TY - JOUR
T1 - 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
AU - Zhao, Yuan
AU - Ling, Zhiqiang
AU - Hao, Yubin
AU - Pang, Xiaowu
AU - Han, Xianlin
AU - Califano, Joseph A.
AU - Shan, Liang
AU - Gu, Xinbin
N1 - Funding Information:
We thank Mr. Wei Sha and Ms. Dan Zhang for technical support, and Dr. Paul Wang for help in animal imagingThis work was supported in part by NCI/NIH P20CA118770, NIDCR/NIH 1R15DE025138-01, NIDCR/NIH 1R25DE025778-01, and NIH/NCRR/RCMI/4, G12 RR003048 at Howard University
PY - 2017
Y1 - 2017
N2 - 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-22miR124) and a mock vector-transfected subline (JHU-22vec) by transfecting the mimic of miR-124 into JHU-22 cancer cells. Restored expression of miR-124 in JHU-22miR124 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-22miR124 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 sitemutated 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 antiapoptotic Bcl-2 and Bcl-xL, and enhanced expression of cytochrome c and caspase proteins in JHU-22miR124 compared with JHU-22vec 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.
AB - 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-22miR124) and a mock vector-transfected subline (JHU-22vec) by transfecting the mimic of miR-124 into JHU-22 cancer cells. Restored expression of miR-124 in JHU-22miR124 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-22miR124 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 sitemutated 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 antiapoptotic Bcl-2 and Bcl-xL, and enhanced expression of cytochrome c and caspase proteins in JHU-22miR124 compared with JHU-22vec 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.
KW - BcL-2 family
KW - Ceramide
KW - Head and neck cancer
KW - MiR-124
KW - Sphingosine kinase 1
UR - http://www.scopus.com/inward/record.url?scp=85017566441&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85017566441&partnerID=8YFLogxK
U2 - 10.18632/oncotarget.15334
DO - 10.18632/oncotarget.15334
M3 - Article
C2 - 28212569
AN - SCOPUS:85017566441
SN - 1949-2553
VL - 8
SP - 25005
EP - 25020
JO - Oncotarget
JF - Oncotarget
IS - 15
ER -