Mus81基因沉默对MDA-MB-231乳腺癌细胞增殖、凋亡及裸鼠成瘤能力的影响

目的:分析甲磺酸盐及紫外线敏感性81号基因(Mus81)在乳腺癌组织中的表达水平,观察Mus81基因敲减对三阴性乳腺癌细胞MDA-MB-231增殖、凋亡和裸鼠移植瘤形成能力的影响。方法:从TCGA数据库下载乳腺癌样本基因表达数据,应用perl及R软件整理数据筛选出每个样本Mus81基因表达量。通过慢病毒介导的小干扰RNA(short interfering RNA,siRNA)技术构建Mus81基因敲减的MDA-MB-231乳腺癌细胞系(即Mus81敲减组)和阴性对照组MDA-MB-231乳腺癌细胞系,以实时定量PCR法检测Mus81基因的敲减效率,再以MTT检测实验、克隆形成实验、细胞流式检测及实时定量PCR法检测两组MDA-MB-231细胞的生长、增殖、细胞周期分布、凋亡水平及Mus81下游基因表达水平。最后,向BALB/c裸鼠右侧腋下注射Mus81基因敲减组和阴性对照组MDA-MB-231乳腺癌细胞,观察Mus81基因沉默对MDA-MB-231细胞在裸鼠中成瘤能力的影响。结果:Mus81基因在乳腺癌组织中的平均表达量明显高于癌旁组织(P<0.05)。Mus81基因敲减组MDA-MB-231细胞中Mus81基因的表达水平明显低于阴性对照组(P<0.05),Mus81基因的敲减效率达70.7%。较之阴性对照组,Mus81基因敲减组MDA-MB-231细胞的生长速度明显减缓(P<0.05);形成的细胞克隆数也明显下降(P<0.05);细胞凋亡水平、G2/M期细胞比例则明显升高(P<0.05)。STC2等Mus81下游基因的表达水平在两组MDA-MB-231细胞间也有明显差异(P<0.05)。裸鼠成瘤实验显示,Mus81基因敲减组形成的裸鼠瘤体体积和重量均明显低于阴性对照组(P<0.05)。结论:Mus81基因在乳腺癌组织中的表达明显升高,且可能通过调控STC2等下游基因的表达促进三阴性乳腺癌细胞的生长增殖及裸鼠体内成瘤能力并抑制其凋亡,提示其可能是一个潜在的乳腺癌治疗靶点。

Effects of Mus81 gene knockdown on proliferation and apoptosis of MDA-MB-231 breast cancer cell and tumorigenicity in nude mice

Objective:To understand the expression level of Mus81 gene in breast cancer tissue,and to observe the effect of Mus81 gene knockdown on the proliferation,apoptosis and the ability of transplanted tumor formation of triple negative MDA-MB-231 breast cancer cell.Methods:Download the gene expression data of breast cancer samples from the TCGA database,and the expression of Mus81 gene in each sample was screened by perl and R software.By using lentivirus mediated short interfering RNA（siRNA) technology,we constructed the MDA-MB-231 human breast cancer cells line（Mus81 knockdown group) and negative control group MDA-MB-231 breast cancer cells line,real-time quantitative PCR was used to detect the knockdown efficiency of Mus81 gene,and then MTT detection experiment,flow cytometry,colony forming assay,cell flow cytometry,and real-time quantitative PCR were used to detect the growth,proliferation,cell cycle distribution,apoptosis level and Mus81 downstream gene expression of the two groups of MDA-MB-231 cells level.Finally,MDA-MB-231 breast cancer cells in the Mus81 knockout group and the negative control group were injected into the right axilla of BALB/c nude mice to observe the effect of Mus81 gene silencing on the tumor-forming ability of MDA-MB-231 cells in nude mice.Results:The average expression of Mus81 gene in breast cancer tissues was significantly higher than that in adjacent tissues（P＜0.05).The expression level of Mus81 gene in MDA-MB-231 cells in the Mus81 knockdown group was significantly lower than that in the negative control group（P＜0.05),and the knockout efficiency of Mus81 gene was 70.7%.Compared with the negative control group,the growth rate of MDA-MB-231 cells in Mus81 knockdown group was significantly slower（P<0.05).The number of cell clones was also significantly decreased（P<0.05).The level of apoptosis and the proportion of cells in G2/M phase were significantly increased（P<0.05).The expression levels of Mus81 downstream genes such as STC2 in MDA-MB-231 cells were also significantly different between the two groups（P<0.05).Tumorigenesis experiment in nude mice showed that the volume and weight of the tumor formed in the knockout group were significantly lower than that in the negative control group（P<0.05).Conclusion:The expression of Mus81 gene was significantly increased in breast cancer tissues,and it may promote the growth and proliferation of triple negative breast cancer cells,tumor-forming ability and inhibit apoptosis in nude mice by regulating the expression of STC2 and other downstream genes,suggesting that Mus81 may be a potential therapeutic target for breast cancer.