BECN1影响三阴性乳腺癌对多西他赛敏感性的实验研究

Study of BECN1 affects the sensitivity of triple negative breast cancer to docetaxel

目的:探讨沉默BECN1后,三阴性乳腺癌(TNBC)MDA-MB-231细胞对多西他赛敏感性变化。方法:将MDA-MB-231细胞分为干扰组、阴性对照组(NC组)和空白组,NC组与干扰组分别使用含有阴性对照慢病毒、BECN1干扰慢病毒的完全培养基培养进行转染处理,空白组不做处理。实时荧光定量PCR检测各组细胞BECN1 mRNA的表达。蛋白免疫印迹反应(Western blot, WB)检测各组细胞BECN1蛋白的表达。CCK-8法检测多西他赛处理各组细胞后的半抑制浓度(IC50)。流式细胞术检测多西他赛干预48 h后各组细胞的凋亡率。结果:荧光显微镜下可观察干扰组、NC组干扰慢病毒感染效率均达95%以上。实时荧光定量PCR结果显示,与空白组及NC组比较,干扰组BECN1 mRNA的表达显著降低(P<0.05)。WB实验结果显示,与空白组及NC组比较,干扰组BECN1蛋白的表达显著降低(P<0.05)。CCK-8结果显示,多西他赛干预48 h后,与空白组及NC组比较,干扰组IC50显著减小(P<0.05),多西他赛呈浓度依赖性抑制MDA-MB-231细胞的增殖,在同一浓度下,干扰组细胞增殖抑制率高于空白组及NC组(P<0.05)。流式细胞术结果显示,0.1μg/mL多西他赛干预48 h后,与空白组及NC组比较,干扰组细胞凋亡率明显减小(P<0.05)。结论:通过RNA干扰沉默BECN1基因能提高多西他赛对MDA-MB-231细胞的增殖抑制作用,但减弱了多西他赛诱导三阴性乳腺癌细胞MDA-MB-231凋亡效果,提示BECN1有可能成为治疗三阴性乳腺癌细胞化疗耐药的一个重要靶点。

Objective:To investigate the change in the sensitivity of triple negative breast cancer(TNBC)MDAMB-231 cells to docetaxel after silenceof BECN1.Methods:MDA-MB-231 cells were divided into interference group,negative control group(NC group),and control group.NC group and interference group were transfected with complete culture medium containing negative control lentivirus or BECN1 interference lentivirus,respectively,and control group was received no treated.Real-time fluorescence quantitative PCR was used to detect the expression of BECN1 mRNA in each group of cells.WB was used to detect the expression of BECN1 protein in each group of cells.The CCK-8 method was used to detect the half maximal inhibitory concentration(IC50)of cells thattreated with docetaxel.Flow cytometry was used to detect the apoptosis rate of each group of cells after48 hours of docetaxel intervention.Results:Under the fluorescence microscope,it was observed that the interference efficiency of interfering lentivirus in the interference group and NC group were more than 95%.Real-time fluorescence quantitative PCR results showed that compared with the control group and the NC group,the expression of BECN1 mRNA in the interference group was significantly reduced(P<0.05).WB results showed that compared with the control group and the NC group,the expression of BECN1 protein in the interference group was significantly reduced(P<0.05).CCK-8 results showed that compared with the control group and the NC group,the IC50 of the interference group was significantly reduced after 48 hours of docetaxel intervention(P>0.05),and docetaxel showed a concentration-dependent inhibition of MDA-MB-231 proliferation.At the same concentration,the cell proliferation inhibition rate of the interference group was higher than that of the control group and the NC group(P<0.05).Flow cytometry results showed that compared with the control group and the NC group,the apoptosis rate of the interference group was significantly reduced after 48 hours of 0.1μg/ml docetaxel intervention(P<0.05).Conclusion:Silencing the BECN1 gene by RNA interference can increase the proliferation inhibitory effect of docetaxel on MDA-MB-231 cells,andreduces the effect of docetaxel-induced apoptosis of triple negative breast cancer cells MDA-MB-231,suggesting that BECN1 may become an important target for the treatment of triple negative breast cancer cell chemoresistance.