钙调蛋白小亚基Capn4对依托铂甙诱导的鼻咽癌细胞CNE2 DNA损伤修复的作用

Effect of Capn4 on DNA damage repair of nasopharyngeal carcinoma cell CNE2 induced by VP-16

目的研究钙调蛋白小亚基Capn4对依托铂甙(VP-16)诱导的鼻咽癌细胞CNE2 DNA损伤修复中的作用及可能影响的修复通路。方法构建shRNA下调Capn4表达的CNE2细胞并以Western blot法验证;高内涵检测γ-H2AX在CNE2细胞及下调Capn4表达的CNE2细胞中的平均荧光强度,分析下调Capn4对VP-16诱导的CNE2细胞DNA损伤修复的作用;NHEJ(非同源重组修复)通路特异性的质粒EJ5-GFP用Fugen6 HD转入CNE2细胞及下调Capn4的CNE2细胞,用高内涵仪检测GFP的阳性率,观察下调Canp4对VP-16诱导的鼻咽癌细胞DNA损伤NHEJ修复通路的影响。结果 shRNA确实下调Capn4在鼻咽癌细胞中的表达。2.5μmol/L VP-16处理CNE2^(Vector)细胞和CNE2^(Capn4(-))细胞后6 h和12 h,CNE2^(Capn4(-))的损伤都较空载体的CNE2^(Vector)细胞高;下调Capn4表达对VP-16造成的CNE2细胞DNA损伤修复有抑制作用(P<0.01)。2.5μmol/L VP-16作用后,CNE2^(Capn4(-))细胞与CNE2^(Vector)细胞相比较GFP平均荧光强度明显更低;下调CNE2表达能够抑制DNA损伤后NHEJ通路的修复(P<0.01)。结论在鼻咽癌细胞中下调Capn4的表达能抑制VP-16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 VP-16 and its possible pathways. Methods The Capn4 knockdown CNE2 cells using shRNA were constructed and further identified by Western blot. Array Scan High-Content Systems were used to analyze the mean fluorescence intensity of γ-H2 AX in CNE2 cells and CNE2 cells with down-regulation of Capn4. Non-homologous end joining（ NHEJ） pathway-specific plasmid EJ5-GFP was transfected into CNE2 cells and CNE2 cells with down-regulated Capn4 by Fugen6 HD. The positive rate of GFP was detected by Array Scan HighContent Systems. And the effect of Canp4 down-regulation on VP-16-induced DNA damage NHEJ pathway in CNE2 cells was observed.Results The shRNA inhibited Capn4 expression in CNE2 cells. After treated with 2. 5 μmol/L VP-16 for 6 h and 12 h,the damages of CNE2^（Capn4（-））cells were higher than those of CNE2^（Vector）cells. Down-regulation of Capn4 expression inhibited the DNA damage of CNE2 cells induced by VP-16（ P 〈0. 01）. Compared with CNE2^（Vector）cells,the GFP average fluorescence intensity of the CNE2^（Capn4（-））cells after treated with 2. 5 μmol/L VP-16 was significantly lower. Down-regulation of Capn4 expression inhibited the repair of NHEJ pathway in CNE2 cells after DNA damage（ P〈 0. 01）. Conclusion Down-regulation of Capn4 expression can inhibit radiation-induced DNA damage repair,which may be achieved by inhibition of NHEJ pathway.