微小RNA-134靶向叉头盒蛋白M1对喉鳞癌细胞顺铂耐药性的影响

Effect of miR-134 targeting forkhead box protein M1 on cisplatin resistance of laryngeal squamous cell carcinoma cells

目的探讨微小RNA-134(miR-134)靶向叉头盒蛋白M1(FOXM1)对喉鳞癌细胞顺铂(DDP)耐药株FD-LSC-1/DDP的DDP耐药性的影响。方法体外培养NP69、FD-LSC-1、FD-LSC-1/DDP细胞,用实时荧光定量逆转录聚合酶链反应(qRT-PCR)法检测NP69、FD-LSC-1、FD-LSC-1/DDP细胞中miR-134、FOXM1 mRNA表达水平。将FD-LSC-1/DDP细胞分为空白对照组、miR-134-mimics-NC组(阴性对照组)、miR-134-mimics组(过表达miR-134)、miR-134-mimics+pcDNA组(共转染miR-134-mimics和pcDNA)、miR-134-mimics+pcDNA-FOXM1组(共转染miR-134-mimics和pcDNA-FOXM1)。倒置荧光显微镜下观察转染效果;以空白对照组、miR-134-mimics-NC组、miR-134-mimics组FD-LSC-1/DDP细胞为研究对象,以qRT-PCR法检测细胞中miR-134表达;以噻唑蓝(MTT)法检测细胞增殖能力及对DDP的耐药性;以流式细胞仪检测细胞凋亡率;以蛋白质印迹法检测FOXM1、增殖细胞核抗原(PCNA)、细胞增殖相关核抗原(Ki-67)、B淋巴细胞瘤基因-2(Bcl-2)、胱天蛋白酶-3(caspase-3)、Bcl-2相关X蛋白(Bax)蛋白表达。用TargetScan数据库预测miR-134与FOXM1的靶向关系,通过双荧光素酶报告基因实验加以验证并检测miR-134对FOXM1的调控作用。结果与NP69细胞比较,FD-LSC-1细胞中miR-134表达水平显著降低,FOXM1 mRNA表达水平显著升高(P<0.05);与FD-LSC-1细胞比较,FD-LSC-1/DDP细胞miR-134表达水平显著降低、FOXM1 mRNA表达水平显著升高(P<0.05)。空白对照组、miR-134-mimics-NC组、miR-134-mimics组的miR-134表达水平分别为1.02±0.15,1.01±0.15,1.57±0.23,药物半数抑制浓度(IC_(50))分别为(24.25±2.05),(23.67±1.78),(8.64±1.57)μg·mL^(-1),细胞增殖抑制率分别为(17.65±2.66)%,(17.63±2.65)%,(45.87±6.88)%,凋亡率分别为(15.09±2.26)%,(14.97±2.25)%,(38.77±5.82)%,miR-134-mimics组与空白对照组、miR-134-mimics-NC组比较,差异均有统计学意义(均P<0.05)。与空白对照组、miR-134-mimics-NC组比较,miR-134-mimics组FD-LSC-1/DDP细胞Bax、caspase-3蛋白表达均显著升高,PCNA、Ki-67、Bcl-2蛋白表达水平均显著降低(均P<0.05)。TargetScan数据库预测显示FOXM1是miR-134的潜在靶基因,双荧光素酶报告基因实验证实二者存在靶向关系;上调FOXM1逆转了miR-134过表达对FD-LSC-1/DDP细胞顺铂耐药性的作用。结论过表达miR-134能提高FD-LSC-1/DDP细胞化疗敏感性,可能是通过靶向下调FOXM1表达实现的。

Objective To investigate the effect of forkhead box protein M1(FOXM1)targeted by microRNA-134(miR-134)on cisplatin(DDP)resistance of DDP resistant laryngeal squamous cell carcinoma cell line FD-LSC-1/DDP.Methods NP69,FD-LSC-1 and FD-LSC-1/DDP cells were cultured in vitro,and the expression level of miR-134,FOXM1 mRNA in FD-LSC-1 and FD-LSC-1/DDP cells were detected by real-time fluorescent quantitative polymerase chain reaction(qRT-PCR).FD-LSC-1/DDP cells were divided into blank control group,miR-134-mimics-NC group(negative control group),miR-134-mimics group(miR-134 overexpression group),miR-134-mimics+pcDNA group(co-transfection of miR-134-mimics and pcDNA),miR-134-mimics+pcDNA-FOXM1 group(co-transfection of miR-134-mimics and pcDNA-FOXM1).The transfection effect was observed under inverted fluorescence microscope.Blank control group,miR-134-mimics-NC group,miR-134-mimics group FD-LSC-1/DDP cells were selected as the research objects;qRT-PCR method was used to detect the expression of miR-134 in the cells;methyl thiazolyl tetrazolium method was used to detect cell proliferation and resistance to DDP;flow cytometry was used to detect cell apoptosis rate;Western blotting was used to detect FOXM1,proliferating cell nuclear antigen(PCNA),cell proliferation-associated nuclear antigen(Ki-67),B lymphoma gene-2(Bcl-2),caspase 3,Bcl-2 related X protein(Bax)protein expression.The target relationship between miR-134 and FOXM1 were predicted by TargetScan database;the regulatory effect of miR-134 on FOXM1 was detected and verified by double luciferase reporter gene experiment.Results Compared with NP69 cells,the expression level of miR-134 in FD-LSC-1 cells was significantly reduced,and the expression level of FOXM1 mRNA was significantly increased(P<0.05);compared with FD-LSC-1 cells,the expression level of miR-134 in FD-LSC-1/DDP cells was significantly reduced,and the expression level of FOXM1 mRNA was significantly increased(all P<0.05).The expression levels of miR-134 in blank control group,miR-134-mimics-NC group,and miR-134-mimics group were 1.02±0.15,1.01±0.15,1.57±0.23,respectively;the median inhibitory concentration(IC_(50))of the drug were(24.25±2.05),(23.67±1.78),(8.64±1.57)μg·mL^(-1),respectively;cell proliferation inhibition rate were(17.65±2.66)%,(17.63±2.65)%,(45.87±6.88)%,respectively;apoptosis rate were(15.09±2.26)%,(14.97±2.25)%,(38.77±5.82)%,respectively;the differences between miR-134-mimics group and blank control group or miR-134-mimics-NC group were statistically significant(all P<0.05).Compared with blank control group and miR-134-mimics-NC group,the expression of Bax and caspase-3 protein were significantly increased;PCNA,Ki-67,Bcl-2 protein expression level were significantly reduced in FD-LSC-1/DDP cells in the miR-134-mimics group(all P<0.05).TargetScan database prediction showed that FOXM1 was a potential target gene of miR-134,and double luciferase reporter gene experiment confirmed that FOXM1 and FOXM1 were targeted;up-regulation of FOXM1 reversed the effect of miR-134 overexpression on the cisplatin resistance of FD-LSC-1/DDP cells.Conclusion Overexpression of miR-134 can enhance the chemosensitivity of FD-LSC-1/DDP cells,which may be achieved by targetingly down-regulating the expression of FOXM1.