Yang Xing-Xiao, Ma Ming, Sang Mei-Xiang, Wang Xue-Xiao, Song Heng, Liu Zhi-Kun, Zhu Shu-Chai
Department of Radiation Oncology, The Fourth Hospital of Hebei Medical University, Shijiazhuang, Hebei 050011, P.R. China.
Department of Clinical Laboratory, The Fourth Hospital of Hebei Medical University, Shijiazhuang, Hebei 050011, P.R. China.
Int J Oncol. 2016 May;48(5):1985-96. doi: 10.3892/ijo.2016.3404. Epub 2016 Feb 19.
Radiotherapy has been widely used for the treatment of cancer patients, especially for esophageal cancer patients. Ring finger protein 2 (RNF2) plays an important role in promoting the growth of cancer cells after exposure to irradiation. The present study aims to characterize the proliferative effects of RNF2 on cancer cells, and its mechanisms on the growth of esophageal cancer cells. We demonstrate that expression of RNF2 was markedly upregulated in esophageal cancer cell lines and surgically resected cancer specimens. In addition, RNF2 expression level is positively correlated with the presence of tumor size, lymph node metastases and negatively correlated with patient survival rates, suggesting that it plays an important role in the progression of esophageal cancer. Furthermore, the expression of RNF2 at both mRNA and protein levels in esophageal cancer ECA109 and TE13 cells was detected by real-time PCR and western blot assay after shRNA targeting RNF2. Co-immunoprecipitation (Co-IP) assay and western blot analysis were employed to detect the interaction between RNF2 and r-H2AX, H2AK119ub, and the expression of proteins associated with cell cycle and apoptosis, respectively. We used flow cytometry assay to analyze cell cycle and apoptosis of transfected cells, and further examined cellular growth in vitro and in vivo. shRNA targeting RNF2 gene and protein downregulated RNF2 expression after transfection for 24 h. The proliferation of tumor cells in RNF2-shRNA group was suppressed after radiotherapy. In addition, the interaction of RNF2, H2AK119ub, r-H2AX was increased after exposure to IR, followed by increasing apoptosis rates and inducing the arrest at G0/G1 phase after irradiation and shRNA targeting RNF2. Expression of the short-hairpin RNA is also correlated with the upregulation of p16 and Bax, and the downregulation of cyclin D2, cyclin-dependent kinase (CDK)-4, H2AX and Bcl-2. RNF2 gene knockdown induces radiosensitivity of esophageal cancer cells in vitro and significantly inhibits the growth of tumor cells. The mechanisms include inducing the cell cycle arrest at G0/G1 phase and promoting apoptosis.
放射疗法已被广泛用于癌症患者的治疗，尤其是食管癌患者。无名指蛋白2（RNF2）在癌细胞受到照射后促进其生长方面发挥着重要作用。本研究旨在表征RNF2对癌细胞的增殖作用及其对食管癌细胞生长的机制。我们证明，RNF2在食管癌细胞系和手术切除的癌组织标本中表达明显上调。此外，RNF2表达水平与肿瘤大小、淋巴结转移呈正相关，与患者生存率呈负相关，表明其在食管癌进展中起重要作用。此外，在靶向RNF2的短发夹RNA（shRNA）处理后，通过实时PCR和蛋白质印迹法检测食管癌细胞ECA109和TE13中RNF2在mRNA和蛋白质水平的表达。采用免疫共沉淀（Co-IP）分析和蛋白质印迹分析分别检测RNF2与r-H2AX、H2AK119ub之间的相互作用以及与细胞周期和凋亡相关蛋白的表达。我们使用流式细胞术分析转染细胞的细胞周期和凋亡，并进一步检测体外和体内细胞生长情况。靶向RNF2基因和蛋白质的shRNA在转染24小时后下调了RNF2表达。放射治疗后，RNF2-shRNA组肿瘤细胞的增殖受到抑制。此外，照射后RNF2、H2AK119ub、r-H2AX之间的相互作用增加，随后凋亡率增加，并在照射和靶向RNF2的shRNA处理后诱导细胞停滞在G0/G1期。短发夹RNA的表达还与p16和Bax的上调以及细胞周期蛋白D2、细胞周期蛋白依赖性激酶（CDK）-4、H2AX和Bcl-2的下调相关。RNF2基因敲低可诱导食管癌细胞在体外的放射敏感性，并显著抑制肿瘤细胞的生长。其机制包括诱导细胞周期停滞在G0/G1期和促进凋亡。
