The lncRNA Gm15622 stimulates SREBP-1c expression and hepatic lipid accumulation by sponging the miR-742-3p in mice

Abstract

Excessive lipid deposition is a hallmark of NAFLD. Although much has been learned about the enzymes and metabolites involved in NAFLD, few studies have focused on the role of long noncoding RNAs (lncRNAs) in hepatic lipid accumulation. Here, using in vitro and in vivo models of NAFLD, we found that the lncRNA Gm15622 is highly expressed in the liver of obese mice fed a HFD and in murine liver (AML-12) cells treated with free fatty acids. Investigating the molecular mechanism in the liver-enriched expression of Gm15622 and its effects on lipid accumulation in hepatocytes and on NAFLD pathogenesis, we found that Gm15622 acts as a sponge for the microRNA miR-742-3p. This sponging activity increased the expression of the transcriptional regulator SREBP-1c and promoted lipid accumulation in the liver of the HFD mice and AML-12 cells. Moreover, further results indicated that metformin suppresses Gm15622 and alleviates NAFLD-associated lipid deposition in mice. In conclusion, we have identified an lncRNA Gm15622/miR-742-3p/SREBP-1c regulatory circuit associated with NAFLD in mice, a finding that significantly advances our insight into how lipid metabolism and accumulation are altered in this metabolic disorder. Our results also suggest that Gm15622 may be a potential therapeutic target for managing NAFLD. Excessive lipid deposition is a hallmark of NAFLD. Although much has been learned about the enzymes and metabolites involved in NAFLD, few studies have focused on the role of long noncoding RNAs (lncRNAs) in hepatic lipid accumulation. Here, using in vitro and in vivo models of NAFLD, we found that the lncRNA Gm15622 is highly expressed in the liver of obese mice fed a HFD and in murine liver (AML-12) cells treated with free fatty acids. Investigating the molecular mechanism in the liver-enriched expression of Gm15622 and its effects on lipid accumulation in hepatocytes and on NAFLD pathogenesis, we found that Gm15622 acts as a sponge for the microRNA miR-742-3p. This sponging activity increased the expression of the transcriptional regulator SREBP-1c and promoted lipid accumulation in the liver of the HFD mice and AML-12 cells. Moreover, further results indicated that metformin suppresses Gm15622 and alleviates NAFLD-associated lipid deposition in mice. In conclusion, we have identified an lncRNA Gm15622/miR-742-3p/SREBP-1c regulatory circuit associated with NAFLD in mice, a finding that significantly advances our insight into how lipid metabolism and accumulation are altered in this metabolic disorder. Our results also suggest that Gm15622 may be a potential therapeutic target for managing NAFLD. alanine aminotransferase aspartate aminotransferase Cell Counting Kit-8 long noncoding RNA normal diet total cholesterol NAFLD is a metabolic syndrome with a widespread histological distribution, from simple hepatic steatosis to NASH with or without fibrosis. NASH may eventually develop into cirrhosis and hepatocellular carcinoma (1Bessone F. Razori M.V. Roma M.G. Molecular pathways of nonalcoholic fatty liver disease development and progression.Cell. Mol. Life Sci. 2019; 76: 99-128Crossref PubMed Scopus (241) Google Scholar). A World Health Organization survey found that the number of obese people in the world has nearly tripled since 1975 (https://www.who.int/health-topics/obesity#tab=tab_1). With the rising rate of obesity and type 2 diabetes, NAFLD has become one of the most prominent liver diseases in the world, with an incidence rate of 25% (2Dai W. Ye L. Liu A. Wen S.W. Deng J. Wu X. Lai Z. Prevalence of nonalcoholic fatty liver disease in patients with type 2 diabetes mellitus: a meta-analysis.Medicine (Baltimore). 2017; 96: e8179Crossref PubMed Scopus (149) Google Scholar). The highest incidence rate is in the Middle East and South America at between 30% and 35%, followed by Asia, where the incidence is between 25% and 30% (3Younossi Z. Anstee Q.M. Marietti M. Hardy T. Henry L. Eslam M. George J. Bugianesi E. Global burden of NAFLD and NASH: trends, predictions, risk factors and prevention.Nat. Rev. Gastroenterol. Hepatol. 2018; 15: 11-20Crossref PubMed Scopus (2385) Google Scholar). The major causes of NAFLD include the metabolic diseases associated with type 2 diabetes, genetic predisposition, environmental factors, and gender (3Younossi Z. Anstee Q.M. Marietti M. Hardy T. Henry L. Eslam M. George J. Bugianesi E. Global burden of NAFLD and NASH: trends, predictions, risk factors and prevention.Nat. Rev. Gastroenterol. Hepatol. 2018; 15: 11-20Crossref PubMed Scopus (2385) Google Scholar). Therefore, timely intervention of NAFLD is of critical importance for alleviating the public health burden. Lipids obtained from the diet or synthesized by the liver and stored in adipose tissue are in an equilibrium state in terms of synthesis and energy utilization (4Nguyen P. Leray V. Diez M. Serisier S. Le Bloc'h J. Siliart B. Dumon H. Liver lipid metabolism.J. Anim. Physiol. Anim. Nutr. (Berl.). 2008; 92: 272-283Crossref PubMed Scopus (520) Google Scholar). Chylomicrons transport food lipids to the liver either via the low density lipoprotein receptor or in the form of FFAs that enter liver cells via CD36 (5Kennedy D.J. Kuchibhotla S.D. Guy E. Park Y.M. Nimako G. Vanegas D. Morton R.E. Febbraio M. Dietary cholesterol plays a role in CD36-mediated atherogenesis in LDLR-knockout mice.Arterioscler. Thromb. Vasc. Biol. 2009; 29: 1481-1487Crossref PubMed Scopus (45) Google Scholar). The de novo synthesis of lipids is mainly regulated by transcription factors, such as carbohydrate reactive element-binding protein and SREBP, which control the transcription levels of the rate-limiting enzyme, acetyl-CoA carboxylase, and FASN (6Dentin R. Pegorier J.P. Benhamed F. Foufelle F. Ferre P. Fauveau V. Magnuson M.A. Girard J. Postic C. Hepatic glucokinase is required for the synergistic action of ChREBP and SREBP-1c on glycolytic and lipogenic gene expression.J. Biol. Chem. 2004; 279: 20314-20326Abstract Full Text Full Text PDF PubMed Scopus (346) Google Scholar). Excessive lipids in the liver are transported out of the liver by ATP-binding cassette transporters. If this delicate balance is disrupted, lipid metabolism in the liver can be perturbed, leading to disease (7Wlcek K. Stieger B. ATP-binding cassette transporters in liver.Biofactors. 2014; 40: 188-198Crossref PubMed Scopus (28) Google Scholar, 8Liu Q. Bengmark S. Qu S. The role of hepatic fat accumulation in pathogenesis of non-alcoholic fatty liver disease (NAFLD).Lipids Health Dis. 2010; 9: 42Crossref PubMed Scopus (141) Google Scholar). Therefore, the regulation of lipid homeostasis is crucial. Recently, noncoding RNAs have been shown to be involved in the regulation of lipid homeostasis. Long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) are potential diagnostic markers and targets for the treatment of NAFLD (9Chen Y. Huang H. Xu C. Yu C. Li Y. Long non-coding RNA profiling in a non-alcoholic fatty liver disease rodent model: new insight into pathogenesis.Int. J. Mol. Sci. 2017; 18: E21Crossref PubMed Scopus (52) Google Scholar, 10Szabo G. Csak T. Role of microRNAs in NAFLD/NASH.Dig. Dis. Sci. 2016; 61: 1314-1324Crossref PubMed Scopus (65) Google Scholar). For example, lncRNA-ak012226 is related to lipid accumulation in NAFLD (11Chen X. Xu Y. Zhao D. Chen T. Gu C. Yu G. Chen K. Zhong Y. He J. Liu S. et al.LncRNA-AK012226 is involved in fat accumulation in db/db mice fatty liver and non-alcoholic fatty liver disease cell model.Front. Pharmacol. 2018; 9: 888Crossref PubMed Scopus (21) Google Scholar), while miR-150 deficiency can ameliorate hepatic steatosis and insulin resistance in NAFLD (12Zhuge B. Li G. MiR-150 deficiency ameliorated hepatosteatosis and insulin resistance in nonalcoholic fatty liver disease via targeting CASP8 and FADD-like apoptosis regulator.Biochem. Biophys. Res. Commun. 2017; 494: 687-692Crossref PubMed Scopus (13) Google Scholar). Furthermore, the lncRNA NEAT1/miR-140 axis exacerbates NAFLD by interrupting AMPK/SREBP-1 signaling (13Sun Y. Song Y. Liu C. Geng J. LncRNA NEAT1-MicroRNA-140 axis exacerbates nonalcoholic fatty liver through interrupting AMPK/SREBP-1 signaling.Biochem. Biophys. Res. Commun. 2019; 516: 584-590Crossref PubMed Scopus (27) Google Scholar). Thus, targeting functional lncRNAs and/or miRNAs is an effective strategy to treat NAFLD. Metformin is a first-line drug in the treatment of type 2 diabetes (14Sanchez-Rangel E. Inzucchi S.E. Metformin: clinical use in type 2 diabetes.Diabetologia. 2017; 60: 1586-1593Crossref PubMed Scopus (233) Google Scholar). In recent years, various studies have demonstrated that in addition to treating type 2 diabetes, it also has anti-cancer properties (15Coyle C. Cafferty F.H. Vale C. Langley R.E. Metformin as an adjuvant treatment for cancer: a systematic review and meta-analysis.Ann. Oncol. 2016; 27: 2184-2195Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar), can reverse pulmonary fibrosis, and can ameliorate NAFLD (16Gamad N. Malik S. Suchal K. Vasisht S. Tomar A. Arava S. Arya D.S. Bhatia J. Metformin alleviates bleomycin-induced pulmonary fibrosis in rats: pharmacological effects and molecular mechanisms.Biomed. Pharmacother. 2018; 97: 1544-1553Crossref PubMed Scopus (40) Google Scholar, 17Rouabhia S. Milic N. Abenavoli L. Metformin in the treatment of non-alcoholic fatty liver disease: safety, efficacy and mechanism.Expert Rev. Gastroenterol. Hepatol. 2014; 8: 343-349Crossref PubMed Scopus (35) Google Scholar). Although several studies have explored the molecular mechanism of metformin, its role in the pathological process of NAFLD needs to be further elucidated. In this study, we found that Gm15622 was significantly upregulated in the liver of HFD-induced obese mice. Upregulated Gm15622 significantly increased lipid accumulation, whereas silencing Gm15622 significantly reduced lipid accumulation in AML-12 cells. Mechanistically, Gm15622 positively modulated the expression of SREBP-1c by acting as a miRNA sponge for miRNA-742-3p. We also found that metformin could target Gm15622 to regulate lipid accumulation in HFD mice. Therefore, our study provides new insights into the molecular function of the Gm15622/miR-742-3p/SREBP-1c signaling pathway in the pathogenesis of NAFLD and highlights the potential of Gm15622 as a new therapeutic target for NAFLD. All animal procedures conformed to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH publication 85-23, revised in 1996) and the approved regulations set by the Laboratory Animal Care Committee at Nanjing Normal University (permit 2090658, issued 20 April 2008). Twenty-four male C57BL/6J mice (6–8 weeks old) were purchased from the Model Animal Research Center of Nanjing University (Nanjing, Jiangsu, China). The mice were housed in a room with a 12:12 h light/dark cycle and controlled and with free to and of mice were into were normal diet Research and were fed a HFD weeks mice were to mice with HFD with HFD with metformin and HFD with metformin Park Metformin and number and expression in the of obese PubMed Scopus Google Scholar). Metformin was in and to mice by for of were to control mice. was weeks of metformin the mice were for the of the mice were with an of 20 of liver were in and stored at for A of liver was in for and China). were from the of mice and into at were at for were and stored at for aminotransferase aspartate aminotransferase and total cholesterol levels in and in liver were using of Jiangsu, China). protein in cell were by the of AML-12 cells were in a of and with and For AML-12 cells were with 2 metformin in and FFAs of and of and of were in a and 20 and 20 were to the to of and for In the control of and were for AML-12 cells were at a density of cells in or at cells cell in a at Cell were using to the A and an of were and synthesized by China). Cell was in using a Counting Kit-8 Jiangsu, China). 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Gm15622 was liver-enriched and the HFD Gm15622 We that Gm15622 regulate involved in lipid the between Gm15622 expression and involved in lipid we a Gm15622 and synthesized a Gm15622 were into AML-12 cells and h and were to in and protein levels of lipid synthesis of Gm15622 for SREBP-1c was upregulated at and protein whereas FASN was upregulated at the of expression of SREBP-1c and FASN was at and protein levels Gm15622 can regulate the expression of SREBP-1c and FASN in we that Gm15622 was in the and we found that the Gm15622 a the also that SREBP-1c is a target gene for results indicated that Gm15622 regulate the expression of SREBP-1c by with miRNA-742-3p. Therefore, we the of in and AML-12 cells. The of was with the expression of Gm15622 or SREBP-1c we the Gm15622 or SREBP-1c the and the Gm15622 or SREBP-1c into the of the gene for of The that of reduced Gm15622 and SREBP-1c expression in the with that in the control and that this was the was the of to the Gm15622 or SREBP-1c further the between and the SREBP-1c was with and Gm15622 and by Gm15622 could the of SREBP-1c expression by with of Gm15622 increased the protein of SREBP-1c in AML-12 and this was by of Gm15622 protein levels of was by Therefore, results demonstrated that Gm15622 upregulated the expression of SREBP-1c by acting as a sponge of miR-742-3p. The expression levels of and FASN in AML-12 cells were by treatment with metformin, and The reduced and FASN with metformin the We explored how metformin SREBP-1c and the of metformin in AML-12 cells was to metformin that metformin at a of 2 or cell h We 2 metformin for h as a for AML-12 cells. We expression of and FASN of AML-12 cells with FFAs and 2 The of Gm15622 and the expression of SREBP-1c and FASN at and protein levels were while the of was increased in AML-12 cells with FFAs and metformin that metformin can the in lipid accumulation by FFAs metformin may lipid accumulation and expression of lipid in AML-12 cells by Gm15622 or miR-742-3p. the molecular mechanism by which metformin lipid accumulation through we the and synthesized a Gm15622 Gm15622 was in AML-12 cells for cells were treated with metformin for Gm15622 increased the expression of SREBP-1c and FASN at and protein and the of miR-742-3p. Metformin treatment the expression of SREBP-1c and FASN at and protein levels Gm15622 was in AML-12 cells Metformin treatment of Gm15622 AML-12 cells for h the expression of SREBP-1c and FASN at and protein and increased the of miR-742-3p. Metformin treatment could the expression of SREBP-1c and FASN at and protein levels Gm15622 was out in AML-12 cells results that metformin SREBP-1c and FASN expression levels in vitro by targeting the role of metformin in we mice that been fed a HFD for weeks and with of In the weeks of metformin the obesity of control mice In the of obese mice treated with metformin at was and a of metformin HFD-induced obesity the of metformin on obese mice, the lipid was of a HFD for weeks to in and levels while metformin such in a and are of liver and we found that metformin the HFD-induced in levels of and that metformin a that mice fed HFD of a lipid accumulation and hepatic while metformin and of liver also that metformin HFD-induced hepatic fat accumulation HFD significantly increased the of liver to and the hepatic levels of and liver were by mice a in hepatic expression levels of a in the of miR-742-3p. metformin in expression in a the regulatory role of metformin in lipogenic gene we protein levels in liver and found a to that of Metformin of the hepatic protein levels of SREBP-1c and FASN in a In conclusion, we found that metformin HFD-induced and hepatic lipid accumulation in mice. effects were by the Gm15622/miR-742-3p/SREBP-1c axis Excessive accumulation of lipids by lipid metabolism in the liver is the of NAFLD Q. Bengmark S. Qu S. The role of hepatic fat accumulation in pathogenesis of non-alcoholic fatty liver disease (NAFLD).Lipids Health Dis. 2010; 9: 42Crossref PubMed Scopus (141) Google Scholar). NAFLD causes hepatocellular carcinoma is also a of metabolic syndrome with type 2 diabetes, and (1Bessone F. Razori M.V. Roma M.G. Molecular pathways of nonalcoholic fatty liver disease development and progression.Cell. Mol. Life Sci. 2019; 76: 99-128Crossref PubMed Scopus (241) Google Scholar, W. Ye L. Liu A. Wen S.W. Deng J. Wu X. Lai Z. Prevalence of nonalcoholic fatty liver disease in patients with type 2 diabetes mellitus: a meta-analysis.Medicine (Baltimore). 2017; 96: e8179Crossref PubMed Scopus (149) Google Scholar). Therefore, it a clinical and burden on patients and Although the molecular mechanism of NAFLD has been lncRNAs can be as potential targets for the and treatment of NAFLD (9Chen Y. Huang H. Xu C. Yu C. Li Y. Long non-coding RNA profiling in a non-alcoholic fatty liver disease rodent model: new insight into pathogenesis.Int. J. Mol. Sci. 2017; 18: E21Crossref PubMed Scopus (52) Google Scholar, Y. J. Z. Li H. into the pathogenesis, and of nonalcoholic fatty liver Sci. 2018; Google Scholar). In the study, increased expression of the lncRNA Gm15622 in HFD-induced NAFLD was We found Gm15622 to be of sponging the of a transcription that the expression of that control fatty lipid and cholesterol of Gm15622 can of SREBP-1c hepatic lipid accumulation. Gm15622 can SREBP-1c expression and lipid accumulation in the and demonstrated that Gm15622 can regulate the axis to lipid accumulation in vitro and in the that Gm15622 acts as a and may as a therapeutic target for NAFLD. lncRNAs are involved in the regulation of a of and in lncRNA expression can be related to the development of various diseases T. S. of long non-coding RNAs in Biol. 2017; PubMed Scopus Google Scholar, M. J. J. Long noncoding RNAs in metabolic syndrome related 2016; PubMed Scopus Google Scholar). In and M. J. J. Long noncoding RNAs in metabolic syndrome related 2016; PubMed Scopus Google the lncRNAs that to type 2 diabetes, and as as of action and Long noncoding RNAs as a in hepatocellular 9: Scholar). studies have shown that such as and in the pathological process of NAFLD N. Y. Li and lipid in via 2018; Google Scholar, P. Huang Liu LncRNA as a in hepatic by to with 2019; Full Text Full Text PDF PubMed Scopus Google Scholar). Although lncRNAs have been identified as potential therapeutic targets in NAFLD, lipid accumulation is the most of NAFLD Y. J. Z. Li H. into the pathogenesis, and of nonalcoholic fatty liver Sci. 2018; Google Scholar, B. X. Huang Y. Z. Y. W. D. and non-coding gene regulatory the in liver 2017; Google Scholar). Therefore, for lncRNAs related to lipid accumulation is to the of NAFLD. In this study, we focused on which de novo synthesis of lipids by the expression of Liu et G. and hepatic by axis in non-alcoholic fatty liver found that of can liver fat by the In et X. Chen Zhao Li et pathway is for the regulation of cholesterol Res. 2014; Full Text Full Text PDF PubMed Scopus Google found that lipid by the expression of accumulation in the liver can be by either increased or the function of Gm15622 and the potential of Gm15622 for treating NAFLD, we to and we a Gm15622 of Gm15622 reduced the expression of while of Gm15622 the in vitro and we that Gm15622 can positively regulate SREBP-1c expression in a Thus, we that SREBP-1c is a target of Gm15622 in NAFLD. lncRNAs in the and have of which can be into long noncoding RNA and Sci. 2016; Full Text Full Text PDF PubMed Scopus Google Scholar). we to that Gm15622 is in the and the to that Gm15622 is an that Gm15622 may be involved in regulation in the studies have demonstrated that lncRNAs are involved in regulation by acting as miRNA to regulate the expression of miRNA targets C. Deng Q. Li Y. Huang T. S. of by in Res. 2017; Google Scholar, L. Liu N. of RNA as a for 2018; PubMed Scopus Google Scholar). We to that Gm15622 a target for and can with miR-742-3p. In SREBP-1c is a target gene of miR-742-3p. we found that of Gm15622 increased the expression of which was by of miR-742-3p. of Gm15622 reduced the expression of which was by of Therefore, we that Gm15622 acts as a in AML-12 cells to regulate SREBP-1c expression by sponging which may be a mechanism by which Gm15622 acts as a critical regulator in NAFLD. pathological have been for NAFLD E. F. fatty liver disease pathogenesis, and on drug enzymes and Rev. 2017; PubMed Scopus Google Scholar). for NAFLD are in the development and the most such as and are in clinical N. A. J. A. et or for nonalcoholic J. 2010; PubMed Scopus Google Scholar, V. S. P. P. L. M. J. M. S. et an of the and of nonalcoholic without fibrosis 2016; Full Text Full Text PDF PubMed Scopus Google Scholar). Metformin is the drug for treating diabetes it can also the pathological process of (15Coyle C. Cafferty F.H. Vale C. Langley R.E. Metformin as an adjuvant treatment for cancer: a systematic review and meta-analysis.Ann. Oncol. 2016; 27: 2184-2195Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar, N. Malik S. Suchal K. Vasisht S. Tomar A. Arava S. Arya D.S. Bhatia J. Metformin alleviates bleomycin-induced pulmonary fibrosis in rats: pharmacological effects and molecular mechanisms.Biomed. Pharmacother. 2018; 97: 1544-1553Crossref PubMed Scopus (40) Google Scholar). For example, Chen et S.D. Huang Metformin of cell carcinoma via a signaling 2017; PubMed Scopus Google found that metformin can be as a new drug for cell and et C. S. He Z. X. Chen R. Chen J. a first-line drug for type 2 diabetes the sponge to and in J. Pharmacol. 2018; PubMed Scopus Google that metformin can and of cells by the Metformin treatment also HFD-induced hepatic steatosis S. Milic N. Abenavoli L. Metformin in the treatment of non-alcoholic fatty liver disease: safety, efficacy and mechanism.Expert Rev. Gastroenterol. Hepatol. 2014; 8: 343-349Crossref PubMed Scopus (35) Google Scholar), in et Zhao L. Xu of metformin with on lipid accumulation and metabolism in mice fed with 2018; PubMed Scopus Google found that metformin with the expression of and in HFD mice. studies the potential of metformin for NAFLD treatment and that the molecular mechanism of metformin action in NAFLD Metformin can the and of by lncRNA C. S. He Z. X. Chen R. Chen J. a first-line drug for type 2 diabetes the sponge to and in J. Pharmacol. 2018; PubMed Scopus Google metformin may a role in NAFLD by targeting In this study, we that metformin can liver lipid accumulation and and in HFD mice, we also found that Gm15622 can be in NAFLD by a new molecular mechanism for metformin in the treatment of NAFLD. The liver is the most metabolic and has effects The liver is a metabolic and a of metabolic to in of metabolism 2017; PubMed Scopus (21) Google and Gm15622 plays an role in The of lncRNAs as of gene expression has altered our of the pathological of NAFLD. In Gm15622 is an that lipid metabolism in the results how Gm15622 lipid accumulation in the liver by targeting the also that Gm15622 is a target for metformin treatment of NAFLD. studies are required in and patients to the between Gm15622 and NAFLD disease The from for the of a of this with

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