| Objective: To explore the effect and mechanism of human BMP9 on the proliferation and migration of human bladder cancer cells.Methods: First,the difference of BMP9 expression in bladder cancer tissues and adjacent tissues were queried in bladder cancer related databases.Next,the effect of BMP9 on the proliferation and migration of bladder cancer cells were observed.The expression of BMP9 gene and protein in bladder cancer cells was detected by RT-PCR and Western-blot,respectively.The bladder cancer BIU-87 cells(low BMP9 expression)and T24(high BMP9 expression)were transfected with BMP9 overexpressed adenovirus(AdBMP9)and BMP9 interfering adenovirus(AdsiBMP9)respectively,the high BMP9 expression(BIU-87/AdBMP9)and low BMP9 expression(T24/Adsi BMP9)cells were constructed.The overexpressed adenovirus and interfering adenovirus vector were used to set the control group respectively(BIU-87/AdGFP and T24/AdRFP),and each group contained blank control with no adenovirus.To test whether the over expression and interfering cell models were successfully established,RT-PCR and Western-blot tests were used to detect the changes of BMP9 RNA and protein levels in bladder cancer cells.The MTT test,colony formation test and EDU proliferation test were used to determine the proliferation of bladder cancer cells,and the scratch healing test and MTT migration test were used to detect the migration of bladder cancer cells.The lncRNA which relate to the proliferation and migration of bladder cancer were detected by RT-PCR in bladder cancer BIU-87 cells after transfected with AdBMP9.As the most obviously changed one,lncRNA UCA1 was choosen to do the next study.The small interference RNA of UCA1 were constructed.The AdBMP9 and siUCA1 s were co-transfected into BIU-87 cells to construct the BIU-87/AdBMP9+siUCA1 cell model.The proliferation change was detected by MTT test,colony formation test and EDU proliferation test,and the migration change of BIU-87 cells was detected by scratch healing test and Transwell migration test.The Western-blot test were used to detect the BMP9 related signaling pathway protein AKT,cell proliferation marker PCNA and cell migration marker MMP9 in BIU-87/AdBMP9+siUCA1 cells.After adding AKT inhibitor LY294002 to BIU-87/AdBMP9 cells,the expression change of lncRNA UCA1 was detected by RT-PCR test,and the function of AKT signaling pathway in the BMP9-UCA1 axis was verified.Finally,we validated the effect of BMP9 on bladder cancer cells in animal models.The BIU-87/AdBMP9+siUCA1 and BIU-87/AdBMP9 cells were subcutaneously transplanted into the male nude mice,respectively.After 20 days,the tumor was collected and embedded with paraffin and sliced.The tissue sections were stained with HE and immunohistochemical to observe the tumor formation and the expression of p-AKT,MMP9 and PCNA.Results: The expression of BMP9 in T24 cells of bladder cancer was higher than that of bladder cancer cell BIU-87.MTT test,colony formation test and EDU proliferation test showed that BMP9 could promote the proliferation of bladder cancer cells,and the wound healing test and Transwell migration test showed that BMP9 could promote the migration of bladder cells.The mechanism study showed that BMP9 could promote the expression of lncRNA UCA1 in bladder cancer cells.MTT test,colony formation test and EDU proliferation test showed that the promoting effect of BMP9 on the proliferation of bladder cancer cells was reduced,and scratch healing test and Transwell migration test showed that the migration of BIU-87 cells was rescued too in BIU-87/AdBMP9+siUCA1 cells.Western-blot test showed that the proliferation marker PCNA and migration marker MMP9 of the BIU-87 cells of bladder cancer cells were obviously up-regulated after overexpression of BMP9,and the up regulation was significantly reduced after interference with UCA1.Western-blot results also showed that the AKT signaling pathway of BIU-87 cells in bladder cancer cells was activated after the expression of BMP9;RT-PCR showed that the promotion of BMP9 on UCA1 was reduced after treat with AKT inhibitor LY294002.In vivo,the tumor growth was significantly increased by BMP9,and the promotion effect was significantly reduced after interfering with UCA1.The HE staining results showed that the subcutaneous transplant of bladder cancer cells were successfully growth to tumor;immunohistochemical test showed that BMP9 could increase p-AKT,PCNA and MMP9 levels in bladder cancer cells in vivo.Conclusion: BMP9-p-AKT-UCA1 axis could promote the proliferation and migration of bladder cancer cells. |
