Sun F Z, Wang J S, Li C X, Wang T, Zhang J Y, Zhou Y T, Wang H J, Qian H L
National Key Laboratory of Molecular Oncology, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medicine College, Beijing 100021, China.
Zhonghua Zhong Liu Za Zhi. 2022 Sep 23;44(9):962-967. doi: 10.3760/cma.j.cn112152-20200929-00866.
To determine the effect of tumor metastasis-associated gene 1 (MTA1) on the sensitivity of HeLa cells to radiotherapy, and to clarify its molecular mechanism. The transcriptome differences between MTA1 knocked down Hela cells and control cells were analyzed, and the differentially expressed genes (DEGs) was used to perform Gene-Set Enrichment Analysis (GSEA) and Gene Ontology (GO) cluster analysis. Flow cytometry was used to detect apoptosis in MTA1-overexpressed HeLa cells and control cells before and after 10 Gy X-ray irradiation. Cloning formation assay and real-time cellular analysis (RTCA) were used to monitor the cell proliferation before and after 2 Gy X-ray irradiation. To dissect the underlying molecular mechanisms of MTA1 affecting the sensitivity of radiotherapy, the proteins encoded by the DEGs were selected to construct a protein-protein interaction network, the expression of γ-H2AX was detected by immunofluorescence assay, and the expression levels of γ-H2AX, β-CHK2, PARP and cleaved caspase 3 were measured by western blot. By transcriptome sequencing analysis, we obtained 649 DEGs, of which 402 genes were up-regulated in MTA1 knockdown HeLa cells and 247 genes were down-regulated. GSEA results showed that DEGs associated with MTA1 were significantly enriched in cellular responses to DNA damage repair processes. The results of flow cytometry showed that the apoptosis rate of MTA1 over-expression group (15.67±0.81)% after 10 Gy X-ray irradiation was significantly lower than that of the control group [(40.27±2.73)%, <0.001]. After 2 Gy X-ray irradiation, the proliferation capacity of HeLa cells overexpressing MTA1 was higher than that of control cells (=0.024). The numbers of colon in MTA1 over-expression group before and after 2 Gy X-ray irradiation were (176±7) and (137±7) respectively, higher than (134±4) and (75±4) in control HeLa cells (<0.05). The results of immunofluorescence assay showed that there was no significant expression of γ-H2AX in MTA1 overexpressed and control HeLa cells without X-ray irradiation. Western blot results showed that the expression level of β-CHK2 in MTA1-overexpressing HeLa cells (1.04±0.06) was higher than that in control HeLa cells (0.58±0.25, =0.036) after 10 Gy X-ray irradiation. The expression levels of γ-H2AX, PARP, and cleaved caspase 3 were 0.52±0.13, 0.52±0.22, and 0.63±0.18, respectively, in HeLa cells overexpressing MTA1, which were lower than 0.87±0.06, 0.78±0.12 and 0.90±0.12 in control cells (>0.05). This study showed that MTA1 is significantly associated with radiosensitivity in cervical cancer HeLa cells. MTA1 over-expression obviously reduces the sensitivity of cervical cancer cells to X-ray irradiation. Mechanism studies initially indicate that MTA1 reduces the radiosensitivity of cervical cancer cells by inhibiting cleaved caspase 3 to suppress apoptosis and increasing β-CHK2 to promote DNA repair.
为确定肿瘤转移相关基因1（MTA1）对HeLa细胞放疗敏感性的影响，并阐明其分子机制。分析MTA1敲低的Hela细胞与对照细胞之间的转录组差异，使用差异表达基因（DEG）进行基因集富集分析（GSEA）和基因本体（GO）聚类分析。采用流式细胞术检测10 Gy X射线照射前后MTA1过表达的HeLa细胞和对照细胞的凋亡情况。采用克隆形成试验和实时细胞分析（RTCA）监测2 Gy X射线照射前后细胞的增殖情况。为剖析MTA1影响放疗敏感性的潜在分子机制，选择DEG编码的蛋白质构建蛋白质-蛋白质相互作用网络，通过免疫荧光试验检测γ-H2AX的表达，并通过蛋白质免疫印迹法检测γ-H2AX、β-CHK2、PARP和裂解的caspase 3的表达水平。通过转录组测序分析，我们获得了649个DEG，其中402个基因在MTA1敲低的HeLa细胞中上调，247个基因下调。GSEA结果显示，与MTA1相关的DEG在细胞对DNA损伤修复过程的反应中显著富集。流式细胞术结果显示，10 Gy X射线照射后，MTA1过表达组的凋亡率为（15.67±0.81）%，显著低于对照组[（
