RECQ1沉默对口腔鳞癌细胞增殖、侵袭及上皮间质转化的影响

Effects of RECQ1 silencing on proliferation,invasion and epithelial mesenchymal transition of oral squamous cell carcinoma

目的:探讨沉默DNA解螺旋酶RECQ1基因对口腔鳞癌细胞增殖、迁移及上皮间质转化(EMT)的影响。方法:体外培养人正常口腔角质上皮HOK细胞、口腔鳞癌Cal27细胞,采用实时定量RT-PCR与Western Blot检测RECQ1 mRNA及蛋白表达水平。采用RNA干扰法靶向沉默Cal27细胞RECQ1基因,实验分为5组:si-RECQ1组(si-RECQ1转染至Cal27细胞)、si-RECQ1+转化生长因子β1(TGF-β1)组(RECQ1沉默后加入TGF-β1)、si-NC组、TGF-β1组及空白对照组。采用CCK-8法检测各组细胞增殖抑制率,Transwell法检测细胞侵袭能力,细胞划痕实验检测细胞迁移能力,采用实时定量RT-PCR与Western Blot检测RECQ1及上皮性钙黏附蛋白(E-cadherin)、N钙黏附蛋白(N-cadherin)、波形蛋白(Vimentin)等EMT相关蛋白表达。结果:与HOK细胞相比,Cal27细胞RECQ1 mRNA及蛋白表达水平均显著升高(P<0.05);与空白对照组、si-NC组相比,si-RECQ1组Cal27细胞中RECQ1 mRNA及蛋白表达水平均显著降低(P<0.05);与空白对照组、si-NC组、si-RECQ1+TGF-β1组相比,si-RECQ1组Cal27细胞增殖抑制率、E-cadherin mRNA及蛋白表达水平均显著升高,而TGF-β1组显著降低(P<0.05),且si-RECQ1组Cal27细胞侵袭数量、迁移距离、Vimentin、N-cadherin mRNA及蛋白表达水平均显著降低,而TGF-β1组显著升高(P<0.05)。结论:RECQ1沉默后明显抑制口腔鳞癌细胞增殖、迁移及侵袭,其机制可能是通过抑制EMT过程而实现。

Objective:To investigate the effects of silencing RECQ1 gene on the proliferation,migration,and epithelial mesenchymal transition(EMT)of oral squamous cell carcinoma.Methods:Human normal oral keratinocytes HOK cells and oral squamous cell carcinoma Cal27 cells were cultured in vitro,and the expressions of RECQ1 mRNA and protein were detected by qRT-PCR and Western Blot.RNAi was used in targeted silence RECQ1 gene in oral squamous cell carcinoma Cal27 cells.The experiment was divided into five groups:si-RECQ1 group(si-RECQ1 transfected into Cal27 cells),si-RECQ1+transforming growth factorβ1(TGF-β1)group(added TGF-β1 after RECQ1 silence),si-NC group,TGF-β1 group and blank control group.CCK-8 method was used to detect the inhibition rate of cell proliferation in each group.Transwell assay was used to detect the invasive ability of cells.Cell migration ability was measured by cell scratch test.The expressions of RECQ1 and EMT-related proteins,including E-cadherin,N-cadherin and Vimentin,were detected by qRT-PCR and Western Blot.Results:Compared with HOK cells,the expression of RECQ1 mRNA and protein in Cal27 cells increased significantly(P<0.05);compared with the blank control group and the si-NC group,the expressions of RECQ1 mRNA and protein in Cal27 cells in the si-RECQ1 group decreased significantly(P<0.05);compared with blank control group,si-NC group and si-RECQ1+TGF-β1 group,the proliferation inhibition rate,E-cadherin mRNA and protein expression levels of Cal27 cells in si-RECQ1 group were increased significantly,while TGF-β1 group decreased significantly(P<0.05),and Cal27 cell invasion number,migration distance,Vimentin,N-cadherin mRNA and protein expression levels in si-RECQ1 group decreased significantly,but TGF-β1 group increased significantly(P<0.05).Conclusions:RECQ1 silencing can significantly inhibit the proliferation,migration and invasion of oral squamous cell carcinoma,which may be achieved by inhibiting the EMT process.