Downregulation of microRNA‑199a‑5p attenuates hypoxia/reoxygenation‑induced cytotoxicity in cardiomyocytes by targeting the HIF‑1α‑GSK3β‑mPTP axis

Liu,Da‑Wei; Zhang,Ya‑Nan; Hu,Hai‑Juan; Zhang,Pu‑Qiang; Cui,Wei

doi:10.3892/mmr.2019.10197

Downregulation of microRNA‑199a‑5p attenuates hypoxia/reoxygenation‑induced cytotoxicity in cardiomyocytes by targeting the HIF‑1α‑GSK3β‑mPTP axis

Authors:
- Da‑Wei Liu
- Ya‑Nan Zhang
- Hai‑Juan Hu
- Pu‑Qiang Zhang
- Wei Cui
View Affiliations

Affiliations: Department of Cardiology, Second Hospital of Hebei Medical University and Hebei Institute of Cardiovascular Research, Shijiazhuang, Hebei 050011, P.R. China
Published online on: April 25, 2019 https://doi.org/10.3892/mmr.2019.10197
Pages: 5335-5344
Copyright: © Liu et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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

MicroRNAs (miRs) have been identified as critical regulatory molecules in myocardial ischemia/reperfusion injury; however, the exact expression profile of miR‑199a‑5p in reperfusion injury and the underlying pathogenic mechanisms remain unclear. In the present study, it was revealed that miR‑199a‑5p expression was significantly increased in the plasma of patients with acute myocardial infarction and in a H9c2 cell model of oxygen‑glucose deprivation and reperfusion (OGD/R) via reverse transcription‑quantitative PCR. H9c2 cells were transfected with miR‑199a‑5p mimic or inhibitor, or short interfering RNA (siRNA) specific to hypoxia‑inducible factor‑1α (HIF‑1α). MTS, lactate dehydrogenase (LDH), TUNEL staining and flow cytometry assays were performed to determine the proliferation, LDH activity, apoptosis and mitochondrial membrane potential (ΔΨm) of H9c2 cells, respectively. The overexpression of miR‑199a‑5p in the OGD/R cell model significantly decreased the viability and increased the lactate dehydrogenase leakage of cells; whereas knockdown of miR‑199‑5p induced the opposing effects. Additionally, inhibition of miR‑199‑5p significantly attenuated OGD/R‑induced alterations to the mitochondrial transmembrane potential (ΔΨm) and increases in the apoptosis of cells. Furthermore, the overexpression or knockdown of miR‑199a‑5p decreased or increased the expression of HIF‑1α and phosphorylation of glycogen synthase kinase 3β (GSK3β) in OGD/R‑treated H9c2 cells. Additionally, siRNA‑mediated downregulation of HIF‑1α decreased phosphorylated (p)‑GSK3β (Ser9) levels and reversed the protective effects of miR‑199a‑5p inhibition on OGD/R‑injured H9c2 cells. Similarly, treatment with LiCl (a specific inhibitor of p‑GSK3β) also attenuated the protective effects of miR‑199a‑5p knockdown on OGD/R‑injured H9c2 cells. Mechanistic studies revealed that HIF‑1α was a target of miR‑199a‑5p, and that HIF‑1α downregulation suppressed the expression of p‑GSK3β in OGD/R‑injured H9c2 cells. Furthermore, an miR‑199a‑5p inhibitor increased the interaction between p‑GSK3β and adenine nucleotide transferase (ANT), which was decreased by OGD/R. Additionally, miR‑199a‑5p inhibitor reduced the OGD/R‑induced interaction between ANT and cyclophilin D (Cyp‑D), potentially leading to the increased mitochondrial membrane potential in inhibitor‑transfected OGD/R‑injured H9c2 cells. Collectively, the present study identified a novel regulatory pathway in which the upregulation of miR‑199a‑5p reduced the expression of HIF‑1α and p‑GSK3β, and potentially suppresses the interaction between p‑GSK3β and ANT, thus promoting the interaction between ANT and Cyp‑D and potentially inducing cytotoxicity in OGD/R‑injured H9c2 cells.

View Figures

View References

1	Hausenloy DJ and Yellon DM: Targeting myocardial reperfusion injury-the search continues. N Engl J Med. 373:1073–1075. 2015. View Article : Google Scholar : PubMed/NCBI
2	Pagliaro P, Moro F, Tullio F, Perrelli MG and Penna C: Cardioprotective pathways during reperfusion: Focus on redox signaling and other modalities of cell signaling. Antioxid Redox Signal. 14:833–850. 2011. View Article : Google Scholar : PubMed/NCBI
3	Murry CE, Jennings RB and Reimer KA: Preconditioning with ischemia: A delay of lethal cell injury in ischemic myocardium. Circulation. 74:1124–1136. 1986. View Article : Google Scholar : PubMed/NCBI
4	Yue R, Xia X, Jiang J, Yang D, Han Y, Chen X, Cai Y, Li L, Wang WE and Zeng C: Mitochondrial DNA oxidative damage contributes to cardiomyocyte Ischemia/Reperfusion-injury in rats: Cardioprotective role of lycopene. J Cell Physiol. 230:2128–2141. 2015. View Article : Google Scholar : PubMed/NCBI
5	Kaczorowski DJ, Nakao A, McCurry KR and Billiar TR: Toll-like receptors and myocardial ischemia/reperfusion, inflammation, and injury. Curr Cardiol Rev. 5:196–202. 2009. View Article : Google Scholar : PubMed/NCBI
6	Prasad A, Stone GW, Holmes DR and Gersh B: Reperfusion injury, microvascular dysfunction, and cardioprotection: The ‘dark side’ of reperfusion. Circulation. 120:2105–2112. 2009. View Article : Google Scholar : PubMed/NCBI
7	Laina A, Gatsiou A, Georgiopoulos G, Stamatelopoulos K and Stellos K: RNA therapeutics in cardiovascular precision medicine. Front Physiol. 9:9532018. View Article : Google Scholar : PubMed/NCBI
8	Zhu H and Fan GC: Role of microRNAs in the reperfused myocardium towards post-infarct remodelling. Cardiovasc Res. 94:284–292. 2012. View Article : Google Scholar : PubMed/NCBI
9	Bian B, Yu XF, Wang GQ and Teng TM: Role of miRNA-1 in regulating connexin 43 in ischemia-reperfusion heart injury: A rat model. Cardiovasc Pathol. 27:37–42. 2017. View Article : Google Scholar : PubMed/NCBI
10	Chen X, Zhang L, Su T, Li H, Huang Q, Wu D, Yang C and Han Z: Kinetics of plasma microRNA-499 expression in acute myocardial infarction. J Thorac Dis. 7:890–896. 2015.PubMed/NCBI
11	Zhang XH, Zheng B, Han M, Miao SB and Wen JK: Synthetic retinoid Am80 inhibits interaction of KLF5 with RAR alpha through inducing KLF5 dephosphorylation mediated by the PI3K/Akt signaling in vascular smooth muscle cells. FEBS Lett. 583:1231–1236. 2009. View Article : Google Scholar : PubMed/NCBI
12	Song CL, Liu B, Diao HY, Shi YF, Zhang JC, Li YX, Liu N, Yu YP, Wang G, Wang JP and Li Q: Down-regulation of microRNA-320 suppresses cardiomyocyte apoptosis and protects against myocardial ischemia and reperfusion injury by targeting IGF-1. Oncotarget. 7:39740–39757. 2016.PubMed/NCBI
13	Su S, Luo, Liu X, Liu J, Peng F, Fang C and Li B: miR-494 up-regulates the PI3K/Akt pathway via targetting PTEN and attenuates hepatic ischemia/reperfusion injury in a rat model. Biosci Rep. 37(pii): BSR201707982017. View Article : Google Scholar : PubMed/NCBI
14	Liu Y, Liu G, Zhang H and Wang J: MiRNA-199a-5p influences pulmonary artery hypertension via downregulating Smad3. Biochem Biophys Res Commun. 473:859–866. 2016. View Article : Google Scholar : PubMed/NCBI
15	Wang D, Li Z, Zhang Y, Wang G, Wei M, Hu Y, Ma S, Jiang Y, Che N, Wang X, et al: Targeting of microRNA-199a-5p protects against pilocarpine-induced status epilepticus and seizure damage via SIRT1-p53 cascade. Epilepsia. 57:706–716. 2016. View Article : Google Scholar : PubMed/NCBI
16	Zuo Y, Wang Y, Hu H and Cui W: Atorvastatin protects myocardium against ischemia-reperfusion injury through inhibiting miR-199a-5p. Cell Physiol Biochem. 39:1021–1030. 2016. View Article : Google Scholar : PubMed/NCBI
17	Schnitzer SE, Schmid T, Zhou J, Eisenbrand G and Brüne B: Inhibition of GSK3beta by indirubins restores HIF-1alpha accumulation under prolonged periods of hypoxia/anoxia. FEBS Lett. 579:529–533. 2005. View Article : Google Scholar : PubMed/NCBI
18	Yang X, Lei S, Long J, Liu X and Wu Q: MicroRNA-199a-5p inhibits tumor proliferation in melanoma by mediating hif-1α. Mol Med Rep. 13:5241–5249. 2016. View Article : Google Scholar : PubMed/NCBI
19	Rane S, He M, Sayed D, Vashistha H, Malhotra A, Sadoshima J, Vatner DE, Vatner SF and Abdellatif M: Downregulation of miR-199a derepresses hypoxia-inducible factor-1alpha and Sirtuin 1 and recapitulates hypoxia preconditioning in cardiac myocytes. Circ Res. 104:879–886. 2009. View Article : Google Scholar : PubMed/NCBI
20	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 25:402–408. 2001. View Article : Google Scholar : PubMed/NCBI
21	Nishihara M, Miura T, Miki T, Tanno M, Yano T, Naitoh K, Ohori K, Hotta H, Terashima Y and Shimamoto K: Modulation of the mitochondrial permeability transition pore complex in GSK-3β-mediated myocardial protection. J Mol Cell Cardiol. 43:564–570. 2007. View Article : Google Scholar : PubMed/NCBI
22	Chen L, Cai P, Cheng Z, Zhang Z and Fang J: Pharmacological postconditioning with atorvastatin calcium attenuates myocardial ischemia/reperfusion injury in diabetic rats by phosphorylating GSK3β. Exp Ther Med. 14:25–34. 2017. View Article : Google Scholar : PubMed/NCBI
23	Gyulkhandanyan AV, Mutlu A, Freedman J and Leytin V: Mitochondrial permeability transition pore (MPTP)-dependent and -independent pathways of mitochondrial membrane depolarization, cell shrinkage and microparticle formation during platelet apoptosis. Br J Haematol. 169:142–150. 2015. View Article : Google Scholar : PubMed/NCBI
24	Sivaraman V and Yellon DM: Pharmacologic therapy that simulates conditioning for cardiac ischemic/reperfusion injury. J Cardiovasc Pharmacol Ther. 19:83–96. 2014. View Article : Google Scholar : PubMed/NCBI
25	Heggermont WA, Papageorgiou AP, Quaegebeur A, Deckx S, Carai P, Verhesen W, Eelen G, Schoors S, van Leeuwen R, Alekseev S, et al: Inhibition of MicroRNA-146a and overexpression of its target dihydrolipoyl succinyltransferase protect against pressure overload-induced cardiac hypertrophy and dysfunction. Circulation. 136:747–761. 2017. View Article : Google Scholar : PubMed/NCBI
26	Danielson LS, Park DS, Rotllan N, Chamorro-Jorganes A, Guijarro MV, Fernandez-Hernando C, Fishman GI, Phoon CK and Hernando E: Cardiovascular dysregulation of miR-17-92 causes a lethal hypertrophic cardiomyopathy and arrhythmogenesis. FASEB J. 27:1460–1467. 2013. View Article : Google Scholar : PubMed/NCBI
27	Verjans R, Peters T, Beaumont FJ, van Leeuwen R, van Herwaarden T, Verhesen W, Munts C, Bijnen M, Henkens M, Diez J, et al: MicroRNA-221/222 family counteracts myocardial fibrosis in pressure overload-induced heart failure. Hypertension. 71:280–288. 2018. View Article : Google Scholar : PubMed/NCBI
28	Godwin JG, Ge X, Stephan K, Jurisch A, Tullius SG and Iacomini J: Identification of a microRNA signature of renal ischemia reperfusion injury. Proc Natl Acad Sci USA. 107:14339–14344. 2010. View Article : Google Scholar : PubMed/NCBI
29	Gomez L, Paillard M, Thibault H, Derumeaux G and Ovize M: Inhibition of GSK3beta by postconditioning is required to prevent opening of the mitochondrial permeability transition pore during reperfusion. Circulation. 117:2761–2768. 2008. View Article : Google Scholar : PubMed/NCBI
30	Zhai P, Sciarretta S, Galeotti J, Volpe M and Sadoshima J: Differential roles of GSK-3β during myocardial ischemia and ischemia/reperfusion. Circ Res. 109:502–511. 2011. View Article : Google Scholar : PubMed/NCBI
31	Nguyen T, Wong R, Wang G, Gucek M, Steenbergen C and Murphy E: Acute inhibition of GSK causes mitochondrial remodeling. Am J Physiol Heart Circ Physiol. 302:H2439–H2445. 2012. View Article : Google Scholar : PubMed/NCBI
32	Wu J, Ke X, Fu W, Gao X, Zhang H, Wang W, Ma N, Zhao M, Hao X and Zhang Z: Inhibition of hypoxia-induced retinal angiogenesis by specnuezhenide, an effective constituent of ligustrum lucidum Ait., through suppression of the HIF-1α/VEGF signaling pathway. Molecules. 21(pii): E17562016. View Article : Google Scholar : PubMed/NCBI
33	Wang S, Zhang F, Zhao G, Cheng Y, Wu T, Wu B and Zhang YE: Mitochondrial PKC-ε deficiency promotes I/R-mediated myocardial injury via GSK3β-dependent mitochondrial permeability transition pore opening. J Cell Mol Med. 2:2009–2021. 2017. View Article : Google Scholar
34	Flügel D, Görlach A and Kietzmann T: GSK-3β regulates cell growth, migration, and angiogenesis via Fbw7 and USP28-dependent degradation of HIF-1α. Blood. 119:1292–1301. 2012. View Article : Google Scholar : PubMed/NCBI
35	Huang X, Zuo L, Lv Y, Chen C, Yang Y, Xin H, Li Y and Qian Y: Asiatic acid attenuates myocardial ischemia/reperfusion injury via Akt/GSK-3β/HIF-1α signaling in rat H9c2 cardiomyocytes. Molecules. 21:E12482016. View Article : Google Scholar : PubMed/NCBI
36	Juhaszova M, Wang S, Zorov DB, Nuss HB, Gleichmann M, Mattson MP and Sollott SJ: The identity and regulation of the mitochondrial permeability transition pore: Where the known meets the unknown. Ann NY Acad Sci. 1123:197–212. 2010. View Article : Google Scholar
37	Zhu H, Ding Y, Xu X, Li M, Fang Y, Gao B, Mao H, Tong G, Zhou L and Huang J: Prostaglandin E1 protects coronary microvascular function via the glycogen synthase kinase 3β-mitochondrial permeability transition pore pathway in rat hearts subjected to sodium laurate-induced coronary microembolization. Am J Transl Res. 9:2520–2534. 2017.PubMed/NCBI
38	Obame FN, Plin-Mercier C, Assaly R, Zini R, Dubois-Randé JL, Berdeaux A and Morin D: Cardioprotective effect of morphine and a blocker of glycogen synthase kinase 3 beta, SB216763 [3-(2,4-dichlorophenyl)-4(1-methyl-1H-indol-3-yl)- 1H- pyrrole-2,5-dione], via inhibition of the mitochondrial permeability transition pore. J Pharmacol Exp Ther. 326:252–258. 2008. View Article : Google Scholar : PubMed/NCBI