Skip to main content

Advertisement

SpringerLink
Log in

Post-transcriptional regulation of MMP2 mRNA by its interaction with miR-20a and Nucleolin in breast cancer cell lines

  • Original Article
  • Published:
Molecular Biology Reports Aims and scope Submit manuscript

Abstract

Matrix-metalloproteinase-2 (MMP2) is a foremost MMP, governing invasion of breast cancer cells during metastasis. miR-20a was reported to induce mesenchymal to epithelial transition in MDA-MB-231 cells and its endogenous expression varies directly with invasiveness of breast cancer cells. The inverse and direct correlation of invasiveness with miR-20a and Nucleolin respectively led us to study the post-transcriptional regulation of MMP2 by miR-20a and mRNA stabilizing protein, Nucleolin. Thus, understanding the mechanism of its regulation will enable modification of the invasion potential. MMP2 was found to be higher in MDA-MB-231 than MCF-7 cells both at RNA and protein levels. RNA–protein co-immunoprecipitation assay with Argonaute 2 revealed that MMP2 undergoes miRNA-mediated post-transcriptional regulation. miR-20a decreased MMP2 expression as well as its enzymatic activity as found by zymogram assay. Reporter assay showed that miR-20a directly binds to its putative binding site in MMP2 3′-UTR as per in silico prediction. miR-20a additionally impeded MMP2 mRNA stability, and binding of stabilizing trans-factor Nucleolin to its 3′-UTR was confirmed by RNA–protein co-immunoprecipitation assay. Partial down-regulation of Nucleolin by Si-RNA resulted in the downregulation of MMP2 and Nucleolin over-expression rescued the inhibitory effect of miR-20a on MMP2 expression. Delineating the mechanism of post-transcriptional regulation of MMP2, two of its potent regulators, miR-20a and Nucleolin were identified. It was established for the first time that MMP2 is a direct target of miR-20a. The results also elucidated that Nucleolin binds to MMP2 3′ UTR and its abundance affects MMP2 expression.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

Data Availability

All the raw data and material used in the published article should be available.

Abbreviations

MMP2:

Matrix-metalloproteinase 2

miR:

MicroRNA

UTR:

Untranslated region

Ago2:

Argonaute2

RISC:

RNAi induced silencing complex

Nuc:

Nucleolin

IP:

Immunoprecipitate

TNBC:

Triple negative breast cancer

References

  1. Fanjul-Fernández M, Folgueras AR, Cabrera S, López-Otín C (2010) Matrix metalloproteinases: evolution, gene regulation and functional analysis in mouse models. Biochim Biophys Acta (BBA) 1803(1):3–19. https://doi.org/10.1016/j.bbamcr.2009.07.004

    Article  CAS  Google Scholar 

  2. Dong W, Li H, Zhang Y, Yang H, Guo M, Li L, Liu T (2011) Matrix metalloproteinase 2 promotes cell growth and invasion in colorectal cancer. Acta Biochim Biophys Sin 43(11):840–848

    Article  CAS  PubMed  Google Scholar 

  3. Webb AH, Gao BT, Goldsmith ZK, Irvine AS, Saleh N, Lee RP, Lendermon JB, Bheemreddy R, Zhang Q, Brennan RC, Johnson D, Steinle JJ, Wilson MW, Morales-Tirado VM (2017) Inhibition of MMP-2 and MMP-9 decreases cellular migration, and angiogenesis in in vitro models of retinoblastoma. BMC Cancer 17(1):434–434. https://doi.org/10.1186/s12885-017-3418-y

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Yu C-F, Chen F-H, Lu M-H, Hong J-H, Chiang C-S (2017) Dual roles of tumour cells-derived matrix metalloproteinase 2 on brain tumour growth and invasion. Br J Cancer 117(12):1828–1836. https://doi.org/10.1038/bjc.2017.362

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Kazes I, Il E, Sraer J-D, Hatmi M, Nguyen G (2000) Platelet release of trimolecular complex components MT1-MMP/TIMP2/MMP2: involvement in MMP2 activation and platelet aggregation. Blood 96(9):3064

    Article  CAS  PubMed  Google Scholar 

  6. Chen J-S, Huang X-h, Wang Q, Huang J-Q, Zhang L-j, Chen X-L, Lei J, Cheng Z-X (2013) Sonic hedgehog signaling pathway induces cell migration and invasion through focal adhesion kinase/AKT signaling-mediated activation of matrix metalloproteinase (MMP)-2 and MMP-9 in liver cancer. Carcinogenesis 34(1):10–19

    Article  CAS  PubMed  Google Scholar 

  7. Adya R, Tan BK, Punn A, Chen J, Randeva HS (2008) Visfatin induces human endothelial VEGF and MMP-2/9 production via MAPK and PI3K/Akt signalling pathways: novel insights into visfatin-induced angiogenesis. Cardiovasc Res 78(2):356–365

    Article  CAS  PubMed  Google Scholar 

  8. Kim Y, Lee Y-S, Choe J, Lee H, Kim Y-M, Jeoung D (2008) CD44-epidermal growth factor receptor interaction mediates hyaluronic acid-promoted cell motility by activating protein kinase c signaling involving Akt, Rac1, Phox, reactive oxygen species, focal adhesion kinase, and MMP-2. J Biol Chem 283(33):22513–22528

    Article  CAS  PubMed  Google Scholar 

  9. Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116(2):281–297. https://doi.org/10.1016/S0092-8674(04)00045-5

    Article  CAS  PubMed  Google Scholar 

  10. Svoronos AA, Engelman DM, Slack FJ (2016) OncomiR or tumor suppressor? The duplicity of MicroRNAs in cancer. Cancer Res 76(13):3666–3670. https://doi.org/10.1158/0008-5472.can-16-0359

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Li L, Li H (2013) Role of microRNA-mediated MMP regulation in the treatment and diagnosis of malignant tumors. Cancer Biol Ther 14(9):796–805. https://doi.org/10.4161/cbt.25936

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Wang H, Zhu Y, Zhao M, Wu C, Zhang P, Tang L, Zhang H, Chen X, Yang Y, Liu G (2013) miRNA-29c suppresses lung cancer cell adhesion to extracellular matrix and metastasis by targeting integrin β1 and matrix metalloproteinase2 (MMP2). PLoS ONE 8(8):e70192

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Wang Q, Tang H, Yin S, Dong C (2013) Downregulation of microRNA-138 enhances the proliferation, migration and invasion of cholangiocarcinoma cells through the upregulation of RhoC/p-ERK/MMP-2/MMP-9. Oncol Rep 29(5):2046–2052

    Article  CAS  PubMed  Google Scholar 

  14. Dai Y, Xia W, Song T, Su X, Li J, Li S, Chen Y, Wang W, Ding H, Liu X, Li H, Zhao Q, Shao N (2013) MicroRNA-200b is overexpressed in endometrial adenocarcinomas and enhances MMP2 activity by downregulating TIMP2 in human endometrial cancer cell line HEC-1A cells. Nucleic ACID Ther 23(1):29–34. https://doi.org/10.1089/nat.2012.0385

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Lu L, Xue X, Lan J, Gao Y, Xiong Z, Zhang H, Jiang W, Song W, Zhi Q (2014) MicroRNA-29a upregulates MMP2 in oral squamous cell carcinoma to promote cancer invasion and anti-apoptosis. Biomed Pharmacother 68(1):13–19. https://doi.org/10.1016/j.biopha.2013.10.005

    Article  CAS  PubMed  Google Scholar 

  16. Mendell JT (2008) miRiad roles for the miR-17-92 cluster in development and disease. Cell 133(2):217–222. https://doi.org/10.1016/j.cell.2008.04.001

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Bray F, McCarron P, Parkin DM (2004) The changing global patterns of female breast cancer incidence and mortality. Breast Cancer Res 6(6):229–239. https://doi.org/10.1186/bcr932

    Article  PubMed  PubMed Central  Google Scholar 

  18. Yu Z, Wang C, Wang M, Li Z, Casimiro MC, Liu M, Wu K, Whittle J, Ju X, Hyslop T, McCue P, Pestell RG (2008) A cyclin D1/microRNA 17/20 regulatory feedback loop in control of breast cancer cell proliferation. J Cell Biol 182(3):509–517. https://doi.org/10.1083/jcb.200801079

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Kim K, Chadalapaka G, Lee SO, Yamada D, Sastre-Garau X, Defossez PA, Park YY, Lee JS, Safe S (2011) Identification of oncogenic microRNA-17-92/ZBTB4/specificity protein axis in breast cancer. Oncogene 31:1034. https://doi.org/10.1038/onc.2011.296

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. De S, Das S, Mukherjee S, Das S, Sengupta S (2017) Establishment of twist-1 and TGFBR2 as direct targets of microRNA-20a in mesenchymal to epithelial transition of breast cancer cell-line MDA-MB-231. Exp Cell Res 361(1):85–92. https://doi.org/10.1016/j.yexcr.2017.10.005

    Article  CAS  PubMed  Google Scholar 

  21. Xu T, Jing C, Shi Y, Miao R, Peng L, Kong S, Ma Y, Li L (2015) microRNA-20a enhances the epithelial-to-mesenchymal transition of colorectal cancer cells by modulating matrix metalloproteinases. Exp Ther Med 10(2):683–688. https://doi.org/10.3892/etm.2015.2538

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Zhao S, Yao D, Chen J, Ding N, Ren F (2015) MiR-20a promotes cervical cancer proliferation and metastasis in vitro and in vivo. PLoS ONE 10(3):e0120905

    Article  PubMed  PubMed Central  Google Scholar 

  23. Van Tubergen EA, Banerjee R, Liu M, Broek RV, Light E, Kuo S, Feinberg SE, Willis AL, Wolf G, Carey T, Bradford C, Prince M, Worden FP, Kirkwood KL, Silva NJ (2013) Inactivation or loss of TTP promotes invasion in head and neck cancer via transcript stabilization and secretion of MMP9, MMP2, and IL-6. Clin Cancer Res 19(5):1169. https://doi.org/10.1158/1078-0432.ccr-12-2927

    Article  PubMed  PubMed Central  Google Scholar 

  24. Pichiorri F, Palmieri D, De Luca L, Consiglio J, You J, Rocci A, Talabere T, Piovan C, Lagana A, Cascione L, Guan J, Gasparini P, Balatti V, Nuovo G, Coppola V, Hofmeister CC, Marcucci G, Byrd JC, Volinia S, Shapiro CL, Freitas MA, Croce CM (2013) In vivo NCL targeting affects breast cancer aggressiveness through miRNA regulation. J Exp Med 210(5):951–968. https://doi.org/10.1084/jem.20120950

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Jordan M, Schallhorn A, Wurm FM (1996) Transfecting mammalian cells: optimization of critical parameters affecting calcium-phosphate precipitate formation. Nucleic Acids Res 24(4):596–601

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Dasgupta P, Bandyopadhyay SS (2013) Role of di-allyl disulfide, a garlic component in NF-κB mediated transient G2-M phase arrest and apoptosis in human leukemic cell-lines. Nutr Cancer 65(4):611–622. https://doi.org/10.1080/01635581.2013.776090

    Article  CAS  PubMed  Google Scholar 

  27. Niranjanakumari S, Lasda E, Brazas R, Garcia-Blanco MA (2002) Reversible cross-linking combined with immunoprecipitation to study RNA–protein interactions in vivo. Methods 26(2):182–190. https://doi.org/10.1016/S1046-2023(02)00021-X

    Article  CAS  PubMed  Google Scholar 

  28. Stamenkovic I (2000) Matrix metalloproteinases in tumor invasion and metastasis. Semin Cancer Biol 10(6):415–433. https://doi.org/10.1006/scbi.2000.0379

    Article  CAS  PubMed  Google Scholar 

  29. Bridge KS, Shah KM, Li Y, Foxler DE, Wong SCK, Miller DC, Davidson KM, Foster JG, Rose R, Hodgkinson MR, Ribeiro PS, Aboobaker AA, Yashiro K, Wang X, Graves PR, Plevin MJ, Lagos D, Sharp TV (2017) Argonaute utilization for miRNA silencing is determined by phosphorylation-dependent recruitment of LIM-domain-containing proteins. Cell Rep 20(1):173–187. https://doi.org/10.1016/j.celrep.2017.06.027

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Diederichs S, Haber DA (2007) Dual role for argonautes in MicroRNA processing and posttranscriptional regulation of MicroRNA expression. Cell 131(6):1097–1108. https://doi.org/10.1016/j.cell.2007.10.032

    Article  CAS  PubMed  Google Scholar 

  31. Lee P-T, Liao P-C, Chang W-C, Tseng JT (2007) Epidermal growth factor increases the interaction between nucleolin and heterogeneous nuclear ribonucleoprotein K/poly(C) binding protein 1 complex to regulate the gastrin mRNA Turnover. Mol Biol Cell 18(12):5004–5013. https://doi.org/10.1091/mbc.e07-04-0384

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Saha S, Chakraborty A, Bandyopadhyay SS (2015) Stabilization of oncostatin-M mRNA by binding of Nucleolin to a GC-Rich element in its 3′UTR. J Cell Biochem 117(4):988–999. https://doi.org/10.1002/jcb.25384

    Article  CAS  Google Scholar 

  33. Kim JH, Jeon S, Shin BA (2017) MicroRNA-29 family suppresses the invasion of HT1080 human fibrosarcoma cells by regulating matrix metalloproteinase 2 expression. Chonnam Med J 53(2):161–167. https://doi.org/10.4068/cmj.2017.53.2.161

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Hu Y, Li Y, Wu C, Zhou L, Han X, Wang Q, Xie X, Zhou Y, Du Z (2017) MicroRNA-140-5p inhibits cell proliferation and invasion by regulating VEGFA/MMP2 signaling in glioma. Tumor Biol 39(4):1010428317697558. https://doi.org/10.1177/1010428317697558

    Article  CAS  Google Scholar 

  35. Hu J, Ni S, Cao Y, Zhang T, Wu T, Yin X, Lang Y, Lu H (2016) The angiogenic effect of microRNA-21 targeting TIMP3 through the regulation of MMP2 and MMP9. PLoS ONE 11(2):e0149537

    Article  PubMed  PubMed Central  Google Scholar 

  36. Ghisolfi-Nieto L, Joseph G, Puvion-Dutilleul F, Amalric F, Bouvet P (1996) Nucleolin is a sequence-specific RNA-binding protein: characterization of targets on pre-ribosomal RNA. J Mol Biol 260(1):34–53

    Article  CAS  PubMed  Google Scholar 

  37. Otake Y, Soundararajan S, Sengupta TK, Kio EA, Smith JC, Pineda-Roman M, Stuart RK, Spicer EK, Fernandes DJ (2007) Overexpression of nucleolin in chronic lymphocytic leukemia cells induces stabilization of bcl2 mRNA. Blood 109(7):3069–3075. https://doi.org/10.1182/blood-2006-08-043257

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Abdelmohsen K, Tominaga K, Lee EK, Srikantan S, Kang M-J, Kim MM, Selimyan R, Martindale JL, Yang X, Carrier F, Zhan M, Becker KG, Gorospe M (2011) Enhanced translation by Nucleolin via G-rich elements in coding and non-coding regions of target mRNAs. Nucleic Acids Res 39(19):8513–8530. https://doi.org/10.1093/nar/gkr488

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Fähling M, Steege A, Perlewitz A, Nafz B, Mrowka R, Persson PB, Thiele BJ (2005) Role of nucleolin in posttranscriptional control of MMP-9 expression. Biochim Biophys Acta (BBA) 1731(1):32–40. https://doi.org/10.1016/j.bbaexp.2005.08.005

    Article  CAS  Google Scholar 

  40. Yang Y, Yang C, Zhang J (2015) C23 protein meditates bone morphogenetic protein-2-mediated EMT via up-regulation of Erk1/2 and Akt in gastric cancer. Med Oncol 32(3):76. https://doi.org/10.1007/s12032-015-0547-5

    Article  CAS  PubMed  Google Scholar 

  41. Hsu TI, Lin SC, Lu PS, Chang WC, Hung CY, Yeh YM, Su WC, Liao PC, Hung JJ (2014) MMP7-mediated cleavage of nucleolin at Asp255 induces MMP9 expression to promote tumor malignancy. Oncogene 34:826. https://doi.org/10.1038/onc.2014.22

    Article  CAS  PubMed  Google Scholar 

  42. Wolfson E, Solomon S, Schmukler E, Goldshmit Y, Pinkas-Kramarski R (2018) Nucleolin and ErbB2 inhibition reduces tumorigenicity of ErbB2-positive breast cancer. Cell Death Dis 9(2):47. https://doi.org/10.1038/s41419-017-0067-7

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Hovanessian AG, Puvion-Dutilleul F, Nisole S, Svab J, Perret E, Deng J-S, Krust B (2000) The cell-surface-expressed nucleolin is associated with the actin cytoskeleton. Exp Cell Res 261(2):312–328. https://doi.org/10.1006/excr.2000.5071

    Article  CAS  PubMed  Google Scholar 

  44. Berger CM, Gaume X, Bouvet P (2015) The roles of nucleolin subcellular localization in cancer. Biochimie 113:78–85. https://doi.org/10.1016/j.biochi.2015.03.023

    Article  CAS  PubMed  Google Scholar 

  45. Jia W, Yao Z, Zhao J, Guan Q, Gao L (2017) New perspectives of physiological and pathological functions of nucleolin (NCL). Life Sci 186:1–10. https://doi.org/10.1016/j.lfs.2017.07.025

    Article  CAS  PubMed  Google Scholar 

  46. Zaidi SHE, Malter JS (1995) Nucleolin and heterogeneous nuclear ribonucleoprotein C proteins specifically interact with the 3′-untranslated region of amyloid protein precursor mRNA. J Biol Chem 270(29):17292–17298. https://doi.org/10.1074/jbc.270.29.17292

    Article  CAS  PubMed  Google Scholar 

  47. Chen C-Y, Gherzi R, Andersen JS, Gaietta G, Jürchott K, Royer H-D, Mann M, Karin M (2000) Nucleolin and YB-1 are required for JNK-mediated interleukin-2 mRNA stabilization during T-cell activation. Genes Dev 14(10):1236–1248. https://doi.org/10.1101/gad.14.10.1236

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Sengupta TK, Bandyopadhyay S, Fernandes DJ, Spicer EK (2004) Identification of nucleolin as an AU-rich element binding protein involved in bcl-2 mRNA stabilization. J Biol Chem 279(12):10855–10863. https://doi.org/10.1074/jbc.M309111200

    Article  CAS  PubMed  Google Scholar 

  49. Abdelmohsen K, Gorospe M (2012) RNA-binding protein nucleolin in disease. RNA Biol 9(6):799–808. https://doi.org/10.4161/rna.19718

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

The authors wish to thank Prof. Tapas K. Sengupta (IISER, Kolkata, India) for pc-Nuc plasmid. We like to acknowledge the instrumental facilities funded by DST-FIST and UGC-CAS to the Department of Biophysics, Molecular Biology and Bioinformatics, University of Calcutta. This work was supported by fund from University Grants Commission, India [UGCF.5-1/2015/CAS-I (SAP-II)]. We acknowledge CSIR for providing the fellowship to S Das and S De.

Author information

Authors and Affiliations

  1. Department of Biophysics, Molecular Biology and Bioinformatics, University of Calcutta, 92 A.P.C. Road, Kolkata, 700009, India

    Sayantani Das, Soumasree De & Sumita Sengupta

Authors
  1. Sayantani Das
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Soumasree De
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sumita Sengupta
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

S Das performed most of the experiments, statistical analysis and wrote the manuscript. S De performed some of the experiments. SS conceived the idea, wrote and corrected the manuscript.

Corresponding author

Correspondence to Sumita Sengupta.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 160 KB)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Das, S., De, S. & Sengupta, S. Post-transcriptional regulation of MMP2 mRNA by its interaction with miR-20a and Nucleolin in breast cancer cell lines. Mol Biol Rep 48, 2315–2324 (2021). https://doi.org/10.1007/s11033-021-06261-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11033-021-06261-9

Keywords

Search

Navigation