Silencing of miRNA-148a by hypermethylation activates the integrin-mediated signaling pathway in nasopharyngeal carcinoma

// Hsin-Pai Li 1,2,3,* , Hsin-Yi Huang 1,* , Yi-Ru Lai 1 , Jing-Xuan Huang 1 , Kai-Ping Chang 6 , Chuen Hsueh 2,4,5 and Yu-Sun Chang 1,2,3 1 Graduate Institute of Biomedical Sciences, Chang Gung University, Taoyuan, Taiwan, Republic of China (ROC) 2 Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan, Republic of China (ROC) 3 Department of Microbiology and Immunology, School of Medicine, Chang Gung University, Taoyuan, Taiwan, Republic of China (ROC) 4 Pathology Core, Chang Gung University, Taoyuan, Taiwan, Republic of China (ROC) 5 Department of Pathology, Chang Gung Memorial Hospital at Lin-Kou, Taoyuan, Taiwan, ROC 6 Otolaryngology-Head and Neck Surgery, Chang Gung Memorial Hospital at Lin-Kou, Taoyuan, Taiwan, ROC * These authors contributed equally to this work Correspondence: Hsin-Pai Li , email: // Keywords : miR-148a, DNA methylation, migration, nasopharyngeal carcinoma, integrin-signaling pathway Received : May 22, 2014 Accepted : July 31, 2014 Published : July 31, 2014 Abstract MicroRNAs (miRNAs) play a pivotal role in carcinogenesis by suppressing oncogenes or tumor suppressor genes. Various studies have identified numerous miRNAs and their diverse targets; however, the consequences of dysregulated miRNAs in nasopharyngeal carcinoma (NPC) remain unclear. For this study, we found that miR-148a is downregulated through hypermethylation in NPC biopsies and NPC cell lines compared with adjacent normal and NP cells respectively. Promoter assays demonstrated that upstream stimulatory factor 1 (USF1) is a crucial transcription factor that activates miR-148a promoter activity. EMSA assays confirmed that purified USF1 binds better toward the unmethylated than the methylated CG-containing USF1 consensus probe. The ectopic expression of miR-148a inhibits cell migration in NPC cells through the suppression of integrin-mediated signaling by targeting VAV2, WASL and ROCK1. Biochemical and functional assays provided supporting evidence that these 3 genes are the downstream targets of miR-148a in NPC cells. Furthermore, immunohistochemical staining and Western blotting analysis revealed that the 3 oncogenic targets of miR-148a were overexpressed in NPC biopsies, suggesting that the inactivation of miR-148a caused by DNA methylation promotes NPC progression. Overall, our findings revealed that miR-148a can act as tumor suppressor miRNA and serve as a biomarker as well as a therapeutic target for NPC.