miR-194对非小细胞肺癌A549/DDP细胞顺铂耐药性的影响

目的:探讨microRNA-194(miR-194)对非小细胞肺癌A549/DDP细胞顺铂耐药性的影响及其可能的作用机制?方法:采用实时荧光定量PCR(qRT-PCR)检测非小细胞肺癌耐药细胞A549/DDP及其亲本细胞株A549中miR-194的表达差异,并在A549/DDP细胞中转染miR-194-inhibitor后检测miR-194的表达变化;应用MTT法?克隆形成实验?流式细胞术检测转染后A549/DDP细胞对DDP的敏感性?细胞增殖能力及凋亡变化;Western blot检测转染后A549/DDP细胞中Bax和Bcl-2的表达变化?结果:miR-194在耐药细胞A549/DDP中的表达量显著高于其亲本细胞株A549(P < 0.05),A549/DDP在转染miR-194-inhibitor 24 h后miR-194的表达水平较对照组显著下降(P < 0.05)?抑制miR-194表达后,相对于对照组,DDP对转染miR-194-inhibitor的A549/DDP细胞的半数抑制浓度(half inhibition concentration,IC50)减低(P < 0.05),转染miR-194-inhibitor的A549/DDP细胞增殖能力减弱,经DDP处理后凋亡细胞增多(P < 0.05)?Western blot结果显示,与对照组相比,转染miR-194-inhibitor的A549/DDP细胞Bax表达水平升高,Bcl-2表达水平降低?结论:miR-194可能通过抑制细胞凋亡,上调Bcl-2蛋白及下调Bax蛋白表达而增加A549/DDP细胞对DDP的耐药性,抑制miR-194的表达可逆转A549/DDP细胞的耐药性?

Effect of microRNA-194 on cisplatin resistance of non-small cell lung cancer A549/DDP cell lines

Objective:To explore the effect of microRNA-194 (miR-194) on cisplatin resistance of non-small cell lung cancer (NSCLC) A549/DDP cell lines and its related mechanisms. Methods:The real-time fluorescent quantitative PCR (qRT-PCR) was performed to detect miR-194 expression differences between NSCLC A549/DDP cells and A549 cells. MiR-194 expression differences were detected after MiR-194-inhibitor transfected into A549/DDP cells. MMT assay,colony formation and flow-cytometric analysis were sequentially performed to detect transfected A549/DDP cells to DDP drug sensitivity,cell proliferation ability,and apoptosis on treatment with DDP. The protein expressions of Bax and Bcl-2 in transfected A549/DDP cells were detected by Western blot. Results:The level of miR-194 in A549/DDP cells was significantly higher than that in A549 cells (P < 0.05). The miR-194 level in A549/DDP cells was significantly lower than that in A549/DDP cells after transfected with miR-194 inhibitor (P < 0.05). The inhibited expression of miR-194 could produce following effects: compared to the control group,the half inhibition concentration (IC50) of DDP in A549/DDP with miR-194-inhibitor cells was decreased (P < 0.05),proliferation ability of A549/DDP with miR-194-inhibitor cells was diminished (P < 0.05),apoptosis after treatment with DDP was increased (P < 0.05). Western blotting results showed that,the expression of Bcl-2 was down-regulated and the expression of Bax was up-regulated in A549/DDP/miR-194-inhibitor cells than those in the control group. Conclusion:miR-194 increases the resistance of A549/DDP cells to DDP through inhibition of apoptosis,up-regulating Bcl-2 protein expression and down-regulating Bax. The inhibition of miR-194 expression can reverse the resistance of A549/DDP cells to DDP.