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.
Similar content being viewed by others
References
Balch CM et al (2013) Age as a prognostic factor in patients with localized melanoma and regional metastases. Ann Surg Oncol 20:3961–3968. doi:10.1245/s10434-013-3100-9
Bhatia S, Thompson JA (2012) Systemic therapy for metastatic melanoma in 2012: dawn of a new era. J Natl Compr Cancer Netw JNCCN 10:403–412
Bhullar RP, Bhullar A, Vanaki SS, Puranik RS, Sudhakara M, Kamat MS (2012) Primary melanoma of oral mucosa: A case report and review of literature. Dent Res J 9:353–356
Boyle GM et al (2011) Melanoma cell invasiveness is regulated by miR-211 suppression of the BRN2 transcription factor. Pigment Cell Melanoma Res 24:525–537. doi:10.1111/j.1755-148X.2011.00849.x
Cancer Facts and Figures (2013). American Cancer Society. http://www.cancer.org/acs/groups/content/@epidemiologysurveilance/documents/document/acspc-036845.pdf
Cao K et al (2014a) SiRNA-mediated flotillin-2 (Flot2) downregulation inhibits cell proliferation, migration, and invasion in gastric carcinoma cells. Oncol Res 21:271–279. doi:10.3727/096504014X13946737557031
Cao M, Li Y, Lu H, Meng Q, Wang L, Cai L, Dong X (2014b) miR-23a-mediated migration/invasion is rescued by its target, IRS-1, in non-small cell lung cancer cells. J Cancer Res Clin Oncol. doi:10.1007/s00432-014-1725-0
Chakraborty C, George Priya Doss C, Bandyopadhyay S (2013) miRNAs in insulin resistance and diabetes-associated pancreatic cancer: the ‘minute and miracle’ molecule moving as a monitor in the ‘genomic galaxy’ Curr Drug Targets 14:1110–1117
Chen Y, Zhu X, Zhang X, Liu B, Huang L (2010) Nanoparticles modified with tumor-targeting scFv deliver siRNA and miRNA for cancer therapy. Mol Therapy J Am Soc Gene Therapy 18:1650–1656. doi:10.1038/mt.2010.136
Choi SE et al (2013) Elevated microRNA-34a in obesity reduces NAD + levels and SIRT1 activity by directly targeting NAMPT. Aging Cell 12:1062–1072. doi:10.1111/acel.12135
Doherty SD, Prieto VG, George S, Hazarika P, Duvic M (2006) High flotillin-2 expression is associated with lymph node metastasis and Breslow depth in melanoma. Melanoma Res 16:461–463. doi:10.1097/01.cmr.0000222592.75858.20
Eichelser C, Flesch-Janys D, Chang-Claude J, Pantel K, Schwarzenbach H (2013) Deregulated serum concentrations of circulating cell-free microRNAs miR-17, miR-34a, miR-155, and miR-373 in human breast cancer development and progression. Clin Chem 59:1489–1496. doi:10.1373/clinchem.2013.205161
Garofalo M et al (2013) MiR-34a/c-Dependent PDGFR-alpha/beta downregulation inhibits tumorigenesis and enhances TRAIL-induced apoptosis in lung cancer. PLoS ONE 8:e67581. doi:10.1371/journal.pone.0067581
Gocze K et al (2013) Unique microRNA expression profiles in cervical cancer. Anticancer Res 33:2561–2567
Greenberg E et al (2011) Regulation of cancer aggressive features in melanoma cells by microRNAs. PLoS ONE 6:e18936. doi:10.1371/journal.pone.0018936
Hazarika P et al (2004) Up-regulation of Flotillin-2 is associated with melanoma progression and modulates expression of the thrombin receptor protease activated receptor 1. Cancer Res 64:7361–7369. doi:10.1158/0008-5472.can-04-0823
He L et al (2005) A microRNA polycistron as a potential human oncogene. Nature 435:828–833. doi:10.1038/nature03552
He L et al (2007a) A microRNA component of the p53 tumour suppressor network. Nature 447:1130–1134. doi:10.1038/nature05939
He X, He L, Hannon GJ (2007b) The guardian’s little helper: microRNAs in the p53 tumor suppressor network. Cancer Res 67:11099–11101. doi:10.1158/0008-5472.CAN-07-2672
Heinemann A et al (2012) Tumor suppressive microRNAs miR-34a/c control cancer cell expression of ULBP2, a stress-induced ligand of the natural killer cell receptor NKG2D. Cancer Res 72:460–471. doi:10.1158/0008-5472.CAN-11-1977
Iorio MV, Croce CM (2012) MicroRNA dysregulation in cancer: diagnostics, monitoring and therapeutics. A comprehensive review. EMBO Mol Med 4:143–159. doi:10.1002/emmm.201100209
Kahr HS, Mejlgaard E, Lund B (2013) Primary malignant melanoma of the vagina. Ugeskr Laeger 175:133–134
Kim HR, Roe JS, Lee JE, Cho EJ, Youn HD (2013) p53 regulates glucose metabolism by miR-34a. Biochem biophysical research Commun 437:225–231. doi:10.1016/j.bbrc.2013.06.043
Levati L et al (2011) MicroRNA-155 targets the SKI gene in human melanoma cell lines. Pigment Cell Melanoma Res 24:538–550. doi:10.1111/j.1755-148X.2011.00857.x
Lim LP et al (2005) Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs. Nature 433:769–773. doi:10.1038/nature03315
Lodygin D et al. (2008) Inactivation of miR-34a by aberrant CpG methylation in multiple types of cancer. Cell cycle (Georgetown, Tex) 7:2591–2600
Tarasov V et al (2007) Differential regulation of microRNAs by p53 revealed by massively parallel sequencing: miR-34a is a p53 target that induces apoptosis and G1-arrest. Cell Cycle (Georgetown, Tex) 6:1586–1593
Lu C, Rak JW, Kobayashi H, Kerbel RS (1993) Increased resistance to oncostatin M-induced growth inhibition of human melanoma cell lines derived from advanced-stage lesions. Cancer Res 53:2708–2711
Lu R et al (2014) miR-145 functions as tumor suppressor and targets two oncogenes, ANGPT2 and NEDD9, in renal cell carcinoma. J Cancer Res Clin Oncol 140:387–397. doi:10.1007/s00432-013-1577-z
Malaga-Trillo E, Laessing U, Lang DM, Meyer A, Stuermer CA (2002) Evolution of duplicated reggie genes in zebrafish and goldfish. J Mol Evol 54:235–245. doi:10.1007/s00239-001-0005-1
Matsumoto S et al (2013) Circulating p53-responsive microRNAs are predictive indicators of heart failure after acute myocardial infarction. Circ Res 113:322–326. doi:10.1161/CIRCRESAHA.113.301209
Nemlich Y et al (2013) MicroRNA-mediated loss of ADAR1 in metastatic melanoma promotes tumor growth. J Clin Investig 123:2703–2718. doi:10.1172/JCI62980
Nesca V et al (2013) Identification of particular groups of microRNAs that positively or negatively impact on beta cell function in obese models of type 2 diabetes. Diabetologia 56:2203–2212. doi:10.1007/s00125-013-2993-y
Pritchard CC, Cheng HH, Tewari M (2012) MicroRNA profiling: approaches and considerations. Nat Rev Genet 13:358–369. doi:10.1038/nrg3198
Rivera-Milla E, Stuermer CA, Malaga-Trillo E (2006) Ancient origin of reggie (flotillin), reggie-like, and other lipid-raft proteins: convergent evolution of the SPFH domain. Cell Mol Life Sci CMLS 63:343–357. doi:10.1007/s00018-005-5434-3
Saleiban A, Faxalv L, Claesson K, Jonsson JI, Osman A (2014) miR-20b regulates expression of proteinase-activated receptor-1 (PAR-1) thrombin receptor in melanoma cells. Pigment Cell Melanoma Res 27:431–441. doi:10.1111/pcmr.12217
Satzger I, Mattern A, Kuettler U, Weinspach D, Voelker B, Kapp A, Gutzmer R (2010) MicroRNA-15b represents an independent prognostic parameter and is correlated with tumor cell proliferation and apoptosis in malignant melanoma. Int J Cancer J Int Cancer 126:2553–2562. doi:10.1002/ijc.24960
Shang JX, Zhang D, Zhang JT, Zhao JZ, Zhang Y (2013) Melanocytic neoplasms of central nervous system analysis. Zhonghua yi xue za zhi 93:34–36
Siegel R et al (2012) Cancer treatment and survivorship statistics, 2012. CA Cancer J Clin 62:220–241. doi:10.3322/caac.21149
Siegel R, Ma J, Zou Z, Jemal A (2014) Cancer statistics, 2014. CA Cancer J Clin 64:9–29. doi:10.3322/caac.21208
Spagnolo F, Caltabiano G, Queirolo P (2012) Uveal melanoma. Cancer Treat Rev 38:549–553. doi:10.1016/j.ctrv.2012.01.002
Villares GJ, Zigler M, Bar-Eli M (2011) The emerging role of the thrombin receptor (PAR-1) in melanoma metastasis—a possible therapeutic target. Oncotarget 2:8–17
Volonte D, Galbiati F, Li S, Nishiyama K, Okamoto T, Lisanti MP (1999) Flotillins/cavatellins are differentially expressed in cells and tissues and form a hetero-oligomeric complex with caveolins in vivo. Characterization and epitope-mapping of a novel flotillin-1 monoclonal antibody probe. J Biol Chem 274:12702–12709
Xiao GH, Luo CQ, Tang GM, Zhou JD (2005) [Human endostatin gene transfected adult skin melanoma cells]. Zhong nan da xue xue bao Yi xue ban= J Central South Univ Med Sci 30:677–681
Xu D et al (2012) Let-7b and microRNA-199a inhibit the proliferation of B16F10 melanoma cells. Oncol Lett 4:941–946. doi:10.3892/ol.2012.878
Yamazaki H et al (2012) Overexpression of the miR-34 family suppresses invasive growth of malignant melanoma with the wild-type p53 gene. Exp Ther Med 3:793–796. doi:10.3892/etm.2012.497
Yan D et al (2009) MicroRNA-34a inhibits uveal melanoma cell proliferation and migration through downregulation of c-Met. Invest Ophthalmol Vis Sci 50:1559–1565. doi:10.1167/iovs.08-2681
Zhao F, Zhang J, Liu YS, Li L, He YL (2011) Research advances on flotillins. Virol J 8:479. doi:10.1186/1743-422X-8-479
Zigler M, Kamiya T, Brantley EC, Villares GJ, Bar-Eli M (2011) PAR-1 and thrombin: the ties that bind the microenvironment to melanoma metastasis. Cancer Res 71:6561–6566. doi:10.1158/0008-5472.can-11-1432
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.
Conflict of interest
We declare that we have no conflict of interest.
Author information
Authors and Affiliations
Corresponding authors
Electronic supplementary material
Below is the link to the electronic supplementary material.
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)
Rights and permissions
About this article
Cite this article
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
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00432-014-1874-1