miR-30a对卵巢癌细胞PTEN/PI3K/AKT/mTOR自噬通路及顺铂耐药性的影响

Effects of miR-30a on PTEN/PI3K/AKT/mTOR autophagy pathway and cisplatin resistance of ovarian cancer cells

目的:探究miR-30a对人卵巢癌SKOV3细胞自噬及顺铂耐药性的影响,并初步研究其作用机制。方法:体外培养人卵巢癌SKOV3细胞和人卵巢癌细胞(耐药)细胞(SKOV3/DDP);将SKOV3/DDP细胞随机分为对照组、miR-30a阴性对照(NC)组和miR-30a模拟(mimics)组,SKOV3细胞作为正常组。实时荧光定量PCR(RT-qPCR)法检测各组细胞miR-30a表达情况;CCK-8法检测各组细胞顺铂耐药性;Annexin V-FITC/PI法检测各组细胞凋亡情况;蛋白印迹分析法检测各组细胞微管相关蛋白轻链3 I/II(LC3I/II)、p62、磷酸酶和张力蛋白同源物(PTEN)、磷酸化磷脂酰肌醇-3-激酶(p-PI3K)、磷酸化丝氨酸/苏氨酸蛋白激酶B(p-AKT)、磷酸化哺乳动物雷帕霉素靶蛋白(p-mTOR)表达情况;双荧光素酶报告基因检测系统验证miR-30a与PTEN的靶向关系。结果:与对照组和miR-30a NC组相比,miR-30a mimics组SKOV3/DDP细胞miR-30a表达水平、凋亡率、p62、p-PI3K、p-AKT及p-mTOR水平显著升高(P<0.05),IC 50值、LC3II、PTEN蛋白表达水平及LC3II/LC3I比值显著降低(P<0.05);与正常组相比,对照组SKOV3/DDP细胞上述各指标变化趋势完全相反;mirbase数据库预测显示,miR-30a与PTEN mRNA 3'UTR区有结合位点,与PTEN-3'UTR-WT+miR-30a NC组比较,PTEN-3'UTR-WT+miR-30a inhibitor组荧光素酶活性降低(P<0.05)。结论:miR-30a可能通过靶向抑制PTEN表达,激活PI3K/AKT/mTOR信号通路调控人卵巢癌细胞自噬,降低卵巢癌细胞的顺铂耐药性。

Objective:To explore the effects of miR-30 a on autophagy and cisplatin resistance of human ovarian cancer SKOV3 cells,and to study its mechanism.Methods:Human ovarian cancer SKOV3 cells and human ovarian cancer(drug resistant) cells(SKOV3/DDP) were cultured in vitro.SKOV3/DDP cells were randomly divided into control group,miR-30 a negative control(NC) group and miR-30 a mimics(mimics) group,and SKOV3 cells as normal group.The expression of miR-30 a mRNA was detected by real-time fluorescence quantification(RT-qPCR).CCK-8 method was used to detect cisplatin resistance.Apoptosis was detected by Annexin V-FITC/PI.In addition,Western blot was used to detect the expressions of microtubule associated protein light chain 3 I/II(LC3 I/II),p62,phosphatase and tension homologues(PTEN),phosphorylated phosphatidylinositol-3-kinase(p-PI3 K),phosphorylated serine/threonine protein kinase B(p-AKT),phosphorylated mammalian target of rapamycin(p-mTOR),in each group.Dual luciferase reporter gene detection system was used to verify the targeting relationship between miR-30 a and PTEN.Results:Compared with those in the control group and miR-30 a NC group,the expression level of miR-30 a,apoptosis rate,p62,p-PI3 K,p-AKT and p-mTOR in SKOV3/DDP cells were significantly higher(P<0.05),and the IC50 value,LC3 II,PTEN protein expression level and LC3 II/LC3 I value were significantly lower(P<0.05).Compared with the normal group,the change trend of the above indicators of SKOV3/DDP cells in the control group is completely opposite.The mirbase database predicted that miR-30 a and PTEN mRNA had a binding site in the 3’ UTR region.Compared with PTEN-3’ UTR-WT+miR-30 a NC group,PTEN-3’ UTR-WT+miR-30 a inhibitor group fluorescence Enzyme activity decreased(P<0.05).Conclusion:Overexpression of miR-30 a may activate PI3 K/AKT signaling pathway,regulate autophagy of human ovarian cancer cells,and reduce cisplatin resistance of ovarian cancer cells by targeted down-regulating the expression of PTEN.