LncRNA SORY4-IT1 Sponges MiR-101-3p To Promote Proliferation And Metastasis Of Bladder Cancer Cells Through Up-regulating EZH2

Bladder cancer is one of the most common urological malignancies world-wide,the recommended maintenance schedules from transurethral resection to radiation therapy and systemic chemotherapy at present are effective only in a subset of patients.Therefore,to explore new detailed mechanisms and molecular pathways activated in bladder cancer is essential in developing novel diagnosis and treatment options for anticancer therapy in bladder cancer.Emerging evidences have indicated that long non-coding RNAs(LncRNAs)play vital roles in cancer development and progression in several cancers,and previous studies have suggested that over-expression of SPRY4-IT1 predicated poor prognosis and promoted tumor progress in several cancers.However,the underlying mechanism of SPRY4-IT1 in bladder cancer remains unknown.Therefore,in our study,we aim to explore the expression level of SPRY4-IT1 in bladder cancers and cell lines,and the effect of SPRY4-IT1 on biological behaviors of bladder cancer cells.By in-vitro and in-vivo experiments,we sought to reveal the underlying mechanism of SPRY4-IT1 in bladder cancer cells.Our research is composed of following three parts:Part I Research of expression of SPRY4-IT1 in bladder cancers and cell lines and the effect of SPRY4-IT1 on biological behaviors of bladder cancer cellsObjective:To investigate the SPRY4-IT1 expression levels of bladder cancer samples,adjacent histologically normal bladder tissues and cell lines.We aim to reveal the relation between expression levels of SPRY4-IT1 and clinical parameters,and the effect of SPRY4-IT1 on biological behaviors of bladder cancer cells.Methods:qRT-PCR analysis was used to investigate the SPRY4-IT1 expression levels in 60 pairs of bladder cancer samples and adjacent histologically normal bladder tissues and cell lines.shRNAs targeting SPRY4-IT1 were adopted to knockdown the expression of SPRY4-IT1 and to explore the effect of SPRY4-IT1 knockdown on the cell apoptosis,cell proliferation,cell migration and invasion ability of bladder cancer.Results:SPRY4-IT1 expression was significantly up-regulated(P<0.01)in 75.0%(45/60)of cancerous tissues compared with normal tissues.SPRY4-IT1 expression levels in bladder cancer significantly associated with high cancer grade,lymph node metastasis and distant metastasis.Significantly increased percentage of apoptotic cells in shSPRY4-IT1-transfected bladder cancer cells compared with negative cells.The cell cycle assay demonstrated that SPRY4-IT1 knockdown induced cell cycle arrest at G0/G1 phase in UMUC3 and T24T cells compared with negative control.Migration and invasion of UMUC3 and T24T cells was significantly reduced following down-regulation of SPRY4-IT1 expression detected by transwell assaysConclusion:SPRY4-IT1 functions as an oncogene and play a critical role in bladder cancer progression.Knockdown of SPRY4-IT1 inhibits cell proliferation,migration,and invasion,and promotes apoptosis of bladder cancer cells.Partn ? Research on underlying mechanism of SPRY4-IT1 in bladder cancer cellsObjective:We aim to explore the underlying mechanism of the effect of SPRY4-IT1 on biological roles of bladder cancer cells.Methods:Through searching in online bioinformatics database(FINDTAR3,http://bio.sz.tsinghua.edu.cn/and RNAhybrid),we ought to find out the miRNAs which have putative binding sites with SPRY4-IT1.Subsequently,we applied the biotin-labeled pulldown system to further explore which miRNAs that SPRY4-IT1 could directly interact with.qRT-PCR and Westewn Blot assays were adopted to detect the expression level changes of the miR-101-3p and its downstream genes in T24T and UMUC3 cells with transfection of shSPRY4-IT1 or GV-144-SPRY4-IT1 treatment,to find out the detailed pathway through which SPRY4-IT1 may play its oncogenic role.Human EZH2 3'-UTR fragment containing putative binding sites for miR-101-3p reporter vector and the EZH2 promoter reporter vector were adopted for Dual-luciferase Reporter Assays to confirm the effect of miR-101-3p on EZH2 in the T24T and UMUC3 cells transfected with shSPRY4-IT1 or GV-144-SPRY4-IT1.Rescue experiments were used to further confirm the miRNA sponge role of SPRY4-IT in bladder cancer cell and detailed pathway.Result:RNA pulldown experiment demonstrated that SPRY4-IT1 could directly sponge miR-101-3p specifically.Western Blot assay indicted that Knockdown of SPRY4-IT1 increases the expression of miR-101-3p and E-cadherin,and inhibits the expression of EZH2.The Dual-luciferase Reporter Assays indicated the relative EZH2-3'-UTR luciferase activity was significantly reduced in the cells stably transfected with shSPRY4-IT1.And overexpression of SPRY4-IT1 increased the luciferase activity in UMUC3 and T24T cells(Fig.4G).However,transfection of shSPRY4-IT1 or GV-144-SPRY4-IT1 treatment only slightly changed promoter activity of EZH2 in UMUC3 and T24T cells.Knockdown of miR-101-3p with anti-miR-101-3p inhibitor partly reversed cell apoptosis,cell migration and invasion ability induced by SPRY4-IT1 shRNA.Additionally,we also found that down-regulation of miR-101-3p efficiently reversed the suppression of EZH2 protein level induced by SPRY4-IT1 shRNA in UMUC3 cells.Conclusion:SPRY4-IT1 could function as miR-101-3p sponge to up-regulate the expression of EZH2 at miR-101-3p-mediated posttranscriptional level,thus,to promote the bladder cancer cell proliferation and metastasis.Part ? Research the effect of SPRY4-IT1 on biological roles of bladder cancer cells in vivoObjective:To further determine the anti-tumorigenesis potential of SPRY4-IT1 inhibition in vivo,and further confirm the SPRY4-IT1/miR-101-3p/EZH2/E-cadherin pathway in bladder cancer.Methods:For the tumor xenografts experiments,stable T24T cells(3×106,200?l)transfected with SPRY4-IT1 shRNA or negative control were subcutaneously injected into mice,At 24 days post injection,mice were euthanized,and the tumors were excised,photographed and tissue sections were obtained for further immunohistochemical staining.For the tail vein injections experiment,stable T24T cells(2×106,200?l)transfected with SPRY4-IT1 shRNA or negative control were injected into the tail veins of six mice,which were euthanized 8 weeks after injection.The lungs were excised,photographed,and visible tumor nods on the lung surface were counted.Results:ShSPRY4-IT1 injected mice showed a reduction in tumor volume and weight at the end of the experiment compared with the control groups.Histologic analysis and of resected tumor tissues suggested SPRY4-IT1 expression was positively correlated with EZH2 and negatively associated with miR-101-3p as well as E-cadherin in both control and SPRY4-IT1 inhibition cohorts.In tail vein injection experiment,data showed that Inhibition of SPRY4-IT1 reduced the number of lung metastatic nodules compared with the negative control group.Conclusion:These results further verified the role of SPRY4-IT1 in tumor proliferation and metastasis in vivo,and provided more evidence for therapeutic strategy targeting SPRY4-IT1 in bladder cancer treatment.