钙蛋白酶小亚基Capn4在鼻咽癌细胞CNE2中DNA损伤修复的作用
Effect of Capn4 in repairing DNA damage of nasopharyngeal carcinoma cell CNE2
目的 研究钙蛋白酶小亚基Capn4对放射所致的鼻咽癌细胞CNE2在DNA损伤修复中的作用及可能影响的修复通路。 方法 选取鼻咽癌细胞CNE2与293T细胞在37℃、5％CO2条件下培养，Western blot法检测Capn4在不同细胞中蛋白表达水平；构建shRNA下调Capn4表达的CNE2细胞并以Western blot法验证；高内涵细胞成像分析仪分析γ-H2AX在放射后的CNE2细胞及下调Capn4表达的CNE2细胞中的平均荧光强度；非同源末端连接（NHEJ）通路特异性的质粒EJ5-GFP用Fugen6 HD转入空载的CNE2细胞及下调Capn4的CNE2细胞，用高内涵细胞成像分析仪检测GFP的阳性率，观察下调Capn4对放射诱导的鼻咽癌细胞DNA损伤NHEJ修复通路的影响。 结果 Capn4在鼻咽癌细胞系中表达明显高于正常细胞（P<0.01）；shRNA确实下调Capn4在鼻咽癌细胞中的表达；下调Capn4表达对放射造成的CNE2细胞DNA损伤修复有抑制作用（P<0.01）；下调Capn4表达能够抑制CNE2细胞DNA损伤后NHEJ通路的修复（P<0.01）。 结论 Capn4在鼻咽癌细胞CNE2中高表达，下调Capn4的表达能抑制放射所致的DNA损伤修复，这种作用可能是通过抑制NHEJ通路实现的。
Objective To investigate the effect of Capn4 on the repair of nasopharyngeal carcinoma cell line CNE2 induced by radiation and its possible pathways. Methods The protein levels of Capn4 in CNE2 and 293T cells were examined by Western blot. The Capn4 knockdown cells using shRNA was constructed in CNE2 cells and further identified by Western blot. Highly Connected Cell Imaging Analyzer was used to analyze the Mean Fluorescence Intensity of γ-H2AX in CNE2 cells after radiation and CNE2 cells with down-regulation of Capn4. Non-homologous end joining (NHEJ) pathway-specific plasmids EJ5-GFP were transfected into CNE2 cells with Fugen6 HD and CNE2 cells with downregulation of Capn4. The positive rate of GFP was detected by Highly Connected Cell Imaging Analyzer. The downregulation of Canp4 on NHEJ repair pathway of DNA damage was induced by radiation influences. Results The expression levels of Capn4 were significantly higher in CNE2 cells than in 293T cells. The shRNA inhibited Capn4 expression in CNE2 cells. Downregulation of Capn4 expression could inhibit the DNA damage of CNE2 cells induced by radiation（P<0.01）. Downregulation of Capn4 expression could inhibit the repair of NHEJ pathway in CNE2 cells after DNA damage（P<0.01）. Conclusion Capn4 is highly expressed in nasopharyngeal carcinoma CNE2 cells. Downregulation of Capn4 expression can inhibit radiation-induced DNA damage repair, which may be achieved by inhibition of NHEJ pathway.
/ Capn4 / 鼻咽癌 / 放射 / shRNA / NHEJ通路
Capn4； Nasopharyngeal carcinoma； / Radiation； / shRNA； / NHEJ pathway
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