TAB182维持RPA2 mRNA稳定性促进DNA同源重组修复的研究

TAB182 promotes DNA homologous recombination repair by stabilizing RPA2 mRNA

目的探讨TAB182促进同源重组(HR)修复的相关调控分子及机制。方法利用shRNA敲低人乳腺癌MCF-7细胞中的TAB182基因,TAB182敲低的MCF-7细胞为沉默组,使用shRNA阴性对照物的MCF-7细胞为TAB182阴性对照组。通过RNA测序分析,筛选与TAB182相关差异表达的HR修复基因。qRT-PCR验证相关基因的mRNA表达,Western blot验证相关蛋白的表达。RAD51和BrdU免疫荧光检测DNA断裂末端形成的3′单链DNA(ssDNA);流式细胞术检测细胞周期阻滞和细胞凋亡;集落形成实验检测细胞的放射敏感性。结果RNA测序分析和qRT-PCR验证结果一致,TAB182沉默组细胞中RPA2的mRNA表达降低(t=17.97,P<0.05)。相比于TAB182阴性对照组细胞,TAB182沉默组细胞RPA2在蛋白水平也显著减少。相比于TAB182阴性对照组,TAB182沉默组细胞中RAD51焦点(foci)的表达显著降低;3′ssDNA结合蛋白标志物BrdU在TAB182沉默组细胞的细胞核中foci形成显著减少。在放射菌素D作用后的第4、8、12 h,相比于TAB182阴性对照组,TAB182沉默组细胞RPA2 mRNA的衰减加快(t=5.37、3.79、3.69,P<0.05)。相比于TAB182阴性对照组,TAB182沉默组细胞放射敏感性增加(t=3.48,P<0.05);且TAB182沉默组放射诱发的凋亡率显著增加(t=11.05,P<0.05)、照射后24 h的周期阻滞时间延长(t=8.40,P<0.01)。结论TAB182通过维持RPA2 mRNA的稳定性,促进RPA2的表达,在DSBs的同源重组修复通路中发挥作用。TAB182的缺失可增强肿瘤细胞的放射敏感性。

Objective To investigate the regulating molecules and acting mechanism of TAB182 in HR pathway.Methods TAB182 in human breast cancer MCF-7 cells was knocked down by shRNA strategy,the TAB182 knockdown MCF-7 as the TAB182 knockdown group,and the MCF-7 cell using the shRNA negative control as the TAB182 negative control group.RNA sequencing and qRT-PCR were performed to screen and verify the differentially expressed genes of HR pathway related to TAB182 depression.Western blot was used to detect protein expression.Immunofluorescence staining of nuclear RAD51 and BrdU was used to check the 3'ssDNA formation by the end resection.The cell cycle arrest and apoptosis were measured by flow cytometry.Cloning formation assay was used to evaluate the sensitivity TAB182-knockdown cells to radiation.Results Both quantitative RNA sequencing and qRT-PCR assays showed that TAB182-knockdown significantly decreased the mRNA expression of RPA2(t=17.97,P<0.05).Compared with the TAB182 negative control group,the protein level of RPA2,the number of RAD51 foci,and the 3'ssDNA-binding nuclear protein marker BrdU in TAB182-knockdown cells were significantly reduced.At 4,8,and 12 h after actinomycin D treatment,the attenuation of RPA2 mRNA in the TAB182-knockdown cells was accelerated(t=5.37,3.79,3.69,P<0.05).Compared with the TAB182 negative control group,the radiosensitivity and radiation-induced apoptosis in the TAB182-knockdown group were increased(t=3.48,11.05,P<0.05),and at 24 h after irradiation,the cell cycle block time was prolonged(t=8.40,P<0.01).Conclusions TAB182 plays a role in maintaining RPA2 mRNA stability,thereby promoting HR repair.TAB182 knockdown cells are highly sensitive to ionizing radiation.