环状RNA CDR1as靶向抑制微小RNA-135a-5p调控人肺动脉平滑肌细胞增殖、迁移和侵袭的机制

Mechanism of circular RNA CDR1as regulating proliferation,migration and invasion of human pulmonary artery smooth muscle cells by targeting inhibition of microRNA-135a-5p

目的探讨环状RNA小脑变性相关蛋白1反义转录物(circCDR1as)对人肺动脉平滑肌细胞(HPASMC)增殖、迁移和侵袭的影响及其机制。方法体外培养HPASMC,缺氧诱导后干扰circCDR1as表达、抑制或过表达微小RNA-135a-5p(miR-135a-5p)以及过表达Janus激酶2(JAK2)。实时荧光定量聚合酶链反应(RT-qPCR)检测细胞中circCDR1as、miR-135a-5p表达;蛋白质印迹法(Western blot)检测细胞中JAK2、信号转导和转录激活因子3(STAT3)蛋白表达。组间差异比较采用t检验。结果缺氧组circCDR1as表达水平和磷酸化JAK2(p-JAK2)/JAK2、磷酸化STAT3(p-STAT3)/STAT3比值高于常氧组(1.98±0.07比1.01±0.05、0.62±0.06比0.18±0.04、0.56±0.05比0.15±0.03),miR-135a-5p表达水平低于常氧组(0.52±0.05比1.00±0.04),差异有统计学意义(t=27.620、14.946、17.223、18.362,P<0.05)。si-circCDR1as组circCDR1as表达水平、p-JAK2/JAK2、p-STAT3/STAT3比值和细胞活力、迁移细胞数、侵袭细胞数低于si-NC组(0.41±0.05比1.02±0.06、0.41±0.05比0.61±0.05、0.38±0.03比0.54±0.04、0.42±0.04比0.73±0.06、75.64±17.95比145.38±26.42、68.96±16.82比121.05±24.80),miR-135a-5p表达水平高于si-NC组(1.73±0.06比1.03±0.05),差异有统计学意义(t=19.131、6.928、7.838、10.530、5.348、4.258、21.954,P<0.05)。结论circCDR1as通过靶向抑制miR-135a-5p表达激活JAK2/STAT3信号通路,促进缺氧诱导的HPASMC增殖、迁移和侵袭。

Objective To investigate the effect of circular RNA cerebellar degeneration-related protein 1 antisense(circCDR1as)on the proliferation,migration and invasion of human pulmonary artery smooth muscle cells(HPASMCs)and its mechanism.Methods HPASMCs were cultured in vitro,and after hypoxia induction,the expression of circCDR1as was interfered,microRNA-135a-5p(miR-135a-5p)was inhibited or overexpressed and Janus kinase 2(JAK2)was overexpressed in cells.The expression of circCDR1as and miR-135a-5p in cells was detected by real-time quantitative polymerase chain reaction(RT-qPCR).The protein expression of JAK2,signal transducer and activator of transcription 3(STAT3)in cells was detected by Western blotting.Results The expression levels of circCDR1as and the ratio of phosphorylated JAK2(p-JAK2)/JAK2 and phosphorylated STAT3(p-STAT3)/STAT3 in hypoxic group were significantly higher than those in normoxia group(1.98±0.07 vs.1.01±0.05,0.62±0.06 vs.0.18±0.04,0.56±0.05 vs.0.15±0.03),and the expression level of miR-135a-5p was significantly lower than that in normoxia group(0.52±0.05 vs.1.00±0.04,t=27.620,14.946,17.223,18.362,P<0.05).The expression level of circCDR1as,p-JAK2/JAK2,p-STAT3/STAT3 ratio and cell viability,number of migrating cells,and number of invasive cells in si-circCDR1as group were significantly lower than those in si-NC group(0.41±0.05 vs.1.02±0.06,0.41±0.05 vs.0.61±0.05,0.38±0.03 vs.0.54±0.04,0.42±0.04 vs.0.73±0.06,75.64±17.95 vs.145.38±26.42,68.96±16.82 vs.121.05±24.80),and the expression level of miR-135a-5p was significantly higher than that in si-NC group(1.73±0.06 vs.1.03±0.05,t=19.131,6.928,7.838,10.530,5.348,4.258,21.954,P<0.05).Conclusion CircCDR1as can activate JAK2/STAT3 signal pathway by targeting the inhibition of miR-135a-5p expression,and promote the proliferation,migration and invasion of HPASMCs induced by hypoxia.