| Objective:(1)To detect differentially expressed lncRNAs and mRNAs of type 2 diabetes mellitus by using gene chip and bioinformatics technique and explore the role of lncRNAs in the pathogenesis of type 2 diabetes mellitus.(2)Screened the specific high expression of lnc RNAs according to FC(abs)value>2,P≤0.01.Finally,the MEG3、MALAT1 and MIAT gene were selected in this study on the basis of published studies.(3)The deep understanding of endothelial lncRNAs may provide novel biomarkers or therapeutic targets for diabetic complications.Therefore,it is important to increase our understanding of the underlying molecular mechanisms of diabetes-induces endothelial dysfunctions and identify novel potential therapeutic targets for preventing or reducing diabetic complications.Till now,there has been no study revealing the underlying molecular mechanisms associated with MEG3 induction and how it could play a key role in high glucose-induces HUVCEs dysfunction.Therefore,the aim of the present study was to investigate whether MEG3 was potential regulators and molecular biomarker of high glucose-induced endothelial dysfunction.Methods:(1)5 patients with type 2 diabetes mellitus and 5 non-type 2 diabetes mellitus were selected,then drew the blood,blood samples were extracted to RNA.The expression of lncRNAs and mRNAs were analyzed by gene microarray.Lnc RNAs and mRNAs expression profiles were analyzed by bioinformatics tools,including Pathway Disease and gene ontology(GO)analysis.The differentially expressed lnc RNAs in peripheral blood was detected.Moreover,we further analyzed the relevant pathways regulated by differentially expressed genes,as well as lncRNAs participating in the development of type 2 diabetes mellitus.The gene co-expression network was established so as to predict the biological function may be involved in the differential expression of 1ncRNAs.(2)To mensure the expression level of MEG3、MALAT1 and MIAT gene in peripheral blood of type 2 diabetes mellitus and non-type 2 diabetes mellitus,to analyze the association with the risk factors of type 2 diabetes mellitus,to investigate the potential significance in the diagnosis of type 2 diabetes mellitus.In the present studies,200 type 2 diabetes mellitus and 200 non-type 2 diabetes mellitus were enrolled.The expression levels of MEG3、MALAT1 and MIAT gene were qualified by quantitative real-time polymerase chain reaction(qRT-PCR).A receiver operating characteristic(ROC)curve was adopted to explore the value of the expression of MEG3、MALAT1 and MIAT gene in the diagnosis of type 2 diabetes mellitus.(3)Lentivirus vector of MEG3-specific small interfering RNA(siRNA)and scrambled(Scr)siRNA were transfected into HUVECs.qRT-PCR was used to detect the expression of inflammatory cytokines,TGFβ signaling pathway and Wnt/β-catenin signaling pathway related genes.The percentage of apoptotic cells was measured by flow cytometry.Cell viability was determined through MTT assay.Detection of apoptosis related protein,TGFβ signaling pathway and Wnt/β-catenin signaling pathway related proteins by Western blot assay.Results:(1)A total of 68 lncRNAs were screened,which 44 were up-regulated and 24 were down-regulated in peripheral blood of type 2 diabetes mellitus.A total of 74 mRNAs were screened,which 56 were up-regulated and 18 were down-regulated in peripheral blood of type 2 diabetes mellitus.(2)Signal pathway enrichment contained synaptic vesicle cycle、ovarian steroidogenesis、micro RNAs in cancer、metabolism of xenobiotics by cytochrome P450 and chemical carcinogenesis.Enrichment of the disease included keutel syndrome、 hereditary mixed polyposis syndrome、other humoral immunodeficiencies、cerebrotendinous xanthomatosis、retinitis pigmentosa(RP).(3)In biological process,GO Term enrichment in the differentially expressed mRNAs may involve in detection of visible light、detection of light stimulus、regulation of bone mineralization、regulation of biomineral tissue development and anatomical structure arrangement.In cellular component,GO Term enrichment in the differentially expressed mRNAs may involve in endocytic vesicle membrane、cytoplasmic vesicle membrane、vesicle membrane、neuron projection and neuron part.In molecular function,GO Term enrichment in the differentially expressed mRNAs may involve in lipoprotein transporter activity、cholesterol 26-hydroxylase activity、 cholestanetriol 26-monooxygenase activity、tubulin-glutamic acid ligase activity and structural constituent of bone.(4)The expression level of MEG3 was significantly lower in T2 DM group than in control group,which showed a significant difference(P<0.001).The expression level of MALAT1 was significantly higher in T2 DM group than in control group,which showed a significant difference(P<0.001).The expression level of MIAT was significantly higher in T2 DM group than in control group,which showed a significant difference(P<0.001).(5)For total participants and male of T2 DM group,the plasma glucose levels was lower in MEG3 high expression group than in MEG3 low expression group,which showed a significant difference(P<0.05);For female of control group,the plasma glucose levels was lower in MEG3 high expression group than in MEG3 low expression group,which showed a significant difference(P<0.05).For total participants,male and female of T2 DM group,the plasma glucose levels was higher in MALAT1 high expression group than in MALAT1 low expression group,which showed a significant difference(P<0.05);For total participants,female of control group,the plasma glucose levels was higher in MALAT1 high expression group than in MALAT1 low expression group,which showed a significant difference(P<0.05).For total participants,male and female of T2 DM group,the plasma glucose levels was higher in MIAT high expression group than in MIAT low expression group,which showed a significant difference(P<0.05);For male of T2 DM group,the DBP levels was lower in MIAT high expression group than in MIAT low expression group,which showed a significant difference(P<0.05);For female of control group,the plasma glucose levels was higher in MIAT high expression group than in MIAT low expression group,which showed a significant difference(P<0.05).(6)For total participants and male of T2 DM group,the expression levels of MEG3 was significantly negative correlated with the plasma glucose levels,which showed a significant difference(P<0.05);For female of T2 DM group,the expression levels of MEG3 was significantly positive correlated with the plasma LDL levels,which showed a significant difference(P<0.05);For total participants and female of T2 DM group,the expression levels of MALAT1 was significantly positive correlated with the plasma glucose,TC,LDL levels,which showed a significant difference(P<0.05);For male of T2 DM group,the expression levels of MALAT1 was significantly positive correlated with the plasma glucose levels,which showed a significant difference(P<0.05).For total participants of control group,the expression levels of MALAT1 was significantly positive correlated with the plasma glucose levels,which showed a significant difference(P<0.05);For female of control group,the expression levels of MALAT1 was significantly positive correlated with the plasma glucose and SBP levels,which showed a significant difference(P<0.05);For total participants,male and female of T2 DM group,the expression levels of MIAT was significantly positive correlated with the plasma glucose levels,which showed a significant difference(P<0.05);For total participants and male of control group,the expression levels of MIAT was significantly positive correlated with the SBP levels,which showed a significant difference(P<0.05).(7)The area under the receiver operating characteristic curve of MEG3 expression level was 0.818(95%CI: 0.777-0.859,P<0.001)with 94.9% of sensitivity and 94.4% of specificity;The area under the receiver operating characteristic curve of MALAT1 expression level was 0.838(95%CI: 0.723-0.854,P<0.001)with 95.5% of sensitivity and 95.2% of specificity;The area under the receiver operating characteristic curve of MIAT expression level was 0.805(95%CI: 0.666-0.944,P=0.001)with 95.0% of sensitivity and 95.0% of specificity.(8)Compared with HUVECs cultured in 48,72 and 96 hours normal sugar(Control,5 mM D-glucose),after high glucose(HG,30 mM D-glucose)stimulated 48,72,96 hours,the expression level of MEG3 decreased in a time-dependent manner.(9)The expression level of MEG3 was significantly lower in HG and HG+MEG3siRNA group than in control group,which showed a significant difference(P<0.05);The expression level of MEG3 was significantly lower in HG+MEG3siRNA group than in HG group,which showed a significant difference(P<0.05).The expression level of α-SMA,VEGF,TNF-α and IL-6 were significantly higher in HG and HG+MEG3siRNA group than in control group,which showed a significant difference(P<0.05);The expression level of α-SMA,VEGF,TNF-α and IL-6 were significantly higher in HG+MEG3siRNA group than in HG group,which showed a significant difference(P<0.05).(10)The expression level of TGFβ1,SMAD2 and SMAD7 were significantly higher in HG and HG+MEG3siRNA group than in control group,which showed a significant difference(P<0.05);The expression level of TGFβ1,SMAD2 and SMAD7 were significantly higher in HG+MEG3siRNA group than in HG group,which showed a significant difference(P<0.05).(11)The expression level of β-catenin and Cyclin D1 were significantly higher in HG and HG+MEG3siRNA group than in control group,which showed a significant difference(P<0.05);The expression level of β-catenin and Cyclin D1 were significantly higher in HG+MEG3siRNA group than in HG group,which showed a significant difference(P<0.05).The expression level of TCF7L2 was significantly lower in HG and HG+MEG3siRNA group than in control group,which showed a significant difference(P<0.05);The expression level of TCF7L2 was significantly lower in HG+MEG3siRNA group than in HG group,which showed a significant difference(P<0.05).(12)Compared with HUVECs cultured in 48,72 and 96 hours,the OD value of cell were significantly lower in HG and HG+MEG3siRNA group than in control group,which showed a significant difference(P<0.05);the OD value of cell were significantly higher in HG+MEG3siRNA group than in HG group,which showed a significant difference(P<0.05).(13)The apoptotic rate of cell were significantly higher in HG and HG+MEG3siRNA group than in control group,which showed a significant difference(P<0.05);the apoptotic rate of cell were significantly lower in HG+MEG3siRNA group than in HG group,which showed a significant difference(P<0.05).(14)The expression level of Bax、Caspase 3 and P53 protein were significantly higher in HG group than in control group,which showed a significant difference(P<0.05);The expression level of Bax、Caspase 3 and P53 protein were significantly lower in HG+MEG3siRNA group than in HG group,which showed a significant difference(P<0.05).The expression level of Bcl-2 protein was significantly lower in HG group than in control group,which showed a significant difference(P<0.05);The expression level of Bcl-2 protein was significantly higher in HG+MEG3siRNA group than in HG group,which showed a significant difference(P<0.05).(15)The expression level of SMAD2 and β-catenin protein were significantly higher in HG group than in control group,which showed a significant difference(P<0.05);The expression level of SMAD2 and β-catenin protein was significantly higher in HG+MEG3siRNA group than in HG group,which showed a significant difference(P<0.05).Conclusion:(1)In type 2 diabetes mellitus,lncRNAs and mRNAs are differentially expressed,suggesting that these differentially expressed lncRNAs were may involved in processes for the development of type 2 diabetes mellitus.LncRNAs were regarded as possible potential targets for clinical diagnosis and treatment.Through further biological function information exploration,the expression of differentially expressed lnc RNAs were involved in many signal pathways and diseases and a variety of biological functions.Provided the theoretical basis for the follow-up research of type 2 diabetes mellitus.(2)MEG3 expression level in peripheral blood of type 2 diabetes mellitus patients was significantly decreased.MALAT1 and MIAT expression levels in peripheral blood of type 2 diabetes mellitus patients were significantly increased.ROC curve indicated that MEG3,MALAT1 and MIAT were regarded as a potential biomarker in diagnosing type 2 diabetes mellitus patients.(3)This study demonstrates the involvement of lnc RNA MEG3 in high glucose-induced endothelial dysfunction.MEG3 is significantly downregulated in endothelial cell model of hyperglycemia.In addition,MEG3 knockdown could exacerbate inflammatory damage in endothelial cell.Interestingly,MEG3 knockdown in HUVECs significantly induces proliferation and inhibits apoptosis by upregulating Bcl-2 and downregulating Bax,caspase-3 and P53.It should be noted that MEG3 knockdown could activate TGF-β signaling pathway via upregulating TGF-β1,SMAD2 and SMAD7 as well as SMAD2 protein,activate Wnt/β-catenin signaling pathway via upregulating β-catenin and Cyclin D1 as well as β-catenin protein and downregulating TCF7L2.Our results indicated that MEG3 can be regarded as the novel therapeutic target and molecular biomarker for high glucose-induced endothelial dysfunction. |
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