The Study Of SPAG9/HEF1/Rac1 Pathway's Influences On EMT Process In Bladder Transitional Cell Carcinoma

Objective:Aiming to explore SPAG9 influences on bladder transitional cancer cells invasion and migration and other biological behaviors.Further studying on the mechanisms of bladder cancer and looking for novel therapeutic target on treating bladder cancer.Methods:1?Using quantitive real-time PCR and immunohistochemistry to detect SPAG9 and HEF1 expression in bladder transitional cancer cells and adjacent non-cancerous tissue quantitatively and qualitatively.2 ? Constructing specific knockdown or overexpression vectors of SPAG9 and HEF1,building transfected T24 and 5637 cell lines using RNA interference,studying the influence of different SPAG9/HEF1 expression on BTCC's invasion and migration properties via Transwell assay.3 ?Building xenograft model with nude mice to study the effects of variation expression of SPAG9 on HEF1 protein in vivo.Using Western Blot technology to study SPAG9's induction on HEF1 in vitro.4?Searching papers to select specific EMT related protein,detecting different expression of SPAG9/HEF1's influence on EMT proteins with Western Blot.5?Using Western Blot to evaluate HEF1 expression's influence on Rac1 signaling pathway.Determining interaction between HEF1 and Rac1 with co-transfection combination and Western Blot.6 ? Statistical analysis on clinical parameters of bladder cancer specimens such as age,tumor size and tumor grade combined with specimens' SPAG9 and HEF1 expression.Summarizing the mutual tendency between SPAG9 and HEF1.Results:1?SPAG9 and HEF1 are found to significantly overexpressed in bladder tumor specimens compared with its adjacent non-cancerous tissue after statistical analysis(P<0.005).High expression of SPAG9 was observed in 61.8%(84/136)of all caseswhereas HEF1 expression was 69.1%(94/136).We also observed that the proportion of overexpression of SPAG9 and HEF1 in low grade bladder cancer was relatively higher than in high grade.High SPAG9 expression in low grade was 88.8%(48/54)in comparison of 63.4%(52/82)in high grade(P<0.001).High HEF1 expression in low grade was 83.3%(45/54)in comparison of 68.3%(56/82)in high grade(P<0.05).SPAG9 and HEF1 expression were both significantly associated with clinical parameters as tumor size,muscle invasion and tumor grade.2 ? SPAG9 could significantly induce HEF1 expression both in vivo and in vitro.We evaluated the expression of HEF1 with shSPAG9 and pcDNA3.1-SPAG9 transfected T24 and 5637 cell lines,HEF1 expression was severely reduced after SPAG9 knockdown,whereas overexpression of SPAG9 could result in an obvious elevation in HEF1 relative mRNA level which is 3.6 times high than its control group(P<0.01).Western Blot results showed that knockdown or overexpression of SPAG9 could significantly silence or enhance HEF1 protein level(P<0.01).Immunohistochemistry on nude mice model confirmed that tumor originated from shSPAG9 transfected cells showed a decreasing expression of HEF1 compared with control group.Tumor originated from pcDNA3.1-SPAG9 exhibited an elevated expression of HEF1 compared to control group.3 ? Transwell assay performed on T24 and 5637 cell lines transfected with shSPAG9 or pcDNA3.1-SPAG9 revealed that overexpression of SPAG9 could contribute to the malignant migration/invasion properties of BTCC 2 or 3.5 times high than its control group,whereas the effects of SPAG9 knockdown on cells' migration and invasion showed otherwise(P<0.05).Western Blot analysis on EMT proteins showed that knockdown of SPAG9 could elevate E-cadherin protein level combined with a decrease protein level on N-cadherin?Vimentin and MMP-2.Overexpressed SPAG9 could inhibit E-cadherin level along with an upregulated N-cadherin ?Vimentin and MMP-2 protein level(P<0.05).4?Similar knockdown vectors(shHEF1)and overexpression vectors(pcDNA3.1-HEF1)were constructed,Transwell assay and Western Blot were deployed to explore the influences of different HEF1 expressionon cellular invasion and migration and EMT related proteins.Like SPAG9,compared with control group,shHEF1 could attenuate cellular invasion and migration as well as elevating E-cadherin expression and decreasing N-cadherin ? Vimentin,MMP-2protein level.However,pcDNA3.1-HEF1 acts the opposite from shHEF1 on migration,invasion and EMT protein level(P<0.05).Also,knocking down HEF1 could result in Rac1 deactivation(GTP-Rac1 decrease)whereas upregulating HEF1 could enhance GTP-Rac1 level.5 ? Transfection combinations of shSPAG9+pcDNA3.1-HEF1 and pcDNA3.1-SPAG9+ shHEF1 was each carried out.Downregulation of HEF1 attenuated the potency of invasion and migration lifted by pcDNA3.1-SPAG9,whereas upregulation of HEF1 restored T24 and 5637 cells' aggressive phenotype and lifts the decreasing HEF1 protein level induced by shSPAG9.6?After extracting proteins from xenograft model and T24/5637 cell lines transfected with shSPAG9 and pcDNA3.1-SPAG9,we found that shSPAG9 could inhibit the activation of Rac1 and overexpression of SPAG9 could elevate GTP-Rac1 protein level.We designed two transfection combinations: shSPAG9 +pcDNA3.1-HEF1 and pcDNA3.1-SPAG9 + shHEF1.Overexpression of HEF1 could rescue the inactivation of Rac1 caused by shSPAG9,whereas inhibition of HEF1 could attenuate the activation of Rac1 caused by pcDNA3.1-SPAG9.Conclusion:SPAG9 could influence HEF1 expression and induced EMT process in bladder transitional cell carcinoma(TCC)through Rac1 signalling pathway.As to in which binding sites that SPAG9 combined to HEF1 gene and specific mechanisms in SPAG9-HEF1 regulation needs further investigation.