Identification of FLOT2 as a novel target for microRNA-34a in melanoma.
OBJECTIVE
To confirm whether flotillin 2 (FLOT2) is a direct target of miR-34a and miR-34a/FLOT2 pathway plays a key role in melanoma proliferation and metastasis.


METHODS
First, miR-34a and FLOT2 expressions were both detected in human tissues and cell lines by qRT-PCR. Then, after transfection of mimics/inhibitor of miR-34a into melanoma cell lines, MTT, colony formation, scratch migration assays and transwell invasion assays were performed to evaluate the impact of miR-34a on cell proliferation and metastasis. Western blot, qRT-RCR and dual luciferase reporter gene assays were carried out to confirm whether FLOT2 is a direct target gene of miR-34a. In functional recovery experiments, proliferation and metastasis ability of WM35 and WM451 was tested after being co-transfected with miR-34a inhibitor/si-FLOT2 or miR-34a mimics/FLOT2 cDNA to confirm that FLOT2 is downregulated by miR-34a.


RESULTS
The miR-34a significantly lower-expressed in metastasis melanoma tissues compared to in situ melanoma, nevi and normal skin whereas FLOT2 has an opposite trend. The level of miR-34a and FLOT2 in different melanoma cell lines was also tested and found that metastatic melanoma cell lines has lower miR-34a expression and higher FLOT2 expression compare to in situ melanoma cell line. MiR-34a overexpression profoundly inhibits WM451 cell proliferation and metastasis, whereas miR-34a reduction had a promoting effect to proliferation and metastasis of WM35. Results of Western blot, qRT-RCR and dual luciferase reporter gene assays revealed that FLOT2 is a direct target gene of miR-34a. Furthermore, overexpression/blockage of FLOT2 could attenuate effect of miR-34a overexpression/inhibition which indicated miR-34a suppresses melanoma biological behavior partially through FLOT2 inhibition.


CONCLUSIONS
Our study confirmed that miR-34a is involved in the tumor inhibition of melanoma by directly targeting FLOT2 gene. This finding provides potential novel strategies for therapeutic interventions of melanoma.
Department of Plastic and Reconstructive Surgery, Third Xiangya Hospital of Central South University, Changsha, 410013, China.
Rui
R
Liu
Huiqing
H
Xie
Chengqun
C
Luo
Zizi
Z
Chen
Xiao
X
Zhou
Kun
K
Xia
Xiang
X
Chen
Ming
M
Zhou
Peiguo
P
Cao
Ke
K
Cao
Jianda
J
Zhou
D015153:Blotting, Western; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D015536:Down-Regulation / Q000187:drug effects; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D008545:Melanoma / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology*; D008565:Membrane Proteins / Q000187:drug effects / Q000378:metabolism*; D035683:MicroRNAs / Q000378:metabolism* / Q000494:pharmacology; D058990:Molecular Targeted Therapy / Q000379:methods; D060888:Real-Time Polymerase Chain Reaction; D012878:Skin Neoplasms; D015854:Up-Regulation / Q000187:drug effects; D000096142:Cutaneous Malignant Melanoma
C522425:MIRN34 microRNA, human; D008565:Membrane Proteins; D035683:MicroRNAs; C106354:flotillins