MicroRNA-34a inhibits uveal melanoma cell proliferation and migration through downregulation of c-Met.
OBJECTIVE
MicroRNAs (miRNAs) are endogenously expressed, noncoding, small RNAs that inhibit protein translation through binding to target mRNAs. Recent studies have demonstrated that miRNAs can regulate tumor cell proliferation and migration. MicroRNA-34a (miR-34a), a potential key effector of the p53 tumor-suppressor gene, was studied as a potential tumor suppressor in uveal melanoma.


METHODS
Northern blot analysis was performed to detect the expression level of miR-34a in uveal melanoma cells and melanocytes. Subsequently, melanoma cell proliferation and migration were examined by MTS cell proliferation and transwell migration assays, respectively. The target of miR-34a was predicted by bioinformatics and confirmed using a luciferase assay. In addition, expression of c-Met and cell cycle-related proteins was determined by Western blotting and immunofluorescence after the introduction of miR-34a.


RESULTS
miR-34a is actively expressed in melanocytes but not in uveal melanoma cells based on Northern blot analysis. Transfection of miR-34a into uveal melanoma cells led to a significant decrease in cell growth and migration. After identification of two putative miR-34a binding sites within the 3' UTR of the human c-Met mRNA, miR-34a was shown to suppress luciferase activity using HEK293 cells with a luciferase reporter construct containing the binding sites. miR-34a was confirmed to downregulate the expression of c-Met protein by Western blotting and immunofluorescence. Furthermore, the introduction of miR-34a downregulated phosphorylated Akt and cell cycle-related proteins.


CONCLUSIONS
These results demonstrate that miR-34a acts as a tumor suppressor in uveal melanoma cell proliferation and migration through the downregulation of c-Met.
Yan
Dongsheng
D
School of Ophthalmology and Optometry, Eye Hospital, Wenzhou Medical College, Wenzhou, Zhejiang, People's Republic of China.
Zhou
Xiangtian
X
Chen
Xiaoyan
X
Hu
Dan-Ning
DN
Dong
Xiang Da
XD
Wang
Jiao
J
Lu
Fan
F
Tu
LiLi
L
Qu
Jia
J
D015152:Blotting, Northern; D015153:Blotting, Western; D018797:Cell Cycle Proteins / Q000378:metabolism; D002465:Cell Movement / Q000502:physiology*; D049109:Cell Proliferation; D015536:Down-Regulation; D015972:Gene Expression Regulation, Neoplastic / Q000502:physiology; D006801:Humans; D008544:Melanocytes / Q000378:metabolism; D008545:Melanoma / Q000378:metabolism / Q000473:pathology*; D035683:MicroRNAs / Q000502:physiology*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D019859:Proto-Oncogene Proteins c-met / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured; D014604:Uveal Neoplasms / Q000378:metabolism / Q000473:pathology*
D018797:Cell Cycle Proteins; C522425:MIRN34 microRNA, human; D035683:MicroRNAs; D019859:Proto-Oncogene Proteins c-met; D051057:Proto-Oncogene Proteins c-akt