The Mechanisms Of ZIC1 Gene In The Regulation Of Lung Metastasis And Peritoneal Implantation Metastasis In Gastric Cancer Cells

Gastric cancer (GC) remains one of the most frequent cancers in the world, and the second mortality leading cause of cancer death in China. The patients of gastric cancer are mainly at advanced stage when they are diagnosed, and most of them have had metastasized. Chemotherapy is usually applied to these patients and targeted agents contribute to the effect of chemotherapy. Thus, identification of potential molecular targets of gastric cancer metastasis will be of great importance in the view of cancer therapy in future.ZIC1 is one of potential tumor suppressor gene in gastric cancer which was screened and validated by our previous research. ZIC1, one of five ZIC family genes, encodes zinc finger transcription factors which contain C2H2 zinc finger domains. Recent studies have indicated that ZIC1 is not only playing an important role in a variety of developmental processes, but also involved in the development and progression of many human cancers including endometrial cancer, thyroid cancer, medulloblastoma, malignant mesenchymal tumor and gastroenteric tumor. Our previous in vitro experiments have found that ZIC1 could suppress cell proliferation, migration and invasion of gastric cancer cells. However, these biological effects have not been clarified in vivo experiments.In this study, we designed in vivo experiments including tumor xenografts and lung, peritoneal metastasis in nude mice to evaluate the effect of ZIC1 on tumor growth and metastasis in gastric cancer. Furthermore, we identify multiple downstream targets related to tumor metastasis by ectopic expression of ZIC1 by cDNA microarray analysis.Material and Methods1. The tumor formation effect of ZIC1 in nude mice.① SGC7901 gastric cancer cells was stably transfected with pFlag-CMV4 Vector and pFlag-CMV4-ZIC1.② Subcutaneous xenografts of gastric cancer cells in nude mice was established and tumor formation was evaluated.2. The effect of ZIC1 on gastirc cancer lung metastasis and peritoneal implantation.① BGC823 gastric cancer cells was stably transfected with lentivirus-GFP Vector and lentivirus-GFP ZIC1.② Lung metastasis was observed after gastric cancer cells were transplanted into nude mice through the lateral tail vein.③ Peritoneal implantation metastasis was evaluated after stable transfected cells were injected into the abdominal cavity of nude mice.3. Screening and validation of the downstream targets related with the metastasis by overexpression of ZIC1 in gastric cancer cells.① Differential expression genes after ectopic expression of ZIC1 in BGC823 cell lines were identified by cDNA microarray and the expression levels of seven potential targets which were related with metastasis were validated by Q-PCR.② The expression levels of EPB41L3 were validated in BGC-Vector and BGC-ZIC1 cell by RT-PCR.③ The expression levels of EPB41L3 were detected in seven gastric cancer cell lines and GES-1 cell by Q-PCR.Results1. ZIC1 has no significant effect on the xenograft nude mice of gastric cancers.① Stabe ectopic expression of ZIC1 in SGC7901 cells were esbalished.② Ectopic expression of ZIC1 didn’t affect the tumor formation in xenograft mice.2. Ectopic expression of ZIC1 suppresses the lung metastasis and peritoneal implantation of gastric cancers.① Ectopic expression of ZIC1 in BGC823 cell were generated with lentivirus-GFP ZIC1.② Ectopic expression of ZIC1 significantly suppress the lung metastasis of gastric cancer in mice and improve the survival time (p<0.05).③ Ectopic expression of ZIC1 suppress peritoneal implantation metastasis of gastric cancer (p<0.05).3、ZIC1 modulates multiple downstream genes related with cell metastasis.① cDNA microarray demonstrated that 289 genes are up-regulated while 304 genes are down-regulated by exogenous expression of ZIC1 in BGC823 gastric cancer cells. The expression levels of seven potential targets involved with metastasis were validated by Q-PCR, which were consistent with the microarray results. Among those genes, the expression of EPB41L3 was most significantly upregulated..② RT-PCR analysis validated the expression of EPB41L3 was upregulated by forced expression of ZIC1.③ The expression of EPB41L3 mRNA was downregulated in gastric cancer cells when compared to GES-1 cell.Conclusions1. Ectopic expression of ZIC1 can suppress the lung metastasis and peritoneal implantation of gastric cancer in nude mice, while not affect the tumor’s formation of gastric cancer.2. ZIC1 regulated multiple downstream genes involved in gastric cancer metastasis. EPB41L3 was one of most significant upregulated gene, which potentially transcriptional regulator of ZIC1 on the role of gastric cancer metastasis.