FTY720通过miR-494/MST1抑制结肠癌细胞并增加吉西他滨敏感性的分子机制

背景结肠癌在我国恶性肿瘤中发病率和死亡率均居第5位,化疗是其主要治疗方式,有研究表明免疫抑制剂FT Y720对癌症细胞增殖起一定的抑制作用,抑制剂联合化疗药物可提高癌症治疗效果.本研究使用免疫抑制剂FTY720与吉西他滨联合处理结肠癌细胞,并探索miR-494/哺乳动物Ste20样激酶1 (mammalian Ste20-like kinase 1,MST1)对在此过程中对结肠癌细胞增殖和凋亡的影响,以期为结肠癌的治疗提供新的思路.目的研究FT Y720和吉西他滨对结肠癌细胞存活率和凋亡的影响和潜在的分子机制.方法用0.0001μg/mL、0.001μg/mL、0.01μg/mL、0.1μg/mL、1μg/mL的吉西他滨和2.5μmol/L、5μmol/L、7.5μmol/L、10μmol/L、12.5μmol/L的FTY720处理结肠癌SW1116细胞,CCK8法和流式细胞术检测SW1116细胞存活率和凋亡率,实时定量聚合酶链式反应检测miR-494和MST1 mRNA的含量,Western blot检测MST1、p21和Caspase-3蛋白表达水平,双荧光素酶报告系统验证miR-494与MST1的调控关系.结果吉西他滨(0.0001μg/mL、0.001μg/mL、0.01μg/mL、0.1μg/mL、1μg/mL)和FTY720 (2.5μmol/L、5μmol/L、7.5μmol/L、10μmol/L、12.5μmol/L)均可降低结肠癌SW1116细胞的存活率,且具有浓度依赖性,根据结果选取抑制率约为50%的0.1μg/mL吉西他滨和10μmol/L的FTY720进行后续实验,吉西他滨和FT Y720均可抑制细胞存活并促进细胞凋亡,且联合使用比单独使用效果更好;过表达miR-494可逆转FTY720、吉西他滨对SW1116细胞存活率和凋亡的作用;miR-494靶向调控MST1;抑制MST1可逆转FTY720和吉西他滨对SW1116细胞存活率和凋亡的影响.结论FTY720和吉西他滨通过miR-494/MST1抑制SW1116细胞存活,促进细胞凋亡.FTY720和吉西他滨对结肠癌SW1116具有抑制作用,且联合使用效果更佳.

FTY720 inhibits colon cancer cell survival and increases their sensitivity to gemcitabine through the miR-494/MST1 pathway

BACKGROUND Colon cancer ranks 5 th in both incidence and mortality among malignant tumors in China.Chemotherapy is the main treatment method.Studies have shown that immunosuppressive agent FTY720 has a certain inhibitory effect on cancer cell proliferation.Inhibitors combined with chemotherapy drugs can improve the therapeutic effect of cancer.In this study,immunosuppressive agent FTY720 and gemcitabine were used together to treat colon cancer cells,and the role of miR-494/mammalian Ste20-like kinase 1(MST1) in the proliferation and apoptosis of colon cancer cells was explored,with an aim to provide a new treatment for colon cancer.AIM To investigate the effects of FTY720 and gemcitabine on the survival and apoptosis of colon cancer cells and the potential molecular mechanisms involved.METHODS Colon cancer SW1116 cells were treated with gemcitabine at concentrations of 0.0001 μg/mL,0.001 μg/mL,0.01 μg/mL,0.1 μg/mL,and 1 μg/mL and FTY720 at concentrations of 2.5 μmol/L,5 μmol/L,7.5 μmol/L,10 μmol/L,and 12.5 μmol/L.CCK8 assay and flow cytometry were applied to detect the survival rate and apoptosis rate of SW1116 cells.Quantitative real-time polymerase chain reaction was used to measure the levels of miR-494 and MST1 mRNA.Western blot was carried out to detect the expression levels of MST1,p21,and Caspase-3 proteins.Dual-luciferase reporter assay was performed to verify the relationship between miR-494 and MST1.RESUL TS Gemcitabine and FTY720 reduced the survival rate of colon carcinoma SW1116 cells in a concentration dependent manner.According to the results,0.1 μg/mL gemcitabine and 10 μmol/L FTY720 with an inhibition rate of about 50% were selected for subsequent experiments.Gemcitabine and FTY720 both inhibited cell survival and promoted cell apoptosis,and their combined use was better than the single use.Overexpression of miR-494 reversed the effects of FTY720 and gemcitabine on survival and apoptosis in SW1116 cells.MiR-494 targeted and regulated MST1.Inhibition of MST1 reversed the effects of FTY720 and gemcitabine on the survival and apoptosis in SW1116 cells.CONCLUSION FTY720 and gemcitabine inhibit SW1116 cell survival and promote apoptosis through the miR-494/MST1 pathway.The combination of FTY720 and gemcitabine has more significantly inhibitory effects on the survival and apoptosis of SW1116 cells.