Skip to main content

Advertisement

SpringerLink
Log in

The effect of bta-miR-1296 on the proliferation and extracellular matrix synthesis of bovine mammary fibroblasts

  • Published:
In Vitro Cellular & Developmental Biology - Animal Aims and scope Submit manuscript

Abstract

Mammary fibrosis in dairy cows is a chronic condition caused by mastitis, and can lead to serious culling of dairy cows resulting in huge economic losses in the dairy industry. MicroRNAs (miRNAs) exert an important role in regulating mammary gland health in dairy cows. This study investigated whether exosomal miRNAs in mammary epithelial cells can regulate the proliferation of bovine mammary fibroblasts (BMFBs) in mastitis. Liposome transfection technology was used to construct a cellular model of the overexpression and inhibition of miRNAs. The STarMir software, dual luciferase reporter gene test, real-time quantitative PCR (qRT-PCR), a Cell Counting Kit-8 (CCK-8), and a Western Blot and plate clone formation test were used to investigate the mechanism by which bta-miR-1296 regulates the proliferation of BMFBs. Target gene prediction results revealed that glutamate-ammonia ligase was a direct target gene by which bta-miR-1296 regulates cell proliferation. It was found that bta-miR-1296 significantly inhibited the proliferation of BMFBs. After BMFBs were transfected with a bta-miR-1296 mimic, mRNA expression in the extracellular matrix (ECM), α-smooth muscle actin (α-SMA), collagen type I alpha 1 chain (COL1α1) and collagen type III alpha 1 chain (COL3α1), and various cell growth factors (basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor-BB (PDGF-BB), and transforming growth factor-β1 (TGF-β1)) were down-regulated, and the expressions of α-SMA, COL1α1, COL3α1, phospho-extracellular regulated protein kinases, phospho-protein kinase B, TGF-β1, and phospho-Smad family member3 proteins were inhibited. In conclusion, bta-miR-1296 can inhibit the proliferation of BMFBs and the synthesis of ECM in BMFBs, thus affecting the occurrence and development of mammary fibrosis in dairy cows and laying the foundation for further studies to clarify the regulatory mechanism of mammary fibrosis.

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

Figure 1.
Figure 2.
Figure 3.
Figure 4.
Figure 5.
Figure 6.

Similar content being viewed by others

Data availability

The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

References

  • Alsaweed M, Lai CT, Hartmann PE, Geddes DT, Kakulas F (2016) Human milk miRNAs primarily originate from the mammary gland resulting in unique miRNA profiles of fractionated milk. Sci Rep 6:20680

  • Barratt SL, Blythe T, Ourradi K, Jarrett C, Welsh GI, Bates DO, Millar AB (2018) Effects of hypoxia and hyperoxia on the differential expression of VEGF-A isoforms and receptors in Idiopathic Pulmonary Fibrosis (IPF). Respir Res 19:9

    Article  PubMed  PubMed Central  Google Scholar 

  • Bi Y, Ding Y, Wu J, Miao Z, Wang J, Wang F (2020) Staphylococcus aureus induces mammary gland fibrosis through activating the TLR/NF-kappaB and TLR/AP-1 signaling pathways in mice. Microb Pathog 148:104427

    Article  CAS  PubMed  Google Scholar 

  • Buckley CD (2011) Why does chronic inflammation persist: An unexpected role for fibroblasts. Immunol Lett 138:12–14

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Chen G, He M, Yin Y, Yan T, Cheng W, Huang Z, Zhang L, Zhang H, Liu P, Zhu W, Zhu Y (2017a) miR-1296-5p decreases ERBB2 expression to inhibit the cell proliferation in ERBB2-positive breast cancer. Cancer Cell Int 17:95

    Article  PubMed  PubMed Central  Google Scholar 

  • Chen Q, Yang W, Wang X, Li X, Qi S, Zhang Y, Gao M-Q (2017b) TGF-β1 induces EMT in bovine mammary epithelial cells through the TGFβ1/Smad signaling pathway. Cell Physiol Biochem 43:82–93

    Article  CAS  PubMed  Google Scholar 

  • Choi J-H, Lim K-H, Park E, Kim J-Y, Choi Y-K, Baek K-H (2013) Glutamate-ammonia ligase and reduction of G0 population in PANC-1 cells. J Cell Biochem 114:303–313

    Article  CAS  PubMed  Google Scholar 

  • Deng H, Xie C, Ye Y, Du Z (2020) MicroRNA-1296 expression is associated with prognosis and inhibits cell proliferation and invasion by Wnt signaling in non-small cell lung cancer. Oncol Lett 19:623–630

    CAS  PubMed  Google Scholar 

  • Feng XH, Derynck R (2005) Specificity and versatility in tgf-beta signaling through Smads. Annu Rev Cell Dev Biol 21:659–693

    Article  CAS  PubMed  Google Scholar 

  • Gao Y, Wang Y, Li Y, Xia X, Zhao S, Che Y, Sun Y, Lei L (2016) TGF-beta1 promotes bovine mammary fibroblast proliferation through the ERK 1/2 signalling pathway. Cell Biol Int 40:750–760

    Article  CAS  PubMed  Google Scholar 

  • Gjorevski N, Nelson CM (2011) Integrated morphodynamic signalling of the mammary gland. Nat Rev Mol Cell Biol 12:581–593

    Article  CAS  PubMed  Google Scholar 

  • He G, Ma M, Yang W, Wang H, Zhang Y, Gao M-Q (2017) SDF-1 in mammary fibroblasts of bovine with mastitis induces EMT and inflammatory response of epithelial cells. Int J Biol Sci 13:604–614

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Henderson NC, Rieder F, Wynn TA (2020) Fibrosis: From mechanisms to medicines. Nature 587:555–566

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Iwano M, Plieth D, Danoff TM, Xue C, Okada H, Neilson EG (2002) Evidence that fibroblasts derive from epithelium during tissue fibrosis. J Clin Investig 110:341–350

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Jing-Yao Liu GFM, Lei W-H, Hart CE, Lasky JA, Brody AR (1997) Rapid activation of PDGF-A and -B expression at sites of lung injury in Asbestos-exposed rats. Am J Respir Cell Mol Biol 17:129–140

    Article  Google Scholar 

  • Lee YJ, Han HJ (2010) Troglitazone ameliorates high glucose-induced EMT and dysfunction of SGLTs through PI3K/Akt, GSK-3β, Snail1, and β-catenin in renal proximal tubule cells. Am J Physiol Ren Physiol 298:F1263–F1275

    Article  CAS  Google Scholar 

  • Lyu C-C, Yuan B, Che H-Y, Meng Y, Zheng Y, He Y-T, Ji Z-H, Cong S, Ji X-Y, Jiang H, Zhang J (2022) Musashi2 binds TGFβR1 regulates mastitis viaTGFβ/Smad signaling pathway in dairy cows. Res Square. https://doi.org/10.21203/rs.3.rs-2364816/v1

  • Maseki S, Ijichi K, Tanaka H, Fujii M, Hasegawa Y, Ogawa T, Murakami S, Kondo E, Nakanishi H (2012) Acquisition of EMT phenotype in the gefitinib-resistant cells of a head and neck squamous cell carcinoma cell line through AKT/GSK-3β/snail signalling pathway. Br J Cancer 106:1196–1204

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Massague J, Gomis RR (2006) The logic of TGFbeta signaling. FEBS Lett 580:2811–2820

    Article  CAS  PubMed  Google Scholar 

  • Moustakas A, Heldin C-H (2016) Mechanisms of TGFβ-induced epithelial–mesenchymal transition. J Clin Med 5(7):63

  • Schlessinger J (2004) Common and distinct elements in cellular signaling via EGF and FGF receptors. Science 306(5701):1506–7

    Article  CAS  PubMed  Google Scholar 

  • Scotton CJ, Chambers RC (2007) Molecular targets in pulmonary fibrosis: The myofibroblast in focus. Chest 132:1311–1321

    Article  PubMed  Google Scholar 

  • Seki E, De Minicis S, Osterreicher CH, Kluwe J, Osawa Y, Brenner DA, Schwabe RF (2007) TLR4 enhances TGF-beta signaling and hepatic fibrosis. Nat Med 13:1324–1332

    Article  CAS  PubMed  Google Scholar 

  • Shan X, Wen W, Zhu D, Yan T, Cheng W, Huang Z, Zhang L, Zhang H, Wang T, Zhu W, Zhu Y, Zhu J (2017) miR 1296–5p inhibits the migration and invasion of gastric cancer cells by repressing ERBB2 expression. PLoS One 12:e0170298

    Article  PubMed  PubMed Central  Google Scholar 

  • Tao Y, Ma C, Fan Q, Wang Y, Han T, Sun C (2018) MicroRNA-1296 facilitates proliferation, migration and invasion of colorectal cancer cells by targeting SFPQ. J Cancer 9:2317–2326

    Article  PubMed  PubMed Central  Google Scholar 

  • Thiery JP (2002) Epithelial–mesenchymal transitions in tumour progression. Nat Rev Cancer 2:442–454

    Article  CAS  PubMed  Google Scholar 

  • Vermeulen T, Görg B, Vogl T, Wolf M, Varga G, Toutain A, Paul R, Schliess F, Häussinger D, Häberle J (2008) Glutamine synthetase is essential for proliferation of fetal skin fibroblasts. Arch Biochem Biophys 478:96–102

    Article  CAS  PubMed  Google Scholar 

  • Wang B, Ding W, Zhang M, Li H, Gu Y (2015) Rapamycin attenuates aldosterone-induced tubulointerstitial inflammation and fibrosis. Cell Physiol Biochem 35:116–125

    Article  PubMed  Google Scholar 

  • Wang J, Zhang X, He X, Yang B, Wang H, Shan X, Li C, Sun D, Wu R (2018) LPS-induced reduction of triglyceride synthesis and secretion in dairy cow mammary epithelial cells via decreased SREBP1 expression and activity. J Dairy Res 85:439–444

    Article  CAS  PubMed  Google Scholar 

  • Wang Y, Fan S, Lu J, Zhang Z, Wu D, Wu Z, Zheng Y (2017) GLUL promotes cell proliferation in breast cancer. J Cell Biochem 118:2018–2025

    Article  CAS  PubMed  Google Scholar 

  • Wynn TA, Ramalingam TR (2012) Mechanisms of fibrosis: therapeutic translation for fibrotic disease. Nat Med 18:1028–1040

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Xu H, Zhang T, Hu X, Xie Y, Wu R, Lian S, Wang J (2021) Exosomal miR-193b-5p as a regulator of LPS-induced inflammation in dairy cow mammary epithelial cells. In Vitro Cell Dev Biol Anim 57:695–703

    Article  CAS  PubMed  Google Scholar 

  • Xu Q, Liu X, Liu Z, Zhou Z, Wang Y, Tu J, Li L, Bao H, Yang L, Tu K (2017) MicroRNA-1296 inhibits metastasis and epithelial-mesenchymal transition of hepatocellular carcinoma by targeting SRPK1-mediated PI3K/AKT pathway. Mol Cancer 16:103

    Article  PubMed  PubMed Central  Google Scholar 

  • Yang W, Li X, Qi S, Li X, Zhou K, Qing S, Zhang Y, Gao M-Q (2017) lncRNA H19 is involved in TGF-β1-induced epithelial to mesenchymal transition in bovine epithelial cells through PI3K/AKT Signaling Pathway. PeerJ 5:e3950

  • Zhao H, Feng YL, Liu T, Wang JJ, Yu J (2021) MicroRNAs in organ fibrosis: From molecular mechanisms to potential therapeutic targets. Pathol Res Pract 225:153588

    Article  CAS  PubMed  Google Scholar 

  • Zou X-Z, Liu T, Gong Z-C, Hu C-P, Zhang Z (2017) MicroRNAs-mediated epithelial-mesenchymal transition in fibrotic diseases. Eur J Pharmacol 796:190–206

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by the National Natural Science Foundation of China (31772698, 32172814, 31972747, 32002247, 32172937). We thank International Science Editing (http://www.internationalscienceediting.com) for editing this manuscript.

Author information

Authors and Affiliations

  1. Key Laboratory of Bovine Disease Control in Northeast China, Ministry of Agriculture and Rural Affairs, College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Heilongjiang Province, Daqing, 163319, People’s Republic of China

    Yuejie Yang, Tao Yuan, Rui Wu, Zijian Geng, Shuai Lian & Jianfa Wang

  2. Veterinary Medicine Faculty, Heilongjiang Bayi Agricultural University, No. 2 Xinyang Road, Daqing, People’s Republic of China

    Jianfa Wang

Authors
  1. Yuejie Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tao Yuan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Rui Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Zijian Geng
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Shuai Lian
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jianfa Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

All authors reviewed the manuscript. R. W., S.L. and J.W. designed the experiments. Y.Y., and T.Y. performed the experiments. Y.Y., J.W. and T.Y. help check the whole manuscript for terminology. Y.Y., T.Y., Z.G., and J.W. performed the data analysis and wrote the manuscript.

Corresponding authors

Correspondence to Shuai Lian or Jianfa Wang.

Ethics declarations

Competing interest

All authors declare that they have no conflict of interest.

Supplementary Information

Below is the link to the electronic supplementary material.

Figure S1

Primary culture, purification and identification of BMFBs. (A) At 6 days of culture, BMFBs were observed growing out of the tissue mass (40×). (B) On the bottom 9 days of culture, pebble-like BMECs began to appear (40×). (C) On day 12 of culture, BMFBs had grown all over the cell vial (40×). (D) DAPI-stained nuclei (blue). (E) Expression of waveform protein in BMFBs (green). (F) Merging of D and E. (PNG 2.00 MB)

High resolution image (TIF 3842 KB)

Supplementary file2 (XLSX 210 KB)

Supplementary file3 (XLSX 90 KB)

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Yang, Y., Yuan, T., Wu, R. et al. The effect of bta-miR-1296 on the proliferation and extracellular matrix synthesis of bovine mammary fibroblasts. In Vitro Cell.Dev.Biol.-Animal 60, 183–194 (2024). https://doi.org/10.1007/s11626-024-00851-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11626-024-00851-0

Keywords

Search

Navigation