MicroRNA-375 Suppresses the Tumor Aggressive Phenotypes of Clear Cell Renal Cell Carcinomas through Regulating YWHAZ
Xiang Zhang1, Nai-Dong Xing2, Cheng-Jun Lai3, Rui Liu4, Wei Jiao1, Jue Wang5, Jie Song6, Zhong-Hua Xu1
1 Department of Urology, Qilu Hospital of Shandong University, Jinan, Shandong 250012, China
2 Teaching and Research Institute of Urology, School of Medicine, Shandong University, Jinan, Shandong 250012, China
3 Department of Urology, Qilu Hospital of Shandong University, Jinan, Shandong 250012; Department of Urology, Qingyun People's Hospital, Dezhou, Shandong 253700, China
4 Department of the First Operation Room, Qilu Hospital of Shandong University, Jinan, Shandong 250012, China
5 Central Laboratory, The Second Hospital of Shandong University, Jinan, Shandong 250012, China
6 Department of Urology, Qilu Hospital of Shandong University; Medicaid and Health Care Service Office, Qilu Hospital of Shandong University, Jinan, Shandong 250012, China
Dr. Jie Song
Department of Urology, Qilu Hospital of Shandong University, Wenhua West Road 44#, Jinan, Shandong 250012
Source of Support: None, Conflict of Interest: None
Background: MicroRNAs (miRNAs) are key regulators during tumor initiation and progression. MicroRNA-375 (MiR-375) has been proven to play a tumor-suppressive role in various types of human malignancies; however, its biological role in clear cell renal cell carcinoma (ccRCC) remains unclear. The purpose of this study was to explore the biologic role as well as the underlying mechanism of miR-375 in ccRCC progression.
Methods: Quantitative polymerase chain reaction (qPCR) was applied to test the expression of miR-375 in tissues and cell lines by t-test. Functional experiments were used to investigate the biological role of miR-375 utilizing a gain-of-function strategy. The target of miR-375 was investigated by bioinformatic analysis and further verified by luciferase reporter assay, qPCR, Western blotting, and functional experiments in vitro.
Results: Our study demonstrated that miR-375 was significantly downregulated in ccRCC tissues (cancer vs. normal, 0.804 ± 0.079 vs. 1.784 ± 0.200, t = 5.531 P < 0.0001) and cell lines, and loss of miR-375 expression significantly associated with advanced Fuhrman nuclear grades (Grade III and IV vs. Grade I and II, 1.000 ± 0.099 vs. 1.731 ± 0.189, t = 3.262 P = 0.003). Functional studies demonstrated that miR-375 suppressed ccRCC cell proliferation, migration, and invasion (all P < 0.05 in both 786-O and A498 cell lines). Multiple miRNA target prediction algorithms indicated the well-studied oncogene YWHAZ as a direct target of miR-375, which was further confirmed by the luciferase reporter assay, qPCR, and Western blotting. Moreover, restoration of YWHAZ could rescue the antiproliferation effect of miR-375.
Conclusions: The data provide the solid evidence that miR-375 plays a tumor-suppressive role in ccRCC progression, partially through regulating YWHAZ. This study expands the antitumor profile of miR-375, and supports its role as a potential therapeutic target in ccRCC treatment.
结果：本实验证实miR-375在肾癌临床标本(肾癌组织 比 正常肾组织, 0.804 ± 0.079 比1.784 ± 0.200, P <0.0001) 和细胞系中均显著表达下调，且miR-375的低表达与更高的肾癌Fuhrman细胞核分级显著相关(高核分级 vs. 低核分级, 1.000±0.099 比 1.731±0.189, P =0.003)。功能学实验证实miR-375过表达可显著抑制ccRCC细胞增殖、迁移和侵袭(所有P<0.05)。多种miRNA靶点预测软件提示原癌基因YWHAZ是miR-375的靶基因，我们进一步应用荧光素酶报告实验、qPCR和蛋白印迹实验证实miR-375对YWHAZ的调控。并且，挽救实验证实YWHAZ再表达可逆转miR-375抑制细胞增殖的作用。