Methylation And Aberrant Expression Of Wnt Antagonist SFRP1 In Bladder Cancer

PrefaceWnt signaling pathway plays important roles in proliferation, differentiation, and apoptosis in adult tissues. Aberrant activation of Wnt pathway may induce tumorigenesis. In recent years, upstreams in Wnt pathway involving carcinogenesis have been observed. Overexpression of members of Wnt family, such as Wntl, Wnt2 and Wnt3a, has been found in several human cancers. In addition, aberrant activation of Wnt pathway by downregulation of Wnt antagonists can also induce tumorigenesis. For example, downregulation of Dkk family has been found in several cancers. Recently, expression loss of SFRP1 has been reported in human cancers.Bladder cancer is the most common tumor in urinary system, of which the pathogenesis is not very clear. Studies have shown aberrant Wnt signals are involved in the development of bladder cancer. For instance, Wnt7b was found to be expressed more significantly in superfitial bladder cancers than normal bladder tissues. Wnt5A was also found to be expressed in several bladder cancer cell lines. In addition, Wnt inbitory factor 1 (WIF1) was downregulated in bladder cancer, which led to aberrant activation of Wnt/β-catenin pathway and upregulation of Wnt target gene c-myc and cyclinD1. In this study, we measured the expression status of SFRP1 in bladder cancer and explored the corresponding mechanism, in order to study the roles of SFRP1 in the pathogenesis of bladder cancer.Materials and methods一.Materials45 bladder cancers, and their matched adjacent normal tissue samples were all obtained from the second affiliated hospital, Chinese Medical University. The samples were frozen in liquid nitrogen immediately after surgery. Human bladder cancer cell lines T24,5637 and SCaBER were all purchased from KUNKEN, Shanghai. Trizol and RT-PCR kit were from Takara, Dalian. Methylation detection kit was purchased from GENMED, Shanghai. Antibody of SFRP1 was purchased from SANTA CRUZ, American.二.MethodsHuman bladder cancer cell lines T24,5637 and SCaBER were cultured in RPMI 1640 supplemented with 10% fetal bovine serum, penicillin (100IU/ml), and streptomycin (100μg/ml). DNA was extracted by satured NaCl method and RNA was extracted by Trizol methods. SFRP1 mRNA was detected by RT-PCR. Methylation status of SFRP1 was detected by methylation specific PCR (MSP). SFRP1 protein was determined by Western blotting.三.Statistical analysisMethylation status of SFRP1 between cancer and cancer adjacent tissues were compared by x2 test. Expression status of SFRP1 mRNA between cancer and cancer adjacent tissues were compared by student's t test. Differences were considered statistically significant when P values were less than 0.05.Results一.Methylation and expression of SFRP1 in bladder cancers and matched cancer adjacent tissues.In 45 gastric cancers, methylation of SFRP1 was detected in 28 (62.2%). In matched cancer adjacent tissues,6 (13.3%) were found with SFRP1 methylation. The methylation rate in bladder cancers is significant higher than that in matched cancer adjacent tissues (P<0.01).Expression of SFRP1 mRNA was determined in 10 bladder cancers (5 with SFRP1 methylation) and matched cancer adjacent tissues respectively. Expression levels of SFRP1 mRNA in 5 bladder cancers with SFRP1 methylation were significantly lower than their corresponding adjacent tissues (P＜0.01). There was no difference between SFRP1 expression in 5 bladder cancers without SFRP1 methylation and their corresponding adjacent tissues.二.Methylation and expression of SFRP1 in bladder cancer cell lines SFRP1 was methylated in T24 and 5637, not in SCaBER. SFRP1 mRNA and prtein were detected in SCaBER, but not in T24 and 5637.三.Effects of the demethylating agents,5'-aza-deoxycytidine on the expression of SFRP1 in bladder cancer cell lines6 hours after treating T24 and 5637 with 5'-aza-deoxycytidine, both T24 and 5637expressed SFRP1 mRNA and protein.Conclusion1. SFRP1 is methylated in some bladder cancers.2. SFRP1 is downregulated in some bladder cancers due to methylation.3. SFRP1 methylation and aberrant expression may be involved in the pathogenesis of some bladder cancers via excessive activation of Wnt signal pathway.