Background: To explore the function and the potential molecular mechanism of miR-218 in lung cancer cell.
Methods: The expression of miR-218 mRNA was determined by real-time PCR in lung cancer tissues, adjacent tissues and lung cancer cells. Transwell assay was used to detect the migration and invasion of A549 cell after transfected with Anti-miR-218 or negative control and HC4006 cell after transfected with miR-218 mimics and miR-218 negative control. Targetscan and MiRanda were used to calculate the potential targets of miR-218 and Luciferase reporter assay was performed to identify that the Robo1 was one target genes of miR-218. Transwell assay was used to detect whether miR-218 regulated the invasion of lung cancer cell transfected with anti-miR-218 or negative control via Robo1.
Results: The expression of miR-218 in the lung cancer tissues was significantly lower than that in the adjacent tissues (P<0.05). Inhibition of miR-218 improved the migration and invasion of A549 cell. Overexpression of miR-218 suppressed the migration and invasion of HCC4006 cell. The co-transfection of anti-miR-218 or miR-218 mimics and the Robo1 3'UTR increased or reduced the luciferase activity of Robo1 compared with the control group (P<0.05). Inhibition of miR-218 and Robo1 recovered the invaded cells of A549. Overexpression of miR-218 and inhibition of Robo1 reduced the number of the invased cells of HCC4006. These results suggested that miR-218 banded Robo1 directly and inhibited lung cancer cell invasion by targeting Robo1.
Conclusions: MiR-218 inhibited the migration and invasion of lung cancer cells through regulating Robo1 expression. .
背景与目的 本研究旨在探讨肺癌中miR-218的表达，研究miR-218在肺癌细胞中的功能及其可能的分子机制。方法 应用实时荧光定量 PCR（qRT-PCR）检测15例肺癌组织和15例癌旁组织中miR-218的表达。在肺癌细胞A549中转染miR-218的抑制物（Anti-miR-218），在肺癌细胞HCC4006中转染miR-218的模拟物后，用Transwell实验检测细胞的迁移侵袭能力的变化。用Targetscan和MiRanda软件预测miR-218的可能靶点，转染miR-218的抑制物及模拟物后用qRT-PCR和Western blot检测Robo1的mRNA和蛋白表达水平。用双荧光素酶报告基因方法鉴定miR-218和Robo1的调控关系。用Anti-miR-218、miR-218模拟物或阴性对照与Si-Robo1或Si-NC同时转染细胞，应用Transwell实验检测转染后细胞的侵袭迁移能力的变化。结果 与癌旁组织比较，肺癌组织中miR-218在肺癌组织中表达水平显著降低（P<0.01）。在A549细胞中转染miR-218的抑制物，能够显著降低miR-218的表达，促进了细胞的迁移侵袭。在HCC4006中转染miR-218的模拟物能够显著提高miR-218的表达，同时抑制了细胞的迁移侵袭能力。利用生物信息学预测出在Robo1的3′UTR区有miR-218的结合位点，双荧光素酶报告基因实验进一步证实miR-218能够调控Robo1的转录活性。抑制miR-218能够提高Robo1的表达；过表达miR-218显著降低Robo1的表达，且miR-218能够通过调控Robo1影响细胞的迁移侵袭。结论 MiR-218在肺癌组织中呈现低表达状态，miR-218可能是通过抑制Robo1 的表达抑制肺癌细胞侵袭迁移。.