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NHEJ通路相关蛋白的作用

Effect of calmodulin small subunit Capn4 on DNA repair of NHEJ pathway-associated protein in nasopharyngeal carcinoma cell CNE2 induced by etoposide

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NHEJ通路相关蛋白Ku80及DNApks的作用,以及对裸鼠体内成瘤的影响。方法高内涵分析仪检测空载的CNE2细胞及下调Capn4的CNE2细胞中非同源重组NHEJ修复通路的特异性质粒EJ5-GFP的阳性率;检测空载的CNE2细胞及下调Capn4表达的CNE2细胞在VP16诱导后Ku80的表达;实时荧光定量PCR检测空载的CNE2细胞及下调Capn4表达的CNE2细胞的Ku80转录水平的变化;体内异种移植空载的CNE2细胞及下调Capn4的CNE2细胞后观察VP-16对鼻咽癌肿瘤质量大小的影响及抑瘤率。结果高内涵分析显示下调Capn4的表达能够抑制CNE2细胞DNA损伤后NHEJ修复通路的GFP阳性率(P<0.01);下调Capn4表达后VP16诱导的CNE2细胞NHEJ通路相关蛋白Ku80、DNApks的表达下降(P<0.01);下调Capn4表达能下调裸鼠成瘤后肿瘤的体积与质量(P<0.01)。结论Capn4对NHEJ通路的抑制与通路相关蛋白Ku80、DNApks有关;下调Capn4的表达能够抑制鼻咽癌肿瘤生长、增加鼻咽癌对放化疗的敏感。

Objective To investigate the effect of calmodulin small subunit Capn4 on the DNA repair of non-homologous end-ligation(NHEJ)repair pathway-associated proteins Ku80 and DNApks in nasopharyngeal carcinoma cell line CNE2 induced by etoposide(VP-16)and tumor formation in nude mice.Methods High-content analyzer was used to detect the positive rate of non-homologous recombination-terminal NHEJ-specific plasmid EJ5-GFP in empty CNE2 cells and down-regulated Capn4 CNE2 cells.The expression of Ku80 was detected in empty CNE2 cells and down-regulated Capn4 CNE2 cells after induced by VP-16.Real-time quantitative PCR was used to detect the changes of Ku80 transcription level in empty CNE2 cells and down-regulated Capn4 CNE2 cells.The effects of VP-16 on tumor weight of nasopharyngeal carcinoma and tumor inhibition rate were observed after in vivo xenograft by empty CNE2 cells and down-regulated CNE2 cells of Capn4.Results High-content analysis showed that down-regulation of Capn4 expression inhibited GFP-positive rate of NHEJ repair pathway after DNA damage in CNE2 cells(P<0.01).The down-regulation of Capn4 expression decreased the expression of NPEJ pathway-related proteins Ku80 and DNApks induced by VP16 in CNE2 cells(P<0.01).Down-regulation of Capn4 expression decreased the volume and weight of tumors in nude mice(P<0.01).Conclusion The inhibition of NHEJ pathway by Capn4 is related to the pathway-related proteins Ku80 and DNApks.Down-regulation of Capn4 expression can inhibit the growth of nasopharyngeal carcinoma and increase the sensitivity of nasopharyngeal carcinoma to radiotherapy and chemotherapy.