The Role And Molecular Mechanisms Of MiR-139-5p In Prostate Cancer

Part I Study of clinical relevance:the different expression of miR-139-5p between prostate cancer and benign prostatic hyperplasia or health individualsObjective:MicroRNAs(miRNAs)are a type of short,single stranded non-coding RNA molecules with about 22 nucleotides,which can regulate gene expression in the posttranscriptional level and play an important biological role in various species.Several evidences suggest that the aberrant expression of miRNAs contribute to tumor cell proliferation,apoptosis,metastasis and invasion,as well as the anti-tumor drug resistance.Recent study indicated that miRNAs involved in the occurrence and development of prostate cancer.Therefore,exploring the expression and relative mechanisms of miRNAs is vital for diagnosis and treatment of prostate cancer.In particular,miR-139-5p is one of the most common cancer-associated miRNAs that we interested in,which plays an important role in the pathogenesis of various tumors,however,the role of miR-139-5p in prostate cancer remains unknown.This present study aims to explore the expression of miR-139-5p in the peripheral whole blood of patients with prostate cancer,benign prostatic hyperplasia and in health individuals;further report the relationship between miR-139-5p and clinicopathological parameters of prostate cancer;also evaluate the efficiency of miR-139-5p for the diagnosis of prostate cancer.Methods:1.All peripheral whole blood samples of patients with prostate cancer,benign prostatic hyperplasia and health individuals were obtained from the Department of Urology,Beijing Hospital(Beijing,China)between Mar.2014 and Jul.2016.The demographics and clinical characteristics of them were obtained,including serum prostate specific antigen(PSA),age,the results of MR imaging and pathologic information,and the strategy of treatments and so on.No patients with prostate cancer received androgen deprivation therapy or radiotherapy.After extraction RNAs from peripheral whole blood samples of all participants,differential expressions of miR-139-5p were detected by quantitative Real Time PCR.The application of patient-derived materials was approved by the Research Ethics Committee of Beijing Hospital,and written consent was obtained from all patients.2.The expression of miR-139-5p in patients with different of clinical stage and Gleason score was detected by real time PCR.To further evaluate the relationship between the expression of miR-139-5p and the malignancy degree of prostate cancer.3.Receiver operating characteristics(ROC)curve was performed to test diagnostic efficacy of miR-13 9-5p as a non-invasive biomarker.Results:1.There were no statistical differences between three groups for the patients' age and BMI(P>0.05).Serum PSA levels were significantly higher in patients with prostate cancer compared with that in patients with benign prostatic hyperplasia and health individuals(P<0.001).2.The level of miR-139-5p was examined in the peripheral blood of patients with prostate cancer,benign prostatic hyperplasia and health individuals using real time PCR.The expression of miR-139-5p was significantly up-regulated in the peripheral blood of patients with prostate cancer compared with that of patients with benign prostatic hyperplasia and health individuals(P<0.001).The relative expression of miR-139-5p was 5.3±2.8 for patients with prostate cancer,1.4±0.8 for patients with benign prostatic hyperplasia and 1.0±0.8 for health individuals.3.According to the stratification of prostate cancer patients with different of PSA level,tumor stage and Gleason score,clinicopathological parameters changes are accompanied by up-regulation of miR-139-5p in peripheral blood of prostate cancer patients.Patients with more aggressive tumors exhibited significantly higher miR-13 9-5p expression compared with that of patients with less aggressive tumors(P<0.05).4.Based on an ROC analysis,blood miR-139-5p was able to distinguish prostate cancer patients from healthy controls(AUC:0.915)and patients with benign prostatic hyperplasia(AUC:0.936)(P<0.001).Conclusion:1.The expression of miR-139-5p was significantly higher in peripheral blood of prostate cancer patients than patients with benign prostatic hyperplasia and healthy individuals,which showed a strong association between prostate cancer and miR-139-5p expression.2.The more high-risk stage,the higher expression of the miR-139-5p was found.Much higher expression of peripheral blood miR-139-5p was detected in prostate cancer patients with more advanced stage(pT3/4)and more aggressive tumors(Gleason score>7),suggesting that peripheral blood miR-139-5p may be tightly associated with progression of prostate cancer.3.Peripheral blood miR-139-5p was able to distinguish patients with prostate cancer from patients with benign prostatic hyperplasia and healthy individuals,suggesting miR-139-5p can be a non-invasive biomarker with high specificity and sensitivity for prostate cancer detection.Part II Study of molecular mechanism:miR-139-5p have an effect on PTEN to inhibit prostate cancer cell apoptosisObjective:Growing evidence indicated that miRNAs play an important role in the pathogenesis of prostate cancer.Aberrant expression of miRNAs may contribute to the progression of prostate cancer and the transformation of prostate cancer cells from androgen dependent to castration resistant prostate cancer(CRPC),which is the important cause of death in patients.Previous studies have proved that miR-139-5p expression was closely correlated to the development and progression of prostate cancer,however,the molecular mechanism of miR-139-5p in prostate cancer remains unknown.In this part of study,we intend to explore the effects of miR-139-5p on prostate cancer cells in apoptosis and proliferation via in vitro cell functional study.Related experiment was performed to verify the potential target gene of miR-139-5p,and to explore the specific role and molecular mechanisms by which miR-139-5p was involved in prostate cancer cell apoptosis.Methods1.miR-139-5p mimic and inhibitor were synthesized and transfected into PC3 cells to establish experimental model.2.After establishing experimental model,the expression of miR-139-5p in PC3 cells was detected by real time PCR.The in vitro proliferation of the PC3 cells was measured using the CCK-8 assays.The expression of Bcl2 and Bax was measured by western blot.3.PTEN may be the potential target gene of miR-139-5p in prostate cancer by bioinformatics analysis,then,confirmed by dual luciferase reporter assay,western blot,and immunofluorescence(IF),4.miR-139-5p inhibitor and si-PTEN were transfected into PC3 cells to verify that miR-139-5p can combine with PTEN to directly regulate the expression of Bcl2 and Bax,which can inhibit prostate cancer cells apoptosis and promote the development of prostate cancer,eventually.Results:1.The cell models of high/low miR-139-5p expression were successfully established.2.Transfection with miR-139-5p inhibitor can reduce the activity of prostate cancer cells,and inhibit the expression of Bcl2 and promote the expression of Bax.3.Dual luciferase reporter assay demonstrated that PTEN was the target gene of miR-139-5p.The expression of PTEN was negatively regulated by miR-139-5p.4.Knockdown of PTEN rescued miR-139-5p inhibitor-reduced prostate cancer cells apoptosis.Conclusion:1.miR-139-5p,act as oncogene,can promote the proliferation of PC3 cells and inhibit PC3 cells apoptosis.2.The silence of miR-139-5p expression can promote prostate cancer cells apoptosis.3.PTEN was the direct target gene of miR-139-5p.miR-139-5p can impact the regulation of prostate cancer cells apoptosis via inhibiting PTEN.