Fan Rong, Zhong Jie, Zheng Sichang, Wang Zhengting, Xu Ying, Li Shuyi, Zhou Jie, Yuan Fei
Department of Gastroenterology, Shanghai Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, No. 197, Ruijin Er Rd, Shanghai, 200025, China.
Tumour Biol. 2014 May;35(5):4209-17. doi: 10.1007/s13277-013-1551-z. Epub 2013 Dec 29.
The objectives of this study were to detect the expressions of microRNA-218 (miR-218) in human gastrointestinal stromal tumor (GIST) tissues and cells and explore its effects on the biological features of GIST-T1 cells and the expression of its target gene KIT, so as to provide new insights for GIST treatment. Using quantitative real-time polymerase chain reaction (qRT-PCR), we detected the expressions of miR-218 in the tissues and adjacent tissues of GIST and in the GIST cell lines including GIST882, GIST430, GIST48, and GIST-T1. Forty-eight hours after the miR-218 mimic was transfected into the GIST-T1 cells, the expression of miR-218 in the GIST-T1 cells was detected by qRT-PCR. The effect of miR-218 on the GIST-T1 cell viability was detected using MTT. The effect of miR-218 on the proliferation and apoptosis of GIST-T1 cell was analyzed using flow cytometry. Transwell invasion chamber was applied to detect the effect of miR-218 on the invasion of GIST-T1 cells. KIT was identified to be a target gene of miR-218 by the luciferase reporter enzyme system, and the effect of miR-218 on the expression of KIT protein in cells was determined using Western blotting. As shown by qRT-PCR, compared with that in the GIST adjacent tissue, the expressions of miR-218 in the tumor tissue and GIST cell lines were significantly decreased (P < 0.0001). Compared with the control group, the expression of miR-218 increased significantly in GIST-T1 cells transfected with miR-218 mimic for 48 h (P < 0.01). MTT showed that the cell viability decreased significantly after the overexpression of miR-218 in the GIST-T1 cells (P < 0.01). Flow cytometry showed that the cell proliferation index significantly declined after the overexpression of miR-218 (P < 0.01); meanwhile, the apoptosis of cells also significantly increased (P < 0.01). Detection using the Transwell invasion chamber showed that the number of cells passing through the Transwell chamber significantly dropped after the enhanced expression of miR-218 (P < 0.01). Luciferase reporter gene assay showed that, compared with the control group, the relative luciferase activity significantly declined in the miR-218 mimic transfection group (P < 0.01). Compared with the control group, the expression of KIT protein in the GIST-T1 cells transfected with miR-218 mimic for 48 h significantly decreased (P < 0.01). In conclusion, the expression of miR-218 decreases in human GIST tissue and cell lines. miR-218 can negatively regulate the expression of KIT protein and inhibit the proliferation and invasion of GIST cells. Treatment based on the enhanced expression of miR-218 may be a promising strategy for GIST.
本研究旨在检测微小RNA-218（miR-218）在人胃肠道间质瘤（GIST）组织和细胞中的表达，探讨其对GIST-T1细胞生物学特性及靶基因KIT表达的影响，为GIST治疗提供新的思路。采用定量实时聚合酶链反应（qRT-PCR）检测miR-218在GIST组织及其癌旁组织以及GIST882、GIST430、GIST48和GIST-T1等GIST细胞系中的表达。将miR-218模拟物转染至GIST-T1细胞48小时后，用qRT-PCR检测GIST-T1细胞中miR-218的表达。采用MTT法检测miR-218对GIST-T1细胞活力的影响。运用流式细胞术分析miR-218对GIST-T1细胞增殖和凋亡的影响。应用Transwell侵袭小室检测miR-218对GIST-T1细胞侵袭能力的影响。通过荧光素酶报告酶系统鉴定KIT为miR-218的靶基因，采用蛋白质免疫印迹法检测miR-218对细胞中KIT蛋白表达的影响。qRT-PCR结果显示，与GIST癌旁组织相比，肿瘤组织和GIST细胞系中miR-218的表达显著降低（P＜0.0001）。与对照组相比，转染miR-218模拟物48小时后的GIST-T1细胞中miR-218表达显著升高（P＜0.01）。MTT结果表明，GIST-T1细胞中miR-218过表达后细胞活力显著降低（P＜0.01）。流式细胞术显示，miR-218过表达后细胞增殖指数显著下降（P＜0.01）；同时，细胞凋亡也显著增加（P＜0.01）。Transwell侵袭小室检测显示，miR-218表达增强后穿过Transwell小室的细胞数量显著减少（P＜0.01）。荧光素酶报告基因检测显示，与对照组相比，miR-218模拟物转染组的相对荧光素酶活性显著下降（P＜0.01）。与对照组相比，转染miR-218模拟物48小时后的GIST-T1细胞中KIT蛋白表达显著降低（P＜0.01）。综上所述，miR-218在人GIST组织和细胞系中的表达降低。miR-218可负向调节KIT蛋白表达，抑制GIST细胞的增殖和侵袭。基于增强miR-218表达的治疗可能是GIST一种有前景的治疗策略。
