| Background and Objective:It has been reported that abnormal expression of TGFBR3was found in multiple human tumors,which has an important roles in tumor development. Previous study of our group suggested that the abnormal expression of TGFBR3may play an important role in bladder cancer tissues.But its expression is not be defined and its influence on the biological behavior of bladder cancer has not been reported in bladder cancer cells.The aim of this study is to investigate expression of TGFBR3in bladder cancer cells and then to explore the influence on the biological behavior.Methods:qRT-PCR and Western Blotting were performed to determine the expression of TGFBR3in human normal urothelial cell line SV-HUC-1and human bladder cancer cell lines5637and T24.We choose the high expression cell lines into the next experiment.it was interfered by designed and synthesized siRNA that can decrease the expression of TGFBR3more than75%in mRNA level,to demonstrate the effects of TGFBR3on the biological behavior in vitro.WST-1assay and Colony formation assay were used to examin the growth and activity, The cell cycle distribution was measured by PI staining, Wound closure assay and Migration and Invasion assays were performed to determine the change of motility, migration, invasion.Results:TGFBR3mRNA level and protein level was abnormally increased in T24cell lines compared with SV-HUC-l,but reduced in5637cell lines.The expression of mRNA were3.420±0.0875,1±0.1800,0.620±0.1270(P＜0.05).So we choose T24cells as the tools for RNAi. Designed and synthesized siRNA-R3-1-4can decrease the expression of T24cells in mRNA level. The efficiency of interference were85.48%,89.37%,79.83%,68.78%respectively(P＜0.001). Therefore, siRNA-R3-1-3was selected as the effective siRNA sequences. WST-1assay suggested that compared with siRNA-NC group and Mock group, the growth and activity of siRNA-TGFBR3group were reduced at48h post-transfection(P＜0.01),the difference increased at72h post-transfection(P＜0.001). Colony formation assay demonstrated that the average colony number decreased significantly compared with control groups(P＜0.05).Propidium Iodide staining results showed that cell cycle distribution is similar in siRNA-TGFBR3group and siRNA-NC group and there are no obvious peak sub-G1. Wound closure assay result suggested that the motility of siRNA-TGFBR3group was reduced compared to siRNA-NC group. Migration assays revealed the number of migrated cells were258±12.5,62.7±6in siRNA-NC group and siRNA-TGFBR3group respectively (P＜0.001). The number per field of invasion cells through the Matrigel matrix were158.4±20.8and20.3±8.5in control group and siRNA-TGFBR3group (P＜0.001).Conclusion:1.Our study demonstrated the expression of TGFBR3in bladder cancer cell lines for the first time. TGFBR3mRNA level and protein level was abnormally increased in T24cell lines compared with SV-HUC-l,but reduced in5637cell lines.2.Designed and synthesized four pairs of siRNAs can silence TGFBR3efficiently and specifically. The efficiency of interference was more than75%in mRNA level when we used siRNA-R3-1-3.3.Compared with control groups,transfected with siRNA-TGFBR3,the cell cycle distribution and apoptosis of T24cells had no obvious change, but which can affect the growth, motility,migration and invasion. |
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