EIF4A3-Induced circ-BNIP3 Aggravated Hypoxia-Induced Injury of H9c2 Cells by Targeting miR-27a-3p/BNIP3

Acute myocardial infarction (AMI) results from long-term diminished blood supply diminishment (ischemia) to the heart, and the main reason for ischemia is hypoxia. BCL2 interaction protein 3 (BNIP3) can be upregulated by hypoxia and participates in the mediation of hypoxia-activated apoptosis in cardiac myocyte death. The purpose of this study was to interrogate the mechanism of BNIP3 in hypoxia-activated cardiac myocyte injury. Cell viability and apoptosis were evaluated by Cell counting kit 8 (CCK-8), 5-ethynyl-2’-deoxyuridine (EdU), TdT-mediated dUTP Nick-End Labeling (TUNEL), and caspase-3 activity assays. Molecular interactions were assessed by RNA immunoprecipitation (RIP) and pull-down assays. Gene levels were assessed via quantitative real-time PCR and western blot. BNIP3 expression was upregulated by hypoxia in H9c2 cells. We found that circ-BNIP3 (hsa_circ_0005972), whose annotated gene was BNIP3, was induced by hypoxia and positively regulated BNIP3 expression. Knockdown of BNIP3 or circ-BNIP3 reversed the effect of hypoxia in attenuating H9c2 cell viability and inducing apoptosis. circ-BNIP3 sponged miRNA-27a-3p (miR-27a-3p) to upregulate BNIP3 expression. Moreover, eukaryotic translation initiation factor 4A3 (EIF4A3) bound with the upstream region of the circ-BNIP3 mRNA transcript and induced circ-BNIP3 expression in H9c2 cells. EIF4A3-induced circ-BNIP3 aggravated hypoxia-caused injury of H9c2 cells through targeting miR-27a-3p/BNIP3 pathway, indicating circ-BNIP3 as a new target for relieving hypoxia-induced injury of cardiac myocytes. Acute myocardial infarction (AMI) results from long-term diminished blood supply diminishment (ischemia) to the heart, and the main reason for ischemia is hypoxia. BCL2 interaction protein 3 (BNIP3) can be upregulated by hypoxia and participates in the mediation of hypoxia-activated apoptosis in cardiac myocyte death. The purpose of this study was to interrogate the mechanism of BNIP3 in hypoxia-activated cardiac myocyte injury. Cell viability and apoptosis were evaluated by Cell counting kit 8 (CCK-8), 5-ethynyl-2’-deoxyuridine (EdU), TdT-mediated dUTP Nick-End Labeling (TUNEL), and caspase-3 activity assays. Molecular interactions were assessed by RNA immunoprecipitation (RIP) and pull-down assays. Gene levels were assessed via quantitative real-time PCR and western blot. BNIP3 expression was upregulated by hypoxia in H9c2 cells. We found that circ-BNIP3 (hsa_circ_0005972), whose annotated gene was BNIP3, was induced by hypoxia and positively regulated BNIP3 expression. Knockdown of BNIP3 or circ-BNIP3 reversed the effect of hypoxia in attenuating H9c2 cell viability and inducing apoptosis. circ-BNIP3 sponged miRNA-27a-3p (miR-27a-3p) to upregulate BNIP3 expression. Moreover, eukaryotic translation initiation factor 4A3 (EIF4A3) bound with the upstream region of the circ-BNIP3 mRNA transcript and induced circ-BNIP3 expression in H9c2 cells. EIF4A3-induced circ-BNIP3 aggravated hypoxia-caused injury of H9c2 cells through targeting miR-27a-3p/BNIP3 pathway, indicating circ-BNIP3 as a new target for relieving hypoxia-induced injury of cardiac myocytes. Myocardial infarction (MI), resulting from hypoxia or acute, persistent ischemia, is recognized as a severe manifestation of the coronary artery diseases that can lead to irreversible heart muscle damage and the progression of heart failure.1Kannel W.B. Cupples L.A. Gagnon D.R. Incidence, precursors and prognosis of unrecognized myocardial infarction.Adv. Cardiol. 1990; 37: 202-214Crossref PubMed Google Scholar, 2Roger V.L. Go A.S. Lloyd-Jones D.M. Benjamin E.J. Berry J.D. Borden W.B. Bravata D.M. Dai S. Ford E.S. Fox C.S. et al Executive Summary: Heart Disease and Stroke Statistics—2012 Update.Circulation. 2012; 125: 188-197Crossref PubMed Scopus (1098) Google Scholar, 3Boersma E. Mercado N. Poldermans D. Gardien M. Vos J. Simoons M.L. Acute myocardial infarction.Lancet. 2003; 361: 847-858Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar The major clinical symptoms of MI include severe dyspnea, persistent chest pain, syncope, and fever.4Lippi G. Sanchis-Gomar F. Cervellin G. Chest pain, dyspnea and other symptoms in patients with type 1 and 2 myocardial infarction. A literature review.Int. J. Cardiol. 2016; 215: 20-22Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar,5Coventry L.L. Bremner A.P. Williams T.A. Jacobs I.G. Finn J. Symptoms of myocardial infarction: concordance between paramedic and hospital records.Prehosp. Emerg. Care. 2014; 18: 393-401Crossref PubMed Scopus (9) Google Scholar Although tools for preventing and treating MI have been improved to some extent, MI still contributes to large numbers of deaths globally.6Do R. Stitziel N.O. Won H.-H. Jørgensen A.B. Duga S. Angelica Merlini P. Kiezun A. Farrall M. Goel A. Zuk O. et al.NHLBI Exome Sequencing ProjectExome sequencing identifies rare LDLR and APOA5 alleles conferring risk for myocardial infarction.Nature. 2015; 518: 102-106Crossref PubMed Scopus (409) Google Scholar, 7Fiedler J. Jazbutyte V. Kirchmaier B.C. Gupta S.K. Lorenzen J. Hartmann D. Galuppo P. Kneitz S. Pena J.T. Sohn-Lee C. et al.MicroRNA-24 regulates vascularity after myocardial infarction.Circulation. 2011; 124: 720-730Crossref PubMed Scopus (328) Google Scholar, 8Mythili S. Malathi N. Diagnostic markers of acute myocardial infarction.Biomed. Rep. 2015; 3: 743-748Crossref PubMed Google Scholar, 9Acconcia M.C. Caretta Q. Romeo F. Borzi M. Perrone M.A. Sergi D. Chiarotti F. Calabrese C.M. Sili Scavalli A. Gaudio C. Meta-analyses on intra-aortic balloon pump in cardiogenic shock complicating acute myocardial infarction may provide biased results.Eur. Rev. Med. Pharmacol. Sci. 2018; 22: 2405-2414PubMed Google Scholar Circular RNAs (circRNAs), a newly-identified class among noncoding RNAs (ncRNAs), are characterized by a closed and continuous loop structure lacking in the 5′ end cap or 3′ terminal poly(A) tail.10Nigro J.M. Cho K.R. Fearon E.R. Kern S.E. Ruppert J.M. Oliner J.D. Kinzler K.W. Vogelstein B. Scrambled exons.Cell. 1991; 64: 607-613Abstract Full Text PDF PubMed Scopus (609) Google Scholar,11Granados-Riveron J.T. Aquino-Jarquin G. The complexity of the translation ability of circRNAs. Biochimica et Biophysica Acta (BBA) -.Gene Regulatory Mechanisms. 2016; 1859: 1245-1251Google Scholar circRNAs are more stable than linear RNA and are resistant to the effects of RNA exonucleases, cap removal, or adenylation.12Li Y. Zheng Q. Bao C. Li S. Guo W. Zhao J. Chen D. Gu J. He X. Huang S. Circular RNA is enriched and stable in exosomes: a promising biomarker for cancer diagnosis.Cell Res. 2015; 25: 981-984Crossref PubMed Scopus (1057) Google Scholar Over the past years, studies have revealed the role of circRNAs in regulating cellular biological processes, including apoptosis and proliferation.13Zhong Z. Huang M. Lv M. He Y. Duan C. Zhang L. Chen J. Circular RNA MYLK as a competing endogenous RNA promotes bladder cancer progression through modulating VEGFA/VEGFR2 signaling pathway.Cancer Lett. 2017; 403: 305-317Crossref PubMed Scopus (298) Google Scholar,14He R. Liu P. Xie X. Zhou Y. Liao Q. Xiong W. Li X. Li G. Zeng Z. Tang H. circGFRA1 and GFRA1 act as ceRNAs in triple negative breast cancer by regulating miR-34a.J. Exp. Clin. Cancer Res. 2017; 36: 145Crossref PubMed Scopus (194) Google Scholar Recently, the influences of circRNAs in cardiomyocyte injury have been increasingly revealed. For example, circ-NCX1 mediated ischemic myocardial injury by microRNA-133a-3p (miR-133a-3p).15Li M. Ding W. Tariq M.A. Chang W. Zhang X. Xu W. Hou L. Wang Y. Wang J. A circular transcript of ncx1 gene mediates ischemic myocardial injury by targeting miR-133a-3p.Theranostics. 2018; 8: 5855-5869Crossref PubMed Scopus (104) Google Scholar circRNA autophagy-related circular RNA (ACR) attenuated myocardial ischemia/reperfusion injury through inhibiting autophagy via Pink1/FAM65B pathway.16Zhou L.Y. Zhai M. Huang Y. Xu S. An T. Wang Y.H. Zhang R.C. Liu C.Y. Dong Y.H. Wang M. et al.The circular RNA ACR attenuates myocardial ischemia/reperfusion injury by suppressing autophagy via modulation of the Pink1/ FAM65B pathway.Cell Death Differ. 2018; 26: 1299-1315Crossref PubMed Scopus (83) Google Scholar Hsa_circ_0005972, whose annotated gene was BCL2 interacting protein 3 (BNIP3), is a newly identified circRNA by our study, and it has never been related to MI before. BNIP3 belongs to the BH3-only subfamily of Bcl-2 family proteins, which can heterodimerize and antagonize the activities of prosurvival proteins, including Bcl-XL and Bcl-2, as well as induce cell apoptosis.17Vande Velde C. Cizeau J. Dubik D. Alimonti J. Brown T. Israels S. Hakem R. Greenberg A.H. BNIP3 and genetic control of necrosis-like cell death through the mitochondrial permeability transition pore.Mol. Cell. Biol. 2000; 20: 5454-5468Crossref PubMed Scopus (521) Google Scholar,18Ray R. Chen G. Vande Velde C. Cizeau J. Park J.H. Reed J.C. Gietz R.D. Greenberg A.H. BNIP3 heterodimerizes with Bcl-2/Bcl-X(L) and induces cell death independent of a Bcl-2 homology 3 (BH3) domain at both mitochondrial and nonmitochondrial sites.J. Biol. Chem. 2000; 275: 1439-1448Crossref PubMed Scopus (272) Google Scholar Although BNIP3 expression is unable to be detected in the majority of organs normally, including in heart, it can be activated under hypoxia.17Vande Velde C. Cizeau J. Dubik D. Alimonti J. Brown T. Israels S. Hakem R. Greenberg A.H. BNIP3 and genetic control of necrosis-like cell death through the mitochondrial permeability transition pore.Mol. Cell. Biol. 2000; 20: 5454-5468Crossref PubMed Scopus (521) Google Scholar,19Bruick R.K. Expression of the gene encoding the proapoptotic Nip3 protein is induced by hypoxia.Proc. Natl. Acad. Sci. USA. 2000; 97: 9082-9087Crossref PubMed Scopus (634) Google Scholar Also, it has been reported that BNIP3 protein overexpression in some cultured cell lines can lead to membrane insertion, as well as the initiation of the cell death pathway with necrosis-like features.17Vande Velde C. Cizeau J. Dubik D. Alimonti J. Brown T. Israels S. Hakem R. Greenberg A.H. BNIP3 and genetic control of necrosis-like cell death through the mitochondrial permeability transition pore.Mol. Cell. Biol. 2000; 20: 5454-5468Crossref PubMed Scopus (521) Google Scholar Moreover, a study has pointed out that hypoxia and acidosis can induce BNIP3 to activate cardiac myocyte death.20Kubasiak L.A. Hernandez O.M. Bishopric N.H. Webster K.A. Hypoxia and acidosis activate cardiac myocyte death through the Bcl-2 family protein BNIP3.Proc. Natl. Acad. Sci. USA. 2002; 99: 12825-12830Crossref PubMed Scopus (370) Google Scholar However, the mechanism of BNIP3 in hypoxia-induced injury of cardiomyocytes remains elusive. Also, the relation between BNIP3 and circ-BNIP3 has never been explored before. The purpose of the present study was to uncover the mediation mechanism of BNIP3 in hypoxia-caused injury in H9c2 cells. We confirmed that BNIP3 was increased by hypoxia, and BNIP3 silence ameliorated the hypoxia-activated injury of H9c2 cells. We searched on circBase and first identified that circ-BNIP3 was associated with BNIP3 and had positive regulation on BNIP3 expression. We validated that circ-BNIP3 was upregulated under hypoxia and its knockdown reversed hypoxia-caused injury of H9c2 cells. Mechanistically, we validated that circ-BNIP3 sponged miRNA-27a-3p to upregulate BNIP3. Moreover, we explored the mechanism of circ-BNIP3 upregulation and found that EIF4A3 bound to BNIP3 mRNA at the upstream region of circ-BNIP3 mRNA transcripts and facilitated mRNA splicing and induced circ-BNIP3 expression. At first, we incubated H9c2 cells in the hypoxic incubator to construct hypoxic H9c2 cells. Thereafter, we tested viability and apoptosis of H9c2 cells under hypoxia versus normoxia treatment. Consequently, we observed through CCK-8 that the viability of H9c2 cells was attenuated by hypoxia compared with normoxia (Figure 1A). Also, EdU assay validated that H9c2 cell viability was decreased in response to hypoxia (Figure 1B). These results showed that hypoxia impaired viability of H9c2 cells. Besides, TUNEL assay demonstrated that the apoptotic H9c2 cells increased in presence with hypoxia (Figure 1C). To further evaluate the apoptosis level of H9c2 cells under hypoxia, we determined caspase-3 activity. As a result, hypoxia treatment induced caspase-3 activity in H9c2 cells (Figure 1D). In addition, the protein levels of cleaved-caspase-3/9 and Bax were improved by hypoxia, with the levels of total caspase-3/9 unchanged, whereas the protein level of anti-apoptosis Bcl-2 diminished upon hypoxia (Figure 1E). These data indicated that hypoxia led to H9c2 cell injury by abrogating viability and facilitating apoptosis. Then, we examined expression of BNIP3 under hypoxia in H9c2 cells. Results showed increased BNIP3 mRNA and protein levels under hypoxia in H9c2 cells (Figure 1F), implying the participation of BNIP3 in hypoxia-caused injury of H9c2 cells. Next, we probed the function of BNIP3 in regulating hypoxia-stimulated injury in H9c2 cells. Quantitative real-time PCR and western blot analyses revealed that transfection of short hairpin RNA targeting BNIP3 (sh-BNIP3#1), sh-BNIP3#2, or sh-BNIP3#3 decreased the expression of BNIP3 in hypoxia-treated H9c2 cells (Figure 2A). Because sh-BNIP3#1/2 presented better effect in reducing BNIP3 expression, we used them for subsequent loss-of-function assays. We observed by CCK-8 and EdU assays that hypoxia hampered the viability of H9c2 cells, and such an effect could be counteracted by silencing BNIP3 (Figures 2B and 2C). Additionally, apoptosis of H9c2 cells facilitated by hypoxia treatment was abrogated by BNIP3 knockdown (Figures 2D and 2E). Concordantly, the induced levels of pro-apoptotic cleaved-caspase-3/9 and Bax in hypoxic H9c2 cells were reduced under knockdown of BNIP3, whereas the reduced level of anti-apoptotic Bcl-2 in hypoxic H9c2 cells was recovered by knockdown of BNIP3 (Figure 2F). In sum, the abovementioned data indicated that BNIP3 silence reversed hypoxia-stimulated injury of H9c2 cells. Then, we tried to explore the mechanism underlying the upregulation of BNIP3. It has been reported that circRNA can regulate the expression of its host gene21Li X. Wang J. Zhang C. Lin C. Zhang J. Zhang W. Zhang W. Lu Y. Zheng L. Li X. Circular RNA circITGA7 inhibits colorectal cancer growth and metastasis by modulating the Ras pathway and upregulating transcription of its host gene ITGA7.J. Pathol. 2018; 246: 166-179Crossref PubMed Scopus (115) Google Scholar and participate in hypoxia-induced cell injury.15Li M. Ding W. Tariq M.A. Chang W. Zhang X. Xu W. Hou L. Wang Y. Wang J. A circular transcript of ncx1 gene mediates ischemic myocardial injury by targeting miR-133a-3p.Theranostics. 2018; 8: 5855-5869Crossref PubMed Scopus (104) Google Scholar Therefore, we tried to find the circRNAs related to BNIP3 through browsing circBase (http://www.circbase.org/). Results showed that 3 circRNAs were transcribed from BNIP3 gene, which were hsa_circ_0005972, hsa_circ_0020526, and hsa_circ_0020527 (Figure 3A). To figure out the association of the 3 circRNAs with hypoxia-induced damage in H9c2 cells, we examined their expressions under hypoxia. Results showed that only hsa_circ_0005972 was upregulated responding to hypoxia in H9c2 cells (Figure 3B), indicating that hsa_circ_0005972 might participate in the regulation of hypoxia-induced injury of H9c2 cells. Therefore, we termed it as circ-BNIP3 and further investigated its effect on BNIP3 expression. As presented in Figure S1A, quantitative real-time PCR indicated that circ-BNIP3 expression was overtly increased in response to hypoxia in neonatal rat ventricular cardiomyocyte (NRVM). We confirmed that introducing sh-circ-BNIP3#1/2/3 could impair the inductive effect of hypoxia on circ-BNIP3 expression in H9c2 cells, and sh-circ-BNIP3#1 and 2 were more efficient than sh-circ-BNIP3#3 (Figure 3C), so sh-circ-BNIP3#1/2 were used for later experiments. We found that the hypoxia-induced expression of BNIP3 at mRNA and protein levels was abrogated by circ-BNIP3 silence in H9c2 cells (Figure 3D), which meant that circ-BNIP3 positively regulated BNIP3 expression. Moreover, we tried to confirm the circular feature of circ-BNIP3. We validated through circRNA sequencing that circ-BNIP3 was located at chromosome 10 (chr10): 133784141–133787447 with a spliced length of 493 bp (Figure 3E). Also, circ-BNIP3, rather than BNIP3, cannot be digested by RNase R (Figure 3F, left), confirming that circ-BNIP3 had a structure of covalently closed continuous loop.22Qu S. Zhong Y. Shang R. Zhang X. Song W. Kjems J. Li H. The emerging landscape of circular RNA in life processes.RNA Biol. 2017; 14: 992-999Crossref PubMed Scopus (184) Google Scholar, 23Memczak S. Jens M. Elefsinioti A. Torti F. Krueger J. Rybak A. Maier L. Mackowiak S.D. Gregersen L.H. Munschauer M. et al.Circular RNAs are a large class of animal RNAs with regulatory potency.Nature. 2013; 495: 333-338Crossref PubMed Scopus (3922) Google Scholar, 24Qu S. Yang X. Li X. Wang J. Gao Y. Shang R. Sun W. Dou K. Li H. Circular RNA: A new star of noncoding RNAs.Cancer Lett. 2015; 365: 141-148Crossref PubMed Scopus (933) Google Scholar Moreover, circ-BNIP3 amplified by divergent primers was detectable in cDNA rather than gDNA (Figure 3F, right). We further identified that circ-BNIP3 was located in cytoplasm of H9c2 cells by subcellular fractionation and fluorescence in situ hybridization (FISH) staining analysis (Figures 3G and 3H). These results suggested that circ-BNIP3 was a bona fide circRNA. Then, the biological role of circ-BNIP3 in hypoxia-induced damage of H9c2 cells was evaluated. The inhibitive effect of hypoxia on H9c2 cell viability was abrogated by the depletion of circ-BNIP3 (Figures 4A and 4B ). The hypoxia-induced apoptosis of H9c2 cells was reversed by the knockdown of circ-BNIP3 (Figures 4C and 4D). The induced levels of cleaved-caspase-3/9 and Bax and reduced levels of Bcl-2 under hypoxia could be reversed by silencing circ-BNIP3 (Figure 4E). Taken together, circ-BNIP3 silence reversed hypoxia-induced injury of H9c2 cells. In subsequence, we investigated how circ-BNIP3 regulated BNIP3 expression. Mounting studies have revealed that circRNAs could regulate gene expressions through performing as miRNA sponge, including in regulating cardiomyocyte injury.15Li M. Ding W. Tariq M.A. Chang W. Zhang X. Xu W. Hou L. Wang Y. Wang J. A circular transcript of ncx1 gene mediates ischemic myocardial injury by targeting miR-133a-3p.Theranostics. 2018; 8: 5855-5869Crossref PubMed Scopus (104) Google Scholar,25Zeng Z. Zhou W. Duan L. Zhang J. Lu X. Jin L. Yu Y. Circular RNA circ-VANGL1 as a competing endogenous RNA contributes to bladder cancer progression by regulating miR-605-3p/VANGL1 pathway.J. Cell. Physiol. 2019; 234: 3887-3896Crossref PubMed Scopus (43) Google Scholar Hence, we speculated that circ-BNIP3 regulated BNIP3 expression through sponging miRNA in H9c2 cells. We searched Starbase (http://starbase.sysu.edu.cn/) to identify the shared miRNAs interacting with both circ-BNIP3 and BNIP3 mRNA. As presented in Figure 5A, the intersection of the Venn pattern showed 3 miRNAs shared by circ-BNIP3 and BNIP3 mRNA, which were miR-27a-3p, miR-27b-3p, and miR-128-3p (Figure 5A). Further, we determined the involvement of 3 miRNAs in hypoxia-induced injury in H9c2 cells. Results of quantitative real-time PCR analysis showed that only miR-27a-3p was downregulated upon hypoxia versus normoxia control in H9c2 cells (Figure 5B), indicating the association of miR-27a-3p in hypoxia-induced injury in H9c2 cells. Also, previous studies have reported that miR-27a-3p could aggravate proliferation and hamper apoptosis in cancer cells.26Yuan Kluiver Y.,J. Koerts J. de Jong D. Rutgers B.,F. Abdul Razak R. Terpstra M. Plaat B.E. Nolte I.M. Diepstra A. et al.miR-24-3p Is Overexpressed in Hodgkin Lymphoma and Protects Hodgkin and Reed-Sternberg Cells from Apoptosis.Am. J. Pathol. 2017; 187: 1343-1355Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar Therefore, we selected miR-27a-3p for further investigation. RNA immunoprecipitation (RIP) assay confirmed the enrichment of circ-BNIP3, miR-27a-3p, and BNIP3 mRNA in the precipitates of Ago2 (Figure 5C). Moreover, we obtained the binding sequences on circ-BNIP3 and BNIP3 mRNA for miR-27a-3p from Starbase (Figure 5D). Luciferase reporter assays were conducted in both H9c2 and 293T cells to further investigate the interaction of miR-27a-3p with circ-BNIP3 and BNIP3. Results showed that overexpression of miR-27a-3p resulted in decreased luciferase activity of wild-type (WT)-circ-BNIP3 and WT-BNIP3, instead of mutant (mut)-circ-BNIP3 and mut-BNIP3 (Figure 5E). Additionally, pull-down assay confirmed the abundant expression of circ-BNIP3 and BNIP3 mRNA in miR-27a-3p WT pull-down (Figure 5F). Then, we validated that overexpression of miR-27a-3p enhanced miR-27a-3p expression reduced in H9c2 cells treated with hypoxia (Figure 5G) and that overexpression of miR-27a-3p reversed the inductive effect of hypoxia on BNIP3 expression in H9c2 cells (Figure 5H). These results hinted that circ-BNIP3 sponged miR-27a-3p to upregulate BNIP3 in H9c2 cells. Further, we probed the mechanism of circ-BNIP3 upregulation in H9c2 cells. Previous studies demonstrated that EIF4A3, a key regulator of RNA splicing,27Chan C.C. Dostie J. Diem M.D. Feng W. Mann M. Rappsilber J. Dreyfuss G. eIF4A3 is a novel component of the exon junction complex.RNA. 2004; 10: 200-209Crossref PubMed Scopus (173) Google Scholar induced circ-matrix metallopeptidase 9 (MMP9) expression via binding to the upstream region of MMP9 mRNA and induce circular RNA formation.28Wang R. Zhang S. Chen X. Li N. Li J. Jia R. Pan Y. Liang H. EIF4A3-induced circular RNA MMP9 (circMMP9) acts as a sponge of miR-124 and promotes glioblastoma multiforme cell tumorigenesis.Mol. Cancer. 2018; 17: 166Crossref PubMed Scopus (136) Google Scholar Herein, we found through Circular RNA Interactome (https://circinteractome.nia.nih.gov/) that EIF4A3 had 4 binding sites on the upstream region of BNIP3 mRNA transcript (Figure 6A). The higher level of EIF4A3 was examined in H9c2 and NRVM cells after treated with hypoxia (Figure S1B). We conduced pull-down assay using BNIP3 mRNA and confirmed the enrichment of EIF4A3 protein in the pull-down of BNIP3 mRNA rather than control (Figure 6B). To detect whether EIF4A3 bound at the predicted regions on the BNIP3 mRNA transcript, we carried out RIP assay using EIF4A3 antibody, followed by quantitative real-time PCR detection using the primers designed according to four binding regions (a, b, c, and d). Results confirmed that fragment a, b, c, and d were all enriched in EIF4A3 precipitates (Figure 6C). Also, the RNA constructs containing 1, 2, 4, and 4 EIF4A3 binding sites (s1, s2, s3, and s4) were used for pull-down assay. Western blot analysis after pull-down assay illustrated that the depletion of EIF4A3 sites on circ-BNIP3 mRNA transcript resulted in lessened enrichment of EIF4A3 protein (Figure 6D), further confirmed the requirement of the predicted sites for the binding of EIF4A3. Later, we detected the effect of EIF4A3 on circ-BNIP3 expression. We confirmed the overexpression and knockdown of EIF4A3 in H9c2 cells by quantitative real-time PCR analysis (Figure 6E). We verified that overexpression of EIF4A3 induced circ-BNIP3 expression and that knockdown of EIF4A3 reversed the inductive effect of hypoxia on circ-BNIP3 expression in H9c2 cells (Figure 6F). To conclude, the data above suggested that EIF4A3 induced circ-BNIP3 expression in H9c2 cells. Finally, we carried out rescue assays to probe whether BNIP3 participated in circ-BNIP3-mediated hypoxia-induced injury of H9c2 cells. First, we confirmed that transfection of pcDNA3.1/BNIP3 rescued the silence of BNIP3 caused by circ-BNIP3 knockdown under hypoxia (Figure 7A). CCK-8 and EdU analyses demonstrated that silencing circ-BNIP3 facilitated the viability of hypoxia-treated H9c2 cells, and such an effect was counteracted by overexpressing BNIP3 (Figures 7B and 7C). The apoptosis of H9c2 cells under hypoxia was impeded by circ-BNIP3 depletion, and such an effect was impaired by overexpression of BNIP3 (Figures 7D and 7E). Also, the decreased levels of cleaved-caspase-3/9 and Bax and the increased level of Bcl-2 caused by circ-BNIP3 inhibition in hypoxia were reversed by overexpression of BNIP3 in H9c2 cells (Figure 7F). Therefore, it was suggested that circ-BNIP3 regulated hypoxia-induced injury of H9c2 cells in a BNIP3-dependent manner. MI is known to be a result of hypoxia or acute, persistent ischemia, and can contribute to irreversible heart muscle damage and heart failure.1Kannel W.B. Cupples L.A. Gagnon D.R. Incidence, precursors and prognosis of unrecognized myocardial infarction.Adv. Cardiol. 1990; 37: 202-214Crossref PubMed Google Scholar, 2Roger V.L. Go A.S. Lloyd-Jones D.M. Benjamin E.J. Berry J.D. Borden W.B. Bravata D.M. Dai S. Ford E.S. Fox C.S. et al Executive Summary: Heart Disease and Stroke Statistics—2012 Update.Circulation. 2012; 125: 188-197Crossref PubMed Scopus (1098) Google Scholar, 3Boersma E. Mercado N. Poldermans D. Gardien M. Vos J. Simoons M.L. Acute myocardial infarction.Lancet. 2003; 361: 847-858Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar Statistics revealed that MI caused large numbers of deaths globally.6Do R. Stitziel N.O. Won H.-H. Jørgensen A.B. Duga S. Angelica Merlini P. Kiezun A. Farrall M. Goel A. Zuk O. et al.NHLBI Exome Sequencing ProjectExome sequencing identifies rare LDLR and APOA5 alleles conferring risk for myocardial infarction.Nature. 2015; 518: 102-106Crossref PubMed Scopus (409) Google Scholar,9Acconcia M.C. Caretta Q. Romeo F. Borzi M. Perrone M.A. Sergi D. Chiarotti F. Calabrese C.M. Sili Scavalli A. Gaudio C. Meta-analyses on intra-aortic balloon pump in cardiogenic shock complicating acute myocardial infarction may provide biased results.Eur. Rev. Med. Pharmacol. Sci. 2018; 22: 2405-2414PubMed Google Scholar Therefore, exploring the molecular mechanism underlying the hypoxia-induced injury of cardiomyocytes can potentially benefit the improvement of MI treatment. BNIP3 is reputed as a crucial regulator of apoptosis belonging to the BH3-only subfamily of Bcl-2 family proteins.17Vande Velde C. Cizeau J. Dubik D. Alimonti J. Brown T. Israels S. Hakem R. Greenberg A.H. BNIP3 and genetic control of necrosis-like cell death through the mitochondrial permeability transition pore.Mol. Cell. Biol. 2000; 20: 5454-5468Crossref PubMed Scopus (521) Google Scholar,18Ray R. Chen G. Vande Velde C. Cizeau J. Park J.H. Reed J.C. Gietz R.D. Greenberg A.H. BNIP3 heterodimerizes with Bcl-2/Bcl-X(L) and induces cell death independent of a Bcl-2 homology 3 (BH3) domain at both mitochondrial and nonmitochondrial sites.J. Biol. Chem. 2000; 275: 1439-1448Crossref PubMed Scopus (272) Google Scholar Although in many organs, including heart, BNIP3 expression is reported to be undetectable under normal condition, hypoxia is discovered to be able to induce BNIP3 expression.17Vande Velde C. Cizeau J. Dubik D. Alimonti J. Brown T. Israels S. Hakem R. Greenberg A.H. BNIP3 and genetic control of necrosis-like cell death through the mitochondrial permeability transition pore.Mol. Cell. Biol. 2000; 20: 5454-5468Crossref PubMed Scopus (521) Google Scholar,19Bruick R.K. Expression of the gene encoding the proapoptotic Nip3 protein is induced by hypoxia.Proc. Natl. Acad. Sci. USA. 2000; 97: 9082-9087Crossref PubMed Scopus (634) Google Scholar Mounting studies have revealed that BNIP3 positively regulated hypoxia-induced cell injury. For example, miR-210 targeted BNIP3 to protect rat adrenal gland pheochromocytoma (PC-12) cells from hypoxia-induced injury.29Luan Y. Zhang X. Zhang Y. Dong Y. MicroRNA-210 Protects PC-12 Cells Against Hypoxia-Induced Injury by Targeting BNIP3.Front. Cell. Neurosci. 2017; 11: 285Crossref PubMed Scopus (17) Google Scholar Tetramethylpyrazine alleviated the hypoxia-induced apoptosis of myocardial cells via inhibiting Hypoxia inducible factor (HIF)-1α/c-jun N-terminal kinase (JNK)/p38 signaling and insulin like growth factor binding protein 3 (IGFBP3)/BNIP3 axis, and upregulating PI3K/Akt survival signaling.30Lin K.H. Kuo W.W. Jiang A.Z. Pai P. Lin J.Y. Chen W.K. Day C.H. Shen C.Y. Padma V.V. Huang C.Y. Tetramethylpyrazine Ameliorated Hypoxia-Induced Myocardial Cell Apoptosis via HIF-1α/JNK/p38 and IGFBP3/BNIP3 Inhibition to Upregulate PI3K/Akt Survival Signaling.Cell. Physiol. Biochem. 2015; 36: 334-344Crossref PubMed Scopus (36) Google Scholar Furthermore, it has been reported that hypoxia and acidosis can induce BNIP3 to activate cardiac myocyte death.20Kubasiak L.A. Hernandez O.M. Bishopric N.H. Webster K.A. Hypoxia and acidosis activate cardiac myocyte death through the Bcl-2 family protein BNIP3.Proc. Natl. Acad. Sci. USA. 2002; 99: 12825-12830Crossref PubMed Scopus (370) Google Scholar These findings suggested that BNIP3 is an important regulator of hypoxia-resulted in injury for cardiomyocytes. In concordance, this study confirmed that BNIP3 could be upregulated in H9c2 cells under hypoxia, and depletion of BNIP3 reversed the effect of hypoxia in retarding viability and facilitating apoptosis in H9c2 cells. Furthermore, we explored the mechanism of BNIP3 overexpression in H9c2 cells under hypoxia. Previously, it has been revealed that circRNAs are a novel class of ncRNAs able to regulate the expression of its host gene.21Li X. Wang J. Zhang C. Lin C. Zhang J. Zhang W. Zhang W. Lu Y. Zheng L. Li X. Circular RNA circITGA7 inhibits colorectal cancer growth and metastasis by modulating the Ras pathway and upregulating transcription of its host gene ITGA7.J. Pathol. 2018; 246: 166-179Crossr