摘要
目的探讨RNAi干扰Twist基因对喉鳞状细胞癌Hep-2细胞株增殖、侵袭力及E-钙黏蛋白甲基化的影响。方法培养人喉鳞状细胞癌Hep-2细胞株,分别转染siRNA-Twist(siRNA-Twist组)、阳性对照siRNAGAPDH(阳性对照组)和阴性对照错义链(阴性对照组),利用实时荧光定量PCR技术检测各转染组细胞中Twist和E-cad基因表达,Western blot法检测各转染组细胞中Twist和E-cad蛋白表达,MTT比色法检测各转染组细胞增殖情况,Transwell小室检测细胞迁移和侵袭能力,甲基化特异性聚合酶链式反应(MSP)检测不同转染组细胞中Ecad甲基化表达。结果 siRNA-Twist组细胞中Twist mRNA和Twist蛋白表达量均低于阳性对照组和阴性对照组,差异均具有统计学意义(P<0.05);siRNA-Twist组细胞中E-cad mRNA和E-cad蛋白表达量均高于阳性对照组和阴性对照组,差异均具有统计学意义(P<0.05);与阳性对照组和阴性对照组相比,siRNA-Twist组细胞转染48 h^96 h时细胞增殖能力明显减弱,差异均具有统计学意义(P<0.05);siRNA-Twist组细胞迁移细胞数和侵袭细胞数均低于阳性对照组和阴性对照组,差异均具有统计学意义(P<0.05);MSP检测结果显示,siRNA-Twist组细胞中E-cad甲基化扩增条带呈弱阳性,表达强度显著低于阳性对照组和阴性对照组,而非甲基化扩增条带呈阳性。结论特异性抑制Twist基因表达可能通过影响E-cad甲基化而上调E-cad表达,从而抑制细胞增殖及侵袭能力,有望为喉鳞状细胞癌基因治疗提供新的靶点。
Objective To investigate the effects of Twist gene silenced by RNAi interference on proliferation, invasiveness and methylation of E-cadherin in laryngeal squamous cell carcinoma Hep-2 cells. Methods Human laryngeal squamous cell carcinoma Hep-2 cell lines were cultured, and then transfected with siRNA-Twist （siRNA-Twist group）, positive control siRNA-GAPDH （positive control group） and negative control missense chain （negative control group）. The expressions of Twist and E-cad genes in cells of all 3 groups were detected by real-time fluorescence quantitative PCR. The expressions of Twist and E-cad proteins were detected by Western blot. MTT assay and transwell chamber were adopted to examine cell proliferations, cell migration and invasion respectively. The expression of methylation of E-cad was detected by methylation-specific polymerase chain reaction （MSP）. Results The expressions of Twist mRNA and proteins in siRNA-Twist group were lower than those in the positive control group and negative control group respectively, and the differences were all statistically significant （ all P 〈 0.05）. The expressions of E-cad mRNA and proteins in siRNA-Twist group were higher than those in the positive and negative control groups respectively, the differences were all statistically significant （ all P 〈 0.05 ）. The cell proliferation in siRNA-Twist group was significantly reduced after transfected 48 h - 96 h, the difference was statistically significant （P 〈 0.05）. The migration cells and invasion cells in siRNA-Twist group were lower than those in the positive and negative control groups, the differences were statistically significant （ all P 〈 0.05）. MSP test results showed that the amplified bands of E-cad methylation in the siRNA-Twist group were weakly positive, its expression intensity was significantly lower than those of the positive and negative control groups, while the unmethylation amplified bands were positive. Conclusions Specific inhibition of Twist gene might up-re
出处
《中国耳鼻咽喉颅底外科杂志》
CAS
2016年第6期467-471,共5页
Chinese Journal of Otorhinolaryngology-skull Base Surgery
基金
辽宁省自然科学基金(2013021008)
