Effects of miR‑103a‑3p on the autophagy and apoptosis of cardiomyocytes by regulating Atg5

Zhang,Chenjun; Lu,Jide; Wang,Hairong; Qi,Yuan; Kan,Ying; Ge,Zhiru

doi:10.3892/ijmm.2019.4128

Effects of miR‑103a‑3p on the autophagy and apoptosis of cardiomyocytes by regulating Atg5

Authors:
- Chenjun Zhang
- Jide Lu
- Hairong Wang
- Yuan Qi
- Ying Kan
- Zhiru Ge
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Affiliations: Department of Cardiology, Shanghai Pudong New Area Gongli Hospital, Shanghai 200135, P.R. China
Published online on: March 12, 2019 https://doi.org/10.3892/ijmm.2019.4128
Pages: 1951-1960
Copyright: © Zhang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Autophagy and apoptosis are associated with cardiovascular diseases. Emerging evidence shows that microRNAs (miRs) are critical in the development of pathological processes underlying cardiovascular diseases by regulating the induction of apoptosis and autophagy. The present study aimed to investigate the role of miR‑103a‑3p in cardiomyocyte injury through autophagy and apoptosis. H9c2 cells were cultured under hypoxia and reoxygenation (H/R) conditions and were used to mimic cells under ischemia. The transfection of cells with miR‑103a‑3p (mimics and inhibitors) was performed to examine its function in cardiomyocytes. The expression levels of miR‑103a‑3p were evaluated by reverse transcription‑quantitative polymerase chain reaction analysis. Cell viability was determined using an MTT assay, and the lactate dehydrogenase assay (LDH) was used to investigate cell injury. The expression levels of B‑cell lymphoma 2 (Bcl‑2), Bcl‑2‑associated X protein, Beclin‑1, autophagy‑related 5 (Atg5), cleaved caspase‑3 and cleaved caspase‑9 were detected using western blotting. Immunofluorescence assays were performed to detect the expression of LC3 as a marker of autophagy. The target gene of miR‑103a‑3p was identified using dual‑luciferase reporter assays. The results revealed that the expression levels of miR‑103a‑3p were significantly downregulated in cardiomyocytes under H/R conditions. Injury of the cardiomyocytes was evaluated under H/R conditions. Following transfection of the cells with miR‑103a‑3p inhibitors, cell injury was increased, as determined by LDH and MTT assays. The expression levels of apoptotic proteins were consistent with the results obtained in the LDH and cell viability assays. The induction of autophagy was increased in cells under H/R conditions and cells with miR‑103a‑3p inhibitor transfection, whereas the induction of autophagy was decreased in cells transfected with miR‑103a‑3p mimics. In addition, the data indicated that miR‑103a‑3p directly targeted Atg5, which regulated the induction of autophagy and apoptosis. Taken together, these findings indicate that, following the inhibition of miR‑103a‑3p, Atg5 promotes autophagy and apoptosis in cardiomyocytes by directly targeting Atg5. Therefore, miR‑103a‑3p can be considered a potential therapeutic target for myocardial ischemia.

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1	Benjamin EJ, Blaha MJ, Chiuve SE, Cushman M, Das SR, Deo R, de Ferranti SD, Floyd J, Fornage M, Gillespie C, et al: Heart disease and stroke statistics-2017 update: A report from the american heart association. Circulation. 135. pp. e146–e603. 2017, View Article : Google Scholar
2	Bochaton T and Ovize M: Circadian rhythm and ischaemia-reper-fusion injury. Lancet. 391:8–9. 2018. View Article : Google Scholar
3	Montaigne D, Marechal X, Modine T, Coisne A, Mouton S, Fayad G, Ninni S, Klein C, Ortmans S, Seunes C, et al: Daytime variation of perioperative myocardial injury in cardiac surgery and its prevention by Rev-Erbα antagonism: A single-centre propensity-matched cohort study and a randomised study. Lancet. 391:59–69. 2018. View Article : Google Scholar
4	Pattingre S, Tassa A, Qu X, Garuti R, Liang XH, Mizushima N, Packer M, Schneider MD and Levine B: Bcl-2 antiapoptotic proteins inhibit Beclin 1-dependent autophagy. Cell. 122:927–939. 2005. View Article : Google Scholar : PubMed/NCBI
5	Ghavami S, Gupta S, Ambrose E, Hnatowich M, Freed DH and Dixon IM: Autophagy and heart disease: Implications for cardiac ischemia-reperfusion damage. Curr Mol Med. 14:616–629. 2014. View Article : Google Scholar : PubMed/NCBI
6	Nishida K, Kyoi S, Yamaguchi O, Sadoshima J and Otsu K: The role of autophagy in the heart. Cell Death Differ. 16:31–38. 2009. View Article : Google Scholar
7	Matsui Y, Takagi H, Qu X, Abdellatif M, Sakoda H, Asano T, Levine B and Sadoshima J: Distinct roles of autophagy in the heart during ischemia and reperfusion: Roles of AMP-activated protein kinase and Beclin 1 in mediating autophagy. Circ Res. 100:914–922. 2007. View Article : Google Scholar : PubMed/NCBI
8	Zhu H, Tannous P, Johnstone JL, Kong Y, Shelton JM, Richardson JA, Le V, Levine B, Rothermel BA and Hill JA: Cardiac autophagy is a maladaptive response to hemodynamic stress. J Clin Invest. 117:1782–1793. 2007. View Article : Google Scholar : PubMed/NCBI
9	Lavandero S, Troncoso R, Rothermel BA, Martinet W, Sadoshima J and Hill JA: Cardiovascular autophagy: Concepts, controversies, and perspectives. Autophagy. 9:1455–1466. 2013. View Article : Google Scholar : PubMed/NCBI
10	Moss EG: MicroRNAs: Hidden in the genome. Curr Biol. 12:R138–R140. 2002. View Article : Google Scholar : PubMed/NCBI
11	Ambros V: microRNAs: Tiny regulators with great potential. Cell. 107:823–826. 2001. View Article : Google Scholar
12	Salem M, O'Brien JA, Bernaudo S, Shawer H, Ye G, Brkić J, Amleh A, Vanderhyden BC, Refky B, Yang BB, et al: miRNA-590-3p promotes ovarian cancer growth and metastasis via a novel FOXA2-versican pathway. Cancer Res. 78:4175–4190. 2018. View Article : Google Scholar : PubMed/NCBI
13	Sun F, Yu M, Yu J, Liu Z, Zhou X, Liu Y, Ge X, Gao H, Li M, Jiang X, et al: miR-338-3p functions as a tumor suppressor in gastric cancer by targeting PTP1B. Cell Death Dis. 9:5222018. View Article : Google Scholar : PubMed/NCBI
14	Zitzer NC, Snyder K, Meng X, Taylor PA, Efebera YA, Devine SM, Blazar BR, Garzon R and Ranganathan P: MicroRNA-155 modulates acute graft-versus-host disease by impacting T cell expansion, migration, and effector function. J Immunol. 200:4170–4179. 2018. View Article : Google Scholar : PubMed/NCBI
15	Janaszak-Jasiecka A, Siekierzycka A, Bartoszewska S, Serocki M, Dobrucki LW, Collawn JF, Kalinowski L and Bartoszewski R: eNOS expression and NO release during hypoxia is inhibited by miR-200b in human endothelial cells. Angiogenesis. 21:711–724. 2018. View Article : Google Scholar : PubMed/NCBI
16	Voshall A, Kim EJ, Ma X, Yamasaki T, Moriyama EN and Cerutti H: miRNAs in the alga Chlamydomonas reinhardtii are not phylogenetically conserved and play a limited role in responses to nutrient deprivation. Sci Rep. 7:54622017. View Article : Google Scholar : PubMed/NCBI
17	Wei S, Xue J, Sun B, Zou Z, Chen C, Liu Q and Zhang A: miR-145 via targeting ERCC2 is involved in arsenite-induced DNA damage in human hepatic cells. Toxicol Lett. 295:220–228. 2018. View Article : Google Scholar : PubMed/NCBI
18	Wang X, Ha T, Hu Y, Lu C, Liu L, Zhang X, Kao R, Kalbfleisch J, Williams D and Li C: MicroRNA-214 protects against hypoxia/reoxygenation induced cell damage and myocardial ischemia/reperfusion injury via suppression of PTEN and Bim1 expression. Oncotarget. 7:86926–86936. 2016. View Article : Google Scholar : PubMed/NCBI
19	Bartman CM, Oyama Y, Brodsky K, Khailova L, Walker L, Koeppen M and Eckle T: Intense light-elicited upregulation of miR-21 facilitates glycolysis and cardioprotection through Per2-dependent mechanisms. PLoS One. 12:e01762432017. View Article : Google Scholar : PubMed/NCBI
20	Weber DG, Casjens S, Johnen G, Bryk O, Raiko I, Pesch B, Kollmeier J, Bauer TT and Brüning T: Combination of MiR-103a-3p and mesothelin improves the biomarker performance of malignant mesothelioma diagnosis. PLoS One. 9:e1144832014. View Article : Google Scholar : PubMed/NCBI
21	Zhong Z, Lv M and Chen J: Screening differential circular RNA expression profiles reveals the regulatory role of circTCF25-miR-103a-3p/miR-107-CDK6 pathway in bladder carcinoma. Sci Rep. 6:309192016. View Article : Google Scholar : PubMed/NCBI
22	Hu X, Miao J, Zhang M, Wang X, Wang Z, Han J, Tong D and Huang C: miRNA-103a-3p promotes human gastric cancer cell proliferation by targeting and suppressing ATF7 in vitro. Mol Cells. 41:390–400. 2018.PubMed/NCBI
23	Kang S, Shin KD, Kim JH and Chung T: Autophagy-related (ATG) 11, ATG9 and the phosphatidylinositol 3-kinase control ATG2-mediated formation of autophagosomes in Arabidopsis. Plant Cell Rep. 37:653–664. 2018. View Article : Google Scholar : PubMed/NCBI
24	Mizushima N, Noda T, Yoshimori T, Tanaka Y, Ishii T, George MD, Klionsky DJ, Ohsumi M and Ohsumi Y: A protein conjugation system essential for autophagy. Nature. 395:395–398. 1998. View Article : Google Scholar : PubMed/NCBI
25	Ravikumar B, Imarisio S, Sarkar S, O'Kane CJ and Rubinsztein DC: Rab5 modulates aggregation and toxicity of mutant huntingtin through macroautophagy in cell and fly models of Huntington disease. J Cell Sci. 121:1649–1660. 2008. View Article : Google Scholar : PubMed/NCBI
26	Liang XH, Jackson S, Seaman M, Brown K, Kempkes B, Hibshoosh H and Levine B: Induction of autophagy and inhibition of tumorigenesis by beclin 1. Nature. 402:672–676. 1999. View Article : Google Scholar : PubMed/NCBI
27	Sun T, Li X, Zhang P, Chen WD, Zhang HL, Li DD, Deng R, Qian XJ, Jiao L, Ji J, et al: Acetylation of Beclin 1 inhibits autophagosome maturation and promotes tumour growth. Nat Commun. 6:72152015. View Article : Google Scholar : PubMed/NCBI
28	Nishida Y, Arakawa S, Fujitani K, Yamaguchi H, Mizuta T, Kanaseki T, Komatsu M, Otsu K, Tsujimoto Y and Shimizu S: Discovery of Atg5/Atg7-independent alternative macroau-tophagy. Nature. 461:654–658. 2009. View Article : Google Scholar : PubMed/NCBI
29	Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, Noda T, Kominami E, Ohsumi Y and Yoshimori T: LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J. 19:5720–5728. 2000. View Article : Google Scholar : PubMed/NCBI
30	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
31	Bainey KR and Armstrong PW: Clinical perspectives on reper-fusion injury in acute myocardial infarction. Am Heart J. 167:637–645. 2014. View Article : Google Scholar : PubMed/NCBI
32	Tian ZQ, Jiang H and Lu ZB: MiR-320 regulates cardiomyocyte apoptosis induced by ischemia-reperfusion injury by targeting AKIP1. Cell Mol Biol Lett. 23:412018. View Article : Google Scholar : PubMed/NCBI
33	Di Y, Lei Y, Yu F, Changfeng F, Song W and Xuming M: MicroRNAs expression and function in cerebral ischemia reper-fusion injury. J Mol Neurosci. 53:242–250. 2014. View Article : Google Scholar : PubMed/NCBI
34	Liu X, Deng Y, Xu Y, Jin W and Li H: MicroRNA-223 protects neonatal rat cardiomyocytes and H9c2 cells from hypoxia-induced apoptosis and excessive autophagy via the Akt/mTOR pathway by targeting PARP-1. J Mol Cell Cardiol. 118:133–146. 2018. View Article : Google Scholar : PubMed/NCBI
35	Yu M, Xue Y, Zheng J, Liu X, Yu H, Liu L, Li Z and Liu Y: Linc00152 promotes malignant progression of glioma stem cells by regulating miR-103a-3p/FEZF1/CDC25A pathway. Mol Cancer. 16:1102017. View Article : Google Scholar : PubMed/NCBI
36	Onrat ST, Onrat E, Ercan Onay E, Yalim Z and Avşar A: The genetic determination of the differentiation between ischemic dilated cardiomyopathy and idiopathic dilated cardiomyopathy. Genet Test Mol Biomarkers. 22:644–651. 2018. View Article : Google Scholar : PubMed/NCBI
37	Chen L, Li G, Peng F, Jie X, Dongye G, Cai K, Feng R, Li B, Zeng Q, Lun K, et al: The induction of autophagy against mitochondria-mediated apoptosis in lung cancer cells by a ruthenium (II) imidazole complex. Oncotarget. 7:80716–80734. 2016. View Article : Google Scholar : PubMed/NCBI
38	Zhang Q, Xiang J, Wang X, Liu H, Hu B, Feng M and Fu Q: Beta(2)-adrenoceptor agonist clenbuterol reduces infarct size and myocardial apoptosis after myocardial ischaemia/reperfusion in anaesthetized rats. Br J Pharmacol. 160:1561–1572. 2010. View Article : Google Scholar : PubMed/NCBI
39	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-3beta-mediated myocardial protection. J Mol Cell Cardiol. 43:564–570. 2007. View Article : Google Scholar : PubMed/NCBI
40	Mazel S, Burtrum D and Petrie HT: Regulation of cell division cycle progression by bcl-2 expression: A potential mechanism for inhibition of programmed cell death. J Exp Med. 183:2219–2226. 1996. View Article : Google Scholar : PubMed/NCBI
41	Carlsson SR and Simonsen A: Membrane dynamics in autophagosome biogenesis. J Cell Sci. 128:193–205. 2015. View Article : Google Scholar : PubMed/NCBI
42	Chen C, Chen W, Li Y, Dong Y, Teng X, Nong Z, Pan X, Lv L, Gao Y and Wu G: Hyperbaric oxygen protects against myocardial reperfusion injury via the inhibition of inflammation and the modulation of autophagy. Oncotarget. 8:111522–111534. 2017. View Article : Google Scholar
43	Wu Y, Fan W, Huang D and Sun X: Possible intermediary role of autophagy in serum albumin decrease-associated cardiovascular events among patients with coronary heart disease. Int J Cardiol. 250:642018. View Article : Google Scholar
44	Chen L, Wang FY, Zeng ZY, Cui L, Shen J, Song XW, Li P, Zhao XX and Qin YW: MicroRNA-199a acts as a potential suppressor of cardiomyocyte autophagy through targeting Hspa5. Oncotarget. 8:63825–63834. 2017.PubMed/NCBI
45	Huang Z, Wu S, Kong F, Cai X, Ye B, Shan P and Huang W: MicroRNA-21 protects against cardiac hypoxia/reoxygenation injury by inhibiting excessive autophagy in H9c2 cells via the Akt/mTOR pathway. J Cell Mol Med. 21:467–474. 2017. View Article : Google Scholar
46	Shang YY, Yao M, Zhou ZW, Jian-Cui Li-Xia Hu RY, Yu YY, Qiong-Gao, Biao-Yang, Liu YX, et al: Alisertib promotes apoptosis and autophagy in melanoma through p38 MAPK-mediated aurora a signaling. Oncotarget. 8:107076–107088. 2017. View Article : Google Scholar :
47	Cheng Y and Yang JM: Autophagy and apoptosis: Rivals or mates? Chin J Cancer. 32:103–105. 2013.PubMed/NCBI
48	Malhotra R, Warne JP, Salas E, Xu AW and Debnath J: Loss of Atg12, but not Atg5, in pro-opiomelanocortin neurons exacerbates diet-induced obesity. Autophagy. 11:145–154. 2015.PubMed/NCBI
49	Young MM, Takahashi Y, Khan O, Park S, Hori T, Yun J, Sharma AK, Amin S, Hu CD, Zhang J, et al: Autophagosomal membrane serves as platform for intracellular death-inducing signaling complex (iDISC)-mediated caspase-8 activation and apoptosis. J Biol Chem. 287:12455–12468. 2012. View Article : Google Scholar : PubMed/NCBI
50	Park JH and Shin C: MicroRNA-directed cleavage of targets: Mechanism and experimental approaches. BMB Rep. 47:417–423. 2014. View Article : Google Scholar : PubMed/NCBI
51	Arakawa S, Tsujioka M, Yoshida T, Tajima-Sakurai H, Nishida Y, Matsuoka Y, Yoshino I, Tsujimoto Y and Shimizu S: Role of Atg5-dependent cell death in the embryonic development of Bax/Bak double-knockout mice. Cell Death Differ. 24:1598–1608. 2017. View Article : Google Scholar : PubMed/NCBI
52	Luo S and Rubinsztein DC: Atg5 and Bcl-2 provide novel insights into the interplay between apoptosis and autophagy. Cell Death Differ. 14:1247–1250. 2007. View Article : Google Scholar : PubMed/NCBI