Zhao Wenyue, Zou Jiarui, Wang Bo, Fan Panhong, Mao Jun, Li Jiazhi, Liu Han, Xiao Jing, Ma Wei, Wang Mei, Li Lianhong, Song Bo
Department of Pathology and Forensics, Dalian Medical University, Dalian 116044, China.
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Zhonghua Zhong Liu Za Zhi. 2014 Oct;36(10):739-45.
To investigate the effect of microRNA-140 (miR-140) on the migration and invasion of colorectal cancer (CRC) cells and the possible mechanism.
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 wound-healing and Transwell chamber assay after up-regulating or down-regulating miR-140 or knocking down Smad3.
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. Co-transfection of miR-140 inhibitor and Smad3 siRNA had no significant effect on the Smad3 protein expression and the abilities of cell migration and invasion.
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.
探讨微小RNA-140（miR-140）对结直肠癌（CRC）细胞迁移和侵袭的影响及其可能机制。
分别使用脂质体转染试剂（Oligofectamine或Lipofectamine2000）将miR-140模拟物、miR-140特异性抑制剂或针对Smad3的小干扰RNA（siRNA）转染至人CRC细胞系RKO细胞。采用定量实时聚合酶链反应（实时PCR）检测miR-140和Smad3 mRNA的表达水平。通过蛋白质免疫印迹法分析Smad3蛋白。上调或下调miR-140或敲低Smad3后，采用划痕实验和Transwell小室实验检测体外细胞迁移和侵袭能力。
蛋白质免疫印迹分析显示，上调miR-140后Smad3蛋白水平显著降低（0.04±0.01），对照组为（0.47±0.02，P<0.05），阴性对照组为（0.52±0.06，P<0.05）。实时PCR结果表明，miR-140转染组与阴性对照组Smad3 mRNA水平差异无统计学意义（1.11±0.13比1.00±0.06，P>0.05）。划痕实验显示，与对照组和阴性对照组相比，miR-140显著减弱细胞迁移能力，而与Smad3 siRNA转染组相比差异无统计学意义。在未铺基质胶的Transwell小室实验中，miR-140组穿过小室的细胞数为（76.2±4.4），显著低于对照组（267.1±4.9）和阴性对照组（336.1±5.7）（P<0.05），但miR-140组（76.2±4.4）与Smad3 siRNA组（83.5±7.3）差异无统计学意义。铺有基质胶的Transwell小室实验显示，miR-140组穿过膜的细胞数为（109.5±7.4），显著低于对照组（403.1±5.1）和阴性对照组（392.6±8.4）（P<0.05），Smad3 siRNA转染组有类似结果（138.8±3.6）（P>0.05）。下调miR-140可增加Smad3蛋白表达水平，并部分逆转miR-140介导的细胞迁移和侵袭抑制作用。共转染miR-140抑制剂和Smad3 siRNA对Smad3蛋白表达及细胞迁移和侵袭能力无显著影响。
miR-140在转录后水平调控Smad3表达。miR-140可能通过下调Smad3抑制CRC细胞的迁移和侵袭能力。本研究结果提示，miR-140可能作为肿瘤转移诊断和治疗的潜在生物标志物具有独特潜力。
