Comprehensive profiling of novel microRNA-9 targets and a tumor suppressor role of microRNA-9 via targeting IGF2BP1 in hepatocellular carcinoma

// Jiangbo Zhang 1, 2, * , Jin Cheng 1, 3, * , Zhenzhen Zeng 1, * , Yongfeng Wang 1 , Xiaojun Li 1 , Qing Xie 4 , Junqiao Jia 1 , Ying Yan 1 , Zhengyang Guo 1 , Jian Gao 1 , Mingjie Yao 1 , Xiangmei Chen 1 , Fengmin Lu 1 1 Department of Microbiology & Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100191, China 2 Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Guangzhou 510060, China 3 Institute of Sports Medicine, Beijing Key Laboratory of Sports Injuries, Peking University Third Hospital, Beijing 100191, China 4 Department of Clinical Laboratory, Beijing Shijitan Hospital, Capital Medical University, Beijing 100038, China * These authors have contributed equally to this work Correspondence to: Fengmin Lu, e-mail: lu.fengmin@hsc.pku.edu.cn Xiangmei Chen, e-mail: xm_chen6176@bjmu.edu.cn Keywords: microRNA-9, hepatocellular carcinoma, hypermethylation, IGF2BP1, AKT&ERK Received: May 03, 2015      Accepted: October 09, 2015      Published: October 19, 2015 ABSTRACT MicroRNA-9 (miR-9) dysregulation is implicated in a variety of human malignancies including hepatocellular carcinoma (HCC), but its role remains contradictory. In this study, we explored the expression and methylation status of miR-9 in HCC samples, as well as the tumor-related functions of miR-9 in vitro . Bioinformatics analysis, array-based RNA expression profile, and literature retrieval were used to identify miR-9 targets in HCC. The potential downstream candidates were then validated by luciferase reporter assay, real-time quantitative PCR, and western blot or enzyme linked immunosorbent assay (ELISA). The expression status and clinicopathologic significances of miR-9 target genes in clinical samples were further explored. The results showed that miR-9 was frequently downregulated in primary HCC. Its silencing was largely contributed by a high frequency (42.5%) of mir-9-1 hypermethylation, which was correlated with bigger tumor size ( P = 0.0234). In vitro functional studies revealed that miR-9 restoration retarded HCC cell proliferation and migration. IL-6, AP3B1, TC10, ONECUT2, IGF2BP1, MYO1D, and ANXA2 were confirmed to be miR-9 targets in HCC. Among them, ONECUT2, IGF2BP1, and ANXA2 were confirmed to be aberrantly upregulated in HCC. Moreover, upregulation of ONECUT2, IGF2BP1, and IL-6 were significantly associated with poor post-surgery prognosis ( P = 0.0458, P = 0.0037 and P = 0.0461, respectively). Mechanically, miR-9 plays a tumor suppressive role partially through a functional miR-9/IGF2BP1/AKT&ERK axis. Our study suggests that miR-9 functions as a tumor suppressor in HCC progression by inhibiting a series of target genes, including the newly validated miR-9/IGF2BP1/AKT&ERK axis, thus providing potential therapeutic targets and novel prognostic biomarkers for HCC patients.