沉默BECN1对人三阴性乳腺癌细胞生物学行为的影响

The effect of silencing BECN1 on the biological behavior of human triple-negative breast can⁃cer cells

目的:探讨沉默人三阴性乳腺癌(TNBC)细胞MDA-MB-231中beclin 1(BECN1)基因后其生物学特性的变化。方法:设计3组沉默BECN1基因的慢病毒(干扰组1、干扰组2和干扰组3)以及阴性对照慢病毒(NC组)分别转染MDA-MB-231细胞,空白组不做处理。实时荧光定量PCR(RT-qPCR)检测BECN1 mRNA表达,蛋白免疫印迹(WB)实验检测各组细胞BECN1蛋白与自噬标志蛋白LC3B的表达,筛选出抑制率最高的一组用于后续实验。细胞计数试剂8(CCK-8)检测各组细胞的增殖情况、Transwell实验检测各组细胞的迁移及侵袭能力、流式细胞术检测各组细胞凋亡率与周期分布情况。结果:慢病毒感染细胞后,干扰组2中BECN1的信使核糖核酸(mRNA)与蛋白的表达量最低(P<0.05),WB结果显示干扰组2的LC3B蛋白中LC3-Ⅱ/LC31-Ⅰ的比值明显低于空白组和NC组(均P<0.05),选取干扰组2用于后续实验。沉默BECN1基因后细胞的增殖、迁移和侵袭能力均明显降低(均P<0.05);流式细胞术结果显示干扰组2、NC组与空白组的凋亡率没有明显差异;相比于空白组与NC组,干扰组2处于G0/G1期的比例升高(P<0.05)。结论:BECN1基因沉默可以降低细胞的自噬水平,抑制TNBC MDA-MB-231细胞增殖、迁移与侵袭能力。

Objective:To investigate the changes in the biological characteristics of the human triple-negative breast cancer(TNBC)cells MDA-MB-231 after silencing BECN1 gene.Methods:Three groups of BECN1 gene silencing lentiviruses(interference group 1,interference group 2 and interference group 3)and negative control lentiviruse(NC group)were designed,and transfected into MDA-MB-231 cells.The blank group cells were not treated.Real-time fluorescent quantitative PCR(RT-qPCR)was used to detect the expression of BECN1 mRNA.Western blotting was used to detect the protein expression of BECN1 and autophagy marker LC3B.The lentiviral expression vector of BECN1 gene with the highest inhibition target sequence was screened for subsequent experiments.The proliferation,migration and invasion of cells were evaluated by CCK-8 method and Transwell assay,respectively.The apoptosis and cell cycle were determined by flow cytometry.Results:After lentiviral infection,the mRNA and protein expression levels of BECN1 in interference group 2 were the lowest(P<0.05).WB results showed that the LC3-Ⅱ/LC31-Ⅰratio of LC3B protein in interference group 2 was significantly lower than that in blank group and NC group(P<0.05).Interference group 2 was selected for subsequent experiments.After silencing BECN1 gene,the proliferation,migration and invasion of cells were significantly reduced(P<0.05).There was no significant difference in the apoptotic rate of cells among interference group 2,NC group and blank group.Compared with the blank group and NC group,the proportion of cells in G0/G1 phase was increased in interference group 2(P<0.05).Conclusion:BECN1 gene silencing can reduce the level of autophagy and inhibit the proliferation,migration and invasion of TNBC MDA-MB-231 cells.