CAJ | 학술논문

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.
목적 탐토miR-140대결장암RKO세포천이화침습능력적영향급기조공궤제.방법 장miR-140모의물、miR-140특이성억제물화Smad3소간우RNA(siRNA)등분별통과지질체전염지세포,응용실시형광정량PCR(real-time PCR)검측세포중miR-140화Smad3 mRNA적표체,응용Western blot검측Smad3단백적표체.채용세포화흔실험화Transwell소실모형검측miR-140상조、miR-140하조화Smad3하조대RKO세포천이화침습능력적영향.결과 Western blot결과현시,상조miR-140후,miR-140조중Smad3단백적상대표체수평위0.04±0.01,저우공백대조조(0.47±0.02)화음성대조조(0.52 ±0.06,균P＜0.05).real-time PCR검측결과현시,miR-140조중Smad3 mRNA표체수평위1.11 ±0.13,여음성대조조(1.00±0.06)비교,차이무통계학의의(P＞0.05).세포화흔실험현시,miR-140전염세포적천이능력균저우공백대조조화음성대조조,이여Smad3 siRNA조비교,천이능력무명현개변.Transwell소실천이실험현시,miR-140조적천막세포수위76.2±4.4,저우공백대조조(267.1±4.9)화음성대조조(336.1±5.7,균P＜0.05),이여Smad3 siRNA조(83.5±7.3)비교,차이무통계학의의(P＞0.05).Transwell소실침습실험현시,miR-140조적천막세포수위109.5±7.4,저우공백대조조(403.1±5.1)화음성대조조(392.6±8.4,균P＜0.05);이여Smad3 siRNA조(138.8±3.6)비교,차이무통계학의의(P＞0.05).miR-140하조가사Smad3단백표체증고,부분역전miR-140대세포천이화침습능력적억제작용.miR-140억제제화Smad3 siRNA공전염칙대Smad3단백표체화세포적천이화침습능력무명현영향.결론 miR-140재전록후수평조공Smad3적표체.miR-140억제결장암세포천이화침습능력,가능시통과하조Smad3래실현.miR-140가능작위종류전이진단급치료적잠재후선파점.
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.