Study On The Expression, Functional Mechanism And Prognosis Of MiR-23a-3p In Renal Cell Carcinoma

Objective: Renal cell carcinoma(RCC)is the most common renal malignancy.Because of its atypical early symptoms,it is often found at advanced stage and has a poor prognosis.In recent years,many evidences have shown that miRNA plays an important role in the occurrence and development of tumors.As a member of the miRNA family,miR-23a-3p is abnormally expressed in many tumors.The results of gene microarray showed that the expression of miR-23a-3p was up-regulated in RCC,but there was no verification of the expression of miR-23a-3p in RCC specimens.Therefore,the purpose of this study was to understand the expression,function and potential target genes of miR-23a-3p in RCC,and to explore its role as a new biomarker to predict the prognosis of RCC.Methods: In order to detect the expression level of miR-23a-3p in RCC tissues and cell lines,RT-q PCR was applied in this study.The statistical software was used to analyze the expression of miR-23a-3p between tissues(RCC tissue and its adjacent paired normal tissues)and between cell lines(RCC cell lines and normal renal cell line).In addition,we also used the TCGA database to detect the expression level of miR-23a-3p.In order to detect the function of miR-23a-3p in renal cell lines(786-O,ACHN),this study transiently transfected miR-23a-3p functional analogs or inhibitors to achieve the purpose of changing the expression level of miR-23a-3p in renal cancer cells.In order to detect the function of miR-23a-3p in renal cancer cells,this study used cell activity and proliferation assay(namely MTT and CCK-8 assay),scratch assay,transwell assay,flow cytometry and other methods to detect the ability of miR-23a-3p in the proliferation,invasion and apoptosis of RCC lines(786-O and ACHN).Bioinformatics analysis,RT-q PCR,western blot and luciferase reporter gene detection were used to predict and verify the potential targets of miR-23a-3p.In addition,we also analyzed the relationship between the expression level of miR-23a-3p in 118 cases of formalin fixed paraffin-embedded RCC samples and the clinicopathological characteristics of patients,as well as the relationship between miR-23a-3p and its prognosis.Results: miR-23a-3p was significantly up-regulated in both RCC tissue samples and RCC cell lines compared with normal tissues and normal renal cell line,which was consistent with the result in the TCGA database.MTT assay showed that up-regulation of miR-23a-3p expression significantly increased the activity of renal cancer cells,while down-regulation of miR-23a-3p expression significantly decreased the activity of renal cancer cells.CCK-8 experiment confirmed that the overexpression of miR-23a-3p can significantly enhance the proliferation of renal cell carcinoma cells,while the low expression of mir-23a-3p inhibits the proliferation of cells.In terms of cell migration and invasion,both scratch test and Transwell migration and invasion test showed that when mir-23a-3p was highly expressed,the migration and invasion ability of renal cancer cells was enhanced.On the other hand,when miR-23a-3p expression was low,the migration and invasion ability of renal cancer cells was significantly reduced.Flow cytometry suggested that up-regulation of miR-23a-3p inhibited apoptosis.However,down-regulation of miR-23a-3p did not show significant effect on apoptosis of renal cancer cells.In addition,this study used bioinformatics to predict multiple potential targets of miR-23a-3p,and verified by double luciferase experiment and western blot experiment that proline rich nuclear receptor co-activator 2(PNRC2)may be a potential target of miR-23a-3p.Finally,we collected paraffin samples from 118 pairs of renal cancer patients,and the results showed that the expression level of miR-23a-3p was significantly correlated with the clinical stage of renal cancer(P<0.05).In addition,cox proportional risk regression analysis revealed that high expression of miR-23a-3p was associated with poor prognosis in patients with renal cancer.Conclusion: The results of this study indicated that miR-23a-3p was abnormally expressed in RCC tissues and cell lines,which could play an important biological function in RCC cells by regulating its potential target gene PNRC2.In addition,miR-23a-3p is not only related to the clinical stage of renal cancer,but also can be used as a new biomarker to predict the prognosis of renal cancer.