USP22截短质粒构建鉴定及其在肝细胞肝癌细胞中的功能初探  

Construction and identification of USP22 truncated mutants and primary exploration of its biological function in hepatocellular carcinoma cells

作　　者：谢威文 孙宁[1] 王胜利[1] 王春玉[1] 赵越[1] Xie Weiwen;Sun Ning;Wang Shengli;Wang Chunyu;Zhao Yue(College of Life Science,China Medical University,Key Laboratory of Cell Biology to Ministry of Health,Key Laboratory of Medical Cell Biology to Ministry of Education,Chromatin Biology Laboratory,Liaoning Shenyang 110122,China)

机构地区：[1]中国医科大学生命科学学院,卫生部细胞生物学重点实验室,教育部医学细胞生物学重点实验室,染色质生物学研究室,辽宁沈阳110122

出　　处：《现代肿瘤医学》2020年第12期2000-2005,共6页Journal of Modern Oncology

基　　金：国家自然科学基金面上项目(编号:31871286,81872015);国家自然科学基金青年基金项目(编号:31701102,81702800);沈阳市中青年科技创新人才支持计划(编号:RC170541)。

摘　　要：目的:构建编码ubiquitin specific peptidase 22(USP22)截短蛋白的表达质粒,探索USP22在肝细胞肝癌(hepatocellular carcinoma,HCC)中的作用及其分子机制,并初步解析USP22在HCC细胞系中对细胞增殖及索拉菲尼作用的影响。方法:设计合成针对于人源USP22截短质粒序列的引物,通过PCR的方法以含有USP22全长蛋白编码序列的表达载体作为模板,扩增目的片段,再用限制性内切酶EcoRⅠ和XhoⅠ进行双酶切,连接转化后,将构建好的质粒进行酶切鉴定及测序,证明USP22截短质粒构建成功。将构建好的USP22全长及截短表达质粒转染到HCC细胞系HCC-LM3中,分别进行Western blot和confocal实验检测USP22全长及截短质粒在HCC-LM3细胞中的表达及定位。生物学功能方面,利用MTS法检测USP22全长对HCC细胞增殖及索拉菲尼作用的影响。结果:成功构建了USP22截短表达质粒,并在HCC细胞系HCC-LM3中验证了其蛋白表达;USP22全长及所构建的截短质粒编码蛋白在HCC-LM3中定位于细胞核;此外,生物学功能实验证实USP22促进HCC-LM3细胞增殖,并削弱了索拉菲尼对HCC-LM3细胞生长的抑制作用。结论:在HCC-LM3细胞中,USP22及其截短质粒编码蛋白均定位于细胞核;USP22促进HCC细胞增殖,且可能参与在HCC治疗中的索拉菲尼耐受进程。Objective:To construct and identify ubiquitin specific peptidase 22(USP22)truncated mutants,to explore the biological function and molecular mechanism of USP22 in hepatocellular carcinoma(HCC)cells,and further primarily analyze the effects of USP22 on cell growth/proliferation and sorafenib resistance in HCC.Methods:Two expression plasmids carrying truncated mutants of USP22 were constructed by PCR and gene recombination technology.The USP22 full length and its truncated mutants were transfected into HCC-derived cell line(HCC-LM3).Western blot was performed to identify the protein expression.Confocal immunofluorescence was further performed to detect the subcellular localization of USP22 full length and its truncated mutants in HCC-LM3 cells.In addition,MTS assay was used to examine the influence of USP22 on cell growth/proliferation with or without treatment of sorafenib.Results:The truncated mutants of USP22 were successfully constructed.Our results have demonstrated that the endogenous USP22 protein,the full-length USP22 and the truncated mutants of USP22 were distributed in the nucleus of HCC-LM3 cells.USP22 promoted the cell proliferation in HCC-LM3,and partially reversed the effect of sorafenib on HCC-LM3 proliferation.Conclusion:USP22 and its truncated mutants are finally constructed and distributed in the nucleus of HCC-LM3 cells.Moreover,USP22 participates in promoting the cell growth/proliferation and sorafenib resistance in HCC-derived cell line.Thus,the primary experimental results provide the experimental basis for further investigate the molecular mechanism underlying the biological function of USP22 in HCC.

关 键 词：USP22 截短质粒构建 细胞生长增殖 肝细胞肝癌 索拉菲尼 

分 类 号：R735.7[医药卫生—肿瘤]