Study On Long Non-codiog RNA MEG3 Regulating Liver Gluconeogenesis Through CeRNA Mechanism

Background:The prevalence of diabetes and its complications poses a serious threat to global health.About 425 million adults worldwide have diabetes,and more than 90%of them are type 2 diabetes mellitus(T2DM).Studying the pathogenesis of diabetes and finding effective diabetes control methods are still the focus of current diabetes research.The liver is one of the main target organs for the regulation of insulin and energy metabolism in the body.It controls the release of hepatic glucose by controlling gluconeogenesis and glycogenolysis,and plays an important role in maintaining plasma glucose homeostasis in this way.One of the important pathological basis of T2DM is the abnormal increase of glucose output caused by hepatic gluconeogenesis disorder.Long non-coding RNAs(lncRNAs)are defined as transcripts greater than 200 nucleotides in length and thought to be without any coding potential.LncRNAs play a major role in the regulation of cell metabolism,and their discovery opens up new areas for studying the molecular mechanisms of diabetes.Studies have shown that lncRNAs are involved in many pathophysiological processes that regulate diabetes,such as affecting islet function,playing a role in liver metabolism,regulating glucose metabolism and differentiation in adipose tissue,and so on.However,studies on lncRNAs in diabetes have not received sufficient attention.Aberrant expression of only a small number of IncRNAs in diabetes has been detected so far,and the role of fewer IncRNAs in the development of diabetes has been explored.Therefore,it is a very valuable work to explore the correlation between IncRNAs and the occurrence and development of diabetesAbnormal expression of maternally expressed gene 3(MEG3)plays an important role in the development of T2DM.Our previous study have shown that IncRNA MEG3 overexpression promotes insulin resistance in the liver via increased the expression of forkhead transcription factor 1(FoxOl)and downstream gene glucose-6-phosphatase(G6pc)and phosphoenolpyruvate carboxykinase(PEPCK).However,the specific molecular mechanism of MEG3 promoting gluconeogenesis is still unclear.Therefore,we explored the regulation mechanism of MEG3 on the FoxO1/PEPCK/G6pc signaling pathway in gluconeogenesis in this study.In addition,phosphorylated cAMP-response element binding protein(CREB)activates peroxisome proliferators-activated receptor-? coactivator-1?(PGC-1?),and then activated PGC-la activates the transcription of PEPCK and G6pc,thus enhancing glycogenesis.We also investigated the molecular mechanism by which MEG3 regulates the PGC-1?/PEPCK/G6pc signaling pathway.To explore the regulation and mechanism of MEG3 on hepatic gluconeogenesis,enrich the molecular pathological mechanism of hepatic insulin resistance,and provide a theoretical basis for exploring effective targets for prevention and treatment of hepatic insulin resistancePart ? LncRNA MEG3 promotes hepatic insulin resistance by serving as a competing endogenous RNA of miR-214 to regulate ATF4 expressionObjectives:Many studies have shown that IncRNA can act as a competing endogenous RNA(ceRNA)for certain miRNA to inhibit miRNA's targeted suppression of its target genes.Our previous studies showed that MEG3 can upregulate the activities of FoxO1 and its downstream key gluconeogenesis enzymes,PEPCK and G6pc,thereby promoting liver gluconeogenesis.Studies have shown that activating transcription factor 4(ATF4)can promote the transcriptional activity of FoxO1 to promote gluconeogenesis.Bioinfonnatics analysis found that the partial sequences of miR-214 and MEG3 are complementary,and ATF4 is a potential target gene of miR-214.Therefore,we hypothesized that MEG3 serves as a competing endogenous RNA of miR-214 to facilitate ATF4 expression,resulting in the promotion of FoxO1 expression and its downstream gluconeogenic enzymes G6pc and PEPCK,thereby increasing gluconeogenesis and promoting insulin resistance.Methods:Male C57BL/6 mice were fed a high-fat diet(HFD)or a low-fat diet(LFD)to induce insulin resistance.qRT-PCR was used to detect the expression of MEG3,miR-214,ATF4,FoxO1,PEPCK,and G6pc in mouse liver tissue or primary mouse hepatocytes,and Western blot was used to detect protein levels of ATF4,FoxO1,PEPCK and G6pc in liver tissues and primary liver cells.By transfecting small interfering RNA(siRNA),pcDNA3.1 plasmid,inhibitor or mimic,the regulatory mechanism between MEG3 and miR-214 and ATF4 was further explored.The luciferase reporter system detected the targeting relationship between MEG3 and miR-214,and between miR-214 and ATF4.Si-MEG3 was mediated by lentivirus injection into the tail vein of high-fat diet mice for the purpose of analyzing whether lncRNA MEG3 targeted miR-214 to release its inhibitory effect on ATF4,thereby promoting FoxO1 and its downstream genes expression.Results:1.The expression levels of MEG3 and ATF4 in HFD-fed mice and palmitate-stimulated hepatocytes were up-regulated,while the expression of miR-214 was down-regulated.2.MEG3 served as a ceRNA of miR-214 to facilitate ATF4 expression Overexpression of MEG3 and inhibition of miR-214 upregulated the expression level of MEG3,downregulated the expression level of miR-214,and upregulated the expression level of ATF4 at both messenger RNA(message RNA,mRNA)and protein levels.Conversely,inhibition of MEG3 and overexpression of miR-214 downregulated the expression level of MEG3,upregulated the level of miR-214 and downregulated the expression level of ATF4 at both mRNA and protein levels.MiR-214 mimic's down-regulation of ATF4 expression can be attenuated by MEG3 overexpression,and MEG3's up-regulation of ATF4 expression can be attenuated by miR-214 mimic.In addition,the luciferase reporter gene experiment results showed that miR-214 mimic significantly reduced the luciferase activity in the MEG3-wild-type(Wt)group,but had no significant effect on the luciferase activity in the MEG3-mutated(Mut)group,which confirmed the direct binding between MEG3 and miR-214.Moreover,miR-214 mimic significantly reduced the luciferase activity in the ATF4-Wt group,but had no significant effect on the luciferase activity in the ATF4-Mut group,indicating that ATF4 was the target gene of miR-214.The above results indicated that MEG3 can be served as a ceRNA of miR-214 in combination with miR-214 to upregulate the expression of ATF43.Knocking down MEG3 down-regulated the mRNA and protein levels of FoxO1 and FoxO1 downstream genes PEPCK and G6pc,and this effect could be attenuated by miR-214 inhibitor and ATF4 overexpression.4.In high-fat diet mice,the interference of MEG3 can significantly inhibit liver triglyceride accumulation and restore liver glycogen levels.And the interference of MEG3 can improve the fasting blood glucose level and impaired glucose tolerance,as well as the insulin resistance of high-fat diet mice.In addition,the interference of MEG3 in vivo up-regulated the expression of miR-214 in mouse liver tissue,but down-regulated the expression of ATF4.Conclusions:MEG3 relieves the inhibition of ATF4 by targeting miR-214,thereby promoting the expression of FoxO1 and its downstream genes PEPCK and G6pc,which promotes liver insulin resistance and participates in the occurrence of diabetes.Part ? CREB-upregulated lncRNA MEG3 promotes hepatic gluconeogenesis by regulating miR-302a-3p-CRTC2 axisObjectives:Glucagon stimulates the activation of protein kinase A(PKA),thereby activating and phosphorylating the transcription factor cAMP-response element binding protein(CREB),and phosphorylated CREB activates PGC-la,and activated PGC-la activates the transcription of PEPCK and G6pc,which enhances gluconeogenesis CREB-regulated transcripti on coactivator 2(CRTC2)belongs to the CRTC family and can be combined with the basic leucine zipper structure of CREB to regulate its transcription factor activity.Studies have shown that CREB can bind to the cAMP response element on the MEG3 gene promoter to promote the expression of MEG3 The results of bioinformatics analysis showed that the partial sequences of miR-302a-3p and MEG3 were complementary,and CRTC2 was a potential target gene of miR-302a-3p.Therefore,we speculate that in the process of liver gluconeogenesis,CREB binds to MEG3 promoter to promote MEG3 transcription,and MEG3 competitively binds miR-302a-3p,thereby promoting the expression of miR-302a-3p target gene CRTC2.Methods:We simulated the pathological state of gluconeogenesis in primary hepatocytes isolated from C57BL/6J mice and divided the primary hepatocytes of mice into four groups:control,glucagon,glucagon+H89,a selective blocker of PKA,and glucagon+2-naphthol AS-E phosphate(KG-501),a specific CREB inhibitor.SiRNA oligos specific to CREB(si-CREB),siRNA oligos specific to MEG3(si-MEG3),siRNA oligos specific to PGC-1?(si-PGC-1?),siRNA oligos specific to CRTC2(si-CRTC2),pcDNA3.1-CREB plasmid(CREB),pcDNA3.1-MEG3 plasmid(MEG3),pcDNA3.1-CRTC2 plasmid(CRTC2),miR-302a-3p mimic and miR-302a-3p inhibitor were transfected into mouse primary hepatocytes.Expressions of MEG3,miR-302a-3p,CRTC2,PKA,CREB,PGC-1?,PEPCK and G6pc were determined by qRT-PCR and Western blot analysis.The association among MEG3,miR-302a-3p,and CRTC2 was disclosed by luciferase reporter assay.Results:1.MEG3 was highly expressed in high glucagon-treated mouse primary hepatocytes2.Glucagon stimulated the activation of PKA,thereby activating CREB,which combined with the promoter of MEG3 to promote the transcription of MEG3Glucagon time-dependently led to an increase in p-PKA and p-CREB protein expression levels as well as a decrease in cell viability.H89 and KG-501 were effective in inhibiting high glucagon-treated elevation of MEG3 expression.Silencing CREB significantly reduced the expression of CREB and MEG3,whereas overexpression of CREB resulted in the opposite effect.Luciferase reporter gene assay results indicated that wild-Luc yielded a much higher promoter activity in primary hepatocytes compared with CREB-Luc reporter activities.qRT-PCR results showed that CREB inhibition suppressed the gene expression of MEG3 in primary hepatocytes treated with high glucagon.3.CREB-induced MEG3 upregulation mediated PGC-la-PEPCK/G6Pc pathway in hepatic gluconeogenesisMEG3 knockdown reduced the protein levels of gluconeogenesis genes PGC-1?,PEPCK and G6pc in high glucagon-induced mouse primary hepatocytes,whereas MEG3 overexpression significantly increased the protein levels of PGC-la,PEPCK and G6pc.Treatment with H89,KG-501,si-CREB or si-PGC-la significantly decreased the protein levels of these gluconeogenesis genes induced by MEG3 overexpression.4.MEG3 served as a ceRNA to upregulate CRTC2 by targeting miR-302a-3pHigh glucagon treatment could time-dependently elevate CRTC2 expression at protein levels and decrease the expression of miR-302a-3p in mouse primary hepatocytes.Luciferase experiment results showed that miR-302a-3p mimic significantly reduced the luciferase activity in the MEG3-Wt group,but had no significant effect on the luciferase activity in the MEG3-Mut group,which confirmed the direct binding between MEG3 and miR-302a-3p.In addition,miR-302a-3p mimic can also significantly reduce the luciferase activity in the CRTC2-Wt group,but has no significant effect on the luciferase activity in the CRTC2-Mut group,which indicated that CRTC2 was the target gene of miR-302a-3p.In addition,the overexpression of MEG3 led to a decrease in the expression of miR-302a-3p and an enhancement in CRTC2 expression at mRNA and protein levels.On the contrary,MEG3 knockdown reversed this trend.MiR-302a-3p mimic up-regulated the expression of miR-302a-3p,but down-regulated the expression of MEG3 and CRTC2,while miR-302a-3p inhibitor produced the opposite result.MEG3 overexpression overturned the regulatory effect of miR-302a-3p mimic on CRTC2 and attenuated the knockdown effect of si-CRTC2 on CRTC2.5.MEG3 mediated PGC-1?/PEPCK/G6Pc pathway by regulating miR-302a-3p/CRTC2 axis in hepatic gluconeogenesisMiR-302a-3p mimic and si-CRTC2 attenuated the induction of glucagon on PGC-1?,PEPCK and G6pc protein levels in mouse primary hepatocytes,while MEG3 overexpression overturned the inhibitory effect of miR-302a-3p overexpression and CRTC2 inhibition.Conclusions:Glucagon stimulates PKA activation,thereby activating CREB.CREB binds to the promoter of MEG3 to promote transcription of MEG3.MEG3 served as a ceRNA to upregulate CRTC2 by targeting miR-302a-3p,thereby promoting the expression of the miR-302a-3p target gene CRTC2,thereby increasing PGC-1?/PEPCK/G6pc pathway and promoting gluconeogenesis.