Partial Depletion Of SET8 By Target ShRNAs Effects The Tumor Malignant Phenotype And The Mechanism Research On The Relevance Of Wnt Signaling Pathway In Renal Cell Carcinoma

Objective:To investigate the influence of the tumor malignant phenotype in renal cell carcinoma by Histone methylase SET8 interference, we used the partial depletion of SET8 by target shRNAs to downregulate its expression. Then, we investigate the relationship with the target gene and protein of the Wnt signaling pathway to explore its clinical significance.Method:1. Partial depletion of SET8 by shRNAs influence 786-0 and ACHN cell viability, apoptosis, the cell cycle and the abilities of invasion and metastasis:First, to downregulate the level of SET8 in ccRCC cells, we constructed SET8 interference vector with pLKO.1/shSET8-1 and pLKO.1/shSET8-2 plasmid and transfected them into 786-0 and ACHN cells respectively. The MTT cell viability assay was used to assesse the effection of cell viability by SET8 interference. We also investigated the effect of SET8 on both apoptosis and the cell cycle progression by using flow cytometry analysis. The transwell assay was used to evaluate the effect of SET8 in ccRCC cells.2. The interaction between SET8 and the Wnt target gene:a coimmunoprecipitation(co-IP) assay was assessed the SET8 interaction between SET8 and TCF4, LEF1 in 786-0 and ACHN cells.3. The influence of the transcription of Wnt-activated genes by SET8 partial depletion:Western blot assey was used to imply the change of several protein expressions of Wnt target genes by SET8 interference.Results:1. Compared to the control group, the level of cell apoptosis of both ACHN and 786-0 had no difference after being transfected the SET8 interference vector with pLKO.1/shSET8-1 and pLKO.1/shSET8-2 plasmid (P>0.05). However, the cell proliferation in ccRCC cells was significant inhibited by SET8 interference and SET8 downregulation blocked the cell cycle at S phase (P<0.01). What’s more, partial depletion of SET8 by target shRNAs inhibits ccRCC cell migration and invasion (P<0.01).2. The coimmunoprecipitation(co-IP) assay was showed that SET8 interacted with both TCF4,LEF1 in 786-0 and ACHN cells (P<0.01).3. In Western blot assay, the protein expression of cyclin D1, LEF1, Surivrin and H4K20me-1 were Significantly downregulated by SET8 knockdown compared with control group in both 786-0 and ACHN cells (P<0.01).Conclusion:In addition, shRNA-mediated partial depletion of SET8 reduced 786-0 and ACHN cell viability and arrested them at S phase of the cell cycle, which also inhibited their abilities of invasion and metastasis. What’s more, SET8 could interact with TCF4 and LEF1. In addition, several protein expressions of Wnt target genes were downregulated by SET8 interference.