SET8在肾透明细胞癌中的表达及其对786-O细胞增殖迁移能力的影响

Expression of SET8 in renal cell carcinoma and the effects of SET8 silencing on proliferation and migration of 786-O cells

目的 探讨SET8在肾透明细胞癌组织中表达的临床意义及沉默该基因对人肾透明细胞癌786-O细胞增殖迁移能力的影响.方法 收集2006年12月至2011年12月156例肾切除术后标本,病理类型均为肾透明细胞癌.采用免疫组化染色法检测SET8蛋白在肾透明细胞癌组织中的表达,分析SET8蛋白表达与患者临床病理特征的关系.体外培养肾透明细胞癌786-O细胞,针对人SET8基因设计合成4对候选短发夹RNA（short hairpin RNA,shRNA）序列（shRNA1、shRNA2、shRNA3、shRNA4）,以脂质体Lipofectamine 2000为载体,转染肾透明细胞癌786-O细胞,荧光显微镜下观察转染效率.蛋白质印迹法检测786-O细胞中SET8蛋白的表达,筛选有效shRNA序列.将有效shRNA序列转染786-O细胞,细胞分3组：SET8-shRNA转染组（转染SET8-shRNA）、阴性对照组（转染阴性对照shRNA）、正常对照组（未转染组）.对转染后的786-O细胞采用MTT法检测增殖能力;平板克隆形成实验检测细胞克隆形成能力;划痕实验检测细胞迁移能力.结果 SET8蛋白在肾透明细胞癌组织中阳性表达率为98.7％（154/156）,SET8蛋白高表达率为60.3％（94/156）,SET8蛋白表达高低与肾透明细胞癌患者的肿瘤分期、肿瘤大小及淋巴结转移有关（P均＜ 0.05）;在SET8-shRNA浓度为2μg/L、Lipofectamine 2000为4μl/孔的转染条件下,转染效率约70％;筛选出的最佳干扰序列为SET8-shRNA2,转染48 h沉默效率可达64％.SET8-shRNA2转染组SET8蛋白表达率在转染后48 h显著低于阴性对照组、正常对照组（t=-38.174,P＜0.01;t=-58.766,P＜0.01）.shRNA有效沉默SET8基因表达后,SET8-shRNA转染组786-O细胞增殖、克隆形成和迁移能力均受到显著抑制.结论 SET8蛋白在肾透明细胞癌组织中表达升高可能促进了肿瘤的发生、发展.沉默SET8蛋白表达可有效抑制肾透明细胞癌786-O细胞的肿瘤恶性生物学行为.

Objective To investigate the expression of lysine methyltransferase SET8 protein in the clear cell carcinoma of kidney and the effects of silencing SET8 gene by shRNA on proliferation and migration of human renal cell carcinoma cell line 786-O in vitro.Methods Immunohistochemistry （IHC） was employed to detect the expression of SET8 in 156 tissue samples of clear cell renal cell carcinoma in the study between December,2006 and December,2011.Four pairs of SET8-shRNA were designed and synthesized.Transfection was performed with cationic lipid vectors （LipofectamineTM2000）.The transfection efficiency was observed by fluorescence confocal microscopy.The effecive shRNA sequences were screened by Western blot.After transfection with effective shRNA sequences,786-O cells were divided into 3 groups：（1） SET8-shRNA transfection;（2） negative control transfection;（3） blank control.The assays of methyl thiazolyl tetrazolium （MTT）,colony formation and scratch wound were applied to examine variations in cell proliferation,colony formation and migration.Results The positive rate of SET8 expression was 98.7％ （154/156）,and its expression was closely associated with TNM staging,the size of tumor and lymph node metastasis（all P ＜ 0.05）.The transfection efficiency was around 70％ with a concentration of SET8-shRNA 2 μg/L and lipofectamine 2000 4μl/well.SET8-shRNA2 was chosen as the effective sequences with a suppression ratio up to 64％.At 48h post-transfection,the expression of SET8 protein was significantly lower in SET8-shRNA2 group than that in negative control transfection group and blank control group（t =-38.174,P ＜ 0.01;t =-58.766,P ＜ 0.01）.While SET8 gene was silenced by shRNA in SET8-shRNA transfection group,the proliferation,colony formation and migration ability were decreased markedly.Conclusion SET8 protein plays a significant role in the carcinogenesis and progression of renal cell carcinoma.SET8-shRNA can effectively inhibit the expression of SET8 protein in 786-O cells and thus suppress malignant phenotypes of RCC.