lncRNA FEZF1-AS1 调控miR-363-3p影响LPS诱导的血管内皮细胞活力及凋亡

Effect of lncRNA FEZF1-AS1 on viability and apoptosis of LPS-induced vascular endothelial cells by regulating miR-363-3p expression

目的:探讨长链非编码RNA(lncRNA)FEZF1-AS1调控微小RNA-363-3p(miR-363-3p)影响脂多糖(LPS)诱导的血管内皮细胞活力及凋亡的作用机制。方法:体外培养人脐静脉血管内皮细胞(HUVECs),分别将pcDNA-NC、pcDNA-FEZF1-AS1、anti-miR-NC、anti-miR-363-3p、miR-NC及miR-363-3p mimics转染至HUVECs,并给予LPS刺激24 h;采用RT-qPCR检测FEZF1-AS1和miR-363-3p的表达量;MTT法检测细胞活力;流式细胞术检测细胞凋亡率;双萤光素酶报告基因实验验证FEZF1-AS1与miR-363-3p的靶向调控作用;Western blot法检测细胞周期蛋白D1(cyclin D1)、cleaved caspase-3和Ki67的表达量。结果:与control组比较,LPS组FEZF1-AS1的表达水平显著降低(P<0.05),miR-363-3p的表达水平显著升高(P<0.05);与pcDNA-NC+LPS组比较,pcDNA-FEZF1-AS1+LPS组细胞存活率显著升高(P<0.05),细胞凋亡率显著降低(P<0.05),cyclin D1和Ki67蛋白水平显著升高(P<0.05),cleaved caspase-3蛋白水平显著降低(P<0.05);与anti-miR-NC+LPS组比较,anti-miR-363-3p+LPS组细胞存活率显著升高(P<0.05),细胞凋亡率显著降低(P<0.05),cyclin D1和Ki67蛋白水平显著升高(P<0.05),cleaved caspase-3蛋白水平显著降低(P<0.05);双萤光素酶报告基因实验证实FEZF1-AS1可靶向结合miR-363-3p;与miR-NC+pcD⁃NA-FEZF1-AS1+LPS组比较,miR-363-3p+pcDNA-FEZF1-AS1+LPS组细胞存活率显著降低(P<0.05),细胞凋亡率显著升高(P<0.05),cyclin D1和Ki67蛋白水平显著降低(P<0.05),cleaved caspase-3蛋白水平显著升高(P<0.05)。结论:过表达FEZF1-AS1可抑制miR-363-3p表达从而降低LPS诱导的血管内皮细胞凋亡率,增加其细胞活力。

AIM:To investigate the mechanism of long noncoding RNA(lncRNA)FEZF1-AS1 regulating mi⁃croRNA-363-3p(miR-363-3p)on the viability and apoptosis of lipopolysaocharide(LPS)-induced vascular endothelial cells.METHODS:Human umbilical vein endothelial cells(HUVECs)were cultured in vitro.pcDNA-NC,pcDNAFEZF1-AS1,anti-miR-NC,anti-miR-363-3p,miR-NC and miR-363-3p mimics were transfected into the HUVECs and LPS stimulation was applied for 24 h.RT-qPCR was used to detect the expression of FEZF1-AS1 and miR-363-3p.The cell viability was measured by MTT assay.The apoptotic rate was analyzed by flow cytometry.The dual-luciferase reporter experiment was used to verify the targeted regulation of FEZF1-AS1 and miR-363-3p.Western blot was used to determined the expression of cyclin D1,Ki67 and cleaved caspase-3.RESULTS:Compared with control group,the expression level of FEZF1-AS1 in LPS group was significantly reduced(P<0.05),and the expression level of miR-363-3p was significant⁃ly increased(P<0.05).Compared with pcDNA-NC+LPS group,the cell viability in pcDNA-FEZF1-AS1+LPS group was significantly increased(P<0.05),the apoptotic rate was significantly reduced(P<0.05),the protein levels of cyclin D1 and Ki67 were significantly increased(P<0.05),and the protein level of cleaved caspase-3 was significantly reduced(P<0.05).Compared with anti-miR-NC+LPS group,the cell viability in anti-miR-363-3p+LPS group was significantly in⁃creased(P<0.05),the apoptotic rate was significantly reduced(P<0.05),the protein levels of cyclin D1 and Ki67 were significantly increased(P<0.05),and the protein level of cleaved caspase-3 was significantly reduced(P<0.05).Dualluciferase reporter experiment confirmed that FEZF1-AS1 targeted miR-363-3p.Compared with miR-NC+pcDNA-FEZF1-AS1+LPS group,the cell viability in miR-363-3p+pcDNA-FEZF1-AS1+LPS group was significantly reduced(P<0.05),the apoptotic rate was significantly increased(P<0.05),the protein levels of cyclin D1 and Ki67 were significantly re⁃duced(P<0.05),and the protein level of cleaved caspase-3 was significantly increased(P<0.05).CONCLUSION:Over-expression of FEZF1-AS1 promotes the viability and inhibits apoptosis of LPS induced vascular endothelial cells by inhibiting the expression of miR-363-3p.