| 【Objective】Spalt-like transcription factor-4(SALL4)is over expressed in a variety of solid tumors,which is involved in the occurrence,development,recurrence and metastasis of tumors.However,the study of SALL4 in bladder urothelial carcinoma has not been reported,which is worth further study.The purpose of this study is to investigate the expression of SALL4 in bladder urothelial carcinoma and its effect on the cell proliferation,migration and invasion of bladder urothelial carcinoma,and to preliminarily study the role of SALL4 in the epithelial mesenchymal transition(EMT)of bladder urothelial carcinoma.【Methods】1.Selecting 20 cases of bladder normal urothelial mucosa,170 cases of bladder urothelial carcinoma,including 50 cases of low grade urothelial carcinoma,120 cases of high grade urothelial carcinoma,making tissue chip and completing immunohistochemical staining.The expression of SALL4 protein in different tissues was compared and the relationship between SALL4 protein and clinicopathological characteristics of patients with urothelial carcinoma of bladder was analyzed.2.Different bladder urothelial carcinoma cell lines(T24 and 5637)were cultured.Total protein and RNA samples were extracted.The expression of SALL4 protein and m RNA in different bladder carcinoma cell lines were detected by WB and q PCR.3.We construction SALL4 overexpression plasmid and synthesized SALL4 si RNA fragment,SALL4 overexpression plasmid was transfected in T24 cell line and si RNA was transfected in 5637 cell line,and verified the overexpression and interference efficiency by WB and q PCR.4.Through scratch test,Transwell migration and invasion test and CCK8 proliferation test,the effects of SALL4 overexpression or down-regulation on the proliferation,migration and invasion of bladder cancer cells were detected.5.WB was used to detect the expression of β-catenin and EMT related proteins(E-cadherin,N-cadherin,vimentin and Snail)in bladder cancer cell lines under the condition of over expression or down-regulation of SALL4,and to explore the effect of SALL4 change on the expression of EMT related proteins.【Results】1.SALL4 was not expressed in normal urothelium,but was 10% in low-grade urothelial carcinoma and 49.12% in high-grade urothelial carcinoma(P<0.001)..The expression of SALL4 protein was positively correlated with histological grade,invasion depth,lymph node metastasis and vascular invasion of bladder urothelial carcinoma(P < 0.05).153 cases were followed up,58 of them were SALL4 positive,the mean survival time was 25.6 months(0-91 months),95 of them were SALL4 negative,the mean survival time was 36.2 months(0-92 months).2.The expression of SALL4 protein and m RNA in different bladder urothelial carcinoma cells was different,of which 5637 cell line was highly expressed and T24 cell line was low.T24 cell line was used for over expression study and 5637 cell line for knockdown study.3.We successfully constructed the SALL4 overexpression plasmid and obtained the si RNA fragment of knockdown SALL4.The T24 cell model of SALL4 overexpression and the 5637 cell model of SALL4 knockdown were obtained by cell transfection.4.In CCK8 experiment,compared with the blank control group(NC)and the empty vector group,the cell proliferation ability of SALL4 plasmid overexpression group increased significantly in 24 h,48h and 72h(P < 0.05).Compared with the NC and the si RNA group,the cell proliferation ability of SALL4-si RNA interference group decreased significantly in 72h(P < 0.05).5.In the scratch repair experiment,after 72 h overexpression of SALL4,compared with NC group(118.00 ± 12.12)and the empty Vector group(111.67 ±13.58),the cell migration ability of SALL4 overexpression group(186.67 ± 12.22)increased significantly(P < 0.05).Compared with the NC and si RNA group,the cell migration ability in the SALL4-si RNA interference group decreased significantly(P < 0.05).6.In the Transwell experiment,compared with NC group(128.33±17.21)and empty vector group(137.67±17.21),the migration ability of T24 cells in SALL4 overexpression group(187.33±14.57)was significantly increased(P < 0.05).Compared with NC group(76.67±18.58)and the empty vector group(77.67±9.29),the invasion ability of T24 cells in SALL4 overexpression group(149.00±15.72)was significantly increased(P < 0.05).Compared with NC group(180.67±25.77)and si RNA group(181.67±27.15),the migration ability of 5637 cells in SALL4-si RNA interference group(91.67±13.65)decreased significantly(P < 0.05).Compared with NC group(131.33±8.32)and si RNA group(124.00±14.42),the invasion ability of 5637 cells in SALL4-si RNA interference group(89.00±14.00)decreased significantly(P < 0.05).7.In WB experiment,compared with NC group and empty vector group,the expression of N-cadherin,vimentin,snail and β-Catenin protein in SALL4 overexpression group increased significantly(P < 0.05),while the expression of E-cadherin protein decreased significantly(P < 0.05).Compared with NC group and si RNA group,the expression of N-cadherin,vimentin,and β-Catenin protein in the SALL4-si RNA interference group decreased significantly(P < 0.05),while the expression of E-cadherin increased significantly(P < 0.05).【Conclusion】SALL4 was highly expressed in high-grade urothelial carcinoma.The expression of SALL4 was positively correlated with histological grade,depth of invasion,vascular invasion and lymph node metastasis of bladder cancer,and the survival time was shortened.After overexpression of SALL4 gene,it can promote the proliferation,migration and invasion of bladder cancer cells,increase the expression of β-Catenin,and increase the expression of EMT related proteins;knocking down SALL4 can restrain the proliferation,migration and invasion of bladder cancer cells,and down regulate the expression of β-Catenin and EMT related proteins.The results suggest that SALL4 gene is related to proliferation,invasion and poor prognosis of bladder cancer,and may be related to Wnt / β-catenin signaling pathway regulating EMT pathway.Down regulation of SALL4 gene may provide a new idea for the treatment of bladder cancer. |
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