important public health problem in our country. Epidemiological data of a plurality of people show that the prevalence rate of adult NAFLD in the part of China reaches 20 percent and is in a continuously rising situation. NAFLD can be further developed into nonalcoholic steatohepatitis (Nonalcoholic steatohepatitis, NASH), liver fibrosis and liver cirrhosis, and has very close relationship with hyperlipidemia, type 2 diabetes and cardiovascular and cerebrovascular diseases, thus seriously jeopardizing the physical and mental health of people in China and bringing a heavy economic burden to society. However, due to the complexity of the pathophysiological processes of NAFLD and the ambiguity of pathogenesis, there is still a lack of specific and effective therapeutic drugs in clinic. Therefore, research on pathogenesis of NAFLD, NASH and the like becomes a hotspot of global metabolic disease research, and the effect of key regulatory genes and proteins in liver cells in NAFLD and NASH occurrence and development is revealed, so that scientific basis is provided for screening intervention targets and preparing therapeutic drugs.
NAFLD is characterized by a massive accumulation of triglycerides in hepatocytes, whereas NASH includes, among others, hepatic cell injury, elevated expression of inflammatory and fibrotic factors, and the like. The steady state balance of liver triglyceride content is maintained mainly by two routes of approach and two routes of departure. Wherein, two ways include: (1) the de novo synthetic pathway: the hepatic cell takes acetyl coenzyme A generated by glucose decomposition as a raw material, synthesizes fatty acid through multi-step biochemical reaction, and combines with glycerol to form triglyceride; (2) Free fatty acids produced by the breakdown of fatty tissue triglycerides are released into the blood and taken up by hepatocytes to form triglycerides. And two outgoing routes include: (1) Oxidative decomposition of triglycerides and fatty acids to form ketone bodies; (2) Triglycerides bind to apolipoproteins, forming low density lipoproteins, which are released into the blood. Any of these four pathways, such as increased or decreased fatty acid uptake or breakdown, may lead to accumulation of triglycerides, thereby promoting the development of NAFLD/NASH.
Inside hepatocytes, these four pathways are also subject to fine and complex molecular regulation. It has been shown that the de novo synthesis pathway is mainly regulated by steroid response element binding protein 1c (SREBP 1 c) and its downstream target genes, fatty acid uptake is mediated by fatty acid channel protein CD36 located on the liver cell membrane, and oxidative degradation of fatty acids is catalyzed by acetyl-coa dehydrogenase (Acadm), acetyl-coa oxidase 1 (Acox 1), etc. The level of expression of these genes directly determines the homeostasis of liver triglycerides, for example: several studies have shown that increased expression of CD36 during obesity causes fatty acids to enter hepatocytes continuously, synthesize triglycerides and accumulate in the liver, leading to development of NAFLD/NASH and, at the same time, affecting insulin sensitivity in the body, promoting the development of hyperglycemia, hyperinsulinemia and insulin resistance.
More importantly, most of these genes closely related to lipid metabolism are regulated at the transcriptional level by nuclear receptor superfamily members (Nuclear receptor superfamily). Nuclear receptors are a class of transcription factors abundant in mammals, and total of 49 are available. Previous studies have demonstrated that: nuclear receptor family members play a vital role in the basic life activities of reproduction, development, growth, proliferation, differentiation, etc.; for example, androgen receptor and estrogen receptor determine and maintain sexual differentiation and development of secondary sex characteristics; while thyroxine receptors, glucocorticoid receptors and mineralocorticoid receptors maintain homeostasis in the body. Recent studies have demonstrated that some nuclear receptors also play a positive regulatory role in the homeostasis and imbalance of glycolipid metabolism and are therapeutic targets for clinical drugs, for example: SREBP1c and its target gene are mainly regulated positively by Liver X Receptor (LXR) and negatively by farnesol X receptor (Farnesoid X receptor, FXR). Fatty acid oxidative decomposition related enzymes such as Acadm and Acox1 are positively regulated by activating proliferation receptors alpha (Peroxisome proliferatoractivated receptor alpha and PPARalpha) by peroxidases. The first-line clinical drug, fenofibrate, for the treatment and correction of hypertriglyceridemia is then an agonist of pparα. In addition, the results of two human intervention studies respectively published in 2015 and 2019 Lancet journal show that the agonist of FXR, namely obeticholic acid, can effectively improve liver triglyceride deposition, liver inflammation and insulin sensitivity of NAFLD/NASH patients. Therefore, the metabolic functions of the members of the more nuclear receptor family are found and identified, which is not only helpful for elucidating the pathophysiological mechanisms of NAFLD, NASH and type 2 diabetes, but also lays a theoretical foundation for screening and developing therapeutic drugs for diseases.
NR2F6 (Nuclear receptor subfamily 2,group F,member 6), also known as chicken ovalbumin upstream promoter transcription factor 3 (Chicken ovalbumin upstream promoter transcriptional factor 3, COUP-TF 3), one of the nuclear receptor family members, this subclass contains 3 members: NR2F1, NR2F2 and NR2F6, were identified and cloned by Sophia Y.Tsai and Ming-Jer Tsai professor couples of the American Behler medical college during 1986-1992, with the mouse NR2F6 gene located on chromosome 8 and the human NR2F6 gene located on chromosome 19. NR2F6 proteins have a typical nuclear receptor structure, i.e. comprise: (1) A DNA binding region which binds to the TGACCT base region on the downstream gene promoter to promote the transcription and expression of mRNA of the downstream gene and exert transcription regulation; (2) a ligand binding domain, which binds to the ligand. However, up to now, NR2F6, like the other two NR2F nuclear receptors, has not been defined as an endogenous natural ligand and therefore belongs to the orphan nuclear receptor (Orphan nuclear receptor).
At present, the biological function of NR2F6 is still more blank. The only few studies on NR2F6 function have focused mainly on three aspects:
(1) Development: NR2F6 plays an important role in brain neuron clock rhythms and pain perception.
(2) Immunomodulation: NR2F6 is actively involved in the development of T lymphocytes, regulating the aggregation, activation and function of helper T lymphocytes; whereas inhibition of NR2F6 can enhance the anti-PD-L1/PD-1 tumor immunotherapeutic effect.
(3) NR2F6 is involved in the development of a plurality of malignant tumors such as colon cancer, cervical cancer, non-small cell lung cancer, ovarian cancer and the like.
In the field of metabolic diseases, only one report exists: NR2F6 promotes adipocyte differentiation by inhibiting the expression of cytokine IL-17; in the field of liver diseases, only one study reported: NR2F6 promotes the growth, invasion and migration of liver cancer cells. As can be seen from this, NR2F6, one of the nuclear receptor family members, is actively involved in the regulation of gene transcription and maintenance of normal cell functions, but its role in nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH) and type 2 diabetes has not been reported at home and abroad. Furthermore, there is no specific therapeutic drug for NAFLD/NASH in clinic. Drugs commonly used to improve NAFLD/NASH include: metformin, vitamin E, yi Shanfu, etc.; among them, metformin has the main effect of lowering blood sugar, vitamin E is a broad-spectrum antioxidant, and Yi Shan has the effect of improving liver cell injury. Therefore, the research and development of specific and effective targeted drugs for reducing the content of liver triglyceride and improving NAFLD/NASH is still far from being achieved.
Disclosure of Invention
The applicant of the invention discovers the role of a nuclear receptor NR2F6 gene in liver triglyceride metabolism for the first time internationally, proposes that the NR2F6 gene and a protein coded by the same can become important targets for improving NAFLD, NASH and type 2 diabetes, and mainly aims to provide the application of the NR2F6 gene and the protein coded by the same in the aspects of preparing and screening medicines for treating NAFLD, NASH and type 2 diabetes.
It is another object of the present invention to provide a liver-specific NR2F6 inhibitor for use in the treatment of NAFLD, NASH or type 2 diabetes.
It is still another object of the present invention to provide the use of adenovirus shRNA silencing NR2F6 gene for the preparation and screening of a medicament for the treatment of NAFLD, NASH and type 2 diabetes as a means of ameliorating and treating the above mentioned diseases.
The technical scheme adopted by the invention for achieving the aim of the invention is as follows:
firstly, detecting the expression of NR2F6 genes and proteins in NAFLD mouse model, NASH mouse model and liver tissues of NAFLD/NASH patient; further, by utilizing the over-expression and adenovirus silencing expression technology of the adeno-associated virus, the effect test of NR2F6 in liver triglyceride metabolism and systemic glycolipid metabolism is carried out through a mouse and cell model; finally, experiments such as gene expression analysis, luciferase reporter gene and chromatin co-immunoprecipitation prove that NR2F6 has regulation and control effects on lipid metabolism related genes, so that new clues and intervention targets are provided for NALFD/NASH treatment. The method specifically comprises the following steps:
(1) NAFLD and NASH mice showed increased expression of NR2F6 in liver tissue. The expression of NR2F6 genes and proteins in NAFLD and NASH mouse liver tissues is detected by real-time quantitative PCR and Western immunoblotting, the mRNA sequence of the mouse NR2F6 genes is shown as SEQ ID NO. 1 (wherein, 1 st to 681 st sections are 5'-untranslated region sequences (5' -untranslated region), 682 st to 1851 st sections are base sequences of encoding proteins thereof, 1852 nd to 1854 th sections are stop codons, 1855 th to 2218 th sections are 3'-untranslated region sequences (3' -untranslated region)), and the amino acid sequence of the encoded protein NR2F6 is shown as SEQ ID NO. 2.
(2) The real-time quantitative PCR detection shows that the expression of NR2F6 in liver tissue of NAFLD patient is increased, the mRNA sequence of NR2F6 gene in human body is shown as SEQ ID NO 3 (wherein, the 1 st to 122 th sections are 5'-untranslated region sequences, the 123 rd to 1334 th sections are base sequences of encoding proteins thereof, the 1335 th to 1337 th sections are stop codons, and the 1338 th to 1804 th sections are 3' -untranslated region sequences), and the amino acid sequence of the encoded protein NR2F6 is shown as SEQ ID NO 4.
(3) Constructing an adeno-associated virus (AAV 9) of NR2F6, taking GFP gene as a control, and adopting a tail vein injection method to over-express the NR2F6 gene in a C57BL/6 mouse, so as to clearly express the regulation and control effect of the over-expression NR2F6 on promoting the liver triglyceride deposition of a normal mouse.
(4) An adenovirus (shRNA) for silencing endogenous NR2F6 expression is constructed, the shRNA sequence of the adenovirus is shown as SEQ ID NO. 5, and the expression of NR2F6 genes in liver tissues of db/db obese mice is inhibited by a tail vein injection method, so that the effect of silencing NR2F6 expression in improving liver triglyceride deposition of db/db obese mice is clarified.
(5) The shRNA adenovirus is utilized to silence the expression of NR2F6 genes in liver tissues of obese mice by silencing high-fat diet (HFD), and the effect of the shRNA adenovirus on improving liver triglyceride deposition of the obese mice induced by the high-fat diet is verified.
(6) Models of induction of mouse liver triglyceride deposition, NASH, liver injury upon choline and methionine deficiency (Methionine choline deficient, MCD) diet were constructed, verifying that silencing NR2F6 expression ameliorates choline and methionine deficiency diet-induced liver lipid deposition and injury effects.
(7) The effect of NR2F6 on promoting triglyceride deposition of primary hepatocytes of mice was verified by isolating primary hepatocytes of mice, incubating with palmitic acid, and overexpressing and silenced, respectively, the NR2F6 gene by adenovirus.
(8) The glycometabolism phenotype of NR2F6 over-expressed mice was examined, verifying the effect of over-expression of NR2F6 on hyperglycemia and insulin resistance in normal mice.
(9) The sugar metabolism phenotype of the obese mice after silencing NR2F6 gene is detected, and the effect of silencing expression NR2F6 on improving hyperglycemia and insulin resistance of the obese mice is verified.
(10) The expression of the fatty acid channel protein CD36 was regulated by real-time quantitative PCR detection of NR2F 6.
(11) NR2F6 binds to the CD36 promoter region, promoting its gene transcription. Screening and searching NR2F6 binding sites in mouse and human CD36 gene promoter regions through luciferase reporter genes and point mutation experiments, and verifying the effect of the NR2F6 binding sites in promoting transcription and expression of mRNA of the CD36 gene through chromatin co-immunoprecipitation experiments.
The invention relates to a liver-specific NR2F6 inhibitor for treating NAFLD, NASH or type 2 diabetes, which takes NR2F6 genes with mRNA sequences shown as SEQ ID NO. 1 or SEQ ID NO. 3 and/or protein NR2F6 coded by NR2F6 genes with amino acid sequences shown as SEQ ID NO. 2 or SEQ ID NO. 4 as binding targets.
The invention confirms the important role of NR2F6 gene in regulating liver, whole body fat metabolism and sugar metabolism through in vivo and in vitro experiments for the first time, and clarifies the inherent molecular mechanism thereof, thus not only widening the understanding of NR2F6 gene and protein functions, but also deepening the understanding of pathophysiological processes such as NAFLD, NASH, type 2 diabetes and the like. In addition, in view of the fact that agonists/inhibitors of multiple nuclear receptors have been widely used in clinic, the present invention is directed to liver-specific NR2F6 inhibitors obtained by the important role of NR2F6 gene and its encoded protein NR2F6 in regulating liver, systemic lipid metabolism and glycometabolism, and can provide an important novel target for the treatment of NAFLD, NASH or type 2 diabetes. In addition, the adenovirus shRNA designed aiming at the NR2F6 gene also provides a beneficial intervention direction for NAFLD, NASH or type 2 diabetes.
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FIG. 1 shows increased expression of NR2F6 in liver tissue of NAFLD and NASH mice; wherein: (a-B) real-time quantitative PCR (a) and western immunoblotting (B), the expression of NR2F6 gene and protein in high fat diet induced obese mice (HFD) and normal diet mice (ND), respectively, was examined; (C-D) real-time quantitative PCR (C) and western immunoblotting (D), the expression of NR2F6 gene and protein in leptin-deficient obese mice (ob/ob) and control mice (Lean), respectively, was examined; (E-F) real-time quantitative PCR (E) and Western immunoblotting (F), the expression of NR2F6 gene and protein in leptin receptor deficient obese mice (db/db) and control mice (Lean), respectively, was examined; (G-H) real-time quantitative PCR (G) and Western immunoblotting (H), the NR2F6 gene and protein were tested for the induction of NASH mice (NASH diet) and normal diet mice (ND), respectively, on a high-fat, high-cholesterol and high-fructose diet.
FIG. 2 shows an increase in NR2F6 expression in liver tissue of NAFLD patients; wherein: (A) real-time quantitative PCR experiments, found: compared with normal people, the expression of NR2F6 gene in liver tissue of NAFLD patient is obviously increased; (B) Pearson correlation analysis found that NR2F6 expression and liver triglyceride content exhibited significant positive correlation; the results of (C) immunohistochemical staining confirm: the expression of NR2F6 protein in liver tissue of NAFLD patients was also significantly increased.
FIG. 3 is an illustration of overexpression of NR2F6, promoting hepatic triglyceride deposition in normal mice; wherein: the western blot experiment proves that: the NR2F6 gene is successfully over-expressed in liver tissues; (B-C) mice in the NR2F6 over-expression group had increased liver weight (B) and liver triglyceride content (C) compared to control GFP group mice; (D) The oil red staining result of the liver pathological section shows that a large amount of lipid drops are deposited in liver cells; (E-F) NR2F6 over-expressed mice had increased plasma triglyceride levels (E) and increased free fatty acid levels in the liver (F).
FIG. 4 is a graph of silencing NR2F6 expression, improving liver triglyceride deposition in db/db obese mice; wherein: the western blot experiment proves that: successfully inhibit the expression of NR2F6 gene in liver tissue; (B-F) compared to control mice, the silenced NR2F6 expression group had a reduced liver triglyceride content (B), a reduced number and size of lipid droplets in the liver cells (C), a reduced liver weight (D), a reduced plasma triglyceride content (E), and a reduced free fatty acid content in the liver (F).
FIG. 5 is a graph depicting silencing NR2F6 expression and improving liver triglyceride deposition in high fat diet-induced obese mice; wherein: the western blot experiment proves that: successfully inhibit the expression of NR2F6 gene in liver tissue; (B-E) compared to control mice, the silenced NR2F6 expression group had reduced liver triglyceride content (B), reduced liver weight (C), reduced plasma triglyceride content (D), and reduced free fatty acid content of the liver (E).
FIG. 6 is a graph of silencing NR2F6 expression, improving choline and methionine deficiency diet-induced liver lipid deposition and damage; wherein: (a-B) real-time quantitative PCR (a) and western immunoblotting (B), respectively, detected the expression of NR2F6 gene and protein after choline and methionine deficiency diet (MCD) and adenovirus interference; (C-E) shRNA adenovirus silencing NR2F6 expression, effectively improving MCD diet-induced liver triglyceride deposition (C); reducing the glutamic-pyruvic transaminase content (D) of the blood plasma and the glutamic-pyruvic transaminase content (E) of the blood plasma; (F-G) shRNA adenovirus can effectively reduce the expression of inflammatory and fibrosis genes caused by MCD diet after silencing NR2F6 expression.
FIG. 7 is a graph of NR2F6 promoting triglyceride deposition in primary hepatocytes of mice; wherein: the western blot experiment proves that: the NR2F6 gene was successfully overexpressed in the primary hepatocytes of mice; (B) NR2F6 gene overexpression promotes palmitic acid-induced intracellular triglyceride deposition; the western blot experiment proves that: successfully inhibit the expression of NR2F6 gene in the primary liver cells of mice; (D) Inhibiting the expression of NR2F6 gene improves palmitic acid induced intracellular triglyceride deposition.
FIG. 8 is an overexpression of NR2F6, leading to hyperglycemia and insulin resistance in normal mice; wherein: (A-D) NR2F6 overexpressed mice showed increased blood glucose (A), increased plasma insulin levels (B), impaired glucose tolerance (C), and decreased insulin sensitivity (D) compared to control GFP group mice.
FIG. 9 is a diagram of silencing expression of NR2F6, improving hyperglycemia and insulin resistance in obese mice; wherein: (A-F) compared with the mice in the control group, after the shRNA adenovirus silences NR2F6 gene, blood sugar of the mice in the two groups of obese mice of db/db and HFD is reduced (A-B), plasma insulin content is reduced (C-D), and insulin sensitivity is increased (E-F).
FIG. 10 shows NR2F6 regulation of fatty acid channel protein CD36 expression; wherein: (A-C) screening of real-time quantitative PCR experiments found that: in mouse liver tissues after over-expression of NR2F6, the CD36 gene responsible for fatty acid uptake is obviously up-regulated, while the expression of other channel proteins for fatty acid uptake, fatty acid synthesis, fatty acid oxidation and other related genes are not significantly changed; the results of (D) western blotting experiments showed that: in mouse liver tissues after over-expression of NR2F6, the protein expression of CD36 was significantly increased; the results of (E-F) real-time quantitative PCR experiments and Western blotting experiments showed that: after silencing the expression of NR2F6 in liver tissue of obese mice, mRNA (E) and protein (F) expression of CD36 gene was significantly down-regulated.
FIG. 11 is a diagram showing that NR2F6 binds to the CD36 promoter region, promoting its gene transcription; wherein: (A) Binding sites for NR2F6 are present in both the mouse and human CD36 gene promoter regions; (B-C) luciferase reporter and point mutation experiments, confirm: NR2F6 can bind to this site, upregulating the activity of the mouse (B) and human (C) CD36 gene promoters; the chromatin co-immunoprecipitation experiments (D-F) showed that: NR2F6 can bind to this site (D) and increase acetylation (F) of this region histone H3 by recruiting coactivators SRC-1, etc. (E), thereby promoting mRNA transcription and expression of the CD36 gene.
Detailed Description
The invention will be further described with reference to the drawings and examples.
Mice: c57BL/6 male mice at 8 weeks of age were purchased from Shanghai Laike laboratory animals Co., ltd; leptin-deficient ob/ob mice and leptin receptor-deficient db/db mice were purchased from the university of south Beijing, south Beijing biomedical research institute; high fat Diet (HFD Diet) was purchased from Research Diet, usa, cat: d12492, and C57BL/6 male mice were fed for 12 weeks; high fat high cholesterol high fructose feed (NASH feed) was purchased from Research Diet company, usa, cat: d09100310 and C57BL/6 male mice were given 12 weeks of feeding.
And (3) cells: the primary liver cells of mice are self-isolated and cultured by the subject group using collagenase digestion and adherence method.
Adeno-associated virus (AAV 9) is driven with a thyroid-associated globulin gene promoter; adenovirus shRNA sequences that silence NR2F6 expression are: 5'-GGGACAAGUCCAGUGGAAAGCAUUA-3' the negative control adenovirus shRNA sequences are: 5'-GUUCUCCGAACGUGUCACGUUU-3', the base sequences of which are shown as SEQ ID NO. 5 and SEQ ID NO. 6 respectively, all viruses are injected intravenously from the tail of the mice.
Glucose tolerance and insulin tolerance experiments: glucose was purchased from Sigma-Aldrich, usa, mice were starved for 16 hours (food was removed at 17 pm, starvation started) before the experiment, and 2.0mg/kg glucose was then intraperitoneally injected; insulin was purchased from Eli Lilly company, usa, mice were starved for 6 hours (food was removed at 8 a.m., starvation was started) prior to the experiment, and then 0.75units/kg insulin was intraperitoneally injected; the blood sugar of the mice is detected by a handheld blood glucose meter and a Qiangsheng Hao blood glucose test paper of LifeScan company after blood is squeezed from tail veins.
Triglyceride measurement: the triglyceride content in liver homogenates and plasma were determined by the Biovision company kit, usa, cat: K622. wherein, the liver tissue homogenate is obtained by the steps of the cleavage, homogenate and centrifugation of 5% NP-40 lysate at 100 ℃ and the determination method is carried out according to the instruction of the kit.
Real-time quantitative PCR: RNA extraction of tissues and cells was performed with TRIzol reagent from Thermo-Fisher, cat#: 15596026.cDNA reverse transcription was performed using Promega reverse transcription kit, cat#: A3500. real-time quantitative PCR was performed on a Roche LightCycler 480 machine by the Japanese Takara company SYBR Green Premix Ex Taq kit (cat# DRR 820A). The calculation method of gene expression is 2 -△△Ct The expression of the Rplp0 gene serves as an internal reference.
Western blotting: protein extraction of tissues and cells was performed with RIPA reagent from shanghai bi yun biotechnology limited, cat: P0013B. The antibodies were as follows: NR2F6 (Abcam, cat# ab137496; R & D, cat# PP-N2025-00), CD36 (Abcam, cat# ab 133625), GAPDH (Abcam, cat# ab 181602).
Luciferase reporter gene assay: the CD36 gene promoter was constructed on the PGL4.15 plasmid supplied by Promega, and transfected into HepG2 cells by Thermo-Fisher Lipofectamine 3000 (cat# L3000015). The luciferase reporter assay was determined from the Luciferase Reporter Assay System kit from Promega corporation.
Chromatin co-immunoprecipitation: the experiment was performed on mouse primary liver cells. The antibodies were as follows: NR2F6 (R & D company, cat# PP-N2025-00), SRC1 (Abcam company, cat# ab 2859), or IgG. The degree of acetylation of Histone H3 was accomplished by the Acetyl-Histone H3 ChIP Kit (cat# P-2012) of Epigentek company.
Statistical analysis: all data represent: mean ± standard error, data between the two groups were statistically analyzed by two-tailed t-test.
The technical scheme of the invention is further explained by specific examples.
Example 1
(1) Liver-specific mouse models of overexpression and silencing expression of the NR2F6 gene were established.
NAFLD model: c57BL/6 mice were selected and fed with a High Fat Diet (HFD) for 12 weeks, leptin-deficient ob/ob mice, leptin receptor-deficient db/db mice.
NASH model: c57BL/6 mice were fed with a high-fat, high-cholesterol and high-fructose diet for 12 weeks.
The expression of NR2F6 genes and proteins in NAFLD and NASH mouse liver tissues was detected by real-time quantitative PCR and Western immunoblotting, and the results show that: mRNA and protein expression of NR2F6 gene was significantly increased in liver tissue of the above mice compared to the respective control mice, as shown in FIG. 1.
(2) NAFLD patients showed increased expression of NR2F6 gene in liver tissue. The real-time quantitative PCR experiment shows that: compared with normal people, the expression of NR2F6 gene in liver tissue of NAFLD patient is obviously increased, and the NAFLD patient and the liver triglyceride content show obvious positive correlation; immunohistochemical staining results also demonstrated: the expression of NR2F6 protein in liver tissue of NAFLD patients was also significantly increased, as shown in figure 2.
Example 2
Regulation of overexpression of NR2F6 gene in liver metabolism:
(1) An adeno-associated virus (AAV 9) of NR2F6 is constructed, and the NR2F6 gene is over-expressed in a C57BL/6 mouse by a tail vein injection method by taking Green Fluorescent Protein (GFP) gene as a control. The results show that: compared with the control GFP group mice, the NR2F6 over-expression group mice have increased liver weight and increased liver triglyceride content; the results of oil red staining of pathological sections of liver also show that a large number of lipid droplets are deposited in liver cells; in addition, the mice had increased plasma triglyceride levels and increased free fatty acid levels in the liver, and overexpression of the NR2F6 gene promoted hepatic triglyceride deposition in normal mice, as shown in figure 3.
(2) Overexpression of the NR2F6 gene results in hyperglycemia and insulin resistance in normal mice. The glycometabolism phenotype of NR2F6 overexpressing mice was examined and found: compared with the control GFP group mice, the NR2F6 over-expression mice have the advantages of increased blood sugar, increased plasma insulin content, abnormal glucose tolerance and reduced insulin sensitivity, which suggests that the high expression of NR2F6 can promote the insulin resistance and the glucose metabolism disturbance of organisms, as shown in figure 8.
Example 3
Silencing regulation of expressed NR2F6 gene in liver metabolism:
(1) Silencing NR2F6 gene expression improves liver triglyceride deposition in db/db obese mice. Construction of adenovirus (shRNA) silencing endogenous NR2F6 expression, and suppression of expression of NR2F6 Gene in liver tissue of db/db obese mice by tail vein injection, wherein the dose of adenovirus shRNA is 2X 10 per mouse 9 plaque forming unit. The results show that: the expression group with silenced NR2F6 had reduced liver triglyceride content, reduced number and size of lipid droplets in the liver cells, reduced liver weight, reduced plasma triglyceride content and reduced free fatty acid content of the liver compared to control mice, as shown in fig. 4.
(2) Silencing NR2F6 gene expression improves liver triglyceride deposition in high fat diet-induced obese mice. Using the above method, expression of NR2F6 gene in liver tissue of obese mice was induced by using shRNA adenovirus and silencing High Fat Diet (HFD). The results show that: the expression group silencing NR2F6 has reduced liver triglyceride levels, reduced liver weight, reduced plasma triglyceride levels and reduced liver free fatty acid levels compared to control mice, as shown in figure 5.
(3) Silencing NR2F6 gene expression improves choline and methionine deficiency diet-induced liver lipid deposition and damage. A common model for inducing liver triglyceride deposition, NASH, liver injury in mice in the absence of diet with choline and methionine was constructed. The results show that: after MCD diet, mice had significantly up-regulated expression of the NR2F6 gene in the liver. After the shRNA adenovirus is utilized to silence NR2F6 expression, the liver triglyceride deposition caused by MCD diet can be effectively improved, liver injury indexes (the content of plasma glutamic pyruvic transaminase and glutamic oxaloacetic transaminase) are reduced, the expression of liver inflammation related genes is reduced, and the expression of liver fibrosis related genes is reduced, as shown in figure 6.
(4) Silencing the expressed NR2F6 gene improves hyperglycemia and insulin resistance in obese mice. By detecting the sugar metabolism phenotype of the obese mice after silencing the NR2F6 gene, compared with the control mice, the sugar metabolism and insulin resistance symptoms of the two groups of obese mice, db/db and HFD, are obviously improved after adenovirus shRNA is used for silencing the NR2F6 gene, and the symptoms are expressed as follows: blood glucose reduction, plasma insulin content reduction, insulin sensitivity increase, as shown in fig. 9.
Example 4
NR2F6 promotes triglyceride deposition experiments on primary hepatocytes of mice. Primary liver cells of mice are isolated, incubated with palmitic acid, and the NR2F6 gene is overexpressed and silenced, respectively, by adenovirus. The results show that: after NR2F6 overexpression, intracellular triglyceride levels increase; whereas silencing the expression of the NR2F6 gene is effective against palmitic acid induced triglyceride deposition, as shown in figure 7.
Example 5
NR2F6 regulates expression of the fatty acid channel protein CD 36. Through real-time quantitative PCR experimental screening, the CD36 gene responsible for fatty acid uptake is obviously up-regulated in mouse liver tissues after the NR2F6 is over-expressed, and the expression of other channel proteins, fatty acid synthesis, fatty acid oxidation and other related genes for fatty acid uptake is not obviously changed; after silencing the expression of NR2F6 in liver tissue of obese mice, CD36 expression was significantly down-regulated as shown in FIG. 10.
Example 6
NR2F6 binds to the CD36 promoter region to promote CD36 gene transcription. The mouse and human CD36 gene promoter regions find the binding site of NR2F6, and the luciferase reporter gene and the point mutation experiments prove that NR2F6 can bind to the site to up-regulate the activity of the mouse and human CD36 promoters. Chromatin co-immunoprecipitation experiments showed that: NR2F6 can bind to this site and increase acetylation of this region of histone H3 by recruiting coactivator SRC-1, etc., and can promote mRNA transcription and expression of the CD36 gene, as shown in FIG. 11.
In conclusion, the invention firstly establishes a liver-specific NR2F6 high-expression and silencing expression mouse model through in-vivo and in-vitro experiments to prove that the NR2F6 gene and the encoded protein NR2F6 thereof play important roles in regulating liver, systemic lipid metabolism and glycometabolism, and the NR2F6 is found to have the effects of promoting liver triglyceride deposition, insulin resistance and hepatic cell injury. The liver-specific NR2F6 inhibitor is obtained aiming at the NR2F6 gene and the encoded protein NR2F6, and can provide an important novel target for treating NAFLD, NASH and type 2 diabetes. In addition, adenovirus shRNA designed for NR2F6 gene also provides a beneficial intervention direction for NAFLD, NASH and type 2 diabetes.
The previous description of the embodiments is provided to facilitate a person of ordinary skill in the art in order to make and use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above-described embodiments. Those skilled in the art will appreciate that, in light of the principles of the present invention, improvements and modifications can be made without departing from the scope of the invention.
Sequence listing
<110> Shenzhen City people Hospital
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Claims (3)
1. The application of adenovirus shRNA for silencing NR2F6 gene in preparing medicine for treating non-alcoholic fatty liver disease is characterized in that the shRNA takes mRNA with a sequence shown as SEQ ID NO. 1 or SEQ ID NO. 3 as a binding target.
2. The application of adenovirus shRNA for silencing NR2F6 gene in preparing medicine for treating non-alcoholic steatohepatitis is characterized in that the shRNA takes mRNA with a sequence shown as SEQ ID NO. 1 or SEQ ID NO. 3 as a binding target.
3. The application of adenovirus shRNA for silencing NR2F6 gene in preparing medicine for treating type 2 diabetes is characterized in that the shRNA takes mRNA with a sequence shown as SEQ ID NO. 1 or SEQ ID NO. 3 as a binding target.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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Family Cites Families (4)
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| US20150203846A1 (en) * | 2008-11-14 | 2015-07-23 | Christine Victoria Ichim | Treatment of Myelodysplastic Syndrome by Inhibition of NR2F6 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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Non-Patent Citations (6)
| Title |
|---|
| 2型糖尿病肾虚证基因表达谱研究;秦乐等;《时珍国医国药》;20140320;第25卷(第03期);第738-740页 * |
| Orphan nuclear receptor NR2F6 acts as an essential gatekeeper of Th17 CD4+ T cell effector functions;Natascha Hermann-Kleiter等;《Cell communication and signaling》;20140612;第38卷(第12期);第1-12页 * |
| The Nuclear Orphan Receptor NR2F6 Promotes Hepatic Steatosis through Upregulation of Fatty Acid Transporter CD36;Bing Zhou等;《Advanced Science》;20200921;第07卷(第21期);第1-15页 * |
| 复合辅酶治疗非酒精性脂肪性肝炎的疗效观察;刘安楠等;《实用医学杂志》;20090310;第25卷(第05期);第788-789页 * |
| 糖尿病肾病易感基因的研究进展;贺晓蕾;《国外医学.泌尿系统分册》;20030425(第04期);第431-434页 * |
| 高脂饮食诱导幼年和成年小鼠非酒精性脂肪性肝病的研究;张文烁;《万方学位论文》;20190904;第1-60页 * |
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