Abstract
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 2μg/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. Key words: Lysine methyltransferaseSET8; Clear cell renal cell carcinoma; 786-O cell; Cell proliferation; Colony formation; Migration
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