Study On The Molecular Mechanism By Which CERNA1 Regulates Vascular Endothelial Cell Apoptosis

Background and objectiveLong noncoding RNAs(lncRNAs)are defined as non-protein coding transcripts longer than 200 nucleotides.1ncRNAs could regulate the expression of genes at the epigenetic,transcriptional and post-transcriptional levels.They play important roles in multiple physiological processes such as differentiation,proliferation,apoptosis,invasion and reprogramming of induced pluripotent stem cells.Vascular endothelial cells(VECs),which lie in the innermost of blood vessels,are vulnerable to stimulus.Apoptosis in VECs is closely linked to numerous cardiovascular diseases such as arteriosclerosis,thrombus formation and plaque erosion.Formerly,the investigation on the mechanisms of apoptosis mainly focused on the protein-coding genes.Recently,lncRNAs have caused more and more extensive concern.Yet,there are no reports about apoptosis-related IncRNA in VECsIn our previous work,human umbilical vein endothelial cells(HUVECs)were cultured under the serum and FGF-2-deprived condition to simulate the in vivo ischemic condition.We found that a small molecule,ABO,elevated the viability of HUVECs in the absence of serum and FGF-2.Moreover,it was demonstrated that ABO effectively inhibited oxLDL-induced apoptosis of VECs and atherosclerosis in ApoE-/-mice.These data suggest that ABO is an appropriate molecule for finding new factors that inhibit VECs apoptosis.In conclusion,by using ABO,this study aimed to identify and explore the newly key factors and signal pathway participated in VECs apoptosis,thus provided new targets and effective tools for clinical treatment of cardiovascular diseases.Study contents1.To find the new factor in regulation of VECs apoptosis by ABO.2.To investigate the molecular action mechanism of CERNA1 in VECs apoptosis.3.To study the role of CERNA1 in atherosclerosis(ApoE-/-mice).4.To explore the expression regulation mechanism of CERNA1 in VECs.Methods1.We used human umbilical vein endothelial cells(HUVECs)as the cell model,ApoE-/-mice as the animal model,and used chemical small molecule ABO as the tool to do the related study.2.In this study,HUVECs were cultured under the serum and FGF-2-deprived condition to simulated the in vivo ischemic condition;HUVECs were cultured under oxLDL treatment to simulated the in vivo atherosclerosis condition;And high-fat feeding ApoE-/-mice to establish the animal model of atherosclerosis.3.Over expression and knock-down of genes:(1)Over expression of gene in cells.We constructed the over expression plasmids or miRNA mimics;(2)Knock-down of gene in cells.We designed,synthesized and screened the effective small interfering RNA or miRNA inhibitor;then transfected into cells by liposome.(3)Over expression of CERNA1 in ApoE-/-mice.We constructed the over-expression plasmid and packaged into lentivirus,then tail vein injected it into mice.4.Gene expression detection system:IncRNA,mRNA or miRNAs levels were detected by qPCR;protein and its phosphorylation level were detected by Western Blot and corresponding antibodies.5.The prediction and verification of miRNA target sites:(1)predition miRNA binding site on IncRNA sequence by microinspector database;(2)predition miRNA target gene by miRDB,Targetscan and DIANA LAB database.(3)Verification miRNA binding site by double luciferases report system.6.Detection of CERNA1 subcellular localization:RNA-FISH and RNA extraction from cytoplasmic and nuclear components.7.Detection system of epigenetic modification:(1)Analysis of DNA methylation by bisulfite sequencing PCR(BSP);(2)Analaysis of histone modification by ChIP and specific antibodies to H3K4me3 and H3K9ac.8.Analysis of atherosclerosis:(1)lipid accumulation by oil red O staining and ELISA;(2)immune response by ELISA;(3)plaque stability detected by quantity of smooth muscle,matrix metalloproteinase activity and necrotic core.Results1.Finding the new factor in regulation of VECs apoptosis by ABO:CERNA1.(1)ABO inhibited the serum and FGF-2 starvation-induced apoptosis in VECs.(2)CERNA1 was upregulated by ABO treatment in VECs.(3)Over expression of CERNA1 inhibted VECs apoptosis induced by serum and FGF-2 deprived.(4)CERNA1 located both in cytoplasm and nuclei.2.CERNA1 suppresses apoptosis by targeting miR-4707-5p and miR-4767 in VECs.(1)CERNA1 functioned as an endogenous sponge of miR-4707-5p and miR-4767.(2)miR-4707-5p and miR-4767 promoted apoptosis by targeting and downregulating two apoptosis inhibitors,AP15 and BCL2L12,respectively.(3)CERNA1 promoted the expression of two apoptosis inhibitors,API5 and BCL2L12,by sponging miR-4707-5p and miR-4767.3.CERNA1 positively regulated apoptosis enhancer-BCL2L10 by epigenetic modification pathway in VECs.(1)CERNA1 positively regulated the RNA level of genome adjacent gene BCL2L10.(2)CERNA1 influenced the epigenetic modification on the promoter of BCL2L10(decreased DNA methylation,increased H3K4me3 and H3K9ac modification),then enhanced its expression.(3)BCL2L10 is an apoptosis enhancer in VECs.(4)ABO altered CERNA1 subcellular localization to determine cell fate.4.The study on the role of CERNA1 in atherosclerosis.(1)Over expression of CERNA1 inhibted VECs apoptosis induced by oxLDL.(2)Over expression of CERNA1 in ApoE-/-mice.(3)Over expression of CERNA1 stabilized the atherosclerotic plaque.(4)CERNA1 enhanced the expression of API5 in smooth muscle cells and macrophage,and then suppressed cell apoptosis,thus stabilized plaque.5.ABO upregulated CERNA1 expression by TIA-1.(1)Knock-down TIA-1 decreased the expression of CERNA1 in VECs.(2)ABO enhanced the expression of CERNA1 in a TIA-1 dependent manner.Conclusions1.CERNA1 suppressed VECs apoptosis by competition endogenous RNA pathway:CERNA1 promoted the expression of two apoptosis inhibitors,API5 and BCL2L12 by sponging miR-4707-5p and miR-4767.2.CERNA1 promoted VECs apoptosis by epigenetic regulation pathway:CERNA1 influenced the epigenetic modification on the promoter of apoptosis enhancer-BCL2L10(decreased DNA methylation,increased H3K4me3 and H3K9ac modification),then enhanced its expression.3.ABO determined the cell fate,not only by enhancing CERNA1 expression but also by altering CERNA1 subcellular localization.4.In ApoE-/-mice,over expression of CERNA1 enhanced API5 protein level in smooth muscle cells and macrophage,then suppressed cell apoptosis,thus stabilized plaque.5.ABO increased the expression of CERNA1 by TIA-1.