慢病毒介导的RNA干扰FOXC2表达对皮肤鳞状细胞癌A431细胞功能影响  

Effect of lentivirus mediated RNA interference on FOXC2 expression on the function of cutaneous squamous cell carcinoma A431 cells

作　　者：陈瑛毅[1] CHEN Yingyi(Department of Dermatology,Dalian Dermatosis Hospital,Liaoning Dalian 116001,China)

机构地区：[1]大连市皮肤病医院皮肤科,辽宁大连116001

出　　处：《现代肿瘤医学》2021年第8期1301-1305,共5页Journal of Modern Oncology

基　　金：大连市卫生局科研基金项目(编号:2016-0258X)。

摘　　要：目的:探讨慢病毒介导的RNA干扰叉头框C2(FOXC2)表达对皮肤鳞状细胞癌A431细胞增殖、侵袭和凋亡的影响及其机制。方法:采用实时荧光定量聚合酶链反应(PCR)和免疫印迹法检测人正常永生化上皮Hecate细胞和皮肤鳞状细胞癌A431细胞中FOXC2 mRNA和蛋白的表达水平。将体外培养的A431细胞分为对照组(正常培养)、NC-shRNA组(LV-NC-shRNA慢病毒感染)和FOXC2-shRNA组(LV-FOXC2-shRNA慢病毒感染),采用噻唑蓝(MTT)法检测细胞活力,平板克隆形成实验检测细胞克隆形成能力,Transwell小室实验检测细胞侵袭,流式细胞术检测细胞凋亡,免疫印迹法检测细胞中蛋白激酶B(AKT)、磷酸化(p)-AKT、细胞周期蛋白D1(CyclinD1)、基质金属蛋白酶9(MMP-9)和B淋巴细胞瘤-2基因(Bcl-2)蛋白表达水平。结果:与Hecate细胞比较,A431细胞中FOXC2 mRNA和蛋白表达水平均明显升高(P<0.05)。与对照组比较,NC-shRNA组细胞中FOXC2 mRNA表达水平、细胞存活率、侵袭数目、克隆形成率、细胞凋亡率和FOXC2、p-AKT、CyclinD1、MMP-9、Bcl-2蛋白表达水平差异均无统计学意义(P>0.05);与对照组或NC-shRNA组比较,FOXC2-shRNA组细胞中FOXC2 mRNA表达水平、细胞存活率、侵袭数目、克隆形成率和FOXC2、p-AKT、CyclinD1、MMP-9、Bcl-2蛋白表达水平均明显降低,而细胞凋亡率明显升高(P<0.05)。结论:沉默FOXC2表达可抑制皮肤鳞状细胞癌A431细胞增殖、侵袭并促进细胞凋亡,其作用机制可能与抑制AKT信号通路活化有关。Objective:To investigate the effect of lentivirus mediated RNA interference expression of FOXC2 on proliferation,invasion and apoptosis of cutaneous squamous cell carcinoma A431 cells and mechanism.Methods:The expression of FOXC2 mRNA and protein in human normal immortalized epithelial Hecate cells and cutaneous squamous cell carcinoma A431 cells were detected by real-time fluorescence quantitative PCR and Western blot.A431 cells cultured in vitro were divided into control group(normal culture),NC shRNA group(LV-NC-shRNA lentivirus infection)and FOXC2-shRNA group(LV-FOXC2-shRNA lentivirus infection).Cell viability was detected by MTT method.Cell clonogenesis was detected by plate clonogenesis test.Cell invasion was detected by Transwell chamber test.Cell apoptosis was detected by flow cytometry and the expression levels of AKT,p-AKT,CyclinD1,MMP-9 and Bcl-2 were measured by Western blotting.Results:Compared with Hecate cells,the expression of FOXC2 mRNA and protein in A431 cells increased significantly(P<0.05).Compared with the control group,there was no significant difference in FOXC2 mRNA expression level,cell survival rate,invasion number,clone formation rate,apoptosis rate and the expression levels of FOXC2,p-AKT,CyclinD1,MMP-9,Bcl-2 proteins in NC-shRNA group(P>0.05).Compared with the control group or NC-shRNA group,FOXC2 mRNA expression level,cell survival rate,invasion number and the expression levels of FOXC2,p-AKT,CyclinD1,MMP-9 and Bcl-2 in FOXC2-shRNA group were significantly decreased,while the apoptosis rate was significantly increased(P<0.05).Conclusion:The inhibition of FOXC2 expression can inhibit the proliferation,invasion and promote apoptosis of cutaneous squamous cell carcinoma A431 cells,and its mechanism may be related to the inhibition of AKT signaling pathway activation.

关 键 词：皮肤鳞状细胞癌 叉头框C2 细胞增殖 侵袭 凋亡 蛋白激酶B信号通路 

分 类 号：R739.5[医药卫生—肿瘤]