NPHP3-AS1通过调控miR-30a-5p影响缺氧诱导的心肌细胞增殖及凋亡的研究

NPHP3-AS1 affects cardiomyocyte proliferation and apoptosis induced by hypoxia by regulating miR-30a-5p

目的探讨长链非编码RNA(LncRNA NPHP3-AS1)对缺氧诱导的心肌细胞增殖及凋亡的影响,及其对微小RNA-30a-5p(miR-30a-5p)的调控作用。方法体外培养大鼠心肌细胞H9C2,缺氧处理心肌细胞建立细胞损伤模型,将缺氧处理的心肌细胞作为模型组。同时将正常培养的心肌细胞作为对照组。分别将si-NC、si-NPHP3-AS1、anti-miR-30a-5p、si-NPHP3-AS1与anti-miR-30a-5p转染至心肌细胞,随后进行缺氧处理,分别记作模型组+si-NC组、模型组+si-NPHP3-AS1组、模型组+anti-miR-30a-5p组、模型组+si-NPHP3-AS1+anti-miR-30a-5p组。实时荧光定量聚合酶链反应(qRT-PCR)检测NPHP3-AS1、miR-30a-5p的表达量;应用流式细胞仪检测细胞周期及细胞凋亡率;双荧光素酶报告实验验证NPHP3-AS1、miR-30a-5p的靶向结合关系;蛋白免疫印迹法(Western blot)检测半胱氨酰天冬氨酸特异性蛋白酶3(Caspase3)、增殖标记蛋白细胞增殖核抗原67(Ki67)的表达量。结果缺氧处理后,心肌细胞中NPHP3-AS1的表达量显著升高,G0-G1期细胞比例显著升高,S期细胞比例显著降低,细胞凋亡率显著升高,Caspase3蛋白水平显著升高,Ki67蛋白水平显著降低(P均<0.05);干扰NPHP3-AS1的表达后,G0-G1期细胞比例显著降低,S期细胞比例显著升高,细胞凋亡率显著降低,Caspase3蛋白水平显著降低,Ki67蛋白水平显著升高(P均<0.05);双荧光素酶报告实验证实NPHP3-AS1可靶向结合miR-30a-5p;干扰miR-30a-5p能减弱干扰NPHP3-AS1对缺氧诱导的心肌细胞增殖及凋亡的作用。结论干扰NPHP3-AS1可能通过上调miR-30a-5p的表达从而促进缺氧诱导的心肌细胞增殖及抑制细胞凋亡。

Objective To investigate the effect of long non-coding RNA(LncRNA NPHP3-AS1)on the proliferation and apoptosis of cardiomyocytes induced by hypoxia,and its regulation of microRNA-30a-5p(miR-30a-5p).Methods Rat cardiomyocytes H9C2 were cultured in vitro,and the cardiomyocytes were treated with hypoxia to establish a cell injury model.They were randomly divided into control group,model group,model group+si-NC group,model group+si-NPHP3-AS1 group,model group+anti-miR-30a-5p group,model group+si-NPHP3-AS1+anti-miR-30a-5p group.RT-PCR was used to detect the expression levels of NPHP3-AS1 and miR-30a-5p.Flow cytometry was used to detect the cell cycle and apoptosis rate.The dual luciferase report experiment verified the targeted binding relationship of NPHP3-AS1,miR-30a-5p.Western blot was used to detect the expression of Caspase3,Ki67.Results After hypoxia treatment,the expression of NPHP3-AS1 in cardiomyocytes increased significantly,the proportion of G0-G1 phase cells was significantly increased,the proportion of S phase cells was significantly decreased,the rate of apoptosis was significantly increased,and the level of Caspase3 protein was significantly increased.The Ki67 protein level was significantly reduced(all P<0.05).After interfering with the expression of NPHP3-AS1,the proportion of cells in G0-G1 phase was significantly reduced,the proportion of cells in S phase was significantly increased,the apoptotic rate was significantly reduced,the level of Caspase3 protein was significantly reduced,and the level of Ki67 protein was significantly increased(all P<0.05).The dual luciferase report experiment confirmed that NPHP3-AS1 can target miR-30a-5p.Interfering with miR-30a-5p can attenuate the effect of interfering with NPHP3-AS1 on hypoxia-induced cardiomyocyte proliferation and apoptosis.Conclusion Interference with NPHP3-AS1 may promote hypoxia-induced cardiomyocyte proliferation and inhibit apoptosis by up-regulating the expression of miR-30a-5p.