The Function And Regulatory Mechanism Of NLRC5 In Clear Cell Renal Cell Carcinoma

Renal cell carcinoma(RCC)is the second most common cancer in urological system,representing approximately 3%of all adult malignancies.Clear cell renal cell carcinoma(ccRCC)is the most common histologic subtype of RCC,constituting around 75%of cases.More than 30%of RCC patients present with locally advanced and metastatic disease at the time of diagnosis.The five-year survival rate of advanced ccRCC is poor and may result in metastasis or recurrence.Hence,it is important to gain a better understanding of the underlying molecular mechanisms of malignant ccRCC and identify new efficacious therapeutic strategies.NLRC5,a newly discovered member of the NLR family((NOD like receptors),has been reported to regulate immune responses and inflammation,especially in the development of tumors.Previous studies have shown that NLRC5 promote cell proliferation,migration,and invasion in hepatocellular carcinoma.However,to date,the potential regulatory roles and molecular mechanisms by which NLRC5 affects the development and progression of ccRCC remain largely unknown.In this study,human clinical data from The Cancer Genome Atlas(TCGA)database revealed that NLRC5 is higher in ccRCC tissues compared to in normal tissues.Meanwhile,NLRC5 expression was associated with stage and prognosis in ccRCC patients.And further exploreits effect onthecell proliferation,migration and invasionandthemolecular mechanism of ccRCC.Methods1.Published mRNA expression data in ccRCC and normal kidney tissue was downloaded from TCGA.We used the Student’s t-test to assess the difference between tumour and normal samples.Furthermore,we analysed the relationship between the level of NLRC5 genes mRNA expression in ccRCC and clinicopathologic feature or prognosis.2.Patients with ccRCC were recruited from the First Affiliated Hospital of Anhui Medical University.The expression of NLRC5 protein and mRNA in human ccRCC andnormalkidneytissuesweredetectedbyRT-PCR,westernblotting and immunohistochemical staining.Moreover,expression of NLRC5 was analyzed by RT-PCR and western blotting in ccRCC cell lines and human normal cell line HK-2.3.pEGFP-C3-NLRC5 plasmid or small interfering RNA(siRNA)was performed using Lipofectamine~TMM 2000(Invitrogen)according to the manufacturer’s protocol.Western blotting and RT-PCR were applied to verify the change of NLRC5 expression.4.The function of NLRC5 on ccRCC cells were examined by CCK-8,transwell migration and invasion experiments,flow cytometry and western blotting.Western blotting analysis was performed to assess the role of Wnt/β-catenin signalling pathway in the oncogenic function of NLRC5.5.The nude mice were assigned to the following two groups:NLRC5-shRNA and NC-shRNA.Then,200μl of a 786-O(NLRC5-shRNA/NC-shRNA)cell suspension containing 5×10~6 cells was subcutaneously injected into the right flank of each mouse.Tumour sizes were measured weekly.Eight weeks later,the mice were killed,and the tumours were removed for further assessment.Results1.mRNA levels of NLRC5 are significantly higher in ccRCC tissues compared to those in normal tissues.Patients with higher NLRC5 mRNA expression exhibited significantly worse overall survival.NLRC5 mRNA levels were significantly higher in T3/T4-stage tumours than in T1/T2-stage tumours.2.Western blotting and RT-PCR results confirmed that the protein and mRNA levels of NLRC5 were overexpressed in carcinoma tissues compared to levels in adjacent noncancer tissues.NLRC5 was significantly elevated in ccRCC cell lines in comparison to HK-2 cell levels.3.NLRC5-siRNA induced a clear decrease in NLRC5 protein and mRNA levels in786-O and 769-P cells in comparison with levels induced by NC-siRNA.In contrast,NLRC5 protein and mRNA levels were increased by pEGFP-C3-NLRC5 introduction in 786-O and 769-P cells.4.In vitro,overexpression of NLRC5 increased cell proliferation migration and invasion and activated Wnt/β-catenin signalling pathway.Depletion of NLRC5dramatically attenuated cell proliferation,migration and invasion in vitro and inhibited Wnt/β-catenin signalling pathway.5.Measured volumes of the tumours indicated that NLRC5-depleted cells grew much more slowly than NC cells.Moreover,the weights of the tumours from shNLRC5 mice were lower than those from NC mice.Western blotting showed that NLRC5 knockdown inhibited the expression of MMP9,MMP2,PCNA,β-catenin,c-Myc and cyclin D1compared with levels in NC tumours.ConclusionNLRC5 mediates cell proliferation,migration,and invasion by regulating the Wnt/β-catenin signalling pathway in clear cell renal cell carcinoma.