Huang Yujie, Tian Yonghao, Zhang Wenhao, Liu Ruijuan, Zhang Weifang
Department of Microbiology, School of Basic Medical Sciences, Cheeloo College of Medicine, Shandong University, Jinan, China.
Department of Orthopedics, Qilu Hospital of Shandong University, Jinan, China.
Front Oncol. 2021 Feb 25;11:586771. doi: 10.3389/fonc.2021.586771. eCollection 2021.
HPV-positive (HPV+) cervical cancer cells are more radioresistant compared with HPV-negative (HPV-) cervical cancer cells, but the underlying mechanism is not fully illuminated. Our previous mass spectrometry data showed that Ras-associated binding protein Rab12 was up-regulated by HPV, and this study is to investigate the role of Rab12 in the radioresistance of HPV-positive cervical cancer cells.
CCK-8 assay, colony formation assay, flow cytometry, and Western blot were performed to determine cell proliferation, apoptosis, cell cycle distribution, and protein expressions. DNA damage and repair levels were measured by comet assays and detection of γ-H2AX, XRCC4, and pBRCA1 protein expressions.
Rab12 mRNA and protein expressions were up-regulated in cervical cancer tissues and HPV+ cervical cancer cells. Knockdown of Rab12 enhanced radiosensitivity while overexpression of Rab12 promotes radioresistance. Knockdown of Rab12 alleviated G2/M arrest by decreasing p-Cdc2(Tyr15) after radiation, which was a result of the reduction of p-Cdc25C(Ser216). Rab12 knockdown caused more DNA double-strand breaks (DSBs) and inhibited DNA homologous recombination repair (HRR) after radiation. Instead, overexpression of Rab12 enhanced radioresistance by increasing G2/M arrest, which provided more time for DNA HRR.
Rab12 may serve as a potential therapeutic target to improve clinical treatment outcome of cervical cancer.
与HPV阴性（HPV-）宫颈癌细胞相比，HPV阳性（HPV+）宫颈癌细胞对放疗更具抗性，但其潜在机制尚未完全阐明。我们之前的质谱数据显示，HPV可上调Ras相关结合蛋白Rab12，本研究旨在探讨Rab12在HPV阳性宫颈癌细胞放射抗性中的作用。
采用CCK-8法、集落形成试验、流式细胞术和蛋白质免疫印迹法检测细胞增殖、凋亡、细胞周期分布及蛋白质表达。通过彗星试验及检测γ-H2AX、XRCC4和pBRCA1蛋白表达来测定DNA损伤和修复水平。
Rab12 mRNA和蛋白表达在宫颈癌组织及HPV+宫颈癌细胞中上调。敲低Rab12可增强放射敏感性，而过表达Rab12则促进放射抗性。敲低Rab12可通过降低放疗后p-Cdc2（Tyr15）水平来缓解G2/M期阻滞，这是p-Cdc25C（Ser216）减少的结果。敲低Rab12会导致更多DNA双链断裂（DSB），并抑制放疗后的DNA同源重组修复（HRR）。相反，过表达Rab12通过增加G2/M期阻滞来增强放射抗性，为DNA HRR提供更多时间。
Rab12可能是改善宫颈癌临床治疗效果的潜在治疗靶点。
