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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Acknowledgments
This work was supported by National Natural Science Foundation of China (No. 81372140, 81301688, 81272192, 81171882), PhD. Programs Foundation of Ministry of Education of China (No. 20130162110050 and 20130162120093), Natural Science Foundation of Hunan Province (No. 13JJ4028), Project of the Department of Science and Technology of Hunan Province (No. 2013FJ6003), Program for New Century Excellent Talents in University (NCET-11-0527), Postdoctoral Foundation of Central South University (No. 131425), and 125 Talent Project of the third Xiangya Hospital of Central South University.
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432_2014_1874_MOESM1_ESM.tif
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_MOESM2_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)
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Liu, R., Xie, H., Luo, C. et al. Identification of FLOT2 as a novel target for microRNA-34a in melanoma. J Cancer Res Clin Oncol 141, 993–1006 (2015). https://doi.org/10.1007/s00432-014-1874-1
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DOI: https://doi.org/10.1007/s00432-014-1874-1