miR-29c通过Tiam1抑制人食管鳞状细胞癌侵袭和转移的研究

doi:10.3969/j.issn.1673-8640.2015.06.022

摘要/Abstract

摘要： 目的

研究微小RNA(miR)-29c通过靶向T淋巴瘤侵袭转移诱导因子1(Tiam1)基因对食管鳞状细胞癌(ESCC)细胞侵袭和迁移的影响。

方法

通过细胞划痕和transwell实验观察miR-29c过表达的ESCC细胞侵袭迁移能力的变化;通过免疫印迹法和sensor reporter实验研究miR-29c对Tiam1的调控作用和靶标位点;在细胞“拯救”实验中通过免疫印迹法检测相关蛋白表达量的变化以及用免疫荧光-激光共聚焦显微镜观察细胞形态的改变,研究miR-29c抑制ESCC细胞侵袭迁移的调节机制。

结果

细胞划痕实验和transwell实验显示miR-29c能抑制ESCC细胞的侵袭迁移;通过生物信息学方法预测miR-29c的靶基因是Tiam1;免疫印迹法和sensor reporter检测结果显示miR-29c通过与Tiam1 3'-非翻译区(UTR)结合降解Tiam1;细胞功能“拯救”实验的细胞划痕、免疫印迹法和免疫荧光结果显示miR-29c能抑制Tiam1的表达,导致激活态Rac1-三磷酸鸟苷(GTP)减少,致使细胞运动状态改变,迁移能力减弱;而人工合成的针对Tiam1的特异siRNA分子siTiam1的干涉作用能“拯救”miR-29C对ESCC细胞迁移能力的抑制。

结论

miR-29C通过降解Tiam1 mRNA,下调了Rac1的激活水平,导致细胞运动状态变化,从而抑制ESCC细胞的侵袭迁移。

关键词: 微小RNA-29c, T淋巴瘤侵袭转移诱导因子1, 食管鳞状细胞癌, 侵袭转移

Abstract: Objective

To research microRNA(miR)-29c inhibiting invasion and migration of esophageal squamous cell carcinoma(ESCC) cell by targeting T-lymphom invasion and metastasis 1(Tiam1).

Methods

Invasion and migration capacity changes of ESCC cell with overexpression of miR-29c were observed through cell scratch and transwell experiments. Western blot analysis and sensor reporter assay were used to research the regulation and target site of miR-29c for Tiam1. In shRNA rescue experiments, Western blot analysis was used to detect the expression change. Immunofluorescence-laser scanning confocal microscope method was used to investigate the mechanisms of miR-29c regulating the invasion and migration capacities of ESCC cell.

Results

Cell scratch and transwell experiment results showed that miR-29c inhibited the invasion and migration of ESCC cell. Tiam1 was predicted to be the target gene of miR-29c through bioinformatics methods. Western blot analysis and sensor reporter assay showed that miR-29c degradated Tiam1 through binding Tiam1 3'-untranslated region (UTR). The analysis of shRNA rescue experiments, including cell scratch experiment, Western blot and immunofluorescence analysis, showed that activated Rac1- guanosine triphosphate(GTP) reduced due to miR-29c inhibiting Tiam1 expression, which resulting migration capacity of ESCC cell diminished, while the inter ference of siTiam1 (artificially synthesized siRNA-specific molecule for Tiam1) can "rescue" the migration capacity of ESCC cell.

Conclusions

miR-29c lowers the level of Rac1 activation through degradating Tiam1 mRNA, resulting the change of cell motility status, thereby inhibiting ESCC cell invasion and migration.

Key words: MicroRNA-29c, T-lymphom invasion and metastasis 1, Esophageal squamous cell carcinoma, Invasion and migration

中图分类号:

R393

褚庆华, 张婕, 孙奋勇. miR-29c通过Tiam1抑制人食管鳞状细胞癌侵袭和转移的研究[J]. 检验医学, 2015, 30(6): 635-640.

CHU Qinghua, ZHANG Jie, SUN Fenyong. Study of miR-29c inhibiting invasion and migration of ESCC cell by targeting Tiam1[J]. Laboratory Medicine, 2015, 30(6): 635-640.

图/表 7

图1 细胞划痕实验注 :(a)细胞划痕实验照片(×200);(b)细胞划痕实验量化图

图2 transwell实验注 :(a)miR-29c转染培养36 h后transwell小室下层细胞结晶紫染色观察(×200);(b)transwell实验量化图

图3 免疫印迹法检测Tiam1蛋白表达注 :(a)miR-29c转染培养48h后Tiam1蛋白表达免疫印迹图像;(b)免疫印迹试验量化图

图4 共转染wtTiam1/miR-29c组、共转染mutTiam1/miR-29c组及对照组sensor reporter实验结果

图5 细胞划痕实验注 :(a)共转染miR-29c/空白对照和共转染miR-29c/siTiam1细胞培养36 h后细胞划痕实验对比观察(×200);(b)细胞划痕实验量化图

图6 细胞“拯救”实验免疫印迹法检测胞内蛋白的表达水平注 :(a)细胞培养48 h后Tiam1和Rac1-GTP蛋白表达免疫印迹图;(b)“拯救”实验免疫印迹量化图;1为阴性对照组;2为单独转染miR-29c组;3为单独转染siTiam1组;4为共转染miR-29c/siTiam1组;与阴性对照组比较,*P<0.05

图7 免疫荧光-激光共聚焦显微镜观察结果(×4 000) 注 :(a)空白对照组;(b)单独转染miR-29c组;(c)miR-29c/siTiam1共转染组

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