| As increasingly aging of the population and extensive promotion of screening means(PSA),a significant increase in the morbidity of Pea have threat the health of men in our country.Techniques such as PSA analysis,biopsy and Gleason score have improved the detection and clinical managemen of Pea,but still no novel approaches can exactly monitor the progression and predict its outcome.A great quantity of ectopic expression of newly discovered miRNAs were detected in Pea which offers a promising route for biomarker discovery.Accumulating evidence indicates that miRNAs play critical roles in PCa metastasis and have potential use as prognosis predition and therapeutic targets.MicroRNAs are noncoding RNAs that are important for embryonic stem cell development and epithelial to mesenchymal transition(EMT).In Pea,many miRNAs are now known to modulate various important cancer-related genes,acting as oncogenes(miR-21,miR-182,miR-221,miR-222 et al)or tumour suppressors(miR-205,miR-34a et al).Although dysregulated miR-132/212 have been suggested to be directly involved in the proliferation and invasion of multiple malignancies,Formosa et al pointed to miR-132 as a methylation-silenced miRNA in Pea,the exact role of miR-132/212 in PCa has not yet been fully explored..Objectives:1.Investigating the expression and function of miR-132/212 in the progression of Pca.2.Revealing the role of miR-132/212 in the progression of TGF-?-induced EMT in PCa cells.3.Studying the target gene of miR-132/212 in PCa.Innovation and significances:1?Our results indicate that miR-132/212 inhibit TGF-?-induced EMT in PCa cells.2?Our study identify SOX4 is a direct target of miR-132/212.3?Our study further suggests that miR-132/212 might be a potential biomarker,and targeting the miR-132/212/SOX4 axis might be a promising strategy for the treatment of a subset of metastatic PCa.Methods:1?Explore the expression of miR-132/212 in PCa1.1 RT-qPCR analysis of miR-132/212 expression in PCa 3 cell lines(Vcap,Lncap and RWPE).1.2 RT-qPCR analysis of miR-132/212 expression in PCa tissues.Totally,71 clinical samples of PCa(n=57)and Benign Prostatic Hyperplasia(BPH,n= 14)PCa were analyzed by RT-qPCR,and the relationship between miR-132/212 expression level and clinicopathological variables was detected.1.3 The expression patterns of miR-132/212 were further characterized with GSE21304 dataset.2.Functional characterization of miR-132/212 in PCa cells2.1 MTS assay was utilized to investigate the effect of miR-212/132 on the proliferation of PCa cells.2.2 Transwell assay was applied to study the effect of miR-212/132 on the invasion ability of PCa cells.2.3 Wound healing assay was applied to evaluated the effect of miR-212/132 on the migration ability of PCa cells.2.4 Colony formation assay was applied to detect the effect of miR-212/132 on the colony formation ability of PCa cells.3.Revealing the role of miR-132/212 in the progression of TGF-?-induced EMT in PCa cells.3.1 Under the invert microscope,observing the morphologic changes after stimulation with TGF-? at 5 ng ml-1 for 72h.3.2 The effect of miR-132/212 on TGF-?-induced EMT was evaluated by western blot analysis at protein level.3.3 The effect of miR-132/212 on TGF-?-induced EMT was evaluated by RT-qPCR at mRNA level.4.Identifying SOX4 is a direct target of miR-132/2124.1 With the use of two publicly available algorithms(TargetScan and miRanda)to forcast if SOX4 is a theoretical target gene of miR-132/2124.2 Western blot analysis detecte the effect of SOX4 after transfected miR-132/212 mimics.4.3 Luciferase reporter assay were utilized to identify if SOX4 is the target gene of miR-132/212.5.Analyzing the relationship of the expression of SOX4 with the expression of miR-132/212 in prostatic tissues5.1 Immunohistochemistry(IHC)was used to detect the expression of SOX4 at protein level to analyze the relationship with the expression of miR-132/212.5.2 RT-qPCR was utilized to detect the expression of SOX4 at mRNA level to analyze the relationship with the expression of miR-132/212.5.3 With the use of GSE21032 datasets to investigate the correlation of miR-132/212 and SOX4 transcript level.Results:1?MiR-132/212 are downregulated in human PCa and associated with high Gleason score and distant metastasis1.1 MiR-132 and miR-212 are downregulated in human PCa tissues when compared with benign prostatic hyperplasia tissues(both P<0.05).1.2 Decreased miR-132/212 expression was significantly associated with high Gleason scores(P =0.028/P =0.004)and distant metastasis(P =0.043/0.001).1.3 The expression levels of miR-132/212 in Lncap and Vcap cell lines were significantly lower than those in RWPE cell line(P<0.05)1.4 GSE21304 dataset analysis displayed miR-132 expression was downregulated in high grade PCa tissues compared to low grade tissues.By contrast,no significant correlations were identified between miR-212 expression and high Gleason score in PCa tissues(p>0.05,data not shown)2?Functional characterization of miR-132/212 in PCa cellFunctionally,upregulation of miR-132/212 inhibits the migration and invasive capacity of Vcap and Lncap cells by wound-healing and transwell assays,respectively;the colonies formation were significantly fewer in numbers than the control cells(P<0.05);no significant differences were observed in the level of proliferation in Vcap and Lncap cells after transfection with miR-132/212 mimics(P>0.05).3?MiR-132/212 inhibit TGF-?-induced EMT in PCa cells3.1 In cultured Vcap and Lncap cells after stimulation with TGF-? at 5ng/ml for 72h cells showed an elongated fibroblast-like morphology with scattered distribution.3.2 Overexpression of miR-132/212 could inhibit TGF-?(transforming growth factor-?)-induced EMT in Vcap and Lncap cells at both the mRNA and protein expression levels.MiR-132/212 mimics significantly inhibited TGF-?-induced down-regulation of E-cadherin as well as up-regulation of Vimentin.By contrast,miR-132/212 inhibitors significantly restored TGF-?-induced EMT in PCa cells,compared to those of miR-132/212 mimics,evidenced by downregulation of E-cadherin and up-regulation of Vimentin both at mRNA and protein levels.4?SOX4 is a direct target of miR-132/2124.1 Our analysis with the use of two publicly available algorithms(TargetScan and miRanda)revealed that SOX4 is a theoretical target gene of miR-132/212.4.2 Western blot analysis shows that miR-132/212 mimics dramatically decreased the expression level of SOX4.4.3 The reporter plasmids were co-transfected into 293T cells along with miR-132/212 mimics or negative control,luciferase activity was significantly decreased in the miR-132/212-transfected group compared to the control group(P<0.05).5?MiR-132/212 expression is inversely correlated with SOX4 expression in PCa tissues5.1 SOX4 overexpression by immunohistochemistry(IHC)shows significant inverse correlation with the expression levels of miR-132(P<0.001)and miR-212(p=0.0024),respectively.5.2 The inverse correlation between SOX4 expression and miR-132(rs=0.569,P<0.001),and between SOX4 and miR-212(rs=0.528,P<0.001)was identified by Spearman's rank correlation test,respectively.5.3 The bioinformatics data demonstrated that SOX4 overexpression was inversely correlated with the expression levels of miR-132(R=-0.25,P=0.009).However,no significant correlation was observed between SOX4 overexpression and the expression level of miR-212(R=0.05,P=0.578).Conclusion:Our data suggested that miR-132/212 may act as tumor suppressors in PCa progression through disrupting EMT process by directly targeting SOX4. |
PDF Full Text Request