微小RNA-224靶向Tribbles同源蛋白1调控前列腺癌细胞DU145的迁移和侵袭

Inhibitory effects of microRNA -224 on migration and invasion of human prostate cancer cell line DU145 by targeting Tribbles homologous protein 1

目的观察微小RNA-224（miR-224）对前列腺癌细胞DUl45迁移和侵袭能力的影响以及对靶基因Tribbles同源蛋白1（TRIBl）表达的影响。方法通过慢病毒转染前列腺癌细胞株DU145使其过表达miR-224，利用划痕实验和Transwell小室检测细胞迁移和侵袭能力；采用实时定量聚合酶链反应（Real—timePCR）和Westernblot检测细胞TRIBl表达水平的变化，并通过荧光素酶实验验证miR-224与TRIBl基因的直接调控关系。设计拯救实验并重新利用划痕实验和Transwell小室检测细胞迁移和侵袭。结果划痕实验24h后DUl45—224和DU145-vector组细胞的迁移细胞数目分别为（103．2±20．6）、（189．8±34．7）个，miR-224过表达组细胞迁移能力明显下降（P〈0．01）；Transwell细胞侵袭实验显示，DUl45-224组和DU145-vector组细胞分别为（140．8±13．1）、（283．4±15．4）个，组间差异均有统计学意义（P〈0．01）。过表达miR-224后，DUl45细胞TRIBl的蛋白表达下调（P〈0．05）。荧光素酶报告实验结果显示实验组荧光素酶活性值为0．42±0．06，比对照组（0．97±0．09）显著降低（P〈0．01），提示miR-224能明显抑制TRIB1—3’端非编码区域（3’-UTR）的荧光素酶活性。拯救实验结果表明在miR-224过表达的DUl45细胞中进一步转入高表达TRIBl质粒后，迁移细胞数目为（203．2±31．2）个、侵袭细胞数目为（328．6±25．9）个，对比单纯miR-224过表达DUl45细胞[迁移细胞数目为（73．2-4-17．8）个；侵袭细胞数目为（101．2±13．1）个]，迁移和侵袭能力增强，组间差异均有统计学意义（P〈0．01）。结论过表达miR-224可能通过靶向降低TRIBl基因的蛋白表达，抑制前列腺癌细胞的迁移和侵袭能力。

Objective To explore the influence of microRNA - 224 （ miR - 224 ） on migration and invasion of human prostate cancer cell line DU145 and evaluate the correlation between miR -224 and Tribbles homologous protein 1 （TRIB1）. Methods After transfection of mieroRNA ]entivector into DU145 cells, cell migration and invasion were assessed by wound healing assay and Transwell chamber assay. The mRNA and protein expression level of TRIB1 was evaluated by real - time quantitative polymerase chain re- action（ Real- time PCR） and Western blotting respectively. Luciferase reporter assay was used to confirm the relationship between miR - 224 and TRIB1. Rescue assay was performed to re - investigate the migra- tion and invasion of DU145 cells. Results Wound healing assay results showed that in miR - 224 transfec- tion group and empty piasmid group, the number of migrating DU145 cells was （ 103.2 -＋ 20. 6 ） and （ 189.8 -＋ 34. 7 ）, respectively, showing a significant difference between two groups （P 〈 0. 01 ）. Transwell chamber assay revealed that the number of invasive DU145 cells in miR -224 transfection group （ 140. 8 ＋- 13.1 ） was significantly less than that in control group （ 283.4 -＋ 15.4, P 〈 0. 01 ） , indicating that miR - 224 could suppress migration ability of DU145cells. Eetopic overexpression of miR - 224 caused significant decrease of protein level of TRIB1 （P 〈 0. 05 ）. In addition, luciferase assay showed that the luciferase activity of the DU145 cells ＇after transfection of 3＇ untranslated region （3 ＇ - UTR） - TRIBlwt FLuci vector （0. 42± 0. 06） was reduced as compared with 3＇ - UTR - TRIBlmut FLuci vector group （0. 97 ±0. 09 ）, suggesting the normalized TRIB1 -3＇UTR luciferase activity was significantly reduced by miR - 224 （ P 〈 0. 01 ）. Rescue assay identified that the re - expression of TRIB1 could respectively rescue the tumor suppressive effects of miR -224 on cell migration （203.2 ±31.2 vs. 73.2 ±17.8, P 〈0. 01 ） and invasion （328.6±25.9 vs. 101.2 ±13.1, P〈0. 01） in DU145 cells. Conclusion Overexpressed miR - 224 can inhibit migration and invasion of prostate cancer cells by downregulating TRIB1, indicating its tumor suppressive function in prostate cancer.