Identification of FLOT2 as a novel target for microRNA-34a in melanoma.

Abstract

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. 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. 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. 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.