论文部分内容阅读
Objective: The coiled coil is a superhelical structural protein motif that has been thoroughly investigated in recent years and coiled-coil domain-containing proteins have exhibited a large diversity of function in biological systems(e.g.,gene regulation,cell division,membrane fusion,drug extrusion).The aim of this study was to investigate the critical role of coiled-coil domain-containing protein 34(CCDC34)in bladder carcinogenesis,which has never been reported to date.Methods: Immunohistochemical staining and western blot were used to detect CCDC34 expression in bladder cancers specimens and cell lines.Lentivirus-mediated small interfering RNA(siRNA)against CCDC34 was designed and the silencing effect was examined by quantitative RT-PCR in bladder cancer cell lines T24 and 5637.The biological functions of CCDC34 knockdown on bladder cancer cells were investigated by examining cell proliferation using a high content screening(HCS)assay,BrdU incorporation assay and colony formation assay,as well as cell migration by in vitro wound healing assay.Cell cycle distribution and apoptosis were detected by flow cytometric analysis.The expressions of c-Raf and c-Jun as well as the phosphorylation of MEK,ERK1/2,JNK,P38 and AKT were also measured using Western blot.We further investigated the effect of therapeutic siRNA targeting CCDC34 on T24 xenograft tumor growth in nude mice.Results: CCDC34 level was significantly increased in human bladder cancer tissues and cell lines.CCDC34 knockdown significantly suppressed bladder cancer cells proliferation and migration,and induced cell cycle arrest at G2/M phase and increased apoptosis in vitro.In addition,intratumor delivery of therapeutic siRNA targeting CCDC34 elicited delayed tumor growth of bladder cancer xenograft in nude mice.Moreover,CCDC34 knockdown decreased the phosphorylation of MEK,ERK1/2,JNK,p38 and Akt,and the expressions of c-Raf and c-Jun,indicating MAPK and AKT pathways(ERK/MAPK,p38/MAPK,JNK/MAPK and PI3K/Akt)might be involved in CCDC34 modulation of bladder cancer cell proliferation and migration.Conclusions: Our findings revealed for the first time a potential oncogenic role for CCDC34 in bladder carcinoma pathogenesis and it may serve as a biomarker or even a therapeutic target for bladder cancer.