Loss of expression of Kruppel-like factor 6 in primary hepatocellular carcinoma and hepatoma cell lines

Abstract

KLF6 (Zf9, COPEB), an ubiquitous transcription factor, maps to chromosome 10p. Recently, KLF6 was found to have a more generalized role in tumorigenesis as a candidate tumor suppressor gene for some tumors. However, results from other published studies seem not to be in agreement with data from previous studies. Gene-expression analysis is increasingly important in biological research. Loss of expression is one of the mechanisms to functionally inactivate a tumor suppressor gene. To investigate the expression change of KLF6 gene associated with HCC as a step toward a better understanding of the molecular pathophysiology, and to provide the basis for analysis of KLF6 gene in HCC carcinogenesis. We analyzed the expression of KLF6 mRNA in 26 samples of HCC tissues and hepatoma cell lines(Hep3B and HepG2) detected by Real-Time quantitative RT-PCR (qRT-PCR) and conventional RT-PCR assay. To confirm and extend the data obtained at RNA level, we performed detailed immunoblotting analysis on HCC tissues and hepatoma cell lines using a rabbit polyclonal antibody specific for KLF6. NKLF6 detected by qRT-PCR from HCC and corresponding noncancerous tissues was 0.04+/-0.038 and 0.116+/-0.101, respectively. These data demonstrated that KLF6 mRNA level was significantly decreased in HCC, compared with corresponding noncancerous tissues (t =3.683 , P<0.001). The frequency of Hepatoma Cell Lines with KLF6 down-regulation detected by conventional PT-PCR, seems to be consistent with a previous study using real-time PCR assays in tumor samples. KLF6 expression levels were determined by Western blot. Compared to the matched surrounding tissues, a clear decrease of KLF6 protein levels in tumor tissues was observable (t=13.59, P<0.001). Hepatoma cell lines also showed low-level of KLF6 protein (P<0.01) expression. Immunohistochemistry and immunocytochemistry showed a faint diffused staining in the HCC tissues and hepatoma cell lines, and endogenous KLF6 protein was detected mostly in the cytoplasm. KLF6 gene appeared markedly reduced in HCC tissues and hepatoma cell lines. Frequent down-regulation of KLF6 strongly suggested that it is a candidate of tumor suppressor gene for HCC.