The Molecular Mechanisms Of ZIC1Gene In The Regulation Of Cell Cycle And Migration In Gastric Cancer

We have previously shown that ZIC1(Zinc finger of the cerebellum1) is downregulated in gastric cancer cell lines and tissues. In addition, such downregulation is mediated through promoter DNA hypermethylation by methylation specific PCR analysis. ZIC1. one of five ZIC family genes, encodes transcription factors that contain C2H2zinc finger (ZF) domains. Recent studies have demonstrated that ZIC I is not only involved in a variety of developmental processes, but also implicated in the progression of human cancers including medulloblastoma, endometrial cancer and liposarcoma. However, the molecular mechanisms underlying ZIC1function in the progression of gastric cancer remains unknown.Both regulation of cell proliferation and cell cycle play essential roles in the development of gastric cancer. Sonic hedgehog (Shh) signaling pathway contributes to gastric tumorigenesis. The activation of Shh promotes gastric cancer cell differentiation and proliferation. It is well known that MAPK and PI3K pathways are vital for cell proliferation, and cell cycle transit from G1to S phase in human cancers.In this study, we evaluate the effect on cell proliferation, migration and invasion of gastric cancer cells by ectopic expression of ZIC1, and investigate the regulation of MAPK, PI3K and Shh pathways and their related downstream targets in gastric cancer progression. Furthermore, we identify multiple downstream targets of ZIC1by cDNA microarray analysis.Materials and Methods:1. Gastric cancer cell lines were stably tranfected with pCDNA3.1-ZIC1and pCDNA3.1vectors. MTS and Transwell assays were performed to evaluate the effects on cell proliferation, migration and invasion.2. Cell cycle distributions and cell apoptosis after ectopic expression of ZIC1were detected by flow cytometry analysis. The expression levels of phos-Akt and total Akt, phos-Erk1/2and total Erk1/2,p21Waf1/Cip1, p27Kip1and cyclin D1after ectopic expression of ZIC1in gastric cancer cell lines were detected by Western blot.3. The expression level of Shh after ectopic expression of ZIC1was detected by Western blot. Cyclopamine, a Shh signaling inhibitor, was used to block the activation of Shh pathway. In addition, Western blot was performed to observe the expression of p21Waf1/Cip1and cyclin Dl, and Transwell assay to detect cell migration after administration with cyclopamine.4. Differential expression genes after ectopic expression of ZIC1in MKN28cell lines were identified by cDNA microarray. The expression levels of CDKN2B9, TP53INP1and ARHGAP were validated by Q-PCR.Results:1. Ectopic expression of ZIC1suppresses proliferation, migration and invasion in gastric cancer cells. The cell proliferation was showed significant reduction in a 5-day observation after stable transfected with ZIC1in BGC823cells (p<0.001). Moreover, we observed that ectopic expression of ZICl significantly suppressed cell migration in AGS, BGC823and SGC7901gastric cancer cell lines (p<0.001). Overexpression of ZIC1displayed a significantly lower activity of cellular invasion when compared to those empty vector transfectants in AGS cells (p<0.05).2. ZICl inhibits the MAPK and PI3K pathways in gastric cancer cells and alters cell-cycle distributions in gastric cancer cells. We observed a higher proportion of cells in G1phase in AGS (42.74%) and SGC7901(54.03%) cell lines after ectopic expression of ZIC1, compared to which transfected with a control vector. In addition, we demonstrated that the expression level of cyclin D1protein was reduced while p21Waf1/Cip1and p27Kip1were markedly induced in AGS. BGC823and SGC7901gastric cancer cells transfected with pCDNA3.1-ZIC1when compared to those pCDNA3.1empty vector transfectants. We also found that the phosphorylation levels of Erk1/2and Akt were dramatically suppressed by forced overexpression of ZIC1in AGS, MKN28, BGC823and SGC7901gastric cancer cells. As expected, total expression levels of Erkl/2and Akt maintain unchanged.3. Hedgehog (Hh) signaling pathway is involved in the ZIC1regulation of cell-cycle and cell migration in gastric cancer cells. We found that ectopic expression of ZIC1effectively reduces Shh expression in BGC823and SGC7901cell lines by western blot and Q-PCR analysis (p<0.01). After treated with cyclopamine, the expression of ZICl mRNA in BGC823and SGC7901cells showed no obvious differences, but the expression level of p21was markedly up-regulated and cyclin D1down-regulated in AGS, BGC823and SGC7901gastric cancer cell lines. We also found significant inhibition of cellular migration after administration with cyclopamine (p<0.001).4. Gene expression profile changes by ectopic expression of ZIC1. We conducted an affymatrix oligonucleotide microarray, which revealed that132genes are down-regulated while66genes are up-regulated by exogenous expression of ZIC1in MKN28gastric cancer cells. We validated the expression of CDKN2B, ARHGAP9and TP53INP1by q-PCR analysis, which were consistent with the microarray results.Conclusion:1. ZICl inhibit cell proliferation, migration and invasion in gastric cancer cells.2. ZICl regulates G1/S transit mainly through PI3K and MAPK pathways and their downstream cell-cycle regulator kinases (p21, p27, cyclin D1) in gastric cancer.3. ZIC1could inhibit Shh expression in gastric cancer cells. Inhibition of Shh signaling by administration with cyclopamine suppresses cell migration, and regulates the expression of p21and cyclin D1. ZIC1plays important roles in gastric cancer progression by regulation of the Shh signaling pathway.4. ZIC1potentially regulates multiple downstream genes involved in gastric tumorigenesis.