干扰性小核糖核酸靶向抑制趋化因子受体4基因表达对鼻咽癌细胞增殖和侵袭的影响

Effect of targeted inhibition of the expression of chemokine receptor 4 gene by small interfering RNA on the invasion and proliferation of nasopharyngeal carcinoma cells

目的探讨干扰性小核糖核酸(siRNA)靶向抑制鼻咽癌细胞趋化因子受体4(CXCR4)基因表达对癌细胞增殖、侵袭的影响及机制。方法收集新乡医学院第一附属医院2014年1月至2016年12月保存的鼻咽癌及相应的癌旁组织标本各42例,采用实时荧光定量聚合酶链式反应(RT-PCR)和Western blot法分别检测鼻咽癌及相应的癌旁组织中CXCR4 mRNA及蛋白表达。将人鼻咽癌细胞系CNE-2Z分为空白对照组(细胞未做任何处理)、阴性对照组(细胞转染无义siRNA序列)和CXCR4转染组(细胞转染CXCR4的靶向siRNA序列),转染48 h后采用Western blot法检测各组细胞中CXCR4、基质金属蛋白酶(MMP)-2、MMP-9、β-连环蛋白(β-catenin)和细胞周期素D1(Cyclin D1)蛋白表达;应用细胞计数试剂盒和Transwell小室分别检测细胞的增殖和侵袭能力。结果鼻咽癌及相应的癌旁组织中CXCR4 mRNA表达分别为5.526±0.143、0.953±0.091,蛋白表达分别为0.522±0.047、0.053±0.011,鼻咽癌组织中CXCR4 mRNA及蛋白表达均显著高于癌旁组织(P<0.05)。CXCR4转染组细胞中CXCR4、MMP-2、MMP-9、β-catenin、Cyclin D1蛋白表达及细胞存活率、细胞侵袭数均显著低于空白对照组和阴性对照组(P<0.05),但空白对照组和阴性对照组细胞中CXCR4、MMP-2、MMP-9、β-catenin、Cyclin D1蛋白表达及细胞存活率、细胞侵袭数比较差异均无统计学意义(P>0.05)。结论抑制鼻咽癌CNE-2Z细胞中CXCR4基因表达可显著降低肿瘤细胞的增殖与侵袭能力,其机制可能与下调Wnt/β-catenin信号通路有关。

Objective To investigate the effect and its mechanism of targeted inhibition of the expression of chemokine receptor 4（ CXCR4） gene by small interfering RNA（ siRNA） on the invasion and proliferation of nasopharyngeal carcinoma cells. Methods Forty-two nasopharyngeal carcinoma tissue and its adjacent tissues in the First Affiliated Hospital of Xinxiang Medical University from January 2014 to December 2016 were collected. The expression of CXCR4 mRNA and protein in nasopharyngeal carcinoma tissue and its adjacent tissues were detected by real time-polymerase chain reaction and Western blot.The human nasopharyngeal carcinoma cell line CNE-2 Z were divided into blank control group,negative control group and CXCR4 transfection group. The cells in blank control group were not given any treatment; the cells in negative control group were transfected nonsense siRNA sequence; the cells in CXCR4 transfection group were transfected CXCR4 targeting siRNA sequence. The protein expression of CXCR4,matrix metalloproteinases-2（ MMP-2）,MMP-9,β-catenin,Cyclin D1 were detected by Western bloting after 48 h of transfection. The proliferation and invasion ability of the cells were detected by cell counting kit and Transwell chamber. Results The expression of CXCR4 mRNA in nasopharyngeal carcinoma tissue and adjacent tissues was 5. 526 ± 0. 143,0. 953 ± 0. 091 respectively; the expression of CXCR4 protein in nasopharyngeal carcinoma tissue and adjacent tissues was 0. 522 ± 0. 047,0. 053 ± 0. 011 respectively. The expression of CXCR4 mRNA and protein in nasopharyngeal carcinoma tissue were significantly higher than those in adjacent tissues（ P〈 0. 05）. The protein expression of CXCR4,MMP-2,MMP-9,β-catenin,Cyclin D1 in cells,cell survival rate and the number of cell invasion in CXCR4 transfection group were significantly lower than those in the blank control group and negative control group（ P〈 0. 05）; however,there was no significant difference in above indexes between the blank control group and negative control group（ P〈 0. 05）.Conclusion Inhibiting of CXCR4 gene expression in nasopharyngeal carcinoma CNE-2 Z cells can significantly decrease the proliferation and invasion ability of cancer cells,and the mechanism may be related to down regulation of Wnt/β-catenin signaling pathway.