The Mechanism Of YY1-hnRNPM Signaling Pathway In The Progression Of Prostate Cancer

ObjectiveTo explore the effects of heterogeneous nuclear ribonucleoprotein M(hnRNPM)on the epithelial-mesenchymal transition(EMT)in the progression of prostate cancer(PCa),and further explore the molecular mechanism of its regulation.MethodsThe difference of hnRNPM expression was predicted between normal prostate tissue and prostate cancer tissue using the Oncomine database.RT-qPCR and Western blot were used to detect the difference of hnRNPM expression in clinical samples(prostate cancer and benign prostate hyperplasia),different cell lines(LNCaP,C4-2,PC-3,DU145 and H660),as well as the difference of hnRNPM expression between PCa and adjacent normal prostate tissue samples.The difference of hnRNPM expression in benign prostate hyperplasia(BPH),prostate adenocarcinoma(ADENO)and neuroendocrine prostate cancer(NEPC)was detected by immunohistochemistry(IHC).HnRNPM was knocked down or overexpressed respectively in PCa cell lines by siRNA or shRNA and overexpressed plasmid or overexpressed lentivirus.The cell morphology changes were observed by optical microscope.The changes in cell migration and invasion were observed by scratch assay and transwell assay respectively.Changes of EMT-related markers and transcription factors were detected by RT-qPCR.The transcription factors(TFs)of hnRNPM were predicted by the PROMO and GeneCards databases.YY1 binding sites in the hnRNPM promoter were predicted by the GeneCards,PROMO,LASAGNA,and JASPAR databases.The expression levels of YY1 in normal prostate and PCa tissue samples were predicted using the Oncomine database.The associations between the expression of hnRNPM and YY1 were predicted using The Cancer Genome Atlas(TCGA)database.The difference of YY1expression in BPH,ADENO and NEPC were evaluated by IHC.SiRNA and overexpressed plasmids were used respectively to knock down or over express YY1 in C4-2 or PC-3 cell lines and the expression changes of hnRNPM were detected after 72hours by Western blot.The results showed that the hnRNPM expression was negatively correlated with YY1.After overexpressed YY1 or overexpressed YY1 and hnRNPM,cell invasion was detected by transwell assay,simultaneously,the protein levels of N-cadherin,Vimentin and Twist1 were detected by western blot.For the two predicted binding sites of YY1 in the hnRNPM promoter region by databases,ChIP assays and dual-luciferase reporter assays were performed to further investigate the specific mechanism underlying the effect of YY1 on hnRNPM.The hnRNPM overexpressed lentivirus were transfected in PC-3 and DU145 cell lines,and the hnRNPM overexpressed cell lines were screened using puromycin.The negative control lentivirus,containing luciferase plasmids,were transfected into hnRNPM overexpressed cell lines.The treated PC-3 and DU145 cell lines were subcutaneously planted into nude mice respectively,after 6 weeks the tumors were removed,cut into tumor mass with volume of 1 mm~3 and were implanted in situ in nude mice,after 4-6weeks,the in vivo imaging system was used to observe PCa metastasis.ResultsOncomine database showed that the hnRNPM expression in PCa tissues was lower than that in normal prostate tissues,which was consistent with our results detected in clinical samples.The expression level of hnRNPM in PCa tissues was lower than that in adjacent normal prostate tissues.IHC showed that the expression levels of hnRNPM were significantly decreased in the ADENO and NEPC samples compared with the BPH samples,and the lowest level was detected in the NEPC samples.Additionally,hnRNPM was highly expressed in LNCaP cells,which have the weakest invasive ability of the studied PCa cell lines.Furthermore,the expression of hnRNPM was lowest in H660 cells(NEPC cell line),which have the highest invasive ability of the tested cell lines.The shapes of C4-2 and PC-3 cells were significantly changed after stably knocked down hnRNPM in the C4-2 and stably overexpressed hnRNPM in the PC-3 cells(C4-2 cells changed from a cobblestone-like sheet of cells to spindle-shaped cells,while PC-3 cells underwent the opposite transformation).Simultaneously,cell migration and invasion ability,the EMT related markers and Twist1 were enhanced in C4-2 cells while the opposite results were achieved in PC-3 cells.The database predicted that YY1 might be the upstream transcription factor of hnRNPM,and the Oncomine database showed that the YY1 expression in PCa samples was higher than that in normal prostate samples.According to the TCGA database,the expression of YY1 and hnRNPM was negatively correlated.YY1 expression in ADENO tissue was higher than that in BPH tissues by IHC and more notably,that the expression of YY1was the highest in NEPC,which is the most invasive type of PCa.Overexpression of YY1 in C4-2 cells resulted in decreased expression of hnRNPM.In PC-3 cells,knockdown of YY1 resulted in increased expression of hnRNPM.Overexpression of YY1 promoted the invasion of C4-2 cells,and this effect was partly impaired by the overexpression of hnRNPM.Western blot analysis of C4-2 cells showed that the expression of N-cadherin,Vimentin and Twist1 was increased after the transfection of the YY1 over expression plasmid compared to the transfection of the control vector,but the expression of N-cadherin,Vimentin and Twist1 was decreased to some extent after the co-transfection of the YY1 and hnRNPM plasmids.The results of dual-luciferase reporter assays showed that YY1 could exert transcriptional inhibition effect on hnRNPM through the binding site 2 in hnRNPM promoter predicted by the databases.Animal experiments have shown that overexpression of hnRNPM can reduce the metastasis of PCa.ChIP assays showed that YY1 could bind to both predicted binding site 1 and predicted binding site 2 in the promoter of hnRNPM.Dual-luciferase reporter assay showed that YY1 over expression could significantly inhibit the luciferase activity of the wild-type and mutant YY1 binding site 1 but not that of the mutant binding site 2in the hnRNPM promoter in PC-3 cells.In other words,YY1 exerts a suppressive function on hnRNPM mainly through binding site 2.ConclusionsLow expression of hnRNPM could increase the invasiveness of PCa through the EMT pathway.Overexpression of YY1 could promote EMT by reducing hnRNPM expression.We speculate that hnRNPM may be a biomarker that can assist in assessing PCa aggressiveness.