The oncomir face of microRNA-206: A permanent miR-206 transfection study
Abstract
MiR-206 is a remarkable miRNA because it functions as a suppressor miRNA in rhabdomyosarcoma while at the same time, as previously showed, it can act as an oncomiRNA in SMARCB1 immunonegative soft tissue sarcomas. The aim of this study was to investigate the effect of miR-206 on its several target genes in various human tumorous and normal cell lines. In the current work, we created miR-206-overexpressing cell lines (HT-1080, Caco2, iASC, and SS-iASC) using permanent transfection. mRNA expression of the target genes of miR-206 (SMARCB1, ACTL6A, CCND1, POLA1, NOTCH3, MET, and G6PD) and SMARCB1 protein expression were examined with quantitative real-time polymerase chain reaction, immunoblotting, immunocytochemistry, and flow cytometry. MiRNA inhibition was used to validate our results. We found a diverse silencing effect of miR-206 on its target genes. While an overall tendency of downregulation was noted, expression profiles of individual cell lines showed large variability. Only CCND1 and MET were consistently downregulated. MiR-206 had an antiproliferative effect on a normal human fibroblast cell line. A strong silencing effect of SMARCB1 in miR-206 transfected SS-iASC was most likely caused by the synergic influence of the SS18-SSX1 fusion protein and miR-206. In the same cell line, a moderate decrease of SMARCB1 protein expression could be observed with immunocytochemistry and flow cytometry. In the most comprehensive analysis of miR-206 effects so far, a modest but significant downregulation of miR-206 targets on the mRNA level was confirmed across all cell lines. However, the variability of the effect shows that the action of this miRNA is largely cell context-dependent. Our results also support the conception that the oncomiR effect of miR-206 on SMARCB1 plays an important but not exclusive role in SMARCB1 immunonegative soft tissue sarcomas so it can be considered important in planning the targeted therapy of these tumors in the future.
Impact statement
Mir-206 is a very unique microRNA because it can act as a suppressor miRNA or as an oncomiRNA depending on the tumor tissue. In SMARCB1 negative soft tissue sarcomas miR-206 is overexpressed, so thus in epithelioid and synovial sarcomas it functions as an oncomiRNA. MiR-206 has diverse silencing effects on its target genes. We found that the action of miR-206 is largely cell context dependent. The oncomiR role of miR-206 is crucial but not exclusive in SMARCB1 negative soft tissue sarcomas and miR-206 has an antiproliferative effect on a normal human fibroblast cell line. Expressions of miR-206 targets observed in tumors can only be reproduced in the corresponding tumorous cell lines. This is the first study which examined the permanent effect of miR-206 on its target genes in normal, tumor, and genetically engineered cell lines.
Get full access to this article
View all access and purchase options for this article.
References
1. Reisman D, Glaros S, Thompson EA. The SWI/SNF complex and cancer. Oncogene 2009; 28:1653–68
2. Kalimuthu SN, Chetty R. Gene of the month: SMARCB1. J Clin Pathol 2016; 69:484–9
3. Kohashi K, Oda Y. Oncogenic roles of SMARCB1/INI1 and its deficient tumors. Cancer Sci 2017; 108:547–52
4. Kadoch C, Crabtree GR. Reversible disruption of mSWI/SNF (BAF) complexes by the SS18-SSX oncogenic fusion in synovial sarcoma. Cell 2013; 153:71–85
5. Papp G, Krausz T, Stricker TP, Szendroi M, Sapi Z. SMARCB1 expression in epithelioid sarcoma is regulated by miR-206, miR-381, and miR-671-5p on Both mRNA and protein levels. Genes Chromosomes Cancer 2014; 53:168–76
6. Sapi Z, Papp G, Szendroi M, Papai Z, Plotar V, Krausz T, Fletcher CD. Epigenetic regulation of SMARCB1 By miR-206, -381 and -671-5p is evident in a variety of SMARCB1 immunonegative soft tissue sarcomas, while miR-765 appears specific for epithelioid sarcoma. A miRNA study of 223 soft tissue sarcomas. Genes Chromosomes Cancer 2016; 55:786–802
7. Taulli R, Foglizzo V, Morena D, Coda DM, Ala U, Bersani F, Maestro N, Ponzetto C. Failure to downregulate the BAF53a subunit of the SWI/SNF chromatin remodeling complex contributes to the differentiation block in rhabdomyosarcoma. Oncogene 2014; 33:2354–62
8. Bao YP, Yi Y, Peng LL, Fang J, Liu KB, Li WZ, Luo HS. Roles of microRNA-206 in osteosarcoma pathogenesis and progression. Asian Pac J Cancer Prev 2013; 14:3751–5
9. Wang CQ, Huang YW, Wang SW, Huang YL, Tsai CH, Zhao YM, Huang BF, Xu GH, Fong YC, Tang CH. Amphiregulin enhances VEGF-A production in human chondrosarcoma cells and promotes angiogenesis by inhibiting miR-206 via FAK/c-Src/PKCdelta pathway. Cancer Lett 2017; 385:261–70
10. Tian R, Liu T, Qiao L, Gao M, Li J. Decreased serum microRNA-206 level predicts unfavorable prognosis in patients with melanoma. Int J Clin Exp Pathol 2015; 8:3097–103
11. Ge X, Lyu P, Cao Z, Li J, Guo G, Xia W, Gu Y. Overexpression of miR-206 suppresses glycolysis, proliferation and migration in breast cancer cells via PFKFB3 targeting. Biochem Biophys Res Commun 2015; 463:1115–21
12. Chen QY, Jiao DM, Wu YQ, Chen J, Wang J, Tang XL, Mou H, Hu HZ, Song J, Yan J, Wu LJ, Chen J, Wang Z. MiR-206 inhibits HGF-induced epithelial-mesenchymal transition and angiogenesis in non-small cell lung cancer via c-Met/PI3k/Akt/mTOR pathway. Oncotarget 2016; 7:18247–61
13. Amirouche A, Jahnke VE, Lunde JA, Koulmann N, Freyssenet DG, Jasmin BJ. Muscle-specific microRNA-206 targets multiple components in dystrophic skeletal muscle representing beneficial adaptations. Am J Physiol Cell Physiol 2017; 312:C209–21
14. Cui Y, Xie S, Luan J, Zhou X, Han J. Identification of the receptor tyrosine kinases (RTKs)-oriented functional targets of miR-206 by an antibody-based protein array. FEBS Lett 2015; 589:2131–5
15. Ren XL, He GY, Li XM, Men H, Yi LZ, Lu GF, Xin SN, Wu PX, Li YL, Liao WT, Ding YQ, Liang L. MicroRNA-206 functions as a tumor suppressor in colorectal cancer by targeting FMNL2. J Cancer Res Clin Oncol 2016; 142:581–92
16. Krasteva V, Buscarlet M, Diaz-Tellez A, Bernard MA, Crabtree GR, Lessard JA. The BAF53a subunit of SWI/SNF-like BAF complexes is essential for hemopoietic stem cell function. Blood 2012; 120:4720–32
17. Alteri A, De Vito F, Messina G, Pompili M, Calconi A, Visca P, Mottolese M, Presutti C, Grossi M. Cyclin D1 is a major target of miR-206 in cell differentiation and transformation. Cell Cycle 2013; 12:3781–90
18. Chattopadhyay S, Bielinsky AK. Human Mcm10 regulates the catalytic subunit of DNA polymerase-alpha and prevents DNA damage during replication. Mol Biol Cell 2007; 18:4085–95
19. Zhang Q, Lu C, Fang T, Wang Y, Hu W, Qiao J, Liu B, Liu J, Chen N, Li M, Zhu R. Notch3 functions as a regulator of cell self-renewal by interacting with the beta-catenin pathway in hepatocellular carcinoma. Oncotarget 2015; 6:3669–79
20. Crepaldi T, Bersani F, Scuoppo C, Accornero P, Prunotto C, Taulli R, Forni PE, Leo C, Chiarle R, Griffiths J, Glass DJ, Ponzetto C. Conditional activation of MET in differentiated skeletal muscle induces atrophy. J Biol Chem 2007; 282:6812–22
21. Kuo W, Lin J, Tang TK. Human glucose-6-phosphate dehydrogenase (G6PD) gene transforms NIH 3T3 cells and induces tumors in nude mice. Int J Cancer 2000; 85:857–64
22. Serrano-Gomez SJ, Maziveyi M, Alahari SK. Regulation of epithelial-mesenchymal transition through epigenetic and post-translational modifications. Mol Cancer 2016; 15:18
23. Tatrai P, Szepesi A, Matula Z, Szigeti A, Buchan G, Madi A, Uher F, Nemet K. Combined introduction of Bmi-1 and hTERT immortalizes human adipose tissue-derived stromal cells with low risk of transformation. Biochem Biophys Res Commun 2012; 422:28–35
24. Mihaly D, Matula Z, Changchien YC, Papp G, Tatrai P, Sapi Z. First cloned human immortalized adipose derived mesenchymal stem-cell line with chimeric SS18-SSX1 gene (SS-iASC). Cancer Genet 2017; 216–217:52–60
25. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 2001; 25:402–8
26. Kim HK, Lee YS, Sivaprasad U, Malhotra A, Dutta A. Muscle-specific microRNA miR-206 promotes muscle differentiation. J Cell Biol 2006; 174:677–87
27. Koshizuka K, Hanazawa T, Fukumoto I, Kikkawa N, Matsushita R, Mataki H, Mizuno K, Okamoto Y, Seki N. Dual-receptor (EGFR and c-MET) inhibition by tumor-suppressive miR-1 and miR-206 in head and neck squamous cell carcinoma. J Hum Genet 2017; 62:113–21
28. Amir S, Simion C, Umeh-Garcia M, Krig S, Moss T, Carraway KL 3rd, Sweeney C. Regulation of the T-box transcription factor Tbx3 by the tumour suppressor microRNA-206 in breast cancer. Br J Cancer 2016; 114:1125–34
29. Liu W, Xu C, Wan H, Liu C, Wen C, Lu H, Wan F. MicroRNA-206 overexpression promotes apoptosis, induces cell cycle arrest and inhibits the migration of human hepatocellular carcinoma HepG2 cells. Int J Mol Med 2014; 34:420–8
30. Song G, Zhang Y, Wang L. MicroRNA-206 targets notch3, activates apoptosis, and inhibits tumor cell migration and focus formation. J Biol Chem 2009; 284:31921–7
31. Coda DM, Lingua MF, Morena D, Foglizzo V, Bersani F, Ala U, Ponzetto C, Taulli R. SMYD1 and G6PD modulation are critical events for miR-206-mediated differentiation of rhabdomyosarcoma. Cell Cycle 2015; 14:1389–402
32. Kohashi K, Tanaka Y, Kishimoto H, Yamamoto H, Yamada Y, Taguchi T, Iwamoto Y, Oda Y. Reclassification of rhabdoid tumor and pediatric undifferentiated/unclassified sarcoma with complete loss of SMARCB1/INI1 protein expression: three subtypes of rhabdoid tumor according to their histological features. Mod Pathol 2016; 29:1232–42
33. Modena P, Lualdi E, Facchinetti F, Galli L, Teixeira MR, Pilotti S, Sozzi G. SMARCB1/INI1 tumor suppressor gene is frequently inactivated in epithelioid sarcomas. Cancer Res 2005; 65:4012–9
34. Xiao H, Xiao W, Cao J, Li H, Guan W, Guo X, Chen K, Zheng T, Ye Z, Wang J, Xu H. miR-206 functions as a novel cell cycle regulator and tumor suppressor in clear-cell renal cell carcinoma. Cancer Lett 2016; 374:107–16
35. Taulli R, Bersani F, Foglizzo V, Linari A, Vigna E, Ladanyi M, Tuschl T, Ponzetto C. The muscle-specific microRNA miR-206 blocks human rhabdomyosarcoma growth in xenotransplanted mice by promoting myogenic differentiation. J Clin Invest 2009; 119:2366–78
36. Saito T, Nagai M, Ladanyi M. SYT-SSX1 and SYT-SSX2 interfere with repression of E-cadherin by snail and slug: a potential mechanism for aberrant mesenchymal to epithelial transition in human synovial sarcoma. Cancer Res 2006; 66:6919–27
37. Mukherji S, Ebert MS, Zheng GX, Tsang JS, Sharp PA, van Oudenaarden A. MicroRNAs can generate thresholds in target gene expression. Nat Genet 2011; 43:854–9
Cite article
Cite article
Cite article
OR
Download to reference manager
If you have citation software installed, you can download article citation data to the citation manager of your choice
Information, rights and permissions
Information
Published In
Article first published online: August 15, 2018
Issue published: August 2018
Keywords
PubMed: 30111166
Authors
Authors’ Contributions
All authors participated in the design, interpretation of the studies and analysis of the data and review of the manuscript; DM carried out the miRNA transfection experiments, performed the qRT-PCR experiments, the immunocytochemical reactions and the flow cytometry investigation, collected the results and designed and drafted the manuscript. ZM performed the immunoblotting. AR helped with the preparation of figures. JS performed the statistical analysis. GSz helped with the analysis of flow cytometry. ZS conceived of the study and GP, PT and ZS participated in its design and helped to draft the manuscript. All authors have read and approved the final manuscript.
Metrics and citations
Metrics
Journals metrics
This article was published in Experimental Biology and Medicine.
VIEW ALL JOURNAL METRICSArticle usage*
Total views and downloads: 305
*Article usage tracking started in December 2016
Altmetric
See the impact this article is making through the number of times it’s been read, and the Altmetric Score.
Learn more about the Altmetric Scores
Articles citing this one
Receive email alerts when this article is cited
Web of Science: 8 view articles Opens in new tab
Crossref: 8
- A single session of EMS training induces long-lasting changes in circu...
- Dysregulated expression of microRNAs acts as prognostic and diagnostic...
- Association between microRNA 671 polymorphisms and the susceptibility ...
- Genomic Analysis of Abnormal DNAM Methylation in Parathyroid Tumors
- Targeting microRNA-mediated gene repression limits adipogenic conversi...
- miR ‐188‐5p inhibits proliferation, migration, and invas...
- miR-206 as a prognostic and sensitivity biomarker for platinum chemoth...
- miR‐188‐5p emerges as an oncomiRNA to promote gastric cancer cell prol...
Figures and tables
Figures & Media
Tables
View Options
Get access
Access options
If you have access to journal content via a personal subscription, university, library, employer or society, select from the options below:
loading institutional access options
SEBM members can access this journal content using society membership credentials.
SEBM members can access this journal content using society membership credentials.
Alternatively, view purchase options below:
Purchase 24 hour online access to view and download content.
Access journal content via a DeepDyve subscription or find out more about this option.
