Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Microvesicle-mediated delivery of miR-1343: impact on markers of fibrosis

Abstract

Tissue fibrosis, the development of fibrous connective tissue as a result of injury or damage, is associated with many common diseases and cannot be treated effectively. The complex biological processes accompanying fibrosis often involve aberrant signaling through the transforming growth factor beta (TGF-β) pathway. In the search for mechanisms to repress this signaling, microRNAs have emerged as a novel class of molecules capable of targeting single members of the TGF-β pathway, or the pathway as a whole. We previously identified miR-1343 as a potent repressor of TGF-β signaling and fibrosis through the direct attenuation of both canonical TGF-β receptors. Here, we build upon our previous findings to better characterize the function of endogenous miR-1343 in normal biology and examine the potential role of exogenous miR-1343 as a repressor of TGF-β signaling. CRISPR/Cas9-mediated deletion of miR-1343 from A549 lung epithelial cells impacts several processes and genes implicated in fibrosis and known to be TGF-β pathway effectors. Moreover, the responses are opposite to those we observed previously when miR-1343 was overexpressed in the same cell type. We also show that miR-1343 can be shuttled into exosomes, a type of extracellular vesicle that are exported by cells into the surrounding medium and can be absorbed by distant target cells. miR-1343 delivered into primary lung fibroblasts by exosomes has a measurable function in reducing TGF-β signaling and markers of fibrosis. These results highlight a role for miR-1343 in fine-tuning the TGF-β pathway and suggest its use as a therapeutic in fibrotic disease.

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

References

  1. Akhurst RJ, Hata A (2012) Targeting the TGFβ signalling pathway in disease. Nat Rev Drug Discov 11:790–811

  2. Alvarez-Erviti L, Seow Y, Yin H, Betts C, Lakhal S, Wood MJA (2011) Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes. Nat Biotechnol 29:341–345

  3. Boehm JR, Kutz SM, Sage EH, Staiano-Coico L, Higgins PJ (1999) Growth state-dependent regulation of plasminogen activator inhibitor type-1 gene expression during epithelial cell stimulation by serum and transforming growth factor-beta1. J Cell Physiol 181:96–106

  4. Brigstock DR (2010) Connective tissue growth factor (CCN2, CTGF) and organ fibrosis: lessons from transgenic animals. J Cell Commun Signal 4:1–4

  5. Chen C, Deng B, Qiao M, Zheng R, Chai J, Ding Y, Peng J, Jiang S (2012) Solexa sequencing identification of conserved and novel microRNAs in backfat of large white and Chinese Meishan pigs. PLoS ONE 7:e31426

  6. Chen T, Xi Q-Y, Ye R-S, Cheng X, Qi Q-E, Wang S-B, Shu G, Wang L-N, Zhu X-T, Jiang Q-Y, Zhang Y-L (2014) Exploration of microRNAs in porcine milk exosomes. BMC Genomics 15:100

  7. Chiba M, Kimura M, Asari S (2012) Exosomes secreted from human colorectal cancer cell lines contain mRNAs, microRNAs and natural antisense RNAs, that can transfer into the human hepatoma HepG2 and lung cancer A549 cell lines. Oncol Rep 28:1551–1558

  8. Christopher AF, Kaur RP, Kaur G, Kaur A, Gupta V, Bansal P (2016) MicroRNA therapeutics: discovering novel targets and developing specific therapy. Perspect Clin Res 7:68–74

  9. Cohen P, Rajah R, Rosenbloom J, Herrick DJ (2000) IGFBP-3 mediates TGF-beta1-induced cell growth in human airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 278:L545–L551

  10. Collins SJ, Gallo RC, Gallagher RE (1977) Continuous growth and differentiation of human myeloid leukaemic cells in suspension culture. Nature 270:347–349

  11. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339:819–823

  12. Corvol H, Blackman SM, Boëlle P-Y, Gallins PJ, Pace RG, Stonebraker JR, Accurso FJ, Clement A, Collaco JM, Dang H, Dang AT, Franca A, Gong J, Guillot L, Keenan K, Li W, Lin F et al (2015) Genome-wide association meta-analysis identifies five modifier loci of lung disease severity in cystic fibrosis. Nat Commun 6:8382

  13. Derynck R, Zhang YE (2003) Smad-dependent and Smad-independent pathways in TGF-β family signalling. Nature 425:577–584

  14. Duncan MR, Frazier KS, Abramson S, Williams S, Klapper H, Huang X, Grotendorst GR (1999) Connective tissue growth factor mediates transforming growth factor beta-induced collagen synthesis: down-regulation by cAMP. FASEB J 13:1774–1786

  15. Flynn RS, Mahavadi S, Murthy KS, Grider JR, Kellum JM, Akbari H, Kuemmerle JF (2011) Endogenous IGFBP-3 regulates excess collagen expression in intestinal smooth muscle cells of Crohn’s disease strictures. Inflamm Bowel Dis 17:193–201

  16. Fossum SL, Mutolo MJ, Yang R, Dang H, O’Neal WK, Knowles MR, Leir S-H, Harris A (2014) Ets homologous factor regulates pathways controlling response to injury in airway epithelial cells. Nucleic Acids Res 42:13588–13598

  17. Gallo A, Tandon M, Alevizos I, Illei GG (2012) The majority of microRNAs detectable in serum and saliva is concentrated in exosomes. PLoS ONE 7:e30679

  18. Giard DJ, Aaronson SA, Todaro GJ, Arnstein P, Kersey JH, Dosik H, Parks WP (1973) In vitro cultivation of human tumors: establishment of cell lines derived from a series of solid tumors. J Natl Cancer Inst 51:1417–1423

  19. Gore-Hyer E, Shegogue D, Markiewicz M, Lo S, Hazen-Martin D, Greene EL, Grotendorst G, Trojanowska M (2002) TGF-beta and CTGF have overlapping and distinct fibrogenic effects on human renal cells. Am J Physiol Renal Physiol 283:F707–F716

  20. Grotendorst GR, Rahmanie H, Duncan MR (2004) Combinatorial signaling pathways determine fibroblast proliferation and myofibroblast differentiation. FASEB J 18:469–479

  21. Harding C, Heuser J, Stahl P (1984) Endocytosis and intracellular processing of transferrin and colloidal gold-transferrin in rat reticulocytes: demonstration of a pathway for receptor shedding. Eur J Cell Biol 35:256–263

  22. Huan J, Hornick NI, Shurtleff MJ, Skinner AM, Goloviznina NA, Roberts CT, Kurre P (2013) RNA trafficking by acute myelogenous leukemia exosomes. Cancer Res 73:918–929

  23. Huang DW, Sherman BT, Lempicki RA (2009a) Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res 37:1–13

  24. Huang DW, Sherman BT, Lempicki RA (2009b) Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc 4:44–57

  25. Ivkovic S, Yoon BS, Popoff SN, Safadi FF, Libuda DE, Stephenson RC, Daluiski A, Lyons KM (2003) Connective tissue growth factor coordinates chondrogenesis and angiogenesis during skeletal development. Development 130:2779–2791

  26. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (2012) A programmable dual-RNA-guided DNA Endonuclease in adaptive bacterial immunity. Science 337:816–821

  27. Jinek M, East A, Cheng A, Lin S, Ma E, Doudna J (2013) RNA-programmed genome editing in human cells. eLife 2:e00471

  28. Krützfeldt J, Rajewsky N, Braich R, Rajeev KG, Tuschl T, Manoharan M, Stoffel M (2005) Silencing of microRNAs in vivo with “antagomirs”. Nature 438:685–689

  29. Lai EC (2015) Two decades of miRNA biology: lessons and challenges. RNA 21:675–677

  30. Leask A, Abraham DJ (2004) TGF-beta signaling and the fibrotic response. FASEB J 18:816–827

  31. Li G, Luna C, Qiu J, Epstein DL, Gonzalez P (2009) Alterations in microRNA expression in stress-induced cellular senescence. Mech Ageing Dev 130:731–741

  32. Loeys BL, Chen J, Neptune ER, Judge DP, Podowski M, Holm T, Meyers J, Leitch CC, Katsanis N, Sharifi N, Xu FL, Myers LA, Spevak PJ, Cameron DE, De Backer J, Hellemans J, Chen Y et al (2005) A syndrome of altered cardiovascular, craniofacial, neurocognitive and skeletal development caused by mutations in TGFBR1 or TGFBR2. Nat Genet 37:275–281

  33. Lv L-L, Cao Y, Liu D, Xu M, Liu H, Tang R-N, Ma K-L, Liu B-C (2013) Isolation and quantification of microRNAs from urinary exosomes/microvesicles for biomarker discovery. Int J Biol Sci 9:1021–1031

  34. Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM (2013) RNA-guided human genome engineering via Cas9. Science 339:823–826

  35. Martin JL, Baxter RC (1986) Insulin-like growth factor-binding protein from human plasma Purification and characterization. J Biol Chem 261:8754–8760

  36. Meckes DG, Shair KHY, Marquitz AR, Kung C-P, Edwards RH, Raab-Traub N (2010) Human tumor virus utilizes exosomes for intercellular communication. Proc Natl Acad Sci U S A 107:20370–20375

  37. Miska EA, Alvarez-Saavedra E, Abbott AL, Lau NC, Hellman AB, McGonagle SM, Bartel DP, Ambros VR, Horvitz HR (2007) Most Caenorhabditis Elegans microRNAs are individually not essential for development or viability. PLoS Genet 3:e215

  38. O’Reilly S (2016) MicroRNAs in fibrosis: opportunities and challenges. Arthritis Res Ther 18:11

  39. Ohno S, Takanashi M, Sudo K, Ueda S, Ishikawa A, Matsuyama N, Fujita K, Mizutani T, Ohgi T, Ochiya T, Gotoh N, Kuroda M (2013) Systemically injected Exosomes targeted to EGFR deliver antitumor MicroRNA to breast cancer cells. Mol Ther 21:185–191

  40. Pan BT, Johnstone RM (1983) Fate of the transferrin receptor during maturation of sheep reticulocytes in vitro: selective externalization of the receptor. Cell 33:967–978

  41. Park CY, Choi YS, McManus MT (2010) Analysis of microRNA knockouts in mice. Hum Mol Genet 19:R169–R175

  42. Pegtel DM, Cosmopoulos K, Thorley-Lawson DA, van Eijndhoven MAJ, Hopmans ES, Lindenberg JL, de Gruijl TD, Wurdinger T, Middeldorp JM (2010) Functional delivery of viral miRNAs via exosomes. Proc Natl Acad Sci U S A 107:6328–6333

  43. Pilewski JM, Liu L, Henry AC, Knauer AV, Feghali-Bostwick CA (2005) Insulin-like growth factor binding proteins 3 and 5 are overexpressed in idiopathic pulmonary fibrosis and contribute to extracellular matrix deposition. Am J Pathol 166:399–407

  44. Properzi F, Logozzi M, Fais S (2013) Exosomes: the future of biomarkers in medicine. Biomark Med 7:769–778

  45. Reimand J, Arak T, Adler P, Kolberg L, Reisberg S, Peterson H, Vilo J (2016) G:profiler-a web server for functional interpretation of gene lists (2016 update). Nucleic Acids Res 44:W83–W89

  46. Sandfeld-Paulsen B, Jakobsen KR, Bæk R, Folkersen BH, Rasmussen TR, Meldgaard P, Varming K, Jørgensen MM, Sorensen BS (2016) Exosomal proteins as diagnostic biomarkers in lung cancer. J Thorac Oncol 11:1701–1710

  47. Schedlich LJ, Yenson VM, Baxter RC (2013) TGF-β-induced expression of IGFBP-3 regulates IGF1R signaling in human osteosarcoma cells. Mol Cell Endocrinol 377:56–64

  48. Shi Y, Massagué J (2003) Mechanisms of TGF-beta signaling from cell membrane to the nucleus. Cell 113:685–700

  49. Simone NL, Soule BP, Ly D, Saleh AD, Savage JE, Degraff W, Cook J, Harris CC, Gius D, Mitchell JB (2009) Ionizing radiation-induced oxidative stress alters miRNA expression. PLoS ONE 4:e6377

  50. Skonier J, Neubauer M, Madisen L, Bennett K, Plowman GD, Purchio AF (1992) cDNA cloning and sequence analysis of beta ig-h3, a novel gene induced in a human adenocarcinoma cell line after treatment with transforming growth factor-beta. DNA Cell Biol 11:511–522

  51. Sonnylal S, Shi-Wen X, Leoni P, Naff K, Van Pelt CS, Nakamura H, Leask A, Abraham D, Bou-Gharios G, de Crombrugghe B (2010) Selective expression of connective tissue growth factor in fibroblasts in vivo promotes systemic tissue fibrosis. Arthritis Rheum 62:1523–1532

  52. Stolzenburg LR, Wachtel S, Dang H, Harris A (2016) miR-1343 attenuates pathways of fibrosis by targeting the TGF-β receptors. Biochem J 473:245–256

  53. Trapnell C, Roberts A, Goff L, Pertea G, Kim D, Kelley DR, Pimentel H, Salzberg SL, Rinn JL, Pachter L (2012) Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and cufflinks. Nat Protoc 7:562–578

  54. Umezu T, Ohyashiki K, Kuroda M, Ohyashiki JH (2013) Leukemia cell to endothelial cell communication via exosomal miRNAs. Oncogene 32:2747–2755

  55. Valadi H, Ekström K, Bossios A, Sjöstrand M, Lee JJ, Lötvall JO (2007) Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol 9:654–659

  56. van Rooij E, Sutherland LB, Qi X, Richardson JA, Hill J, Olson EN (2007) Control of stress-dependent cardiac growth and gene expression by a MicroRNA. Science 316:575–579

  57. Wang X, Hao L, Bu H-F, Scott AW, Tian K, Liu F, De Plaen IG, Liu Y, Mirkin CA, Tan X-D (2016) Spherical nucleic acid targeting microRNA-99b enhances intestinal MFG-E8 gene expression and restores enterocyte migration in lipopolysaccharide-induced septic mice. Sci Rep 6:31687

  58. Wright FA, Strug LJ, Doshi VK, Commander CW, Blackman SM, Sun L, Berthiaume Y, Cutler D, Cojocaru A, Collaco JM, Corey M, Dorfman R, Goddard K, Green D, Kent JW, Lange EM, Lee S et al (2011) Genome-wide association and linkage identify modifier loci of lung disease severity in cystic fibrosis at 11p13 and 20q13.2. Nat Genet 43:539–546

  59. Wynn TA (2011) Integrating mechanisms of pulmonary fibrosis. J Exp Med 208:1339–1350

  60. Xie S, Sukkar MB, Issa R, Oltmanns U, Nicholson AG, Chung KF (2004) Regulation of TGF- 1-induced connective tissue growth factor expression in airway smooth muscle cells. AJP Lung Cell Mol Physiol 288:L68–L76

  61. Yoo BH, Bochkareva E, Bochkarev A, Mou T-C, Gray DM (2004) 2′-O-methyl-modified phosphorothioate antisense oligonucleotides have reduced non-specific effects in vitro. Nucleic Acids Res 32:2008–2016

  62. Zhang J, Li S, Li L, Li M, Guo C, Yao J, Mi S (2015) Exosome and Exosomal MicroRNA: trafficking, sorting, and function. Genomics Proteomics Bioinformatics 13:17–24

  63. Zhang Y, Wang Z, Gemeinhart RA (2013) Progress in microRNA delivery. J Control Release 172:962–974

  64. Zhou Q, Li M, Wang X, Li Q, Wang T, Zhu Q, Zhou X, Wang X, Gao X, Li X (2012) Immune-related microRNAs are abundant in breast milk exosomes. Int J Biol Sci 8:118–123

Download references

Acknowledgments

We thank Dr. Scott Randell, Dr. Wanda O’Neal and Lisa Jones (University of North Carolina Marsico Lung Institute) for the primary lung fibroblast cultures and A549 RNA-seq.

Funding

This work was supported by the National Institutes of Health (R01HL117843 to AH; F31HL126458 to LRS) and the Children’s Research Fund.

Author information

Correspondence to Ann Harris.

Electronic supplementary material

ESM 1

(PDF 8918 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Stolzenburg, L.R., Harris, A. Microvesicle-mediated delivery of miR-1343: impact on markers of fibrosis. Cell Tissue Res 371, 325–338 (2018). https://doi.org/10.1007/s00441-017-2697-6

Download citation

Keywords

  • microRNA
  • TGF-β
  • Fibrosis
  • Extracellular vesicle
  • Exosome