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OrfA与E2F5的互作关系及其对肺腺癌细胞增殖的影响
穆 璐, 张潇筠, 韩宝泉,等
西北农林科技大学 动物科技学院
摘要:
【目的】确认大眼狮鲈鱼皮肤肿瘤病毒(WDSV)辅助因子OrfA与转录因子E2F5的互作关系,以及OrfA对肿瘤细胞增殖的影响。【方法】采用重组DNA技术构建orfAE2F5基因过表达载体;通过Western Blot试验,检测对比HEK293T、Hela229宫颈癌细胞、A375恶性黑色素瘤细胞、SMMC-7721肝癌细胞和SPC-A-1肺腺癌细胞中E2F5的表达量;通过酵母双杂交和免疫共沉淀试验,验证OrfA与E2F5的互作关系;采用CCK-8试剂盒和PI单染色法,检测过表达orfA基因对SPC-A-1细胞增殖的影响;采用RT-qPCR,检测过表达orfA基因对SMAD4caspase-3基因表达的影响。【结果】成功构建了orfA与E2F5基因过表达载体pLenti-EF1α-orfA和pLenti-EF1α-E2F5;在5种细胞系中,以SPC-A-1细胞E2F5蛋白表达量最高;酵母双杂交和免疫共沉淀试验结果均证明,OrfA与E2F5可以直接相互作用;过表达orfA基因对SPC-A-1细胞增殖具有显著促进作用,同时能够增强SMAD4基因的表达,抑制caspase-3基因的表达。【结论】WDSV OrfA与E2F5有相互作用,且能促进SPC-A-1肺腺癌细胞的增殖。
关键词:  逆转录病毒周期蛋白(OrfA)  E2F5转录因子  SPC-A-1肺腺癌细胞  细胞增殖
DOI:
分类号:
基金项目:国家自然科学基金项目(31702099,30870119)
Interaction between OrfA and E2F5 and effect of OrfA on lung adenocarcinoma cell proliferation
MU Lu, ZHANG Xiaojun, HAN Baoquan,et al
Abstract:
【Objective】This study verified the interaction between OrfA,a walleye dermal sarcoma virus (WDSV) accessory factor and transcription factor E2F5 and analyzed the effect of OrfA on proliferation of tumor cells.【Method】The overexpression vectors of orfA and E2F5 gene were constructed using recombinant DNA technology.The E2F5 protein expression profile in HEK293T,Hela229 cervical cancer cells,A375 malignant melanoma cells,SMMC-7721 liver cancer cells and SPC-A-1 lung adenocarcinoma cells was detected by Western Blot.The interaction between OrfA and E2F5 was verified using yeast two hybrid and Co-immunoprecipitation (Co-IP) assays.The effect of orfA gene overexpression on proliferation of SPC-A-1 cells was examined by CCK-8 and PI staining assays.The effect of orfA gene overexpression on expression of SMAD4 and caspase-3 genes was examined by RT-qPCR.【Result】The orfA and E2F5 gene overexpression vectors of pLenti-EF1α-orfA and pLenti-EF1α-E2F5 were successfully constructed.The E2F5 protein expression in SPC-A-1 cells was the highest among the five types of human cells.The interaction between OrfA and E2F5 was proved by both yeast two-hybrid and Co-immunoprecipitation assays.The overexpression of orfA gene could promote proliferation of SPC-A-1 cells and expression of SMAD4,but inhibit caspase-3.【Conclusion】WDSV OrfA could interact with E2F5 and promote proliferationof SPC-A-1 lung adenocarcinoma cells.
Key words:  retrovirus cyclin (OrfA)  E2F5 transcription factor  SPC-A-1 lung adenocarcinoma cells  cell proliferation