MicroRNAs Expression In Oxidized Low-density Lipoprotein Treated Human Umbilical Vein Endothelial Cells And The Functional Study Of Let-7C

Objective:1. To investigate the microRNA (miRNA) expression profile during human umbilical vein endothelial cells (HUVECs) apoptosis in response to oxidized low-density lipoprotein (ox-LDL) stimulation.2. To make an initial prediction of target genes regulated by differentially expressed miRNAs and to perform a functional analysis of these target genes through using bioinformatics methods.3. To verify the target gene of let-7c and to elucidate the function and mechanism of let-7c regulating HUVECs apoptosis mediated by ox-LDL.Methods:1. HUVECs were exposed to50μg/ml ox-LDL for0to72h and apoptosis was detected by Annexin V-FITC/PI double staining. Then, the optimal time point was selected to identify apoptosis related miRNAs.2. Using a miRCURYTM LNA miRNA microarray approach, we compared the miRNA expression profile in HUVECs following a48h treatment of ox-LDL (50μg/ml) with those in normal control cells.3. Five miRNAs (hsa-miR-142-3p, the most significantly up-regulated miRNA; hsa-miR-33a, the most significantly down-regulated miRNA; hsa-miR-365and hsa-let-7c, apoptomir; as well as hsa-miR-590-5p, miRNA potentially related to atherosclerosis) were selected to be validated with quantitative real-time PCR (qRT-PCR).4. The target genes regulated by the aberrantly expressed miRNAs were, predicted by online-available software (TargetScan, MICRORNA.ORG and MicroCosm v5). The gene ontology (GO) database and KEGG pathway database were used to analyze the functions of these target genes.5. We chose to further study let-7c because of its known effect on apoptosis. QRT-PCR was applied to determine the expression levels of let-7c during ox-LDL induced HUVECs apoptosis. Since Bcl-xl is one of the predicted target genes for let-7c, HUVECs were transfected with let-7c mimics or transfected with let-7c inhibitor and then subjected to ox-LDL treatment for48h, and human Bcl-xl mRNA and protein levels were quantified by qRT-PCR and Western blotting. Moreover, dual luciferase reporter assay system was utilized to confirm whether let-7c directly binds the3'UTR of Bcl-xl mRNA in HUVECs.6. To investigate the role of let-7c in HUVECs apoptosis, we transfected HUVECs with let-7c mimics or transfected with let-7c inhibitor and then subjected them to ox-LDL treatment. Caspase-3activity assay and flow cytometry (FCM) were used to analyze changes in apoptosis. Further, in order to elucidate whether let-7c regulates HUVECs apoptosis via targeting Bcl-xl, HUVECs were cotransfected with pcDNA3.1-Bcl-xl vector and let-7c mimics, Caspase-3activity assay and FCM were used to analyze apoptotic rate of HUVECs.Results:1. HUVECs were treated by ox-LDL to establish apoptotic model in vitro. Treatment with ox-LDL (50μg/ml) for48h significantly increased the percentage of apoptosis in HUVECs, but with little to no effect on the number of necrotic cells. Therefore, the48h time point was selected to identify apoptosis related miRNAs.2. MiRNA microarray showed that ox-LDL significantly up-regulated four miRNAs (hsa-miR-142-3p, hsa-miR-365, hsa-let-7c, and hsa-miR-1207-3p), reversely, ox-LDL down-regulated eleven miRNAs (hsa-miR-32, hsa-miR-589, hsa-miR-18b, hsa-miR-429, hsa-miR-155, hsa-miR-590-5p, hsa-miR-1197, hsa-miR-222, hsa-miR-374a, hsa-miR-33a, and hsa-miR-375).3. We selected5miRNAs that displayed either an increase or decrease in expression (hsa-miR-142-3p, hsa-miR-33a, hsa-let-7c, hsa-miR-365, and hsa-miR-590-5p) to validate with qRT-PCR. The results confirmed the miRNA microarray data and indicated a positive correlation between the quantities of transcripts measured by both microarray and qRT-PCR assay.4. We identified5205putative targets for the15differentially expressed miRNAs via using computational prediction algorithms (TargetScan, MICRORNA.ORG and MicroCosm v5). Gene Ontology annotation and KEGG pathway enrichment analysis demonstrated that these target genes mainly participate in cell proliferation, apoptosis, cell cycle control, oncogene expression, lipid oxidation, and metabolism biological processes and signaling pathways, all of which are closely related to endothelial apoptosis and atherosclerosis.5. Let-7c expression was gradually up-regulated in apoptotic HUVECs induced by ox-LDL. Over-expression of let-7c in HUVECs reduced Bcl-xl protein expression, in the contrast, Bcl-xl protein expression was increased in ox-LDL treated HUVECs after let-7c inhibitor transfection. Although transfection of let-7c mimics or let-7c inhibitor changed Bcl-xl protein levels, transfection did not change Bcl-xl mRNA expression. Additionally, dual luciferase reporter assay system analysis confirmed that let-7c directly binds the3'UTR of Bcl-xl mRNA in HUVECs.6. Let-7c over-expression enhanced Caspase-3activity and increased apoptotic rate in HUVECs, whereas inhibition of let-7c could partly suppress Caspase-3activty and alleviate apoptotic cell death mediated by ox-LDL. Let-7c triggered endothelial apoptosis in part through repressing Bcl-xl, since forced expression of Bcl-xl blocked the ability of let-7c to induce apoptosis in HUVECs.Conclusion: 1. There is a specific miRNA expression profile in apoptotic HUVECs elicited by ox-LDL.2. The potential target genes of15aberrantly expressed miRNAs participate in the regulation of endothelial apoptosis and pathogenesis of atherosclerosis through multiple biological processes and molecular signaling pathways.3. Bcl-xl is one of the direct target genes for let-7c in HUVECs.4. Let-7c promoted endothelial apoptosis through inhibiting Bcl-xl posttranscriptionally.