| Objective: Renal cell carcinoma(RCC) accounts for approximately 2-3% of adults malignancies, approximately 70% of these are clear cell renal cell carcinoma(ccRCC). RCC is a ‘‘quiet'' disease and typically 20%~30% of these are diagnosed at an advanced stage. As it is known to all, cancer development is a multi-step process involving changes in a variety of genes and signal pathways. Whereas we have no effective predictors or treatment for RCC. SET8 is a newly identified sole lysine methytransferase that specifically catalyzes monomethylation of histone H4 lysine 20. SET8 and the H4K20me1 catalyzed by SET8 are jointly implicated in modulating cell cycle progression, maintaining genomic stability and regulating the transcription of several genes. Recently, studies focusing on its role in tumorgenesis become emerging. It is reported that SET8 is highly expressed in different tumor types, and take part in regulating the expression of several oncogenes. Clinically, SET8 is related with poor outcome of liver and lung cancer,indicating that SET8 is involed in carcinogenesis and progression. Wheras the role of SET8 in ccRCC remains unclear.Thus, in this paper we investigated the correlations between SET8 expression levels and clinicopathological variables, in vitro study we explored the effects of SET8 silencing on biological function of ccRCC 786-O cells and the underlying machanism.Methods:1.Immunohistochemistry(IHC) was employed to detect the expression of SET8 in 156 tissue samples of ccRCC in the study.2.Four pairs of SET8-shRNA were designed and synthesized. Transfection was performed with cationic lipid vectors(lipofectamine 2000). 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 group;(2)negative control transfection group(NC-group);(3)untreated group.3.The effects of SET8 silencing on cell proliferation,migration and invasion were evaluated by Methylthiazolyldiphenyl-tetrazolium bromide(MTT) assay, colony formation, scratch wound and transwell assay, respectively.4.The expression of wnt-related genes—?-catenin, c-myc, MMP7 was measured by RT-PCR.5.Statistical analysis All data were represented as mean±SD. The protein expression and clinicopathological parameters were compared by the ?2 test.Student t test or analysis of variance(ANOVA) was used to compare normally distributed variables. All statistical analyses were performed using the SPSS21.0 software(SPSS Company, Chicago, IL, USA), where P<0.05 was considered statistically significant.Results: 1 Immunohistochemistry results and correlations between SET8 expression levels and clinicopathological variables: The positive rate of SET8 expression was 98.7%(154/156). SET8 overexpression rate was 60.3%(94/156), while 62 cases exhibited a low expression level. A high expression level of SET8 protein was significantly correlated with ccRCC TNM staging(P=0.002), the size of tumor(P=0.003) and lymph node metastasis(P=0.020), while no significant association was found between SET8 expression and the variables of age and gender(all P>0.05); 2 Successfully knockdown of SET8 in 786-O cells: The transfection efficiency was around 70% with a concentration of SET8-sh RNA 2ug/L and lipofectamine 2000 4ul/well.SET8-shRNA2 was chosen as the effective sequences with a suppression ratio up to 64%. At 48 h post-transfection,the expression of SET8 protein was significantly lower in SET8-shRNA2 group than that in NC-shRNA and untreated groups(t=-38.174, P<0.01; t=-58.766, P<0.01); 3Knockdown of SET8 inhibited cell proliferation and reduced colony formation of ccRCC in vitro; 3 MTT assay showed that SET8 knockdown significantly supressed 786-O cell proliferation at 24, 48 hours post-transfection, comparing with the NC-shRNA group and untreated group(P<0.05). The colony formation number in 786-O cells infected by SET8-shRNA, which was lower than that in cells in the NC-shRNA or untreated groups(P<0.05); 4 Knockdown of SET8 inhibited cell migration and invasion of ccRCC in vitro. Downregulation of SET8 significantly supressed the migration capability of 786-O cells(P<0.05). The migration index were 0.098±0.001, 0.290±0.007, 0.293±0.002 in SET8-shRNA, NC-shRNA and untreated groups, respectively. The average cell counts crossing the matrigel-coated membrane in one high power field were significantly lower than that in NC-shRNA and untreated groups(P<0.05). 5 Knockdown of SET8 weakens the expression of wnt target genes— c-myc and MMP-7 expression of ccRCC in vitro: In SET8-shRNA group, the expression of c-myc and MMP-7 mRNA were weakened by SET8-shRNA(P<0.05), but the expression of ?-catenin has not been influenced(P>0.05).Conclusion:1 SET8 was overexpressed in ccRCC and significantly associated with TNM staging, the size of tumor and lymph node metastasis.2 Knockdown of SET8 expression in 786-O cells inhibited cell proliferation, colony formation, migration and invasion ability by mediating the expression of c-myc and MMP-7. SET8 may serve as a therapeutic terget in this disease. |
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