miR-340通过下调CCND1表达增加结直肠癌细胞对5-Fu的耐药

miR-340 enhances chemotherapy resistance of colorectal cancer cells to 5-Fu by down-regulating CCND1 expression

目的:探讨细胞周期蛋白D1(cyclin D1)的编码基因CCND1 miR-340介导的逆转结直肠癌细胞对5-氟尿嘧啶(5-Fu)耐药的机制。方法:采用瞬时转染技术将结直肠癌细胞HCT116、SW480株分别转染si-CCND1和miR340-mimic。应用MTT法检测转染后的结直肠癌细胞对5-Fu敏感性的变化,应用双荧光素酶试验验证CCND1对miR340参与的影响结直肠癌细胞对5-Fu敏感性的影响。结果:瞬时转染si CCND1和过表达miR-340后,结直肠癌HCT116和SW480细胞的IC50值均显著低于对照组(10,10 vs 20μmol/L和20,20 vs 40μmol/L,均P<0.05)。共转染CCND1 3'UTR野生质粒和miR-340 inhibitor的结直肠癌HCT116和SW48细胞荧光素酶的活性显著高于共转染空载体和mimic细胞(P<0.01)。结论:CCND1作为不良因子通过抑制miR340的表达进而发挥增加结直肠癌细胞对5-Fu耐药的作用。

Objective：To investigate the mechanism of coding gene of cell cyclin D1（CCND1） participating in miR-340-induced inhibition of chemotherapy resistance of colorectal cancer cells to 5-fluorouracil （5-Fu）. Methods： Colorectal cancer HCT115 and SW480 cell lines were transfected with si-CCND1and miR340-mimic respectively by transient transfection technology. The sensitivity of cancer cells to 5-Fu after transfection was observed with MTT. The effect of CCND1 on miR-340 mediated suppression of chemotherapy resistance of cancer cells to 5-Fu was validated by double luciferase test. Results： After transient transfection with siCCND1 or over-expression of miR-340, the IC50 values of HCT116 and SW480 cell lines were significantly lower than that of control group [CCND1 silencing： 10, 10vs 20 μmol/L; miR-340 over-expressing： 20, 20 vs 40 μmol/L; all P〈0.05）. After co-transfection with CCND1 3＇UTR wild plasmid and miR-340 inhibitor, the luciferase reporter activity in HCT 116 and SW480 cells were significantly higher than that of empty plasmid transfection group or mimic transfection group （P〈0.01）. Conclusion： CCND1, as an unfavorable factor, enhances the chemo-resistance of colorectal cancer cells to 5-FU by inhibiting the expression of miR-340.