| Colorectal cancer (CRC) is one of the most common cancers and the third leading cause of cancer-related death worldwide. Recently, the morbidity of CRC is increasing in the developing countries. Approximate 1.2 milion of new cases emerge in the world and 0.6 milion of patients with CRC die every year.Up to date, the treatment of CRC combines surgery, chemotherapy and radiation. However, the effect of this treatment is not ideal, which indicates that the deep understanding of cancer biology is critical for the diagnosis and treatment of this disease. In this study, we analysized the role of Factor inhibiting HIF(FIH-1) in CRC and identified an important miRNA which could negatively regulate FIH-1 expression. Based on the above results, the function of this miRNA-31/FIH-1 nexus in CRC was studied. Our study will reveal the contribution of miRNA-31/FIH-1 nexus to CRC development and molecular mechanism of CRC progression.Part Ⅰ:The expression profile of FIH-1 and the role of FIH-1 in CRCObjective:To verify the expression of FIH-1 in CRC and to study the correlation between FIH-1 expression and CRC progression.Methods:Immunohistochemical staining was employed to analysize the expression profile of FIH-1 in CRC. Meanwhile, real-time PCR was performed to analysize the correlation between FIH-1 expression and CRC development.Results:The expression of FIH-1 was down-regulated in CRC, compared to their corresponding adjacent normal tissues. Additionally, FIH-1 expression was correlated with invasion depth, lymphonode metastasis, and distance metastasis.Conclusion:FIH-1 expression is down-regulated in CRC and correlated with CRC development.Part Ⅱ:The miRNA-31/FIH-1 nexus identified in CRCObjective:To identify the miRNA targeting FIH-1 and study the key factor causing the down-regulation of FIH-1 in CRC.Methods:The data from Targetscan and related references were analysized to identify the key miRNA which was correlated with CRC and could regulate FIH-1 expression. Real-time PCR was performed to verify miRNA-31 expression in CRC and the correlation between FIH-1 and miRNA expression was analysized. By using the technique of cell transfection, we verified miRNA-31 regulated FIH-1 expression in CRC cell lines. Through the luciferase reporter assay, we defined the miRNA-31/FIH-1 nexus. Additionally, the correlation beween miRNA expression and CRC development was analysized.Results:The expression of FIH-1 was negatively correlated with miRNA-31 in CRC samples and cell lines. The result of luciferase reporter assay indicated that miRNA-31 directly targeted FIH-1 and FIH-1 was one of the target genes of miRNA-31. Up-regualation of miRNA-31 caused the down-regulation of FIH-1 in CRC and was correlated with the advanced progression and poor prognosis of CRC.Conclusion:FIH-1 expression is negatively correlated with miRNA-31 and FIH-1 is one target gene of miRNA-31. Up-regulation of miRNA-31 causes the down-regulation of FIH-1 in CRC. Additionally, miRNA-31 expression is correlated with the prognosis of CRC.Part Ⅲ:The role of miRNA-31/FIH-1 nexus in CRC cell functionObjective:To study the effect of miRNA-31/FIH-1 nexus in CRC cell function.Methods:Firstly, we used the technique of cell transfection to down-regulate miRNA-31 expression in HCT116 and SW1116 cells. Secondly, MTT assay was employed to verify the effect of miRNA-31/FIH-1 nexus to the cell proliferation. Finally, Transwell assay was performed to test the effect of miRNA-31/FIH-1 nexus in cell migration and invasion.Results:By down-regulating miRNA-31 expression, FIH-1 expression was up-regulated. The MTT assay showed that CRC cell proliferation was decreased by down-regulating miRNA-31 expression(up-regulating FIH-1 expression). Similarly, CRC cell migration and invasion were inhibited by down-regulating miRNA-31 expression(up-regulating FIH-1 expression), which was shown in Transwell assays.Conclusion:The miR-31/FIH-1 nexus is shown to control cell proliferation, migration, and invasion in vitro. Down-regulating miRNA-31 could inhibite CRC cell proliferation, migration, and invasion.Part Ⅳ:The role of miRNA-31/FIH-1 nexus in CRC xenograftsObjective:To study the effect of miRNA-31/FIH-1 nexus in CRC xenografts.Methods:Trough the technique of lentivirus infection, we built the cell line with stable down-regulation of miRNA-31. Then, HCT116 cells with anti-miRNA-31-LV and anti-NC-LV were injected into nude mice subcutaneously to analysize the effect of miRNA-31/FIH-1 nexus to the tumor growth of HCT116 xenografts.Results:Both of tumor volume and weight of HCT116 xenografts with down-regulated miRNA-31 were decreased, compared to the control HCT116 xenografts. FIH-1 expression was increased in HCT116 xenografts with down-regulated miRNA-31.Conclusion:By using HCT116 xenografts assay, we demonstrate that miR-31/FIH-1 nexus could not only control CRC cell proliferation, migration, and invasion but also affect CRC cell xenografts. |
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