miR-140通过下调Smad3表达抑制结肠癌细胞迁移和侵袭能力  

microRNA-140 suppresses the migration and invasion of colorectal cancer cells through targeting Smad3

作　　者：赵文月[1] 邹佳芮[1] 王波[1] 范盼红[1] 毛俊[1] 李嘉芝[1] 刘涵 肖晶 马威 王梅[1] 李连宏[1] 宋波[1] 

机构地区：[1]大连医科大学病理学与法医学教研室,116044 [2]口腔医学院 [3]解剖学教研室

出　　处：《中华肿瘤杂志》2014年第10期739-745,共7页Chinese Journal of Oncology

基　　金：国家自然科学基金（81172052）

摘　　要：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-timePCR）检测细胞中miR-140和Smad3mRNA的表达，应用Westernblot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Westernblot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-timePCR检测结果显示，miR-140组中Smad3mRNA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Smad3siRNA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Smad3siRNA组（83．54-7．3）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109．54-7．4，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Smad3siRNA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Smad3siRNA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。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 wound- healing 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-timePCR 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 ceils. 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. g） in the miR-140 group, significantly lower than that in the control （403.1 ± 5.1 ） and NC （392.6 ± 8. d ） groups

关 键 词：结肠肿瘤 微RNAS SMAD3蛋白 肿瘤转移 肿瘤侵润 

分 类 号：R735.35[医药卫生—肿瘤]