| Part I:The expression, hypomethylation and prognostic significance of CXCL10, TCF7L1, TMEM16J and SLC17A9 in stageⅡandⅢcolorectal cancerObjectives:In previous study, we have identified 4 genes that differently expressed in colorectal cancer (CRC) tissues with recurrence and those without recurrence using gene expression profiling assays. This study was aimed to investigate the relationship between the expression of 4 genes and their methylation status, and their potential roles in predicting prognosis of patients with stageⅡandⅢcolorectal cancer.Methods:To examine mRNA expression of 4 genes, real-time quantitative polymerase chain reaction (qPCR) and reverse transcriptase PCR was performed in 36 colorectal cancer tissues with recurrence and 28 without recurrence, and in 3 CRC-metastasis-derived cell lines (SW620, LoVo, Colo205) and 3 primary-CRC-derived ones (SW480, Caco-2, HCT116). Furthermore, protein was evaluated using immunostaining in 118 paraffin-embedded specimens and Western blot in 6 colorectal cancer cell lines, and the correlations between clinicopathologic factors and disease-free survival time and gene expression were analyzed. In addition, the methylation status of the CpG islands in TCF7L1 promoter was also detected by Bisulfite genomic sequencing (BGS) and methylation-specific polymerase chain reaction (MSP).Results:CXCL10, TMEM16J and SLC17A9 mRNA was down-regulated in both CRC tissues with recurrence and metastasis-derived cell lines but TCF7L1 mRNA was up-regulated. The expression level of TCF7L1 was unrelated with gender, age, tumor grade, lymphvascular or perineural invasion and other parameters. However, it was positively related to disease-free survival time, histological type and depth of invasion (P=0.002,0.038,0.020). The expression level of TMEM16J and SLC17A9 were positively related to TNM stage. CXCL10 expression was significantly associated with gender, age and tumor grade. Moreover, lower CXCL10, TMEM16J and SLC17A9 and higher TCF7L1 expression indicated poorer survival rate, respectively (P＜0.05, log-rank test). Multivariate analysis also showed they were independent prognosticators in CRC. Moreover, it was found that up-expression of TCF7L1 is significantly associated with its promoter CpG island hypomethylation.Conclusion:These findings indicate that up-regulation of TCF7L1 causing by hypomethylation or down-expression of any one of CXCL10, TMEM16J or SLC17A9 might play an important role in metastasis and predict poor prognosis of patients with stageⅡandⅢCRC patients.Chinese Library Classification:R73Part 2:Study on biological functions of human TMEM16J and SLC17A9 genesObjectives:In foregoing study, we have validated the expression TMEM16J and SLC17A9 genes were significantly associated with recurrence of stageⅡandⅢcolorectal cancer. Currently, the data concerning the biological functions of both genes were relatively insufficient, especially in cancer. This study was aimed to further investigate the role of TMEM16J and SLC17A9 genes in tumorigenesis of colorectal cancer.Methods:we designed to determine the impact of over-expression of TMEM16J and SLC17A9 by stable transfection of pcDNA3-gene expression vector on cell growth, cell cycle, apoptosis, cell adhesion and migration as well as invasion in human clorectal cancer lines (SW620, LoVo, HCT116, Caco-2). In addition, to further examine the effects of TMEM16J and SLC17A9 over-expression on tumor growth and metastasis, we performed the in vivo assay using an orthotopic xenograft tumor model in the athymic mice. We examined the primary tumor growth and the metastasis.Results:Over-expression of TMEM16J and SLC17A9 prolong cell doubling time or inhibit growth of colorectal cancer cells lines, including SW620, HCT116, LoVo and Caco-2. This inhibition of cell growth was associated with the regulation of cell cycle, increasing of apoptosis. Over-expression of TMEM16J and SLC17A9 elevated cellular capability of adhesion and reduced capability of invasion and migration in LoVo and HCT116. Additionally, the volume of tumor in nude mice was dramatically decreased by TMEM16J and SLC17A9 over-expression especially in LoVo cells (P<0.05).Conclusion:These findings indicate that the expression of TMEM16J and SLC17A9 decreased in human colorectal cancer cells as invasive and metastatic potential increased, indicating possible involvement of TMEM16J and SLC17A9 in invasive and metastatic phenotypes of colorectal cancer cells. TMEM16J and SLC17A9 have extensive effects on colorectal cancer cells in vitro and in vivo by inhibiting cell growth, redistributing cell cycle, promoting apoptosis, reducing invasion in vitro and metastasis in vivo. Both of them are new anti-oncogenes.
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