Attenuation Of Kr(u|")ppel-like Factor4Facilitates Carcinogenesis By Inducing G1/S Phase Arrest In Clear Cell Renal Cell Carcinoma

Background and ObjectivesKruppel-like factors (KLFs) are members of the zinc finger family of transcription factors that contain three domains of Kruppel-like zinc fingers. This family contains at least16members. Kruppel-like factor4(KLF4), also known as Gut-enriched Kruppel-like factor or epithelial zinc finger, is a member of this family. Kruppel-like factor4is a context-dependent oncogene and tumour-suppressor gene, plays a critical role in regulating diverse array of biological processes. However, the function and detailed mechanisms of KLF4in ccRCC development and progression remain unknown. In the present study, the expression pattern of KLF4was examined in both CCRCC tissues and cell lines. Furthermore, the detailed mechanisms of KLF4in the development and progression of ccRCC were also investigated in vitro and in vivo.Methods and Materials(1) Patient matched ccRCC and adjacent non-neoplastic renal samples were analyzed by western blotting, immunohistochemistry and quantitative real-time polymerase chain reaction (qRT-PCR). The correlation between KLF4expression and clinico-pathological parameters was also analyzed.(2) Genomic DNA from ccRCC tissue and the adjacent normal tissue specimens were used to detect the methylation status of the KLF4promoter by MSP analysis.(3) Western blotting and qRT-PCR were used to investigate the expression of KLF4in the human normal kidney tubule epithelial cell line (HKC) and ccRCC cell lines including786-0,769-P,Caki-2,Caki-l and ACHN. Afterwards,786-0, ACHN based on Lentivirus-based overexpression of KLF4and HKC transfected with siRNA against KLF4were used to porferm further study. MTS, Anchorage-dependent and independent growth,transwell invasion and migration assays, together with wound healling assays were performed to investigate the role of KLF4on cells proliferation and invasion. Flow cytometry was also used to detect the effect of KLF4on cell cycle.(4) In vivo study was finallly performed to conferm the effect of KLF4on ccRCC cells using emale BALB/c nude mice.Results(1) KLF4was inactive in primary ccRCC specimens, KLF4downregulation was significantly correlated with tumor stage and tumor diameter. However, no significant correlation was found between KLF4expressions and age, gender, Fuhrman grade.(2) Hypermethylated KLF4promoter region contributed to the low expression of KLF4in primary ccRCC.(3) KLF4expression was absent and restored KLF4inhibited the proliferation in ccRCC cell lines, as well as the migration and invasion, knowkdown of KLF4in HKC lead to inverse effect on proliferation. Furthermore, the anti-proliferation effect of KLF4was associated with its role in up-regulating p21and down-regulating cyclin D1, leading to cell cycle arrest at the G1/S checkpoint.(4) Overexpression of KLF4significantly inhibited the proliferation of ccRCC cells in vivo.ConclusionsBased on ccRCC tissues, cell lines and animal model, our results suggested that KLF4plays an important suppressive role in the development and progression of ccRCC.