Study On Mutation Or Expression Of The Tumor Suppressor Kruppel-like Factor 6(KLF6) In Hepatocellular Carcinomas And Its Functions

Chronic HBV infection is one of the most important risk factors for hepatocellular carcinoma (HCC) development worldwide. The most majority of HCCs result from chronic HBV infection, and HBV-related HCC represents the primary cause of cancer-related death in our country. Although HBV infection appears to play multiple roles in hepatocellular carcinogenesis,the precise molecular mechanism of HBV-related HCC is not well understood up to date. The development of human cancer is generally thought of as a multistep or multistage process in which external stimuli induce accumulation of genetic alterations in hepatocytes leading to abnormal cell development or proliferation . These genetic alterations is involved in two classes of critical genes, commonly referred to as protooncogenes and tumor suppressor genes (TSG). It is generally believed that oncogenes and tumor suppressor genes contribute to each step of human multistage carcinogenesis. Although the inactivation of several tumor suppressor genes including p53 and Rb, as well as p16^INK4 by mutation , allele loss or promoter hypermethylation have been identified over past several years in HCCs, none of them affect more than 50% of cases. So, it is possible that other tumor suppressors exist and play a role in hepatocarcinogenesis. Identification and functional characterization of TSG that play a critical role in humanHCC development would be a significant contribution toward not only the definition of the molecular pathogenesis of HCC but would also broaden the potential for developing effective treatment of HCC.Kruppel-like factors 6 (KLF6) on chromosome lOp, is a ubiquitously expressed zinc finger transcription factor that is part of a growing family of Kruppel-like factors (KLF). These KLF proteins share a nearly identical carboxy terminal DNA binding domain, but have widely divergent amino terminal activation domains, patterns of expression, and transcriptional targets. The KLF family is broadly involved in differentiation and development, growth-related signal transduction, cell proliferation, apoptosis, and angiogenesis. Recently, it was shown that KLF6 has also the ability to reduce cell proliferation concomitantly with the induction of p21WAFl expression in a p53-independent manner. Interestingly, several independent reports described mutations or downregulation of KLF6 gene in human malignancies such as in prostate cancer, astrocytic gliomas, colorectal cancer and non-small-cell lung cancer, thus KLF6 has been proposed as a potential candidate tumor suppressor gene. All these results suggest that KLF6 gene might play a role in development of human cancer, although the molecular mechanism of KLF6 in cancer is not well known .Generally, a tumor suppressor gene is functionally inactivated, by "two hits" in human cancer due to deletion and an inactivating mutation, in accordance with Knudson's original definition of a tumor suppressor gene. It is believed that deletion or somatic mutation of TSGs may result from genome's instability. Some tumor suppressors related to the development and/or progression of HCC may exist on chromosome 10, because the chromosome 10 was found with high instability in moderately or poorly differentiated HCC. Kremer-Tal's research group reported that mutations or/and loss of heterozygosity (LOH) of KLF6 gene were present in 49%(20/41) of primary HCCs and its functional inactivation could contribute to pathogenesis in HCC, However, a recent study failed to find mutations of KLF6 gene in 71 HCCs, so extensive investigation in both HCC and other human cancers is needed to determine the frequence of KLF6 mutation and its role for hepatocarcinogenesis. Up to date, there is rare published data concerning expressionlevels of KLF6 in HCCs,and no published data focusing on KLF6's role on apoptosis induced by DNA-damage agents. The principal aim of this study was to clarify whether functional inactivation of KLF6 gene by mutation or down-regulation contribute to pathogenesis of HCC, and to explore the implication of KLF6 as a posshible therapeutic way to treat HCC and other human cancers.Part IMutations of the kruppel-like factor 6 (KLF6) gene in hepatocellular carcinoma and its effect of growth suppression on humanhepatocellular carcinoma cell line HepG2AimTo detect frequence of KLF6 gene mutation in hepatocellular carcinoma (HCC) tissues and to characterize its effect of growth suppression on human hepatocellular carcinoma cell line HepG2 Methods' We analyzed DNA isolated from 23 hepatocellular carcinoma tissues and correspondingly adjacent nontumor tissues by polymerase chain reaction (PCR). Direct sequencing were used to establish the incidence of mutation in exon2 of KLF6 gene. Two recombinant vectors expressing wild-type KLF6 (wtKLF6) or mutant KLF6 (mtKLF6) were constructed respectively using molecular clone technology and PCR directed mutagenesis. KLF6-transfected HepG2 cells were established after screened with G418. Loss of growth suppressive function of the HCC-derived KLF6 mutant was characterized by in vitro analyzing alteration of cell cycle and proliferation rate using flow cytometry and MTT assay. Expression of p21WAFl, a possible downstream gene of KLF6, was detected in stable KLF6-transfected HepG2cells. Results1. Analysis of the KLF6 gene revealed that 2 of 23 (8.6%) HCCs harbored KLF6mutations. One of the 2 mutations identified was missense variants caused bysingle-nucleotide substitutions(TGG—GGG) which result in single amino acid substitution of tryptophan (W) by glycine (G) at codon 162(W162G).2. The recombinant vectors which expressed wtKLF6 or mtKLF6 were constructedsuccessfully.3.The wtKLF6 or mtKLF6 overexpressing HepG2 cells were establishedsuccessfully4. MTT assay showed strong inhibition of cell growth in wtKLF6- overexpressingclones but no remarkable inhibition of cell growth in mtKLF6- overexpression clones.Cell cycle analysis revealed that wtKLF6 overexpression increased the percentage ofthe total cell population in the G0-G1 phase, however, percentage of G0-G1 phasecells in mtKLF6-overexpressing HepG2 cells did not significantly differ from thecontrol. wtKLF6 markedly increased levels of p21 protein when compared to controlcells, whereas mtKLF6(W162G) failed to do it.Conclusion1. KLF6 gene is mutated in 8.6% of HCC tissues.2. Missense mutation of KLF6 gene failed to inhibit cell growth, partly due to functional loss of KLF6 inducing P21WAF1 expression.3. Mutation of KLF6 gene may contribute to the pathogenesis of HCC.Part II Expression of the kruppel-like factor 6 (KLF6) gene in hepatocellularcarcinoma and its role in DNA damage-induced apoptosis AimTo detect expression levels of KLF6 gene in hepatocellular carcinoma (HCC) tissues and elucidate its role in DNA-induced apoptosis.MethodsExpression of KLF6 protein was detected in 23 HCC tissues and corresponding nontumorous tissues and hepatocellular carcinoma cell lines by Western blot assay. After HepG2 cells were treated with Cisplatin (5 u- g/ml) 12 hours , subcellular localization of KLF6 protein was examined by immunofluorescence with the anti-KLF6 antibody observed under fluorescence microscopy, and expression of KLF6 was detected by RT-PCR and Western blot. Various concentration of cisplatin were added into the culture media, after 24-72h, the viability of HepG2-KLF6 cells were determined by MTT assay. TUNEL assay were performed to detect apoptosis . Expression of p21WAFl and Caspase-3 were examined by RT-PCR and Western blotfollowing HepG2-KLF6 cells treated with various concentrations of cisplatin for 24h. Results1. 9 of 23 (39.1%) HCC tissues had lower KLF6 protein expression levels than theirnormal countparts or normal liver tissues, 5 of the 9 HCC tissues displayed likely a pattern of KLF6 isoform with the lower molecular weight species, but it was not found in noncancerous tissues. Level of KLF6 protein expression in HepG2 or SMMC-7721 cells was lower than normal liver tissues.2. Nuclear localization of KLF6 protein in HepG2 cell is enhanced following cisplatin (5 u g/ml) treatment 12h. RT-PCR and Western blot assay showed that upregulationof KLF6 in HepG2 cells treated with 5 u g/ml concentration of cisplatin is in a time-dependent manner (6h,12h,24h)3. Cell viability by MTT assay was decreased in a dose- and time-dependent manner in HepG2-KLF6 cells compared to HepG2-vector cells following treatment with cisplatin at 2.5 jag/ml, 5.0 |ag/ml, 10.0 [ag/ml;15.0 u.g/ml concentration for 24h 72h , however, cell viability was increased in HepG2-KLF6 cells compared to HepG2-vector cells after the cells treated with low concentration 1.3 u g/ml) of cisplatin for 24h.4. After exposure to cisplatin at various dosage(5.0 |ug/ml, 10.0 fig/ml, 15.0 ng/ml) for 24h, TUNEL assay by flow cytometry was performed to detect apoptosis. The results showed that HepG2-KLF6 cells had a higher percentage of apoptotic cellscontribute to pathogenesis of HCC.