Mechanism Study Of Linc00458/miR-95-5p/HDAC2 Regulating VSMCs Senescence/calcification Under High Glucose

Objective: Vascular complications of diabetes are the major causes of disability and death in patients with diabetes.High concentration glucose can accelerate the senescence of vascular cells and promote the occurrence of vascular complications of diabetes.After intensive control of blood glucose,the vascular complications of diabetes continue to progress,which may be related to the “hyperglycemia memory”phenomenon caused by previous hyperglycemia.Although epigenetic mechanisms are believed to be the basis of this phenomenon,the molecular mechanisms responsible for “hyperglycemia memory” are still far from being understood.This study aims to explore the role and mechanism of linc00458/miR-95-5p/HDAC2 pathway in regulating vascular smooth muscle cells(VSMCs)senescence/ calcification induced by high glucose,which may serve as effective therapeutic targets to delay the occurrence and development of diabetic vascular complications.Methods: VSMCs were cultured in vitro and induced by high glucose(HG,30 m M)to establish an senescence/calcification model of VSMCs.Adoption of senescence-associated-β-galactosidase(SA-β-Gal)and alizarin red staining were used to detect cell senescence and mineralized nodules.Differentially expressed genes(DEGs)in VSMCs stimulated by HG and normal glucose(NG)were detected by microarray.Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis was performed utilizing the KEGG database,to reveal the biological functions that DEGs may participate in.Protein-protein interaction(PPI)network analysis was performed on DEGs using the online database of the Search Tool for the Retrieval of Interaction Genes(STRING).The expression levels of linc00458,miRNAs and histone deacetylase 2(HDAC2)m RNA were determined by q RT-PCR,and the expression levels of p16,p21,osteocalcin(OCN),Runt-related transcription factor 2(Runx2),HDAC2 and FOXO3 protein were detected by Western blot.Linc00458 overexpression(OV)and knockdown(sh RNA)vectors were transfected to verify the effect of linc00458 expression changes on the expression of aging-related indicators p16,p21 and calcification-related indicators OCN,Runx2.Binding sites of miR-95-5p to linc00458 and HDAC2 3’-UTR were analyzed by bioinformatic analysis.Luciferase reporter assay was used to detect the targeting relationship between miR-95-5p and linc00458,or HDAC2.The biotin-labeled RNA pull-down experiment verified the interaction between miR-95-5p and linc00458,or HDAC2.Chromatin Immunoprecipitation(Ch IP)was used to detect the interaction between HDAC2 and H3K9,H3K27 in the FOXO3 promoter region.Cell proliferation rate was measured by MTT colorimetry.Fluorescent probe DCFH-DA was used to detect intracellular reactive oxygen species(ROS).Mi R-95-5p mimics and inhibitor,sh RNA-linc00458,and si HDAC2 were transfected by lipofectamine 3000 to over-express miR-95-5p and inhibit the expression of miR-95-5p,linc00458,and HDAC2.Results:1.The study found that HG could induce the senescence/calcification of VSMCs,which was proven by the increased percentage of SA-β-gal staining positive cells and the formation of mineralized nodules.In HG induced VSMCs,linc00458 expression was upregulated.Knock down the expression of linc00458 could alleviate the senescence/calcification of VSMCs induced by HG,and reduce the expression of aging-related proteins p16,p21 and osteogenic differentiation markers OCN and Runx2.However,overexpression of linc00458 received the opposite results.Bioinformatics analysis showed that there are binding sites of miR-95-5p to linc00458 and HDAC23’-UTR.The expression of linc00458 and HDAC2 increased and the expression of miR-95-5p decreased in the senescence/calcification model of VSMCs induced by HG.Luciferase reporter assay confirmed that linc00458 and HDAC2 were the target genes of miR-95-5p,and RNA pull down confirmed that miR-95-5p can interact directly with linc00458 and HDAC2,respectively.Linc00458 could inhibit the expression of miR-95-5p through molecular sponge mechanism,thus promoting the expression of target gene HDAC2.Knock down miR-95-5p can counteract the effect of sh RNA-linc00458 on delaying the senescence/calcification of VSMCs induced by HG.2.The microarray analysis showed that HDAC2 was upregulated and FOXO3 was downregulated in HG induced VSMCs,which was validated by q RT-PCR and western blot;KEGG pathway enrichment analysis showed that DEGs were enriched into vascular aging/calcification related pathways such as "cell aging","Wnt signaling pathway",PI3 K Akt signaling pathway "," MAPK signaling pathway ","m TOR signaling pathway "and" lipid and atherosclerosis ";Certain DEGs are enriched into the "FOXO signaling pathway".PPI network analysis suggested that there may be a connection between HDAC2 and FOXO3.HDAC2 knockdown upregulated FOXO3 expression,while HDAC2 overexpression had the opposite effect.Acetylation levels of H3K9 and H3K27 in the FOXO3 promoter region decreased in HG induced VSMCs;The acetylation levels of H3K9 and H3K27 increased after inhibiting HDAC2 expression.HG induced increased intracellular ROS production in VSMCs.CAY10683,a selective inhibitor of HDAC2,could promote FOXO3 expression and reduce intracellular ROS levels;The knockdown of FOXO3 could block the protective effect of CAY10683,resulting in an increase in intracellular ROS levels.CAY10683 reversed the senescence of VSMCs induced by HG,which was indicated by the decrease in numbers of SA-β-gal positive cells and downregulation of p16 and p21.CAY10683 could delay the calcification of VSMCs induced by HG,which was characterized by decreased formation of calcified nodules and downregulated expression of Runx2 and OCN.FOXO3 knockdown blocked the protective effect of CAY10683 on delaying HG induced senescence/calcification of VSMCs.Conclusion: This study shows for the first time that linc00458/miR-95-5p/HDAC2 regulatory axis is involved in regulating the senescence/ calcification of VSMCs induced by HG.Under high glucose environment,HDAC2 down regulates the expression of FOXO3 by influencing the level of H3K9 and H3K27 deacetylation in the FOXO3 promoter region and mediating intracellular oxidative stress;CAY10683 reduced the level of H3K9 and H3K27 deacetylation in the FOXO3 promoter region and reversed the senescence/calcification of VSMCs induced by HG.