| Objective: The pathogenesis and the underlying mechanism of endothelial-mesenchymal transition in prostate cancer remain unclear.Plasmacytoma variant translocation 1(PVT1),a novel long non-coding RNA maps to 8q24.21,and in many tumor studies the up-regulation of PVT1 has already been reported.PVT1 is closely related to tumor cell proliferation,invasion,and metastasis.In this study,we employed a combination of techniques to study the role of PVT1 in prostate cancer,which included bioinformatic analysis,Western blotting and cell migration assays of prostate cancer cell lines.We report that PVT1 promotes prostate cancer invasion and metastasis by modulating EMT.Furthermore,PVT1 can promote EMT by up-regulation of Twist1,a transcription factor associated with EMT.We then confirmed that PVT1 acts as a sponge for mi RNA-186-5p and positively regulates Twist1 by a sponge effect.Therefore,this study has revealed a novel MECHANISM for the promotion of EMT in prostate cancer by PVT1.Our findings suggest that the PVT1/mi R-186/Twist1 regulatory axis may be a new therapeutic target for prostate cancer.Methods: 1.To elucidate the biological significance of PVT1 in prostate cancer,we first analyzed the expression of PVT1 in 52 normal prostate tissues and 499 prostate cancer tissues in the TCGA database.To determine the clinical significance of PVT1,we further evaluated the clinicopathological parameters of PVT1 in prostate cancer.we examined the expression of PVT1 in three prostate cancer cell lines including PC-3,DU145,and 22RV1,and a normal prostate cell line WPMY-1 by q RT-PCR.2.Subsequently,over-expression of the PVT1 cell line was constructed by transfecting the pc DNA3.1-PVT1 vector into PC-3 cells,while the low-expression cell line was constructed by transfecting the si RNA-PVT1 vector into DU145.4.Evaluating the effect of PVT1 on Prostate Cancer Cell Proliferation by Colony formation Assay 5.To determine the impact of PVT1 expression on EMT in PCa,we then examined the expression of EMT markers in over-expressed and poorly expressed PVT1 PCa cells using Western blot and q RT-PCR.6.We used bioinformatics software(micro RNA)to predict the potential mi RNA binding sites in PVT1 q RT-PCR confirmed the expression of mi R-186 was negatively regulated by PVT1 in PCa cells 7.Furthermore,we found a reduction of mi R-186 in prostate cancer patients by mining TCGA databases and the association between mi R-186 and PVT1 in prostate cancer was revealed using Pearson analysis.8.To clarify the relationship between PVT1,mi R-186,and Twist1,the si RNA-PVT1 or pc DNA-PVT1 were co-transfected with a mi R-186-5p inhibitor or mi R-186-5p mimics into PC-3 and DU145 cells.Results: Compared with normal tissues,the expression of PVT1 in prostate cancer increased significantly,Through the analysis of the TCGA database we revealed that expression of PVT1 was not associated with T classification,lymph node stage,M classification,and PSA level.However,high PVT1 expression was significantly associated with age and the Gleason Score.Kaplan-Meier analysis revealed that patients with high PVT1 expression experienced poorer overall survival than patients with low PVT1 expression.In summary,the results suggest that lnc RNA PVT1 promotes tumor cell proliferation,invasion,and metastasis in prostate cancer.This may suggest that lnc RNA PVT1 promotes EMT in PCa.We have shown that Twist1,a key mediator of the EMT in cancer,is up-regulated in PC-3 with over-expressed PVT1 and down-regulated in DU145 with low expression of PVT1.These results suggest that PVT1 induces EMT by inducing upregulation of Twist1 expression..We used bioinformatics software(micro RNA)to predict the potential mi RNA binding sites in PVT1.q RT-PCR confirmed the expression of mi R-186 was negatively regulated by PVT1 in PCa cells.the dual-luciferase reporter assay revealed that over-expression of mi R-186 reduced the luciferase activity of the p MIR luciferase reporter containing PVT1-WT,but not the reporter containing PVT1-MUT.Furthermore,we found a reduction of mi R-186 in prostate cancer patients by mining TCGA databases and the association between mi R-186 and PVT1 in prostate cancer was revealed using Pearson analysis.The results showed that lowexpression of PVT1 down-regulated the expression of Twist1,whereas the expression of Twist1 was up-regulated after co-transfection with mi R-186-5p inhibitor and si RNA-PVT1.Simultaneously,high expression of PVT1 up-regulated the expression of Twist1,whereas the expression of Twist1 was down-regulated after co-transfection with mi R-186-5p mimics and pc DNA-PVT1.Conclusion: In conclusion,our study reveals that PVT1 is up-regulated in prostate cancer and that up-regulation of PVT1 promotes cell proliferation,invasion,and metastasis of prostate cancer.Further exploration revealed that PVT1 up-regulated the expression of Twist1 by adsorbing mi R-186,thus promoting EMT.Therefore,our findings suggest that the PVT1/mi R-186/Twist1 regulatory axis may be a new therapeutic target for prostate cancer research. |
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