Inhibitory Effects And Its Mechanism Of Esophageal Cancer Related Gene 2 (ECRG2) On Proliferation And Metastasis Of Human Cancer Cells

A novel tumor suppressor gene, esophageal cancer related gene2 (ECRG2), was cloned and identified by comparing the differential gene expression between normal esophageal epithelia and esophageal cancer and with the technique of mRNA differential display. The full-length cDNA of ECRG2 gene contains 258 nucleotides of the open reading frame that predicts 85 amino acids. ECRG2 gene expression is down regulated in some malignant tissues such as esophageal carcinoma, colon cancer and brain tumor. So we suspected that ECRG2 gene was probably a tumor suppressor gene. This work focused on the inhibitory effects study of ECRG2 gene on proliferation and metastasis of human cancer cells. In our study, we first designed and synthesized specific primers for ECRG2. Then the ECRG2 cDNA gene was amplified by PCR and cloned into pcDNA3.1(+) vector. The recombinant plasmid pcDNA3.1(+)-ECRG2 was constructed successfully and identified by the sequence analysis. To further investigate the inhibitory functions of ECRG2 in cancer, an eukaryotic expression vector pcDNA3.1(+) carrying ECRG2 ORF was transiently and stably transfected respectively into EC9706 cells and PG cells by Liperfectamine 2000, which were negative for the genes. The effects on proliferation and malignant of ECRG2 over-expression on EC9706 cells were evaluated by growth characteristics such as growth in monolayer culture, anchorage-independent growth in soft agar. ECRG2 over-expressing cells showed a slower growth rate and lower ability of colony formation(18%) compared with those of the vector control cells(55%). Endogenous ECRG2 expression was negatively correlated with neoplastic potential as evidenced by attenuated anchorage-independent growth. In mechanistic studies, we found that p53 protein and p21 protein, the negative regulate factors in cell cycle, could be induced higher expression than those of the vector control cells . In conclusion, these data suggest that ECRG2 might reduced the abilities of proliferation and partly reverse malignant behavior of EC9706 cells by inducing cell differentiation, suppressing cell proliferation through p53 pathway. To evaluated the role of ECRG2 in cancer metastasis, we took use of methods such as scrape-wound-migration assay, transwell migration assay, transwell invasion assay and in vivo tumor metastasis. ECRG2 over-expressing cells showed a slower migration and invasion rate in vivo or in vitro. Zymography and Western blotting results demonstrated that ECRG2 protein cound inhibit the latent MMP-2 transformed into the active MMP-2. Taken together, ECRG2 protein might decrease the abilities of metastasis of PG cells by restraining pro-MMP-2 actived. This finding may be helpful in delineating role and molecular mechanism of ECRG2 in cancer and providing a potential biomedicine for cancer therapy in the future.