The Effect Of MiR-34a-5p On P53/DAPK Signaling Pathway In Human Clear Cell Renal Cell Carcinoma

Objective:DAPK is a 160 kDa serine/threonine kinase widely expressed in various tissues.Since DAPK protein is associated with several major cellular processes such as apoptosis and autophagy,it has long been considered as a tumor suppressor.The expression of DAPK is often downregulated due to CpG island methylation in multiple tumors.Though DAPK promoter methylation in renal cell carcinomas has been reported in several studies,the methylation rate dose not correlate well with reduced DAPK expression.Actually,DAPK expression in human clear cell carcinoma has not been well characterized.Previously,DAPK has been reported to be a transcriptional target of p53 in other cells.Whether the p53-DAPK axis is also valid in clear cell renal cell carcinoma remains to be determined.microRNAs are 19～22 nucleotide long non-coding RNAs.It can bind specificly to the 3’ UTR of target mRNAs to promote the mRNA degradation or to inhibit protein translation.Increasing studies indicated microRNAs are associated with the development and progression of ccRCC,however,whether microRNAs interacted with p53-DAPK signaling pathways in ccRCC remains ambiguous.The present study aims to determine the DAPK expression in clear cell renal cell carcinomas of different stages and grades on both mRNA and protein levels.We further examined the role of DAPK in apoptosis,proliferation and migration.Then we examined the expression of p53 and the correlation between p53 protein expression and DAPK protein levels in clear cell carcinomas and different renal cancer cell lines.Finally,we determined how microRNA interacted with the p53-DAPK axis to affect the biological function in ccRCC.Methods:A total of 61 paired clear cell renal cell carcinoma samples and adjacent normal renal tissues were collected and subjected to quantitative PCR and immunoblot analysis.The DAPK expression in clear cell renal cell carcinoma of different stages and grades were examined and compared to normal renal tissues.Twelve pairs of samples were further analyzed by immunohistochemistry to determine the expression and subcellular location of DAPK protein in clear cell carcinoma and paired normal tissues.Furthermore,DAPK expression vectors,DAPK siRNA/shRNAs were used to alter DAPK expression in different renal cell cancer lines.Flow cytometry,nuclear staining and immunoblot were used to determine the effect of DAPK on the apoptosis of renal cancer cells.Colony formation assays,EdU incorporation assay and RTCA were used to evaluate the role of DAPK in cell proliferation.In vivo studies were also used to determine the effect of DAPK on cell proliferation.Transwell assays and wound healing assays were employed to analyze the effect of DAPK on renal cell carcinomas We then measured the expression of p53 in human renal cancer samples using quantitative PCR and immunoblot.We also examined the correlation of p53 protein expression and DAPK protein levels in human renal cancer samples.Furthermore,quantitative PCR and immunoblot were used to determine whether DNA damaging drugs,p53 expression or p53 activators can induce DAPK transcription.A bioinformatic approach was used to identify the protential regulatory microRNA of DAPK.We also validated the regulation of DAPK mRNA by microRNA and examined the effects of the microRNA on apoptosis and proliferation both in vivo and in vitroResults:Findings of qPCR showed DAPK mRNA expression was lower in renal tumors in comparison to its normal counterparts(P<0.05).On immunoblotting,DAPK protein was lower in G4 tumoral tissues when compared to normal renal tissues or tumors of other grades(P<0.05).On immunohistochemistry,weak DAPK protein expression was detected in both the nuclei and cytoplasm of clear cell renal tumor cells,while in adjacent normal renal tissues,DAPK protein was mainly found in the cytoplasm of kidney tubular epithelial cells.Apoptotic analysis indicated that DAPK induce apoptosis of renal cancer cells in both p53 dependent and independent manner DAPK also inhibited the proliferation of renal cancer cells and affected the migration of renal cancer cells in part though promoting apoptosis.Though the expression of p53 was found to be increased in certain groups of renal cancers,the expression of p53 protein did not correlate well with basal DAPK mRNA levels in human renal cancer samples or renal cancer cell lines.Besides,although DNA damaging drugs,p53 overexpression or p53 activators induced the upregulation of classic downstream effector such as p21,it failed to up-regulate DAPK protein.We hypothesized that microRNA may be involved in this process.Hence,we predicted several microRNAs that have the protential to regulate DAPK through a bioinformatic approach.We identified and validated hsa-miR-34a-5p as a novel repressor of DAPK translation which was also regulated by functional p53.Finally,we found hsa-miR-34a-5p affected the proliferation and apoptosis both in vitro and in vivo via DAPKConclusion:Taken together,these results show that DAPK mainly functions as a negative regulator that acts downstream of p53 in ccRCC.The p53-DAPK axis is suppressed due to increased miR-34a-5p,which is also induced by p53.miR-34 inhibitors can rescue p53-DAPK pathway dysfunction and be used as a potential therapeutic target to improve the treatment and prognosis of ccRCC patients.