The Anti-tumor Role And Mechanism Of MiR-362-3p In Renal Cell Carcinoma

Renal cell carcinoma(RCC)is a relatively common human malignancy,accounting for about 3%of all adult malignancies and 90%of all kidney malignancies.According to the data reported by He et al.,there were 68,000 new cases of kidney cancer and 23,400 deaths in China in 2015.Due to its vascularity,RCC is not sensitive to chemotherapy or radiotherapy.The current main treatment methods are surgical treatment and targeted therapy.Since the precise molecular mechanism of RCC development and progression still remains unclear.In addition,if the pathogenesis of RCC can be elucidated,it is of great significance for clinical diagnosis,treatment and subsequent monitoring.MicroRNAs(miRNAs)are abundant classes of endogenous non-coding small RNAs.miRNAs are about 20-24 nucleotides in length and regulate gene expression at the post-transcriptional level by sequence-specific binding with the 3' untranslated region(3'-UTR)of a target mRNA.Usually it can result in gene silencing through the induction of mRNA degradation or the inhibition of protein translation.Many evidences showed that disordered miRNA expression play an important role in the tumorigenesis and progression of RCC.The purpose of our experiment was exploration of the inhibition effect of miR-362-3p on tumorigenicity of RCC as well as the underlying mechanism.Our main findings are as follows:1)Firstly,miR-362-3p was down-regulated in RCC.For evaluating the expression level of miR-362-3p in RCC,quantitative real-time PCR(qRT-PCR)was used to detect 25 pairs of clinical RCC tissues and adjacent non-cancerous tissues,in addition to three types of RCC cell lines 786-0,ACHN,and Caki-1.We found that the mRNA expression level of miR-362-3p in tumor tissue was generally lower than that in non-cancerous tissue.Studies in RCC cell lines also indicated that the expression of miR-362-3p was significantly down-regulation compared to HK-2 cell line.2)The results of bisulfite genomic sequence(BSP)showed that methylation of miR-362-3p were significantly increased in RCC cell lines(786-0,ACHN,and Caki-1).Expression of miR-362-3p was significantly increased after methylation inhibition with 5-Aza-2 '-deoxycytidine(5-Aza-CdR).3)Upregulation of miR-362-3p inhibited proliferation,colony formation,and cell cycle in RCC cells.Furthermore,Overexpression of miR-362-3p was also able to suppress cell motility and the process of epithelial-mesenchymal transition(EMT).AKT/FOXO3a pathway was the downstream signal of miR-362-3p.4)Two methods were applied to explore the potential direct targets of miR-362-3p:bioinformatics prediction and dual-luciferase reporter assay.Finally,we convinced that SP1 was a direct target of miR-362-3p.5)Our study showed repression of SP1 by siRNA can inhibit cell proliferation,colony formation,cell cycle,and cell motility by regulating the AKT/FOXO3a signaling,which phenocopied the effect of miR-362-3p in RCC cells.In summary,the study showed that miR-362-3p can negatively regulates SP1.It was a noval suppressor in RCC.