MiR-320 Enhances The Sensitivity Of Human Colon Cancer Cells To Chemoradiotherapy In Vitro By Targeting FOXM1

Objective:To evaluate the impacts of mi R-320 on colon cancer biological processes,chemo-resistance to 5-FU and Oxaliplatin and radiation resisitance, and clarify the mechanisms of how mi R-320 reverses drug resistance and radiation resistance in colon cancer cells.Methods:1. The HCT116 colon cancer cell line, which expresses relatively low level of mi R-320, was transfected with mi R-320 mimics, while the HT-29 cell line with relatively high level of mi R-320 was transfected by mi R-320 inhibitor. The transfection effeciency was evaluated under a fluorescence microscope, and the level of mi R-320 was deteced by real-time PCR after transfection.2. The MTT assay and colony formation assay were used to evaluate the cell proliferation after transfection; flow cytometry was used to deteced cell apoptosis and cell cycle distribution; Transwell assay was used to investigate the cell migratory and invasive abilities. The inhibitoy rates of 5-FU and Oxaliplatin were deteced by MTT assay, and the half of the inhibition(IC50) was calculated. The cell viability after radiation was measured by MTT assay.3. Luciferase assay was adopted to investigate whether FOXM1 is a target gene of mi R-320. The m RNA expressions of FOXM1 after transfection were evaluate by the real-time PCR. The protein expressions of FOXM1, β-catenin, Cyclin D1 and c-myc after transfection were evaluate by Western blot.Result:1. The result of real-time PCR analysis indicated that cells in HCT116-mimics group had a higher level of mi R-320 compared with HCT116-NC cells after transfection; while mi R-320 inhibitor decreased mi R-320 expression in HT-29-inhibitor cells.2. Overexpression of mi R-320 led to inhibit HCT116 cell proliferation, colony foramation, cell cycle progression, invasion and hypersensitivity to 5-Fu and Oxaliplatin, but had no effect on cell apoptosis. Also, knockdown of mi R-320 reversed these effects in HT-29 cells.3. The data of luciferase reporter assay showed that upregulation of mi R-320 reduced the luciferase activity, while mutations on the mi R-320 binding sites located in 3’UTR of FOXM1 abolished this effect. Moreover, real-time PCR showed the m RNA level of FOXM1 wasn’t changed after transfection. And Western blot assay verified that overexpression of mi R-320 decreased the expression levels of FOXM1,β-catenin, Cyclin D1 and c-myc, while these protein were all increased after knockdown of mi R-320.Conclusion:1. mi R-320 can inhibit cell proliferation, cell cycle progression, migration,invasion and increase sensitivity to 5-FU and Oxaliplatin.2. FOXM1 is a target of mi R-320, and the mi R-320-FOXM1 axis may play an important role in regulating the resistance to chemotheraypy based on 5-FU and Oxaliplatin and radiation.