| Background and objective: Bladder cancer(BC)is one of the most common malignancies in the world.In the past years,the morbidity and mortality of BC have gradually increased in China.Despite the improvement of diagnostic methods and surgical management,the therapeutic effect of BC is still dissatisfactory,with a lower 5-year survival rate.Therefore,exploring the molecular mechanism underlying BC progression may help in improving the prognosis of patients with BC.Long non-codingRNAs(lncRNAs;?200 nucleotides),an important member of non-codingRNAs(ncRNAs),have attracted extensive attention in term of their involvement in various biological processes.lncRNA cancer susceptibility candidate 9(CASC9)has been reported to play a crucial role in carcinogenesis.Nevertheless,there are very few studies on the function and mechanism of lncRNA CASC9 in BC progression.In this previous study,we investigated the impact of lncRNA CASC9 on BC cell proliferation,migration and invasion in vitro,and the mechanism was also explored.Furthermore,we identified the role of lncRNA CASC9 in BC progression in vivo.Part I: lncRNA CASC9 expression in BCPurpose: To investigate the expression levels of lncRNA CASC9 in BC tissues and cell lines.Methods: 1.Reverse transcription quantitative polymerase chain reaction(qRT-PCR)was used to detect the expression of lncRNA CASC9 in 35 cases of BC tissues and their adjacent normal tissues.2.qRT-PCR was used to detect the expression of lncRNA CASC9 in BC patients with stage T1 or T2/T3/T4.3.qRT-PCR was used to detect the expression of lncRNA CASC9 expression in BC patients with or without metastasis.4.qRT-PCR was used to detect the expression of lncRNA CASC9 expression in BC cell lines and human normal urinary tract epithelial cells.Results: 1.lncRNA CASC9 was upregulated in BC tissues compared with that in their corresponding normal tissues.2.The expression of lncRNA CASC9 was higher in BC patients with stage T2/T3/T4 than that in BC patients with stage T1.3.Higher expression of lncRNA CASC9 was found in BC patients with metastasis compared to BC patients without metastasis.4.lncRNA CASC9 was overexpressed in BC cell lines,especially in EJ and T24 cells,relative to human normal urinary tract epithelial cells.Conclusion: The expression of lncRNA CASC9 was up-regulated in BC tissues and cell lines,and was significantly related to TNM stage and metastasis of BC.Part II: The effect of lncRNA CASC9 on the growth and development of BCPurpose: To explore the role of lncRNA CASC9 in BC progression.Methods: 1.EJ and T24 cells were transfected with shRNA-NC or CASC9 shRNA,and then tested for lncRNA CASC9 expression using qRT-PCR.2.At 48 h after transfection,EJ and T24 cells were assayed for cell viability using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT)assay.3.At 48 h after transfection,EJ and T24 cells were assayed for cell proliferation using colony formation assay.4.The migration and invasion of EJ and T24 cells was evaluated using scratch-healing assay and transwell invasion assay.Results: 1.The downregulation of lncRNA CASC9 in EJ and T24 cells was observed following CASC9 shRNA transfection.2.Transfection of EJ and T24 cells with CASC9 shRNA led to the reduced cell proliferation.3.Transfection of EJ and T24 cells with CASC9 shRNA led to the decreased colony formation.4.Knockdown of lncRNA CASC9 markedly inhibited the migration and invasion of EJ and T24 cells.Conclusion: lncRNA CASC9 regulates cell proliferation,migration,and invasion in vitro.Part III: The mechanism of lncRNA CASC9 involved in the progression of BCPurpose: To clarify the mechanism by which lncRNA CASC9 regulates the proliferation,migration,and invasion of BC cells.Methods: 1.qRT-PCR analysis of signal transducer and activator of transcription 3(STAT3)expression in BC tissues and corresponding normal tissues.2.Correlation analysis of lncRNA CASC9 and STAT3 expression in BC tissues.3.qRT-PCR analysis of STAT3 expression in BC cell lines and human normal urinary tract epithelial cells.4.Chip assay was performed to determine the interaction between lncRNA CASC9 and STAT3.5.qRT-PCR analysis of STAT3 expression in EJ and T24 cells following Vector,STAT3,shRNA-NC or STAT3 shRNA transfection.6.EJ and T24 cells were transfected with Vector,STAT3,shRNA-NC or STAT3 shRNA,and then tested for lncRNA CASC9 expression using qRT-PCR.7.EJ and T24 cells were transfected with Vector,STAT3,shRNA-NC or STAT3 shRNA,and then tested for STAT3 expression using qRT-PCR.8.Western blot and qRT-PCR analysis of phosphatase and tensin homolog(PTEN)and enhancer of zeste homolog 2(EZH2)expression in EJ and T24 cells following EZH2 shRNA or shRNA-NC transfection.9.RIP assay were performed to explore the interaction of lncRNACASC9,and EZH2.10.EJ and T24 cells were transfected with CASC9 shRNA or shRNA-NC,followed by treatment with PTEN shRNA or IGF-1.The proliferation of EJ and T24 cells were detected using MTT assay and colony formation assay.11.Scratch-healing assay and transwell invasion assay were carried out to evaluate the migration and invasion of EJ and T24 cells.Results: 1.STAT3 expression was obviously increased in BC tumors as compared to normal tissues.2.A positive correlation between lncRNA CASC9 and STAT3 expression was also observed in BC tissues.3.The expression of STAT3 was much higher in BC cell lines relative to human normal urinary tract epithelial cells.4.STAT3 directly bound to the promoter region of lncRNA CASC9.5.Compared to shRNA-NC group,the expression level of STAT3 in EJ and T24 cells transfected with STAT3-shRNA was significantly lower(* P < 0.05);meanwhile,the expression level of STAT3 in EJ and T24 cells transfected with STAT3 was significantly higher than that in EJ and T24 cells transfected with Vector.6.Transfection of EJ and T24 cell with STAT3 shRNA significantly reduced the expression level of lncRNA CASC9.On the contrary,overexpression of STAT3 significantly increased the expression of lncRNA CASC9 in EJ and T24 cells.7.In both EJ and T24 cells,the expression of STAT3 was unaltered following CASC9 shRNA or CASC9 transfection.8.The expression level of EZH2 was significantly decreased in EJ and T24 cells transfected with EZH2 shRNA.9.EZH2 silencing significantly increased the mRNA and protein levels of PTEN in EJ and T24 cells.10.lncRNA CASC9 could directly bind to EZH2 in EJ and T24 cells.11.Silencing of lncRNA CASC9 markedly inhibited the proliferation of EJ and T24 cells,which was blocked by PTEN inhibition or IGF-1 treatment.12.lncRNA CASC9 silencing-mediated inhibition of cell migration and invasion was also abrogated following PTEN shRNA transfection or IGF-1 treatment.Conclusions: 1.lncRNA CASC9 is activated by the transcription activator STAT3 in BC cells.2.lncRNA CASC9 silences PTEN by interacting with EZH2.3.PTEN and phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)signaling pathway were involved in lncRNA CASC9-mediated BC progression in vitro.Part IV: The effect of lncRNA CASC9 in the progression of BC in vivoPurpose:To further identify the functional role of lncRNA CASC9 in the progression of BC in vivo.Methods: 1.Xenograft tumor assay was performed to demonstrate the function of lncRNA CASC9 in BC growth.BC cells stably transfected with shRNA-NC or CASC9 shRNA were injected into nude mice.2.The tumor size was monitored every 5 days.3.Xenograft tumors were resected at 30 th day after injection.Tumor weight and body weight in the shRNA-NC and CASC9 shRNA group were weighed at 30 th day after injection.Results: 1.The tumor from CASC9 shRNA-transfecting BC cells was much smaller than that from shRNA-NC-transfecting BC cells.Consistently,the same tendencies were also discovered in tumor weight and body weight.2.There was no significant difference between the body weight of nude mice injected with BC cells stably expressing CASC9 shRNA and that of nude mice injected with BC cells stably expressing shRNA-NC.Conclusions: lncRNA CASC9 promotes BC cell tumorigenesis in vivo.Full text conclusion In our study,we found the increased expression of lncRNA CASC9 in BC tissues and cell lines,and explored the functional role of lncRNA CASC9 in BC progression through altering its expression in vitro and in vivo.Mechanistically,the upstream and downstream regulation of lncRNA CASC9 were analyzed in vitro.Our findings revealed that upregulation of lncRNA CASC9 induced by STAT3 promoted the progression of BC by interacting with EZH2 and affecting the expression of PTEN.Our study represented a novel mechanism for BC progression and indicated that targeting lncRNA CASC9 may be a potential therapeutic approach for BC. |
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