| Objective: Type 2 diabetes mellitus(T2DM)is a systemic disease caused by insulin resistance in the body or decreased insulin secretion from pancreatic beta cells.Chronic complications such as macrovascular and microvascular caused by hyperglycemia have seriously affected human health.At present,studies have suggested that genetic factors,environmental factors and lifestyle are closely related to the occurrence and development of T2 DM,but how these factors play a role has not been determined.Recent studies have shown that long non-coding RNAs(lnc RNAs),as regulatory molecules,play an important role in the occurrence and development of many diseases,such as tumor,Alzheimer’s disease and diabetes mellitus.Compared with tumors,there were relatively few studies on the role of lnc RNA in the occurrence and development of T2 DM.Gene microarray was used to screen and study the differentially expressed lnc RNA and m RNA in peripheral blood leukocytes of T2 DM and healthy control groups,and the differentially expressed profiles of lnc RNA between the two groups were obtained and further verified in clinical samples.We further investigated its correlation with T2 DM related clinical metabolic indicators,and explored its regulation on islet β cell apoptosis,insulin secretion and insulin signaling pathway.This study provides some ideas and basis for the further study of the regulatory role of lnc RNA in the pathogenesis of T2 DM.Methods:1.Gene chip test: Differential expression profiles of lnc RNA and m RNA in peripheral blood leukocytes of T2 DM and healthy controls were obtained by gene microarray assay.Gene Ontology and KEGG Pathway were used to analyze the biological processes,cellular components,molecular functions and signal pathways of differentially co-expressed m RNA.2.Clinical sample verification: Quantitative real-time PCR(q RT-PCR)was used to detect the lnc RNA with obvious differential expression for further verification in newly diagnosed T2 DM patients and healthy controls.The results showed that the expression of lnc RNA HIST1H2AG-6 and lnc RNA AIM1-3 in peripheral blood was consistent with the expression of gene microarray.And the two genes were selected as the study subjects,and their expression levels in peripheral blood leukocytes of T2 DM patients and control group were detected by q RT-PCR.The correlations between the expression levels of the two genes and the metabolic indexes of T2 DM were analyzed.ROC curve and multiple regression analysis were used to analyze the clinical value of abnormal expression of lnc RNA HIST1H2AG-6 and lnc RNA AIM1-3 in the diagnosis of type 2diabetes and the prediction of the risk of diabetes.3.Regulation of lnc RNA HIST1H2AG-6 on islet β cell apoptosis,insulin secretion and insulin signaling pathway: Bioinformatics software was used to analyze the differential expression m RNA associated with lnc RNA HIST1H2AG-6 and lnc RNA AIM1-3,and lnc RNA-m RNA interaction network was constructed.Its potential enrichment signal pathway and biological process were explored.lnc RNA HIST1H2AG-6 overexpression plasmid was constructed and transfected into MIN6 cell line of mouse insulinoma.The effect of overexpression of lnc RNA HIST1H2AG-6 transfected on apoptosis and proliferation of MIN6 cells was analyzed by flow cytometry and CCK-8.Enzyme linked immunosorbent assay(ELISA)was used to detect the response of MIN6 cells to glucose-stimulated insulin secretion(GSIS)after overexpression of lnc RNA HIST1H2AG-6.Meanwhile,q RT-PCR and Westernblot were used to detect the expression of lnc RNA HIST1H2AG-6 on MaFa,PDX-1 and GLUT-2 in the nucleic acid and protein levels of MIN6 cells.Then a lentiviral vector overexpressing lnc RNA HIST1H2AG-6 was constructed to infect mouse islet β cells.The effect of overexpression of lnc RNA HIST1H2AG-6 on apoptosis and proliferation of islet βcells in mice was detected by flow cytometry.The response of mouse islet β cells to GSIS after overexpression of lnc RNA HIST1H2AG-6 was detected by ELISA.At the same time,q RT-PCR and Westernblot were used to detect the expression of overexpressed lnc RNA HIST1H2AG-6 on MaFa,PDX-1 and GLUT-2 in pancreatic beta cells at the nucleic acid and protein levels.Finally,the overexpressed lnc RNA HIST1H2AG-6 plasmid was transfected into MIN6 cells,and the levels of PKA,p-PKA,JNK,p-JNK,ERK,p-ERK,P38,p-p38 were measured by Westernblot.The overexpression of lnc RNA HIST1H2AG-6 inhibited the expression of p-PKA and p-JNK phosphorylation products.After the administration of c AMP/PKA agonist Forksin and JNK agonist Anisomycin,the apoptosis of MIN6 cells was detected by flow cytometry and glucose-stimulated insulin secretion was detected by ELISA.And then the expression of insulin transcriptional regulator MaFa,PDX-1 and GLUT-2 was detected by Westernblot.Results:1.The expression profiles of lnc RNA differentially expressed in peripheral blood leukocytes between newly diagnosed T2 DM patients and healthy controls were screened by lnc RNA microarray detection,and a total of 55 lnc RNAs and 36 m RNAs with differential expression(fold change ≥ 2 and P < 0.05)were obtained.GO analysis results showed that the highest enrichment of GO targets was related to programmed apoptosis,cell response to mechanical stimulation,cell membrane depolarization,phosphorylation process and negative regulation of insulin receptor signaling pathway.KEGG Pathway analysis showed that the signaling pathways of lnc RNA-related m RNA transcripts were mainly concentrated in starch and sucrose metabolism pathways,tumor-related signaling pathways and NOD-like receptor signaling pathways.2.Verification by q RT-PCR in clinical samples showed that the expression of lnc RNA HIST1H2AG-6 was increased in the T2 DM group(P =0.002),and the expression of lnc RNA AIM1-3 was decreased in the T2 DM group(P < 0.001),which was consistent with the expression of gene chip detection.And then the relationship between the differential expression of lnc RNA HIST1H2AG-6,lnc RNA AIM1-3 and various metabolic indexes of diabetes mellitus was analyzed.(1)Correlation analysis showed that lnc RNA HIST1H2AG-6 was positively correlated with LDL-C and UA(P = 0.026,P = 0.021),and negatively correlated with HOMA-β(P = 0.039).lnc RNA AIM1-3 was positively correlated with FIN,HOMA-β and HDL-C(P = 0.003,P < 0.001,P < 0.001 respectively),and was negatively correlated with FPG and Hb A1c(both P <0.001).(2)Multiple regression analysis showed that BMI,LDL-C,HDL-C,lnc RNA HIST1H2AG-6 and lnc RNA AIM1-3 were significantly correlated with T2 DM.The expression of lnc RNA AIM1-3 was negatively correlated with T2DM(β =-2.54,OR =0.071,95% CI 0.029 ～ 0.178,P < 0.001),and the expression of lnc RNA HIST1H2AG-6 was positively correlated with T2DM(β =1.756,OR = 5.791,95% CI2.275 ～ 14.739,P <0.001).(3)ROC curve analysis showed that the AUC of lnc RNA HIST1H2AG-6 was 0.664(95%CI 0.549 ～ 0.780,P = 0.007),and that of lnc RNA AIM1-3 was 0.769(95% CI 0.662～0.875,P <0.001).3.Analysis of m RNA co-expressed with lnc RNA HIST1H2AG-6 and lnc RNA AIM1-3showed that related m RNA were enriched in multiple KEGG pathways including starch and sucrose metabolism,NOD receptor signaling pathways,and MAPK signaling pathways,and enriched in GO pathways including cell metabolism,immune response and signal transduction.Among them,starch and sucrose metabolism and MAPK signaling pathway are closely related to the incidence of T2 DM.4.Regulation of overexpression of lnc RNA HIST1H2AG-6 on MIN6 cells(1)Overexpression of lnc RNA HIST1H2AG-6 transfected plasmid in MIN6 cells promoted apoptosis and decreased proliferation of MIN6 cells compared with the control group.(2)Westernblot results showed that the expression of proapoptotic gene cleaved caspase 3was up-regulated,while the expression of antiapoptotic gene Bcl-2 was down-regulated.(3)In the GSIS test,high concentration glucose stimulated insulin secretion of MIN6 cells,and overexpression of lnc RNA HIST1H2AG-6 decreased insulin secretion capacity of MIN6 cells to glucose stimulation.(4)Overexpression of lnc RNA HIST1H2AG-6 caused decreased expression of insulin transcription regulator gene PDX-1 and GLTU-2 at both nucleic acid and protein levels.5.Regulation of lnc RNA HIST1H2AG-6 overexpression on islet β cells in mice(1)Overexpression of lnc RNA HIST1H2AG-6 promotes apoptosis of primary pancreatic islet β cells in mice(P < 0.05).(2)Overexpression of lnc RNA HIST1H2AG-6 significantly decreased insulin secretion in islet β cells in both WT and db/db mice(P < 0.05,P < 0.01).(3)In both WT and db/db groups,overexpression of lnc RNA HIST1H2AG-6 decreased the expression of MaFa,PDX-1 and GLUT-2 at both nucleic acid and protein levels,and the inhibition was more obvious in db/db mice(P < 0.01,P < 0.001).6.Regulation of lnc RNA HIST1H2AG-6 on diabetes-related signaling pathway(1)Lnc RNA HIST1H2AG-6 inhibited the production of p-PKA and p-JNK,the phosphorylation products of PKA and JNK(P < 0.05,P < 0.01).(2)PKA and JNK agonists can partially reverse the decrease in insulin secretion caused by the overexpression of lnc RNA HIST1H2AG-6.Forskolin attenuates the inhibition of overexpression of lnc RNA HIST1H2AG-6 on MaFa,PDX-1 and GLUT-2 protein levels.Anisomycin partially reversed the MIN6 apoptosis induced by lnc RNA HIST1H2AG-6.Conclusion:1.The differential expression profiles of lnc RNA and m RNA in peripheral blood leukocytes of T2 DM patients and healthy controls were analyzed and screened by gene microarray technology,which helped to better understand the pathogenesis of T2 DM from the perspective of long non-coding genes.2.Go and KEGG Pathway analysis were performed on the m RNA co-expressed with differential lnc RNAs,which laid a theoretical foundation for inferencing the biological functions of lnc RNAs associated with T2 DM and the signaling pathways that may be involved in.3.Two long non-coding RNAs(HIST1H2AG-6 and AIM1-3)with differential expression were screened and obtained,which were closely related to T2 DM and have certain diagnostic values for T2 DM.4.Lnc RNA HIST1H2AG-6 may be involved in the regulation of glucose metabolism by promoting the apoptosis of pancreatic β cells,down-regulating the expressions of MaFa,PDX-1 and Gl UT-2,and negatively regulating GSIS.These effects may be achieved by inhibiting JNK and c AMP/PKA signaling pathways. |
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