MiR-4324通过作用于RacGAP1抑制人RCC细胞的增殖  

Effects of miR-4324 on inhibiting the growth of human renal cancer cells by suppressing RacGAP1

作　　者：张庆松 李文贤[1] 郭磊[1] 刘勇[1] 牛海涛[1] 毛昕[1] ZHANG Qingsong;LI Wenxian;GUO Lei;LIU Yong;NIU Haitao;MAO Xin(Department of Urology,Affiliated Hospital of Qingdao University,Qingdao,Shandong,260000,China)

机构地区：[1]青岛大学附属医院泌尿外科,山东青岛260000

出　　处：《临床泌尿外科杂志》2021年第12期954-960,共7页Journal of Clinical Urology

摘　　要：目的:观察miR-4324对人RCC细胞生长的影响及作用机制。方法:实时荧光定量聚合酶链反应(qPCR)检测RCC组织和癌旁组织中RacGAP1 mRNA和miR-4324的表达;通过细胞瞬时转染的方式在人RCC细胞中转染miR-4324模拟物和过表达RacGAP1质粒;qPCR检测细胞miR-4324潜在靶基因RacGAP1 mRNA的表达,Western印迹法检测RacGAP1蛋白的表达;采用细胞增殖实验检测细胞增殖能力;采用EdU实验检测细胞增殖活力。结果:qPCR结果显示,与肾小管上皮细胞和正常肾组织相比,RCC细胞和RCC组织中miR-4324表达明显降低,RacGAP1 mRNA表达明显增高;与对照组相比,转染miR-4324分别抑制786-O和CAKI-1细胞中RacGAP1 mRNA的表达,Western蛋白印迹检测结果验证了miR-4324对RacGAP1基因表达的调控;而RCC细胞786-O和CAKI-1细胞中共转染miR-4324模拟物和过表达RacGAP1质粒后,细胞中RacGAP1表达恢复。MTT结果显示,与阴性对照组相比,转染miR-4324模拟物后,细胞增殖能力明显下降;共转染miR-4324模拟物和过表达RacGAP1质粒后,细胞增殖能力逐渐增强。EdU结果显示,与阴性对照组相比,转染miR-4324模拟物的细胞EdU阳性细胞百分比明显减低,表明细胞增殖能力明显下降。双荧光素酶报告基因实验结果显示,在RCC细胞786-O和CAKI-1细胞中miR-4324能够与RacGAP1基因3’UTR特定序列结合从而靶向调控RacGAP1基因的表达。结论:miR-4324能通过靶向结合RacGAP1的3’UTR特定序列抑制其表达,从而显著抑制RCC细胞的增殖。Objective: To investigate the effects of miR-4324 on the growth of human renal cell carcinoma and its mechanism. Methods: Transient cell transfection was used to overexpress miR-4324 and RacGAP1 gene in human renal cancer cells, and real-time fluorescent quantitative polymerase chain reaction(qPCR) was used to detect the expression of RacGAP1, a potential target gene of miR-4324. RacGAP1 protein expression was detected by Western blotting, and cell proliferation assay was used to detect cell proliferation ability. EdU experiment was used to detect cell proliferation activity. Results: QPCR results showed that compared with renal tubular epithelial cells and normal kidney tissues, the expression of miR-4324 in renal cancer cells and renal cancer tissues were significantly reduced, while the expression of RacGAP1 mRNA was significantly increased. Compared with the control group, miR-4324 inhibited the expression of RacGAP1 mRNA in 786-O and CAKI-1 cells, respectively. Western blotting results verified the regulation of miR-4324 on the expression of RacGAP1 gene, while co-transfected miR-4324 with RacGAP1 in renal cancer cells, the expression of RacGAP1 was restored. The results of cell proliferation assay showed that the cell proliferation ability decreased significantly after miR-4324 transfection compared with the negative control group. After miR-4324 and RacGAP1 co-transfection, the cell proliferation ability gradually increased. EdU results demonstrated that compared with the negative control group, the percentage of EdU-positive cells in miR-4324 group was significantly reduced. The dual-luciferase reporter assay showed that miR-4324 can bind to the 3’UTR specific sequence of RacGAP1 gene in renal cancer cell 786-O and CAKI-1 cells to target and regulate the expression of RacGAP1 gene. Conclusion: MiR-4324 can inhibit the expression of RacGAP1 by targeting the specific sequence of 3’UTR, thereby significantly inhibiting the growth of renal cancer cells.

关 键 词：miR-4324 RacGAP1基因 3’UTR 肾细胞癌 

分 类 号：R737.ll[医药卫生—肿瘤]