miR-9/Beclin1信号轴在口腔鳞癌细胞株Tca83顺铂耐药表型形成中的实验研究

Experimental study of miR-9/Beclin1 signaling axis in cisplatin resistance phenotype of oral squamous cell carcinoma cell line Tca83

目的探讨下调miR-9表达在口腔鳞癌Tca83细胞对顺铂(DDP)耐药性的影响以及可能的作用机制。方法体外培养口腔鳞癌亲本细胞株Tca83及耐药细胞株Tca83^(DDPR)。实时荧光定量PCR(QPCR)法检测Tca83细胞和Tca83^(DDPR)细胞miR-9和Beclin1 mRNA的表达。分别沉默Tca83细胞中miR-9和Beclin1的表达,设置Tca83细胞空白对照(CTL)组、miR-9 inhibitor组、si-NC1组、miR-9 inhibitor+si-Beclin1组、si-NC2组。MTT法检测不同浓度DDP对上述各组细胞增殖活性的抑制作用,并计算半数抑制浓度(IC_(50))和耐药指数(RI)。采用GFP荧光法观察自噬泡形成。Western blotting检测Beclin1和微管相关蛋白1轻链3-β(LC3-Ⅱ)蛋白的表达。双荧光素酶报告实验分析miR-9与Beclin1的靶向关系。结果与Tca83细胞比较,Tca83^(DDPR)细胞miR-9表达量(0.79±0.08)降低,而Beclin1表达量(1.27±0.14)升高,差异有统计学意义(P<0.05)。miR-9 inhibitor组和miR-9 inhibitor+si-Beclin1组Tca83细胞中miR-9的表达量分别为0.39±0.05和0.37±0.04,明显低于CTL组、si-NC1组、si-NC2组(P<0.05)。miR-9 inhibitor+si-Beclin1组Tca83细胞中Beclin1表达量为0.45±0.06,低于CTL组、si-NC1组、miR-9 inhibitor组、si-NC2组(P<0.05)。MTT法检测,DDP对Tca83细胞和Tca83^(DDPR)细胞IC_(50)分别为27.19μg/ml和475.69μg/ml。Tca83^(DDPR)细胞的RI为17.50。DDP对miR-9 inhibitor组Tca83细胞IC_(50)为284.53μg/ml。miR-9 inhibitor组Tca83细胞的RI为10.46。双荧光素酶报告实验分析,Beclin1是miR-9的下游靶基因。Tca83^(DDPR)细胞自噬泡形成率高于Tca83细胞(P<0.05)。miR-9 inhibitor组Tca83细胞自噬泡形成率明显高于CTL组Tca83细胞(P<0.05)。而miR-9 inhibitor+si-Beclin1组Tca83细胞自噬泡形成率明显减少,与miR-9 inhibitor组比较,差异有统计学意义(P<0.05)。Western blotting结果显示,miR-9 inhibitor组Tca83细胞Beclin1、LC3-Ⅱ蛋白表达量为0.79±0.15和0.41±0.10,高于CTL组和si-NC1组(P<0.05)。miR-9 inhibitor+si-Beclin1组Tca83细胞Beclin1、LC3-Ⅱ蛋白表达量为0.34±0.09和0.16±0.05,低于其他各组(P<0.05)。结论下调Tca83细胞中miR-9表达后,Beclin1表达相应增加,通过增加细胞自噬活性,降低DDP对肿瘤细胞增殖活性的抑制作用,从而诱导Tca83细胞对顺铂的耐药性。

Objective To investigate the effect of down-regulation of miR-9 on cisplatin(DDP)resistance of oral squamous cell carcinoma(OSCC)cells Tca83 and its possible mechanism.Methods Tca83 cells and its DDP-resistance cells Tca83^(DDPR) were cultured in vivo.The expressions of miR-9 and Beclin1 mRNA in Tca83 cells and Tca83^(DDPR) cells were detected by QPCR.Tca83 cells were transfected with miR-9 inhibitor as miR-9 inhibitor group and siRNA Beclin1 as miR-9 inhibitor+si-Beclin1 group.There were also including CTL group,si-NC1 group and si-NC2 group.The proliferations of Tca83 cells and Tca83^(DDPR) cells were detected by MTT assay,and 50%inhibition concentration(IC_(50))and resistance index(RI)to DDP were calculated.The autophagosome formation was observed by GFP fluorescence microscopy.The Beclin1 and microtubule associated protein 1 light chain 3β(LC3-Ⅱ)levels were detected by western blotting.The dual luciferase reporter gene system was used to confirm the correlation of miR-9 and Beclin1.Results Expression of miR-9 in Tca83^(DDPR) cells was 0.79±0.08,lower than that in Tca83 cells(P<0.05),and expression of Beclin1 mRNA in Tca83^(DDPR) cells was 1.27±0.14,higher than that in Tca83 cells(P<0.05).MiR-9 levels of Tca83 cells in miR-9 inhibitor group and miR-9 inhibitor+si-Beclin1 group were 0.39±0.05 and 0.37±0.04,lower than that in CTL group,si-NC 1 group and si-NC 2 group(P<0.05).Beclin1 mRNA level of Tca83 cells in miR-9 inhibitor+si-Beclin1 group were 0.45±0.06,lower than that in CTL group,si-NC1 group,miR-9 inhibitor group and si-NC2 group(P<0.05).IC_(50) of DDP on Tca83 cells and Tca83^(DDPR) cells were respectively 27.19μg/ml and 475.69μg/ml.RI of Tca83^(DDPR) cells was 17.50.IC_(50) of DDP on Tca83 cells in miR-9 inhibitor group was 284.53μg/ml.RI of Tca83 cells in miR-9 inhibitor group was 10.46.Beclin1 might be a target protein of miR-9 which was verified by the dual luciferase reporter gene system.The autophagosome formation rate of Tca83^(DDPR) cells was higher than that of Tca83 cells(P<0.05).Besides,the autophagosome formation rate of Tca83 cells in miR-9 inhibitor group was higher than that of Tca83 cells in CTL group and si-NC1 group(P<0.05);and the autophagosome formation rate of Tca83 cells in miR-9 inhibitor+si-Beclin1 group was lower than that of Tca83 cells in miR-9 inhibitor group(P<0.05).The expression of Beclin1 and LC3-Ⅱproteins in miR-9 inhibitor group were respectively 0.79±0.15 and 0.41±0.10,which were higher than these in CTL group and si-NC1 group(P<0.05).The expression of Beclin1 and LC3-Ⅱproteins in miR-9 inhibitor+si-Beclin1 group were respectively 0.34±0.09 and 0.16±0.05,which were lower than these in the other 4 groups(P<0.05).Conclusion It's suggested that silencing miR-9 significantly make the cisplatin-resistance phenotype of Tca83 cells and inhibited the cytotoxicity of DDP to Tca83 via negative targeting Beclin1 expression which would be related to chemotherapy-induced autophagy.