RNF146真核表达载体的构建及其对非小细胞肺癌细胞系生物学行为的影响  

Construction of RNF146 eukaryotic expression vector and influence of its overexpression on biologic behavior of non-small cell lung cancer cell line

作　　者：高英[1] 惠林萍[2] 宋成洋 徐慧慧[1] 王睿[1] 田大力[3] 邱雪杉[4,5] 王恩华[4,5] 

机构地区：[1]中国医科大学附属第四医院病理科,沈阳110032 [2]中国医科大学附属第四医院中心实验室,沈阳110032 [3]中国医科大学附属第四医院胸外科,沈阳110032 [4]中国医科大学基础医学院病理学教研室 [5]中国医科大学附属第一医院病理科

出　　处：《山西医药杂志》2014年第7期723-726,I0001,共5页Shanxi Medical Journal

基　　金：国家自然科学基金(30973502);辽宁省科技厅科学技术计划(2012225072)

摘　　要：目的构建E3泛素连接酶环指蛋白146(RNF146)基因真核表达载体,研究RNF146对非小细胞肺癌细胞系体外生物学行为的影响。方法采用反转录-聚合酶链反应(RT-PCR)和蛋白印迹法筛选RNF146低表达细胞系。构建pEX4-RNF146真核表达载体,经酶切和测序后瞬时转染LTE细胞,荧光显微镜观察、蛋白印迹法检测转染效率。四甲基偶氮唑盐(MTT)比色实验检测肿瘤细胞体外增殖能力,划痕实验评价肿瘤细胞体外迁移能力,Buyden小室实验检测肿瘤细胞体外侵袭能力。结果酶切和基因测序证实重组质粒pEX4构建正确。MTT比色实验显示转染pEX4-RNF146组细胞增殖速度明显高于转染空载体组和未转染组(P<0.05)。划痕实验显示转染组细胞24h迁移率明显高于空载体组和未转染组(P<0.05)。Buyden小室实验结果表明转染组比空载体组和未转染组穿膜数目明显增多(P<0.05)。结论 RNF146基因转染能促进非小细胞肺癌LTE细胞的体外增殖、迁移和侵袭能力,可能是一个潜在的促肿瘤增殖、侵袭和转移的相关基因。Objective To construct the eukaryotic expression vector pEX4-RNF146 and to investigate the effect of RNF146 overexpression on the biological behaviors of human non-small cell lung cancer （NSCLC）cell line in vitro .Methods RT-PCR and Western blotting were used to screen NSCLC cell line with low RNF146 expres-sion at mRNA and protein levels.Full-length coding sequence of human RNF146 was subcloned into plasmid pEX4 and the recombinant plasmids were transiently transfected into LTE cells after tested by the restriction en-zyme analysis and gene sequencing method.The expression of RNF146 gene was evaluated by fluorescence micro-scope and Western blot.The cell proliferation ability,migratory and invasive ability were detected by MTT,and in addition Wound-healing assay and matrigel invasion assay using Buyden chamber were also carried out respec-tively.Results The results of restriction enzyme analysis and gene sequencing method indicated the correctness of the recombinant pEX4-RNF146 plasmid construction.The proliferation ability of RNF146 overexpression cells by transient transfection were markedly increased in comparing with that of the control cells（P ＆lt;0.05）.Effective up-regulating of RNF146 expression in LTE cells increased the ability of these cells to migrate as measured in the wound-healing assay（P ＆lt;0.05）.The number of cell passing through the matrigel and multipore membrane was al-so increased in the RNF146-transfected cell compared with those of the vector-transfected clones and parental cells （P ＆lt;0.05）.Conclusion Transfected expression of RNF146 promotes cell proliferation,migration and invasive a-bility of LTE cells in vitro .Conclusively,RNF146 may play an important role in promoting the proliferation,in-vasion and metastasis of the cancer cells.

关 键 词：癌 非小细胞肺 泛素蛋白连接酶类 RNF146 增殖 RNF146 

分 类 号：R734.2[医药卫生—肿瘤]