The Anti-tumor Role And Mechanism Of MiR-493-5p In Prostate Cancer

Prostate cancer(PCa)is the most common cancer worldwide and a leading cause of cancer-related death in the United States and in Europe.After the introduction of the prostate-specific antigen(PSA)test,the detection of PCa dramatically increased with a peak in the early 1990s.Today,approximately 85%of men newly diagnosed with PCa present with localized early stage tumors.Despite the significant improvement in early detection due to routine PSA testing,medical and scientific communities are still debating its benefits because there is no consensus regarding whether it effectively reduces the risk of death from the disease.PSA levels are prostate but not cancer specific and may fluctuate due to,for example,infections,inflammation,or benign prostatic hyperplasia(BPH),resulting in high false-positive rates.The poor correlation between PSA levels and disease state leads to unnecessary diagnoses and overtreatment of indolent PCa.Due to the molecular heterogeneity of PCa,the identification and clinical translation of routinely tested disease-and stage-specific molecular markers is a rational approach to potentially expedite PCa diagnosis,prognosis,and treatment response,opening the way to personalized medicine.Beyond proteins and messenger RNAs(mRNAs),which have shown clinical utility in various clinical scenarios,there is a growing interest in the potential utility of microRNAs(miRNAs)as PCa biomarkers.The miRNAs are evolutionarily conserved short(approximately 18-22 nucleotides[nt])noncoding single-stranded RNA molecules that act as posttranscriptional gene regulators.Initially transcribed in the nucleus by RNA polymerase II or III as long primary transcripts(pri-miRNAs),miRNAs are subsequently processed into 70-to 100-nt precursor RNAs(pre-miRNAs)by the microprocessor complex,consisting of the RNase III enzyme Drosha and its interacting partner DGCR8.This initial cleavage is followed by Exportin-5/RanGTP-mediated pre-miRNA translocation to the cytoplasm for further processing into a 19-to 25-nt duplex by the RNase III endonuclease Dicer and TRBP.The final processing by Dicer is likely to culminate in the assembly of the two strands into the RNA-induced silencing complex(RISC);the key component of the RISC complex is an Argonaute protein.Within RISC,miRNAs negatively regulate translation of target mRNAs by altering their stability either through the binding to their 30 untranslated regions(UTR),or,to a lesser extent,to the 50 UTR or to the coding sequence.As a result,the miRNAs can directly target mRNA to degradation in the presence of perfect complementarity or induce translational repression through different mechanisms.It has also been demonstrated that miRNAs not only repress,but in some cases also may be able to activate gene expression directly or indirectly through the interaction with micro-ribonucleo-proteins such as Ago2 and FXR1.Due to the complexity of these regulatory mechanisms,and because each miRNA can modulate the expression of multiple mRNAs and,furthermore,each mRNA may be targeted by several different miRNAs,it is not surprising that miRNAs have been shown to be involved in almost all key cellular processes,such as proliferation,differentiation,migration,apoptosis,and sternness main-tenance.Alterations in the expression of cancer-related miRNAs can be affected by chromosomal rearrangements,promoter methylation,or transcriptional deregulation.Indeed,20-40%of miRNAs are located near CpG islands,confirming their possible epigenetic silencing,especially demonstrated for urologic diseases.The miRNAs are frequently located within fragile chromosomal sites that exhibit DNA amplifications,deletions,or translocations during tumor progression.Some individual miRNAs have been characterized either as tumor suppressors or oncogenes(onco-miRs),depending on the deregulated downstream targets,making them even more interesting and leading to huge numbers of related publications.A growing body of literature,in particular,has investigated the potential use of miRNAs as useful biomarkers for cancer diagnosis,prognosis,and therapy,including VIIPCa,and associated their levels with clinicopathologic parameters.Evaluation of the expression levels of specific miRNAs in clinical prostate tissue samples may be used to detect cancer,predict the cancer prognosis and monitor its evolution,and as markers for therapy selection and response.Our research aimed to explore the inhibition effect to progression effect and molecular mechanism of miR-493-5p in prostate cancer.The main investigations and results are as follows:1)miR-493-5p is down-regulated in prostate cancer compared to the normal patient.To evaluate the expression of miR-493-5p in prostate cancer,quantitative real-time PCR(qRT-PCR)was performed in 59 clinical PCa serum samples and 69 BPH serum samples,as well as in three types of prostate cancer cell lines PC-3,DU-145 and LNCaP.The expression level of miR-493-5p was generally lower in tumor tissue than in non-tumor tissue.The examination of miR-493-5p in more malignant prostate cancer cell lines PC-3?DU-145 also showed significant downregulation compared with LNCaP cell line.2)To better characterize the role of miR-493-5p in prostate cancer,we conducted gain of function(CCK-8,colony formation and Transwell)analysis by transfecting prostate cancer cell lines DU-145,PC-3 with chemically synthesized miR-493-5p mimic.We found that over-expression of miR-493-5p could induce inhibit cells growth and cell motility.Furthermore,Overexpression of miR-493-5p could also inhibit cell motility and induces the EMT by regulating Akt/GSK-3p/Snail signaling.3)Subcutaneous PC-3 xenograft was successfully established in nude mice.Tumor growth was suppressed in miR-493-5p treating group.4)We explored the possible targets of miR-493-5p by searching the online databases.We identified that EGFR,CREB1 and c-MET could be novel direct targets of miR-493-5p.Further qPCR analysis and luciferase assay confirmed that miR-493-5p transcriptionally repressed EGFR,CREB1 and c-MET by interacting with the essential binding sequence located in 3'-UTR.5)Repression of CREB1,c-Met and EGFR by siRNA inhibited cell motility and induced EMT by regulating Akt/GSK-3?/Snail signaling,which phenocopied the effect of miR-493-5p on cell motility and EMT.6)Finally,we combined our results with previously published studies to construct a regulatory network of miR-493-5p on the target genes c-Met,EGFR and CREB1,and found that CREB1 can also indirectly regulate the expression of c-Met.In conclusion,this study indicated that miR-493-5p was a novel suppressor in prostate cancer through its negative regulation of CREB1,EGFR and c-Met.