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01.06.2015 | Original Article – Cancer Research | Ausgabe 6/2015

Journal of Cancer Research and Clinical Oncology 6/2015

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

Zeitschrift:
Journal of Cancer Research and Clinical Oncology > Ausgabe 6/2015
Autoren:
Rui Liu, Huiqing Xie, Chengqun Luo, Zizi Chen, Xiao Zhou, Kun Xia, Xiang Chen, Ming Zhou, Peiguo Cao, Ke Cao, Jianda Zhou
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1007/​s00432-014-1874-1) contains supplementary material, which is available to authorized users.

Abstract

Purpose

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 texted 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.

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Zusatzmaterial
Supplement Figure 1. (a) Sequence of FLOT2-3’-UTR. (b) Possible bind sites of miR-34a to FLOT2-3’-UTR. (c) Primer Sequences of Mutations (TIFF 1303 kb)
432_2014_1874_MOESM1_ESM.tif
Supplement Figure 2. PAR-1 expression (a) Western blot results of PAR-1 in WM451 transfected with siFLOT2 and the negative control. (b) Western blot results of PAR-1 in WM451 transfected with miR-34a-mimics and the negative control. (ns: p > 0.05;*:p < 0.05,**:p < 0.01,***:p < 0.001) (TIFF 471 kb)
432_2014_1874_MOESM2_ESM.tif
Literatur
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