lncRNA BRE-AS1调控Wnt/β-catenin信号通路影响宫颈癌SiHa细胞增殖、侵袭和凋亡的实验研究

Effects of lncRNA BRE-AS1 on SiHa cells in cervical cancer of proliferation, invasion and apoptosis by Wnt/β-catenin signaling pathway

目的:探讨lncRNA BRE-AS1对宫颈癌SiHa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PCR检测人子宫颈上皮永生化细胞系H8和宫颈癌细胞系SiHa中lncRNA BRE-AS1的表达水平。将对数生长期的SiHa细胞分为未转染组(正常培养)、阴性对照组(转染空载体)和lncRNA BRE-AS1组(转染lncRNA BRE-AS1表达载体),采用实时荧光定量PCR检测各细胞中lncRNA BRE-AS1的表达水平,噻唑蓝法检测细胞增殖能力,平板克隆实验细胞克隆形成能力,Transwell小室检测细胞侵袭能力,流式细胞仪检测细胞凋亡能力,免疫印迹法检测细胞中Wnt/β-catenin信号通路相关蛋白β-catenin、细胞周期蛋白D1(Cyclin D1)、基质金属蛋白酶2(MMP-2)和c-Myc的表达。结果:与人子宫颈上皮永生化细胞系H8相比,宫颈癌细胞系SiHa中lncRNA BRE-AS1的表达水平明显降低(P<0.01)。与未转染组相比,lncRNA BRE-AS1组细胞中lncRNA BRE-AS1的表达水平和细胞凋亡率明显升高,且细胞增殖活力、克隆形成率、穿膜细胞数和细胞中β-catenin、c-Myc、Cyclin D1、MMP-2蛋白的表达水平均明显降低(P<0.05);而阴性对照组和未转染组相比,上述指标均未见明显改变(P>0.05)。结论:lncRNA BRE-AS1可抑制宫颈癌SiHa细胞增殖、侵袭并促进其凋亡,其作用机制可能与抑制Wnt/β-catenin信号通路有关。

Objective:To investigate the effects of lncRNA BRE-AS1 on SiHa cells of the proliferation, invasion and apoptosis and its mechanism.Methods:The expression of lncRNA BRE-AS1 in immortalized human cervical epithelial cell line H8 and cervical cancer cell line SiHa was detected by real-time fluorescence quantitative PCR.SiHa cells in logarithmic growth phase were divided into un-transfected group(normal culture),negative control group(transfected empty vector) and lncRNA BRE-AS1 group(transfected with lncRNA BRE-AS1 expression vector).The expression level of lncRNA BRE-AS1 was detected by quantitative PCR of real-time fluorescence, cell proliferation was detected by thiazole blue method, and cell cloning ability was performed by plate cloning experiment plate cloning, cell invasiveness was detected by Transwell chamber, the apoptotic ability was detected by flow cytometry.Flow cytometry and the expression of Wnt/β-catenin signaling pathway-related proteins β-catenin, cyclin D1,matrix metalloproteinase 2(MMP-2) and c-Myc were measured by Western blot.Results:Compared with human cervical epithelial immortalized cell line H8,the expression level of lncRNA BRE-AS1 in cervical cancer cell line SiHa was significantly lower(P<0.01).Compared with the untransfected group, the expression level of lncRNA BRE-AS1 and the apoptotic rate of cells in the lncRNA BRE-AS1 group increased significantly, on the other hand the cell proliferation activity, clone formation rate, the number of transmembrane cells and the expression levels of β-catenin, c-Myc, Cyclin D1 and MMP-2 protein in the cells decreased significantly(P<0.05),while compared with the negative control group and the untransfected group, the above indexes did not change significantly(P>0.05).Conclusion:LncRNA BRE-AS1 can inhibit the proliferation, invasion and promote apoptosis of SiHa cells, and its mechanism may be related to the inhibition of signaling pathway of Wnt/β-catenin.