MiR-124 acts as a tumor suppressor by inhibiting the expression of sphingosine kinase 1 and its downstream signaling in head and neck squamous cell carcinoma

// Yuan Zhao 1 , Zhiqiang Ling 2 , Yubin Hao 1 , Xiaowu Pang 1 , Xianlin Han 3 , Joseph A. Califano 4, * , Liang Shan 1, 5 , Xinbin Gu 1, 6 1 Department of Oral Pathology, College of Dentistry, Howard University, Washington DC, USA 2 Zhejiang Cancer Hospital, Zhejiang Cancer Research Institute, Hangzhou, Zhejiang, China 3 Sanford Burnham Prebys Medical Discovery Institute, Orlando, Florida, USA 4 Department of Otolaryngology, Head and Neck Surgery, Johns Hopkins University, San Diego, California, USA 5 Department of Radiology, College of Medicine, Howard University, Washington DC, USA 6 Cancer Center, Howard University, Washington DC, USA * Current address: Department of Surgery, Division of Otolaryngology, US San Diego Health, San Diego, California, USA Correspondence to: Xinbin Gu, email: xgu@howard.edu Keywords: miR-124, sphingosine kinase 1, ceramide, BcL-2 family, head and neck cancer Received: September 24, 2016     Accepted: January 10, 2017     Published: February 15, 2017 ABSTRACT By analyzing the expression profile of microRNAs in head and neck squamous cell carcinomas (HNSCC), we found that the expression level of miR-124 was 4.59-fold lower in tumors than in normal tissues. To understand its functions, we generated a miR-124-expressing subline (JHU-22 miR124 ) and a mock vector-transfected subline (JHU-22 vec ) by transfecting the mimic of miR-124 into JHU-22 cancer cells. Restored expression of miR-124 in JHU-22 miR124 cells led to reduced cell proliferation, delayed colony formation, and decreased tumor growth, indicating a tumor-suppressive effect of miR-124. Subsequent target search revealed that the 3′-UTR of SphK1 mRNA carries a complementary site for the seed region of miR-124. SphK1 was also detected to be overexpressed in HNSCC cell lines, but down-expressed in JHU-22 miR124 cells and tumor xenografts. These results suggest that SphK1 is a target of miR-124. To confirm this finding, we constructed a 3'-UTR-Luc-SphK1 vector and a binding site-mutated luciferase reporter vector. Co-transfection of 3'-UTR-Luc-SphK1 with miR-124 expression vector exhibited a 9-fold decrease in luciferase activity compared with mutated vector, suggesting that miR-124 inhibits SphK1 activity directly. Further studies on downstream signaling demonstrated accumulation of ceramide, increased expression of the pro-apoptotic Bax, BAD and PARP, decreased expression of the anti-apoptotic Bcl-2 and Bcl-xL, and enhanced expression of cytochrome c and caspase proteins in JHU-22 miR124 compared with JHU-22 vec cells and tumor xenografts. We conclude that miR-124 acts as a tumor suppressor in HNSCC by directly inhibiting SphK1 activity and its downstream signals.