| Colon cancer (CRCs) is one of the most common malignant tumors in digestive diseases system. The development of CRCs is associated with the activation of oncogenes, as well as the inactivation of tumor suppressor genes. There were growing evidences suggested that the inactivation of tumor suppressor genes was closely related to the epigenetic changes, which including DNA methylation,, histone acetylation, and chromatin remodeling and so on. Among these, DNA methylation is considered as one of most important mechanisms. Study on these epigenetic events not only may reveal new pathogenesis of colon cancer, but also contribute to explore novel biomarkers of early detection and prognosis prediction of CRCs.We have previously found that ZIC1 is one of novel tumor suppressor candidates in gastric cancer through cDNA microarray. ZIC1 expression was also found to be silenced or significant downregulation in a panel of colon cancer cell lines, and DNA hypermethylation of ZIC1 promoter was related with its downregulation expression. ZIC1 gene encodes zinc finger transcription factor and plays a crucial role in development of nervous system. Recently, studies have demonstrated that ZIC1 participated in tumor progression, including medulloblastomas, desmoid tumors, liposarcomas and endometrial cancers.In this study, we sought to determine the possible regulation of ZIC1 exprssion and its potential utility of promoter methylation as a biomarker for differenential dignosis between colon cancer and adjacent non-tumor tissues. We evaluate the effect on proliferation, apoptosis and cell cycle of colon cancer cells by ectopic expression of ZIC1, and investigate the signaling pathways of these regulations. Finally, we testify the downstream targets of ZIC1 by cDNA microarray screening and qPCR validation.Materials and Methods:1. ZIC1 mRNA expression of 24 paired colon cancer and adjacent non tumor tissues were analyzed by quantitative RT-PCR (qRT-PCR), and ZIC1 promoter methylation status were detected by methylation specific PCR (MSP) in 40 paired colon cancer and adjacent non-tumor tissues.2. To determine the effect on cell proliferation, we performed colony formation and cell viability assays in HCT116 and HT29 which tranfected with ZICl or empty vector. Meanwhile, we analyzed the effect on cell apoptosis and cell cycle after ectopic exprssion of ZIC1 by flow cytometry analysis.3. We determined the expression of the PI3K/Akt, MEK/Erk pathway kinases (Akt, Erkl/2) and the apoptosis regulatory proteins (Bad, Bcl-xl, Caspase-3) by western blot.4. Microarray studies were employed to identify differential expression of those genes involving in tumorigenesis by ZIC1. We validated 10 candidate target genes in stable transfection cell lines (HCT116 and HT29) by qRT-PCRResult:1. QRT-PCR results showed that the level of ZIC1 mRNA was significantly decreased in colon cancer tissues relative to adjacent non-tumor tissues (Wilcoxon.matched paired tests, p=0.0001,n=24). Furthermore, methylation of ZIC1 gene promoter was frequently detected in primary tumor tissues (85%,34/40), but not in adjacent non-tumor tissues. 2. The number of surviving colonies formed was significantly reduced by overexpression ZIC1 in colon cancer cells (HCT116 and HT29) when compared with the control vector transfectants (p<0.01). Moreover, MTS assay showed that the survival cells were markedly decreased in cells (HCT116 and HT29) transfected with ZIC1 when compared with vector (p<0.01). Ectopic expression of ZIC1 could induce cell apoptosis, while not affect on cell cycle.3. Ectopic expression of ZIC1 could suppress the expression of phosphorylation of Erk1/2 and Akt, as well as phosphorylation of Bad and Bcl-xl, while activate the Bad and cleaved-Caspase-3.4. Results revealed that 337 genes were downregulated and 95 genes were upregulated by ZIC1 in colon cancer cells. Many of these genes have been reported to play important roles in cell proliferation, cell apoptosis, cell migration, transcriptional regulation and signal transduction. We validated the expression pattern in 10 selected genes (CCNA2, IGFBP3, ANGPT2, GADD45B, LAMB2, LAMB3, MALAT1, PNMA2, RPA4, TACSTD2) in colon cancer cells HCT116 and HT29 transfected with ZIC1 by qRT-PCR. These datas were concordant with that obtained from the microarray analysis.Conclusion:1. Downregulation of ZIC1 is associated with promoter DNA hypermethylation in colon cancer tissues.2. ZIC1 could inhibit cell proliferation by inactiviation of PI3K/AKT and MEK/Erk pathways, and induced cell apoptosis by regulating Bcl-xl/Bad/Caspase-3 cascades.3. ZIC1 may participate in the progression of colon cancer by modulating those functional genes involved in cellular activities.
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