miR-223在结肠癌组织中的表达及促进结肠癌细胞迁移侵袭的机制研究

目的:探讨微小RNA-223 (mi R-223)在结肠癌组织中的表达及对结肠癌HT-29细胞侵袭、迁移能力的影响及机制。方法:检测mi R-223在结肠癌组织与癌旁组织中的表达。通过脂质体转染法将mi R-223模拟物(mi R-223 mimics,mi R-223 mimics组)及microRNA无关序列(mi R-223 NC,NC组)转染入结肠癌HT-29细胞。采用Real-time PCR检测转染后细胞中mi R-223和TWIST的表达,Western blot检测TWIST的蛋白表达,Tranwell检测细胞的迁移与侵袭能力。双荧光素酶报告基因检测mi R-223对TWIST基因启动子活性的影响。采用Transwell迁移与侵袭实验检测mi R-223 mimic及Twist si RNA共转染后人结肠癌细胞系HT-29迁移与侵袭能力的变化。结果:与癌旁结肠组织比较,mi R-223在结肠癌组织中呈现明显高表达(P0.05);与空白对照组和mi R-223 NC组比较,转染mi R-223 mimics后的HT-29细胞中的mi R-223表达显著增加(P0.05)。与阴性对照组和空载转染组相比较,mi R-223 mimics转染组穿透的细胞数目明显增加(P0.05),且mi R-223 mimics转染组的细胞侵袭能力显著增强(P0.05)。与mi R-223 NC组和空白对照组比较,转染mi R-223 mimics的HT-29细胞的TWIST基因m RNA和蛋白表达均显著增加(P0.05)。双荧光素酶检验结果显示TWIST为mi R-223的下游靶基因。共转染TWIST si RNA和mi R-223 mimics的结肠癌HT-29细胞的迁移与侵袭能力较单独转染mi R-223 mimics的HT-29细胞显著减弱(P0.05)。结论:mi R-223可能通过上调下游靶基因TWIST水平促进结肠癌HT-29细胞的迁移与侵袭。

Study on the Expression of miR-223 in Colon Cancer and the Mechanism of Promoting the Migration and Invasion of Colon Cancer Cells

ABSTRACT Objective: To investigate the expression of microRNA-223 (miR-223) in colon cancer and its influence and mechanism on the invasion and migration of HT-29 cells. Methods: The expression of miR-223 in colon cancer and adjacent tissues was detected. miR-223 mimics (miR-223 mimics, miR-223 mimics group) and microRNA unrelated sequences(miR-223 NC, NC group) were transfected into colon cancer HT-29 cells by liposome transfection.Detection of miR-223 and TWIST expression in transfected cells by Real- time PCR, Western blot was used to detect the protein expression of TWIST, and Tranwell was used to detect the cell migration and invasion. Double luciferase reporter gene was used to detect the effect of miR-223 on the promoter activity of TWIST gene. Transwell migration and invasion assay was used to detect the migration and invasion of human colon cancer cell line HT-29 transfected with miR-223 mimics and TWIST siRNA. Results: The expression of miR-223 was significantly higher than that of the adjacent colon(P<0.05). Compared with the blank control group and miR-223 NC group, the expression of miR-223 in HT-29 cells transfected with miR-223 mimics increased significantly(P<0.05). Compared with the negative control group and the empty transfection group, the number of cells penetrated by miR-223 mimics transfection group increased significantly (P<0.05), and the cell invasion ability of miR-223 mimics transfection group increased significantly(P<0.05). Compared with miR-223 NC group and blank control group, the expression of TWIST gene mRNA and protein in HT-29 cells transfected with miR-223 mimics increased significantly(P<0.05). Double luciferase analysis showed that TWIST was the downstream target gene of miR-223. The migration and invasion ability of HT-29 cells co transfected with TWIST siRNA and miR-223 mimics was significantly weaker than that of HT-29 cells alone transfected with miR-223 mimics(P<0.05). Conclusion: miR-223 may promote the migration and invasion of HT-29 cells by up regulating the level of downstream target gene TWIST.