Protective effects of miR-25 against hypoxia/reoxygenation‑induced fibrosis and apoptosis of H9c2 cells

Liu,Qifang; Wang,Yongjin; Yang,Tianlun; Wei,Wu

doi:10.3892/ijmm.2016.2702

Protective effects of miR-25 against hypoxia/reoxygenation‑induced fibrosis and apoptosis of H9c2 cells

Authors:
- Qifang Liu
- Yongjin Wang
- Tianlun Yang
- Wu Wei
View Affiliations

Affiliations: Department of Cardiology, Xiangya Hospital, Central South University, Changsha, Hunan 410078, P.R. China, Department of Cardiology, Heping Hospital, Changzhi Medical College, Changzhi, Shanxi 046000, P.R. China
Published online on: August 11, 2016 https://doi.org/10.3892/ijmm.2016.2702
Pages: 1225-1234

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

It has been previously demonstrated that microRNA (miR)-25 plays critical roles in collagen deposition. Ischemia/reperfusion injury to the myocardium results in fibrosis and collagen deposition. However, whether miR-25 is involved in the development of hypoxia/reoxygenation (H/R)‑induced fibrosis in cardiomyocytes or not remains largely unknown. For this purpose, in the present study, cardiomyocyte H9c2 cells were subjected to H/R. The techniques of flow cytometry, western blot analysis and RT-qPCR were used and we observed increases in the cell apoptosis rate and fibrosis as well as blocking of the cell cycle in the G1 phase. Moreover, the expression of miR-25 was downregulated after H/R and high‑mobility group box 1 (HMGB1) expression was increased. We also found that the overexpression of miR-25 under conditions of H/R inhibited fibrosis and cell apoptosis as well as reversing the cell cycle blocking. Additionally, the targeting of HMGB1 by miR-25 was confirmed by a dual‑luciferase reporter gene assay. Moreover, the effects of miR-25 were further enhanced by a transforming growth factor-β1 (TGF-β1)/Smad3 inhibitor, SB431542, as fibrosis was reduced and apoptosis was suppressed. In conclusion, the protective effects of miR-25 against H/R-induced fibrosis and apoptosis H9c2 cells were due to direct targeting of HMGB1 through the downregulation of the TGF-β1/Smad3 signaling pathway.

View Figures

View References

1	Lee SW, Won JY, Kim WJ, Lee J, Kim KH, Youn SW, Kim JY, Lee EJ, Kim YJ, Kim KW and Kim HS: Snail as a potential target molecule in cardiac fibrosis: paracrine action of endothelial cells on fibroblasts through snail and CTGF axis. Mol Ther. 21:1767–1777. 2013. View Article : Google Scholar : PubMed/NCBI
2	Dhooghe B, Noël S, Huaux F and Leal T: Lung inflammation in cystic fibrosis: pathogenesis and novel therapies. Clin Biochem. 47:539–546. 2014. View Article : Google Scholar : PubMed/NCBI
3	Srivastava SP, Koya D and Kanasaki K: MicroRNAs in kidney fibrosis and diabetic nephropathy: roles on EMT and EndMT. BioMed Res Int. 2013:1254692013. View Article : Google Scholar : PubMed/NCBI
4	López-Novoa JM and Nieto MA: Inflammation and EMT: an alliance towards organ fibrosis and cancer progression. EMBO Mol Med. 1:303–314. 2009. View Article : Google Scholar
5	Andersson U and Tracey KJ: HMGB1 is a therapeutic target for sterile inflammation and infection. Annual Rev Immunol. 29:139–162. 2011. View Article : Google Scholar
6	Lynch J, Nolan S, Slattery C, Feighery R, Ryan MP and McMorrow T: High-mobility group box protein 1: a novel mediator of inflammatory-induced renal epithelial-mesenchymal transition. Am J Nephrol. 32:590–602. 2010. View Article : Google Scholar : PubMed/NCBI
7	Andrassy M, Volz HC, Igwe JC, Funke B, Eichberger SN, Kaya Z, Buss S, Autschbach F, Pleger ST, Lukic IK, et al: High-mobility group box-1 in ischemia-reperfusion injury of the heart. Circulation. 117:3216–3226. 2008. View Article : Google Scholar : PubMed/NCBI
8	Roush S and Slack FJ: The let-7 family of microRNAs. Trends Cell Biol. 18:505–516. 2008. View Article : Google Scholar : PubMed/NCBI
9	Zhao J, Tang N, Wu K, Dai W, Ye C, Shi J, Zhang J, Ning B, Zeng X and Lin Y: MiR-21 simultaneously regulates ERK1 signaling in HSC activation and hepatocyte EMT in hepatic fibrosis. PLoS One. 9:e1080052014. View Article : Google Scholar : PubMed/NCBI
10	Brønnum H, Andersen DC, Schneider M, Sandberg MB, Eskildsen T, Nielsen SB, Kalluri R and Sheikh SP: miR-21 promotes fibrogenic epithelial-to-mesenchymal transition of epicardial mesothelial cells involving Programmed Cell Death 4 and Sprouty-1. PLoS One. 8:e562802013. View Article : Google Scholar : PubMed/NCBI
11	Yang Y, Huang JQ, Zhang X and Shen LF: MiR-129-2 functions as a tumor suppressor in glioma cells by targeting HMGB1 and is down-regulated by DNA methylation. Mol Cell Biochem. 404:229–239. 2015. View Article : Google Scholar : PubMed/NCBI
12	Lu F, Zhang J, Ji M, Li P, Du Y, Wang H, Zang S, Ma D, Sun X and Ji C: miR-181b increases drug sensitivity in acute myeloid leukemia via targeting HMGB1 and Mcl-1. Int J Oncol. 45:383–392. 2014.PubMed/NCBI
13	Li X, Wang S, Chen Y, Liu G and Yang X: miR-22 targets the 3′UTR of HMGB1 and inhibits the HMGB1-associated autophagy in osteosarcoma cells during chemotherapy. Tumour Biol. 35:6021–6028. 2014. View Article : Google Scholar : PubMed/NCBI
14	Tang Q, Zhong H, Xie F, Xie J, Chen H and Yao G: Expression of miR-106b-25 induced by salvianolic acid B inhibits epithelial- to-mesenchymal transition in HK-2 cells. Eur J Pharmacol. 741:97–103. 2014. View Article : Google Scholar : PubMed/NCBI
15	Divakaran V, Adrogue J, Ishiyama M, Entman ML, Haudek S, Sivasubramanian N and Mann DL: Adaptive and maladptive effects of SMAD3 signaling in the adult heart after hemodynamic pressure overloading. Circ Heart Fail. 2:633–642. 2009. View Article : Google Scholar : PubMed/NCBI
16	Barallobre-Barreiro J, Didangelos A, Schoendube FA, Drozdov I, Yin X, Fernández-Caggiano M, Willeit P, Puntmann VO, Aldama-López G, Shah AM, et al: Proteomics analysis of cardiac extracellular matrix remodeling in a porcine model of ischemia/reperfusion injury. Circulation. 125:789–802. 2012. View Article : Google Scholar : PubMed/NCBI
17	Chen Z, Hu Z, Lu Z, Cai S, Gu X, Zhuang H, Ruan Z, Xia Z, Irwin MG, Feng D and Zhang L: Differential microRNA profiling in a cellular hypoxia reoxygenation model upon posthypoxic propofol treatment reveals alterations in autophagy signaling network. Oxid Med Cell Longev. 2013:3784842013. View Article : Google Scholar
18	Di Y, Lei Y, Yu F, Changfeng F, Song W and Xuming M: MicroRNA expression and function in cerebral ischemia reperfusion injury. J Mol Neurosci. 53:242–250. 2014. View Article : Google Scholar : PubMed/NCBI
19	Zhang J, Ren JY, Chen H and Han GP: Statins decrease expression of five inflammation-associated microRNAs in the plasma of patients with unstable angina. Beijing Da. Xue Xue Bao. 47:761–768. 2015.In Chinese.
20	Dirkx E, Gladka MM, Philippen LE, Armand AS, Kinet V, Leptidis S, El Azzouzi H, Salic K, Bourajjaj M, da Silva GJ, et al: Nfat and miR-25 cooperate to reactivate the transcription factor Hand2 in heart failure. Nat Cell Biol. 15:1282–1293. 2013. View Article : Google Scholar : PubMed/NCBI
21	Klune JR, Dhupar R, Cardinal J, Billiar TR and Tsung A: HMGB1: endogenous danger signaling. Mol Med. 14:476–484. 2008. View Article : Google Scholar : PubMed/NCBI
22	Tsung A, Klune JR, Zhang X, Jeyabalan G, Cao Z, Peng X, Stolz DB, Geller DA, Rosengart MR and Billiar TR: HMGB1 release induced by liver ischemia involves Toll-like receptor 4-dependent reactive oxygen species production and calcium-mediated signaling. J Exp Med. 204:2913–2923. 2007. View Article : Google Scholar : PubMed/NCBI
23	Xu H, Su Z, Wu J, Yang M, Penninger JM, Martin CM, Kvietys PR and Rui T: The alarmin cytokine, high mobility group box 1, is produced by viable cardiomyocytes and mediates the lipopolysaccharide-induced myocardial dysfunction via a TLR4/phosphatidylinositol 3-kinase gamma pathway. J Immunol. 184:1492–1498. 2010. View Article : Google Scholar
24	Hu X, Jiang H, Bai Q, Zhou X, Xu C, Lu Z, Cui B and Wen H: Increased serum HMGB1 is related to the severity of coronary artery stenosis. Clin Chim Acta. 406:139–142. 2009. View Article : Google Scholar : PubMed/NCBI
25	Andrassy M, Volz HC, Riedle N, Gitsioudis G, Seidel C, Laohachewin D, Zankl AR, Kaya Z, Bierhaus A, Giannitsis E, et al: HMGB1 as a predictor of infarct transmurality and functional recovery in patients with myocardial infarction. J Intern Med. 270:245–253. 2011. View Article : Google Scholar : PubMed/NCBI
26	Zhao D, Wang Y and Xu Y: Decreased serum endogenous secretory receptor for advanced glycation endproducts and increased cleaved receptor for advanced glycation endproducts levels in patients with atrial fibrillation. Int J Cardiol. 158:471–472. 2012. View Article : Google Scholar : PubMed/NCBI
27	Frangogiannis NG, Smith CW and Entman ML: The inflammatory response in myocardial infarction. Cardiovasc Res. 53:31–47. 2002. View Article : Google Scholar
28	Hu X, Fu W and Jiang H: HMGB1: A potential therapeutic target for myocardial ischemia and reperfusion injury. Int J Cardiol. 155:4892012. View Article : Google Scholar : PubMed/NCBI
29	Su Z, Yin J, Wang T, Sun Y, Ni P, Ma R, Zhu H, Zheng D, Shen H, Xu W and Xu H: Up-regulated HMGB1 in EAM directly led to collagen deposition by a PKCβ/Erk1/2-dependent pathway: cardiac fibroblast/myofibroblast may be another source of HMGB1. J Cell Mol Med. 18:1740–1751. 2014. View Article : Google Scholar : PubMed/NCBI
30	Saltis J, Agrotis A and Bobik A: Regulation and interactions of transforming growth factor-beta with cardiovascular cells: implications for development and disease. Clin Exp Pharmacol Physiol. 23:193–200. 1996. View Article : Google Scholar : PubMed/NCBI
31	Petrocca F, Vecchione A and Croce CM: Emerging role of miR-106b-25/miR-17-92 clusters in the control of transforming growth factor beta signaling. Cancer Res. 68:8191–8194. 2008. View Article : Google Scholar : PubMed/NCBI