FOXM1影响人宫颈癌细胞株Hela/DDP顺铂化疗敏感性的作用

Effect of FOXM1 on cisplatin chemosensitivity of human cervical cancer cell line HeLa/DDP

目的探讨叉头盒蛋白M1(FOXM1)对人宫颈癌细胞株Hela/DDP顺铂化疗敏感性的作用。方法将含有正义FOXM1互补的脱氧核糖核酸(cDNA)的真核表达载体、空质粒的真核表达载体采用脂质体转染法转染至人宫颈癌细胞株Hela/DDP,分别记为过表达组、过表达对照组;另构建干扰FOXM1基因表达的重组载体、空载载体,利用脂质体介导将其转染至人宫颈癌细胞株Hela/DDP,分别记为沉默组、沉默对照组;并设置空白组、抑制剂组。上述每组5个复孔,均加入顺铂,抑制剂组另加入硫链丝菌肽,培养48 h。实时逆转录荧光定量聚合酶链反应(RT-qPCR)、蛋白质免疫印迹法(Western Blot)检测各组FOXM1信使核糖核酸(mRNA)及蛋白表达;倒置显微镜观察细胞形态学改变;噻唑蓝(MTT)法检测各组细胞增殖抑制率;流式细胞仪双染法检测各组细胞凋亡率;RT-qPCR、Western Blot检测各组细胞周期蛋白B1(Cyclin B1)、细胞分裂周期因子25B(CDC25B)、存活蛋白(Survivin)、p21 mRNA及蛋白表达。结果与空白组比较,沉默组和抑制剂组FOXM1、Cyclin B1、CDC25B、Survivin mRNA及蛋白表达均下降(P<0.05),过表达组上述指标均升高(P<0.05);与空白组比较,沉默组和抑制剂组增殖抑制率、凋亡率、p21 mRNA及蛋白表达均升高(P<0.05),过表达组上述指标均下降(P<0.05);空白组、沉默对照组、过表达对照组细胞连接紧密、形态均匀、轮廓清楚,过表达组细胞连接致密,沉默组和抑制剂组细胞减少,轮廓不清、形态不均、大小不一,且可见细胞皱缩及细胞碎片。结论FOXM1表达下调可增加人宫颈癌细胞株Hela/DDP顺铂化疗敏感性,FOXM1表达上调则可降低其化疗敏感性,推测与正反馈调节Cyclin B1、CDC25B、Survivin表达,负反馈调节p21表达有关。

Objective To investigate the effect of forkhead box protein M1(FOXM1)on the chemosensitivity of human cervical cancer cell line HeLa/DDP to cisplatin.Methods The eukaryotic expression vector containing sense FOXM1 complementary deoxyribonucleic acid(cDNA)and empty plasmid were transfected into human cervical cancer cell line HeLa/DDP by liposome transfection,which were recorded as over-expression group and over-expression control group respectively.In addition,the recombinant vector and empty vector interfering with FOXM1 gene expression were constructed and transfected into human cervical cancer cell line HeLa/DDP mediated by liposome,which were recorded as silencing group and silencing control group respectively.Blank group and inhibitor group were set.Every group set 5 wells,and cisplatin was added to each of well,and thiostreptomyces peptide was added to the inhibitor group for 48h.The expressions of FOXM1 messenger RNA(mRNA)and protein were detected by real-time reverse transcription fluorescence quantitative polymerase chain reaction(RT-qPCR)and Western blot(WB).The morphological changes were observed by inverted microscope.The inhibition rates of cell proliferation were detected by thiazole blue(MTT).The apoptosis rates were detected by flow cytometry.RT-qPCR and WB were used to detect the expressions of Cyclin B1,CDC25B,survivin,p21 mRNA and proteins.Results Compared with the blank group,the expressions of FOXM1,Cyclin B1,CDC25B,Survivin mRNA and proteins decreased in the silencing group and inhibitor group(P<0.05),and the above indexes increased in the over-expression group(P<0.05).Compared with the blank group,the proliferation inhibition rates,apoptosis rates,p21 mRNA and protein expressions increased in the silencing group and inhibitor group(P<0.05),and the above indexes decreased in the over-expression group(P<0.05).The cells in the blank group,the silencing control group and the over-expression control group were tightly connected,uniform in shape and clear in outline.The cells in the over-expression group were tightly linked.The cells in the silencing group and the inhibitor group were reduced,with unclear outline,uneven in shape and different in size,and cell shrinkage and cell fragments were seen.Conclusion Down regulation of FOXM1 expression can increase the chemosensitivity of human cervical cancer cell line HeLa/DDP to cisplatin,while up regulation of FOXM1 expression can reduce its chemosensitivity.It is speculated that it is related to positive feedback regulating the expressions of Cyclin B1,CDC25B and survivin,and negative feedback regulating the expression of p21.