Objective To investigate the effect of microRNA-140 (miR-140) on the migration and invasion of colorectal cancer (CRC) cells and the possible mechanism.Methods miR-140 mimics,miR-140 specific inhibitor or small interfering RNA (siRNA) against Smad3 were transfected into human CRC cell line RKO cells respectively,using Oligofectamine or Lipofectamine2000.Quantitative real-time PCR (real-time PCR) was used to measure the expression levels of miR-140 and Smad3 mRNA.Smad3 protein was analyzed by Western blot.The in vitro cell migrating and invasive abilities were determined by woundhealing and Transwell chamber assay after up-regulating or down-regulating miR-140 or knocking down Smad3.Results The Western blot assays showed that the Smad3 protein level was significantly reduced after up-regulating miR-140 (0.04 ± 0.01),compared with that of (0.47 ± 0.02,P ＜ 0.05) in the control group and that of (0.52 ± 0.06) in the negative control group (P ＜ 0.05 for both).The results of real-time PCR indicated that no significant difference was found in the levels of Smad3 mRNA between miR-140 transfection and NC groups (1.11 ± 0.13 vs.1.00 ± 0.06,P ＞ 0.05).The wound-healing assay showed that the migrating ability was dramatically attenuated by miR-140 compared with that in the control and NC groups,whereas no significance was found when compared with that of the Smad3 siRNA transfected cells.The number of cells migrating through Transwell chamber without matrigel in the miR-140 group was (76.2 ± 4.4),remarkably lowered than that in the control (267.1 ± 4.9) and NC (336.1 ± 5.7) groups (P ＜ 0.05 for both),but no significant difference between the miR-140 (76.2 ± 4.4) and Smad3 siRNA (83.5 ± 7.3) groups.Transwell chamber with matrigel assay showed that number of cells penetrating through the membrane was (109.5 ±7.4) in the miR-140 group,significantly lower than that in the control (403.1 ± 5.1) and NC (392.6 ± 8.4) groups (P ＜ 0.05 for both),while Smad3 siRNA transfection had a similar effect (138.8 ± 3.6) (P ＞ 0.05).Down-regulation of miR-140 increased the level of smad3 protein expression,and partially reversed the inhibition of the cell migration and invasion mediated by miR-140.Cotransfection of miR-140 inhibitor and Smad3 siRNA had no significant effect on the Smad3 protein expression and the abilities of cell migration and invasion.Conclusions miR-140 regulates the Smad3 expression at the post-transcriptional level,miR-140 suppresses the migrating and invasive abilities of CRC cells,possibly through down-regulation of Smad3.The findings of this study suggest that miR-140 may have a unique potential as a possible biomarker candidate for diagnosis and therapy of tumor metastasis.%目的 探讨miR-140对结肠癌RKO细胞迁移和侵袭能力的影响及其调控机制.方法 将miR-140模拟物、miR-140特异性抑制物和Smad3小干扰RNA(siRNA)等分别通过脂质体转染至细胞,应用实时荧光定量PCR(real-time PCR)检测细胞中miR-140和Smad3 mRNA的表达,应用Western blot检测Smad3蛋白的表达.采用细胞划痕实验和Transwell小室模型检测miR-140上调、miR-140下调和Smad3下调对RKO细胞迁移和侵袭能力的影响.结果 Western blot结果显示,上调miR-140后,miR-140组中Smad3蛋白的相对表达水平为0.04±0.01,低于空白对照组(0.47±0.02)和阴性对照组(0.52 ±0.06,均P＜0.05).real-time PCR检测结果显示,miR-140组中Smad3 mRNA表达水平为1.11 ±0.13,与阴性对照组(1.00±0.06)比较,差异无统计学意义(P＞0.05).细胞划痕实验显示,miR-140转染细胞的迁移能力均低于空白对照组和阴性对照组,而与Smad3 siRNA组比较,迁移能力无明显改变.Transwell小室迁移实验显示,miR-140组的穿膜细胞数为76.2±4.4,低于空白对照组(267.1±4.9)和阴性对照组(336.1±5.7,均P＜0.05),而与Smad3 siRNA组(83.5±7.3)比较,差异无统计学意义(P＞0.05).Transwell小室侵袭实验显示,miR-140组的穿膜细胞数为109.5±7.4,低于空白对照组(403.1±5.1)和阴性对照组(392.6±8.4,均P＜0.05);而与Smad3 siRNA组(138.8±3.6)比较,差异无统计学意义(P＞0.05).miR-140下调可使Smad3蛋白表达增高,部分逆转miR-140对细胞迁移和侵袭能力的抑制作用.miR-140抑制剂和Smad3 siRNA共转染则对Smad3蛋白表达和细胞的迁移和侵袭能力无明显影响.结论 miR-140在转录后水平调控Smad3的表达.miR-140抑制结肠癌细胞迁移和侵袭能力,可能是通过下调Smad3来实现.miR-140可能作为肿瘤转移诊断及治疗的潜在候选靶点.
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