POLQ表达降低对SACC-83细胞DNA损伤敏感性的影响

Study on enhanced sensitivity to DNA damage in POLQ knocking-down salivary of adenoid cystic carcinoma-83 cells

目的:探讨DNA损伤环境中抑制POLQ对唾液腺腺样囊性癌(salivary adenoid cystic carcinoma,SACC)细胞SACC-83增殖及克隆形成能力、细胞周期及DNA损伤修复通路的影响。方法:应用shRNA(short hairpin RNA)瞬时转染方法构建POLQ敲减的SACC-83细胞模型,利用qRT-PCR及Western免疫印迹实验检测POLQ干扰效率。应用不同浓度DNA损伤剂依托泊苷(etoposide,ETP/VP-16-213)诱导SACC-83细胞发生DNA损伤,利用Western免疫印迹实验检测γH2AX的表达水平,评价DNA双链断裂水平。在不同浓度依托泊苷诱导形成的DNA损伤环境中,通过CCK-8细胞增殖实验检测抑制POLQ对SACC-83细胞增殖能力的影响。采用平板克隆实验观察抑制POLQ对SACC-83细胞克隆形成能力的影响,应用流式细胞仪分析抑制POLQ对SACC-83细胞周期的影响,应用Western免疫印迹实验检测抑制POLQ对SACC-83细胞γH2AX、RAD51及PARP1表达水平的影响。采用SPSS 20.0软件包对数据进行统计学分析。结果:应用shRNA瞬时转染方法抑制SACC-83细胞POLQ mRNA及蛋白的表达。依托泊苷以剂量依赖的方式促进SACC-83细胞γH2AX表达。POLQ表达降低可抑制SACC-83细胞增殖能力(P<0.001),且抑制作用随ETP浓度增加而减小。在依托泊苷诱导形成的DNA损伤环境中,POLQ表达降低可抑制SACC-83细胞克隆形成能力(P<0.001),诱导细胞发生S期阻滞(P<0.01)。POLQ通过促进γH2AX(P<0.05)及同源重组(homologous recombination,HR)通路相关蛋白RAD51(P<0.05)、抑制非同源末端连接(alternative non-homologous end joining,altNHEJ)通路相关蛋白PARP1(P<0.01),调节DNA损伤修复。结论:POLQ表达降低,会促进SACC-83细胞对DNA损伤的敏感性。

PURPOSE:To investigate the effects of POLQ inhibition on proliferation,colony formation,cell cycle,DNA damage and repair in salivary adenoid cystic carcinoma-83(SACC-83)cell line.METHODS:POLQ knocking-down SACC-83 cells were constructed using short hairpin RNA(shRNA)transient transfection,and the inhibition efficiency was detected by qRT-PCR and Western blot.DNA damage in SACC-83 cells was induced by different concentration of DNA damage agent etoposide(VP-16-213),and the levels ofγH2AX expression were detected by Western blot to evaluate DNA double-strain breaks.Under different concentration of etoposide-induced DNA damage condition,CCK-8 assay was used to evaluate the effect of POLQ inhibition on cell proliferation in SACC-83 cell line.Under etoposide-induced DNA damage condition,plate colony assay was performed to detect the effect of POLQ inhibition on cell clone formation ability in SACC-83 cell line,and flow cytometry was used to detect the effect of POLQ inhibition on cell cycle in SACC-83 cell line.Furthermore,under etoposide-induced DNA damage condition,Western blot was used to analyze POLQ,γH2AX,RAD51 and PARP1 protein expression.SPSS 20.0 software package was used for statistical analysis.RESULTS:The mRNA and protein expression of POLQ was inhibited by shRNA transient transfection.IncreasedγH2AX in SACC-83 was closely coupled with increased concentrations of etoposide.The results of CCK-8 assay showed that POLQ knocking-down suppressed cell proliferation ability in SACC-83 cell line,and the inhibitory effect was mitigated with increased concentration of etoposide(P<0.001).The result of plate colony assay demonstrated that under etoposide-induced DNA damage condition,compared with the control group,POLQ knocking-down suppressed cell colony ability in SACC-83 cell line(P<0.001).Moreover,the results of flow cytometry demonstrated that under etoposide-induced DNA damage conditions,compared with the control group,POLQ knocking-down induced S phase arrest(P<0.01).Mechanistically,the results of Western blot showed that POLQ regulated DNA damage and repair by promoting expression ofγH2AX(P<0.05)and homologous recombination(HR)pathway-related protein RAD51(P<0.05),respectively,and down-regulating the alternative non-homologous end joining(alt-NHEJ)pathway-related protein PARP1(P<0.01).CONCLUSIONS:POLQ knocking-down promotes the sensitivity of SACC-83 cell line to DNA damage.