肿瘤转移相关基因1对宫颈癌HeLa细胞放疗敏感性的作用及机制
目的:探讨肿瘤转移相关基因1(MTA1)对宫颈癌HeLa细胞放疗敏感性的影响及其分子机制。方法:对MTA1敲降的HeLa细胞及对照HeLa细胞进行转录测序，获得差异表达基因并对其进行基因集富集分析和GO聚类分析。采用流式细胞术检测MTA1过表达的HeLa细胞和对照HeLa细胞在10 Gy X射线照射前后的细胞凋亡情况，克隆形成实验和实时无标记动态细胞分析(RTCA)技术检测细胞的增殖情况。选取差异表达基因编码的蛋白构建蛋白相互作用网络，采用免疫荧光实验检测γ－H2AX的表达情况，采用Western blot法检测γ－H2AX、β－CHK2、PARP和cleaved caspase 3的表达水平...
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| Published in | 中华肿瘤杂志 Vol. 44; no. 9; pp. 962 - 967 |
|---|---|
| Main Authors | , , , , , , , |
| Format | Journal Article |
| Language | Chinese |
| Published |
国家癌症中心 国家肿瘤临床医学研究中心 中国医学科学院北京协和医学院肿瘤医院分子肿瘤学国家重点实验室，北京 100021
23.09.2022
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| Subjects | |
| Online Access | Get full text |
| ISSN | 0253-3766 |
| DOI | 10.3760/cma.j.cn112152-20200929-00866 |
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| Abstract | 目的:探讨肿瘤转移相关基因1(MTA1)对宫颈癌HeLa细胞放疗敏感性的影响及其分子机制。方法:对MTA1敲降的HeLa细胞及对照HeLa细胞进行转录测序，获得差异表达基因并对其进行基因集富集分析和GO聚类分析。采用流式细胞术检测MTA1过表达的HeLa细胞和对照HeLa细胞在10 Gy X射线照射前后的细胞凋亡情况，克隆形成实验和实时无标记动态细胞分析(RTCA)技术检测细胞的增殖情况。选取差异表达基因编码的蛋白构建蛋白相互作用网络，采用免疫荧光实验检测γ－H2AX的表达情况，采用Western blot法检测γ－H2AX、β－CHK2、PARP和cleaved caspase 3的表达水平。结果:转录组测序分析获得差异表达基因649个，其中402个基因在MTA1敲降的HeLa细胞中表达上调，247个基因表达下调。与MTA1相关的差异表达基因富集于DNA损伤修复过程，GO聚类分析提示，DNA损伤修复富集于第2位。流式细胞术检测结果显示，10 Gy X射线照射后，MTA1过表达的HeLa细胞凋亡率[(15.67±0.81)%]低于对照HeLa细胞[(40.27±2.73)%，
P<0.001]。RTCA结果显示，过表达MTA1的HeLa细胞经2 Gy X射线照射后，细胞增殖能力高于对照HeLa细胞(
P＝0.024)。克隆形成实验显示，过表达MTA1的HeLa细胞经2 Gy X射线照射前后的克隆数分别为(176±7)个和(137±7)个，均高于对照细胞[分别为(134±4)个和(75±4)个，均
P<0.05]。免疫荧光实验结果显示，未经X射线照射的MTA1过表达及对照HeLa细胞中的γ－H2AX几乎无表达，经过10 Gy X射线照射后48 h，对照HeLa细胞中可见明显γ－H2AX表达，且MTA1过表达HeLa细胞中γ－H2AX表达相对较低。Western blot结果显示，10 Gy X射线照射后，过表达MTA1的HeLa细胞中β－CHK2的表达水平(1.04±0.06)高于对照HeLa细胞(0.58±0.25,
P＝0.036)，过表达MTA1的HeLa细胞中γ－H2AX、PARP和cleaved caspase 3的表达水平分别为0.52±0.13、0.52±0.22和0.63±0.18，均低于对照HeLa细胞(分别为0.87±0.06，0.78±0.12和0.90±0.12，均
P>0.05)。
结论: |
|---|---|
| AbstractList | 目的:探讨肿瘤转移相关基因1(MTA1)对宫颈癌HeLa细胞放疗敏感性的影响及其分子机制。方法:对MTA1敲降的HeLa细胞及对照HeLa细胞进行转录测序，获得差异表达基因并对其进行基因集富集分析和GO聚类分析。采用流式细胞术检测MTA1过表达的HeLa细胞和对照HeLa细胞在10 Gy X射线照射前后的细胞凋亡情况，克隆形成实验和实时无标记动态细胞分析(RTCA)技术检测细胞的增殖情况。选取差异表达基因编码的蛋白构建蛋白相互作用网络，采用免疫荧光实验检测γ－H2AX的表达情况，采用Western blot法检测γ－H2AX、β－CHK2、PARP和cleaved caspase 3的表达水平。结果:转录组测序分析获得差异表达基因649个，其中402个基因在MTA1敲降的HeLa细胞中表达上调，247个基因表达下调。与MTA1相关的差异表达基因富集于DNA损伤修复过程，GO聚类分析提示，DNA损伤修复富集于第2位。流式细胞术检测结果显示，10 Gy X射线照射后，MTA1过表达的HeLa细胞凋亡率[(15.67±0.81)%]低于对照HeLa细胞[(40.27±2.73)%，
P<0.001]。RTCA结果显示，过表达MTA1的HeLa细胞经2 Gy X射线照射后，细胞增殖能力高于对照HeLa细胞(
P＝0.024)。克隆形成实验显示，过表达MTA1的HeLa细胞经2 Gy X射线照射前后的克隆数分别为(176±7)个和(137±7)个，均高于对照细胞[分别为(134±4)个和(75±4)个，均
P<0.05]。免疫荧光实验结果显示，未经X射线照射的MTA1过表达及对照HeLa细胞中的γ－H2AX几乎无表达，经过10 Gy X射线照射后48 h，对照HeLa细胞中可见明显γ－H2AX表达，且MTA1过表达HeLa细胞中γ－H2AX表达相对较低。Western blot结果显示，10 Gy X射线照射后，过表达MTA1的HeLa细胞中β－CHK2的表达水平(1.04±0.06)高于对照HeLa细胞(0.58±0.25,
P＝0.036)，过表达MTA1的HeLa细胞中γ－H2AX、PARP和cleaved caspase 3的表达水平分别为0.52±0.13、0.52±0.22和0.63±0.18，均低于对照HeLa细胞(分别为0.87±0.06，0.78±0.12和0.90±0.12，均
P>0.05)。
结论: |
| Abstract_FL | Objective: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.Methods: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.Results: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)%,
P<0.001]. After 2 Gy X-ray irradiation, the proliferation capacity of HeLa cells overexpressing MTA1 was higher than that of control cells (
P=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 (
P<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,
P=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 (
P>0.05).
Conclusions: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. |
| Author | 王婷 钱海利 王劲松 李春晓 王海娟 张竞尧 周彦彤 孙芳洲 |
| AuthorAffiliation | 国家癌症中心 国家肿瘤临床医学研究中心 中国医学科学院北京协和医学院肿瘤医院分子肿瘤学国家重点实验室，北京 100021 |
| AuthorAffiliation_xml | – name: 国家癌症中心 国家肿瘤临床医学研究中心 中国医学科学院北京协和医学院肿瘤医院分子肿瘤学国家重点实验室，北京 100021 |
| Author_FL | Zhang Jingyao Wang Ting Wang Jinsong Li Chunxiao Zhou Yantong Sun Fangzhou Wang Haijuan Qian Haili |
| Author_FL_xml | – sequence: 1 fullname: Sun Fangzhou – sequence: 2 fullname: Wang Jinsong – sequence: 3 fullname: Li Chunxiao – sequence: 4 fullname: Wang Ting – sequence: 5 fullname: Zhang Jingyao – sequence: 6 fullname: Zhou Yantong – sequence: 7 fullname: Wang Haijuan – sequence: 8 fullname: Qian Haili |
| Author_xml | – sequence: 1 fullname: 孙芳洲 – sequence: 2 fullname: 王劲松 – sequence: 3 fullname: 李春晓 – sequence: 4 fullname: 王婷 – sequence: 5 fullname: 张竞尧 – sequence: 6 fullname: 周彦彤 – sequence: 7 fullname: 王海娟 – sequence: 8 fullname: 钱海利 |
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| Keywords | Cervical neoplasms 肿瘤转移相关基因1 放疗敏感性 MTA1 Radiosensitivity HeLa cells 宫颈肿瘤 HeLa细胞 |
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| Title | 肿瘤转移相关基因1对宫颈癌HeLa细胞放疗敏感性的作用及机制 |
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