Study On The Role Of Nuclear Transcription Factor Gli1 Of SHH Pathway In HCC

Liver cancer is the sixth most common malignant tumors worldwide, in which hepatocellular carcinoma (HCC) is the main type and has been ranked the 3rd lethal cancer in the global and the 2nd in China. HCC is a multifocal, high invasive and preferred metastasia with poor prognosis disease. HCC is one of the most seriously endangerous disease to the health of human. Despite of the tremendous efforts, it remains unclear that the mechanisms of carcinogenesis inducing the malignant transformation and the fast progression of HCC.Recently, there is the evidence of aberrant activation of several signaling cascades such as Hedgehog (HH), Notch, Wnt and BMP-TGF-β-Activin in tumorigenesis and development of cancer. These signaling pathways are essential for numerous processes during morphogens, controlling growth and patterning in certain tissues of a range of different organisms at different developmental stages. Sonic hedgehog (SHH), one of the Hedgehog pathways, is an evolutionarily conserved signalling pathway for the regulation of patterning and cell fates of endoderm-derived axial organs in Drosophila and vertebrae embryonic development. SHH pathway plays important roles in control many essential tissues and cellular properties such as patterning fields of cells or regulating cell differentiation and proliferation in human embryo development. SHH pathway mainly included four factors: the secreted glycoprotein ligand Shh, the transmembrane protein receptor Ptch, the G-protein coupled phosphoprotein receptor Smo and nuclear transcription factor Gli. Homever, it has been reported that there was an abnormal activation of SHH in an increasing number of solid tumours originate from endoderm, including basal cell carcinoma, small-cell lung carcinoma, pancreatic adenocarcinoma, oesophageal, stomach, biliary tract cancers, prostate cancer, breast cancer, colon cancer and liver cancer. SHH signaling pathway is also responded in the vital process of hepatocyte, from endoderm precursor cell and embryonic stem cell to adult hepatic cell as well as HCC. But as a signaling pathway to promote hepatic cell malignant transformation, the molecular mechanisms of SHH in the carcinogenesis and progression of HCC still remain to be clarified.The Gli homologue family of transcription factors in SHH signaling pathway, Gli1, Gli2 and Gli3, mediate the HH morphogenetic signal by regulating the expression of HH target genes. They have similarly amino acid motifs of highly conserved five tandem zinc fingers and a consensus cysteine-histidine link. This zinc finger motif shows significant sequence to be important in the development process. The Gli proteins family has different functions depending on the difference of construction. The Gli proteins all have C-terminal that is transactivation domain. But Gli2 and Gli3 undergo cleavage generating an N-terminal protein that preferentially accumulates in the nucleus and acts as a repressor of HH target genes, so that Gli2 and Gli3 show strong dominant-negative regulation function in C-terminaHy truncated forms. In particular, Gli1 appears to be a constitutive activator and of various gene expression. Current knowledge has revealed that Gli1 is an upstream factor modulating the expression regulation function of various downstream genes, but there are few studies on how Gli1 regulate downstream gene.Combination the bioinformatics and pertinent literature, we find that oncongene c-myc is extensively involed in the carcinogenesis and progression of various cancers including HCC, and c-myc is one of Gli1 target genes in some kinds of cancer other than HCC. However, there is no specific sub-localization of Gli1 in different cells. It is interesting whether the regulation of Gli1 on c-myc expression plays some role in the development of HCC.Subjecting to the signaling transduction molecule Gli1 as a focal point, we investigated the expression status of molecules of SHH pathway in hepatoma carcinoma cell lines and clinical HCC tissues, and the mechanism of transcriptional regulation of Gli1 to the target gene of c-myc, explored the effects of Gli1 regulating to c-myc on the proliferation, apoptosis and cell function of hepatoma carcinoma cell lines, and finally investigate the change of the biological behaviours of hepatic cancer cells with over- and down- regulation of Gli1 protein expression in vitro and in vivo respectively.In the first part of this study, the expression patterns of Shh, Ptch, Gli1, hTERT and c-myc in HepG2, Hep3B hepatoma carcinoma cells and L02 human normal embry liver cells, and primary human hepatocytes (PHH) were investigated by reverse transcription PCR (RT-PCR), immunocytochemistry, and by real-time PCR, immunohiso-hemistry and Western blot in clinical HCC. The results showed that the SHH signaling was activated in both HCC cell lines and clinical HCC tissues, indicated by the much higher levels of mRNA and protein of SHH pathway constitute monculers, c-myc, hTERT than these in L02, PHH lines, and adjacent non neoplastic live tissues and normal liver tissues.According to above results, it has been proposed that Gli1 is involved in the regulation of c-myc expression. In the second part, a luciferase reporter vector c-myc-luc including its P1 and P2 promoter, Gli1 expression vector and siRNA target Gli1 vector were constructed respectively. Double luciferase reporter system and cotransfection were used. The results showed that in HEK293 and L02 cells that were original no or low expression c-myc, Gli1 expression vector could significantly activate c-myc expression in a dose-dependent manner, but in hep3B cells with high expression level of endogenous Gli1 and c-myc, Gli1 expression vector did not significantly induce the expression of c-myc cotransfection of pGL3-c-mycp plasmid and various doses of Gli1 RNAi vector showed a dose-dependent inhibition of c-myc expression in Hep3B cells. To prove that Gli1 can activate endogenous c-myc and increase cell proliferation, stably express Gli1 cell lines of L02 and siRNA stably inhibited Gli1 cell lines of Hep3B were established, real-time PCR and Western blot were performed to determine the effect of Gli1 on endogenous expression level of c-myc, and WST-8 method was applied to test the cell proliferation. The results showed that endogenous c-myc was activated by Gli1 in L02 cells, and Gli1 has a powerful positive regulation effect on Hep3B cell proliferation.To further elucidate the molecule mechanism of the interaction between Gli1 and c-myc in Hep3B cells, immunoprecipitation and double immunofluorescent staining analysis were used. The results indicated that Gli1 and c-myc colocalize and interact with each other in Hep3B cells.To investigate the influence and mechanism of down-regulation of the Gli1 with SHH signaling specific inhibitor cyclopamine and siRNA on biological behaviours of HCC, serum starvation culture Hep3B cells were treated with cyclopamine, and nude orthotropic transplant tumor model was established and treated with cyclopamine. It was found that cyclopamine inhibited HCC cell proliferation and caused mitochondrial membrane potential collapse, an indicator of early stage of apoptosis, and down-regulation the expression of HCC markerα-fato-protein (AFP). In vivo, inhibition of Gli1 with siRNA and cyclopamine decreased the growth of transplanting HCC tumor, and down regulated the expression of Gli1 and c-myc synergically.Above evidences have suggested, SHH signaling is abnormally activated in HCC. As the nuclear activating factor of SHH pathway, Gli1 could regulate the transcription and translation of c-myc, which is involved in the carcinogenesis and growth of HCC. Blocking Gli1 would induce that down-regulation of c-myc, inhibit the growth of HCC, and prompt the apoptosis of HCC. Gli1 plays some roles in the carcinogenesis, growth and apoptosis of HCC and it may be a new target of hepatocellular carcinoma treatment.