Korean Circ J.  2022 Sep;52(9):680-696. 10.4070/kcj.2021.0252.

CircZNF609 Aggravated Myocardial Ischemia Reperfusion Injury via Mediation of miR-214-3p/PTGS2 Axis

• Tang WQ
• Yang FR
• Chen KM
• Yang H
• Liu Y
• Dou B

• KMID: 2532933
• DOI: http://doi.org/10.4070/kcj.2021.0252

Abstract

Background and Objectives
Circular RNAs were known to play vital role in myocardial ischemia reperfusion injury (MIRI), while the role of CircZNF609 in MIRI remains unclear. This study was aimed to investigate the function of CircZNF609 in MIRI.
Methods
Hypoxia/reoxygenation (H/R) model was established to mimic MIRI in vitro. Quantitative polymerase chain reaction was performed to evaluate gene transcripts. Cellular localization of CircZNF609 and miR-214-3p were visualized by fluorescence in situ hybridization. Cell proliferation was determined by CCK-8. TUNEL assay and flow cytometry were applied to detect apoptosis. Lactate dehydrogenase was determined by commercial kit. ROS was detected by DCFH-DA probe. Direct interaction of indicated molecules was determined by RIP and dual luciferase assays. Western blot was used to quantify protein levels. In vivo model was established to further test the function of CircZNF609 in MIRI.
Results
CircZNF609 was upregulated in H/R model. Inhibition of CircZNF609 alleviated H/R induced apoptosis, ROS generation, restored cell proliferation in cardiomyocytes and human umbilical vein endothelial cells. Mechanically, CircZNF609 directly sponged miR-214-3p to release PTGS2 expression. Functional rescue experiments showed that miR-214-3p/ PTGS2 axis was involved in the function of circZNG609 in H/R model. Furthermore, data in mouse model revealed that knockdown of CircZNF609 significantly reduced the area of myocardial infarction and decreased myocardial cell apoptosis.
Conclusions
CircZNF609 aggravated the progression of MIRI via targeting miR-214-3p/ PTGS2 axis, which suggested CircZNF609 might act as a vital modulator in MIRI.

Keyword

PTGS2; Hypoxia; Ischemia reperfusion injury

Figure

• 

  Figure 1 CircZNF609 was upregulated in H/R model. (A) Human cardiomyocytes were subjected to H/R model as aforementioned. qPCR was performed to detect the expression of CircZNF609. (B) Expression of CircZNF609 in nucleus and cytosol were determined by qPCR. (C) Subcellular location of CircZNF609 was observed by fluorescence in situ hybridization (n=3).H/R = hypoxia/reoxygenation; qPCR = quantitative polymerase chain reaction.*p<0.05; †p<0.01; ‡p<0.001.

• 

  Figure 2 Knockdown of CircZNF609 ameliorated H/R induced myocardial injury. siRNA was transfected to HCMs to knockdown CircZNF609 in HCMs before H/R stimulation. (A) Expression of CircZNF609 was determined by qPCR. (B) Cell proliferation was evaluated by CCK-8 assay. (C) Cytotoxicity was evaluated by LDH assay. (D) ROS generation was detected by a ROS kit. (E) Cell apoptosis was determined by flow cytometry. (F) Cell apoptosis was determined by TUNEL. (G) Protein level of Bcl-2, Bax, cleaved caspase 3 were evaluated by western blots (n=3).HCM = human cardiomyocyte; H/R = hypoxia/reoxygenation; LDH = lactate dehydrogenase; ROS = reactive oxygen species.*p<0.05; †p<0.01; ‡p<0.001.

• 

  Figure 3 CircZNF609 functioned as a sponge of miR-214-3p. (A) Starbase 2.0 was used to predict the potential binding site of miR-214-3p and CircZNF609. (B) RNA immunoprecipitation was performed in HCMs to evaluate the enrichment of CircZNF609 in AGO2-pulled complex. (C) Direct binding of miR-214-3p and CircZNF609 was verified by dual luciferase assay. (D) Subcellular location of CircZNF609 and miR-214-3p in HCMs was observed by FISH. (E) Expression of miR-214-3p was determined by quantitative polymerase chain reaction (n=3).HCM = human cardiomyocyte; MUT = mutant type; WT = wild-type.*p<0.05; †p<0.01; ‡p<0.001.

• 

  Figure 4 miR-214-3p targeted PTGS2. (A) Starbase 2.0 was used to predict the potential binding site of miR-214-3p and PTGS2. (B) Direct binding of miR-214-3p and CircZNF609 in HCMs was verified by dual luciferase assay. (C) mRNA expression of PTGS2 in HCMs was determined by quantitative polymerase chain reaction. (D) Protein expression of PTGS2 was determined by western blot (n=3).HCM = human cardiomyocyte; MUT = mutant type; WT = wild-type.*p<0.05; †p<0.01; ‡p<0.001.

• 

  Figure 5 CircZNF609 aggravated H/R induced myocardial injury via regulating miR-214-3p/PTGS2 axis. HCMs were transfected with si-CircZNF609 or co-transfected si-CircZNF609 with miR-214-3p inhibitor and si-PTGS2. Then the cells were subjected to H/R stimulation. (A) Cell proliferation was evaluated by CCK-8 assay. (B) Cytotoxicity was evaluated by LDH assay. (C) ROS generation was detected by a ROS kit. (D) Cell apoptosis was determined by flow cytometry. (E) Cell apoptosis was determined by TUNEL. (F) Protein level of Bcl-2, Bax, cleaved caspase 3 were evaluated by western blots (n = 3).HCM = human cardiomyocyte; H/R = hypoxia/reoxygenation; LDH = lactate dehydrogenase; ROS = reactive oxygen species.*p<0.05; †p<0.01; ‡p<0.001.

• 

  Figure 6 CircZNF609/miR-214-3p/PTGS2 axis regulated H/R-induced injury in mouse myocardial cells and HUVECs. Primary mouse cardiomyocytes and HUVECs were cultured and divided into five group: control, H/R, H/R+si-CircZNF609, H/R+si-CircZNF609+miR-214-3p inhibitor, H/R+si-CircZNF609+miR-214-3p inhibitor+si-PTGS2. (A) The levels of CircZNF609, miR-214-3p and PTGS2 were detected by quantitative polymerase chain reaction. (B) The cell viability was tested by CCK8 assay. (D) The activity of LDH was determined by commercial kit. (E) The cell apoptosis was investigated by flow cytometry. Three independent experiments were performed in each group.HCM = human cardiomyocyte; H/R = hypoxia/reoxygenation; LDH = lactate dehydrogenase; HUVEC = human umbilical vein endothelial cell.*p<0.05; †p<0.01; ‡p<0.001.

• 

  Figure 7 Knockdown of CircZNF609 alleviated MIRI in vivo. (A) The area of myocardial infarction in mice was determined. (B, C, D) The levels of CircZNF609, miR-214-3p and PTGS2 in myocardial tissues of mice were investigated by quantitative polymerase chain reaction. (E) The apoptosis rate was tested by TUNEL staining. (F) The protein levels of Bax, Bcl-2 and cleaved caspase 3 in myocardial tissues of mice were examined by western blot (n=5).MIRI = myocadiac ischemia reperfusion injury.*p<0.05; †p<0.01; ‡p<0.001.

Reference

1. Xu G, Zhao X, Fu J, Wang X. Resveratrol increase myocardial Nrf2 expression in type 2 diabetic rats and alleviate myocardial ischemia/reperfusion injury (MIRI). Ann Palliat Med. 2019; 8:565–575. PMID: 31865720.

2. Yuan L, Dai X, Fu H, et al. Vaspin protects rats against myocardial ischemia/reperfusion injury (MIRI) through the TLR4/NF-κB signaling pathway. Eur J Pharmacol. 2018; 835:132–139. PMID: 30063916.

3. Shen Y, Liu X, Shi J, Wu X. Involvement of Nrf2 in myocardial ischemia and reperfusion injury. Int J Biol Macromol. 2019; 125:496–502. PMID: 30468811.

4. Zhou LY, Zhai M, Huang Y, et al. The circular RNA ACR attenuates myocardial ischemia/reperfusion injury by suppressing autophagy via modulation of the Pink1/ FAM65B pathway. Cell Death Differ. 2019; 26:1299–1315. PMID: 30349076.

5. Li M, Ding W, Tariq MA, et al. A circular transcript of ncx1 gene mediates ischemic myocardial injury by targeting miR-133a-3p. Theranostics. 2018; 8:5855–5869. PMID: 30613267.

6. Song YF, Zhao L, Wang BC, et al. The circular RNA TLK1 exacerbates myocardial ischemia/reperfusion injury via targeting miR-214/RIPK1 through TNF signaling pathway. Free Radic Biol Med. 2020; 155:69–80. PMID: 32445866.

7. Legnini I, Di Timoteo G, Rossi F, et al. Circ-ZNF609 is a circular RNA that can be translated and functions in myogenesis. Mol Cell. 2017; 66:22–37.e9. PMID: 28344082.

8. Liu C, Yao MD, Li CP, et al. Silencing of circular RNA-ZNF609 ameliorates vascular endothelial dysfunction. Theranostics. 2017; 7:2863–2877. PMID: 28824721.

9. Carnieto A Jr, Dourado PM, Luz PL, Chagas AC. Selective cyclooxygenase-2 inhibition protects against myocardial damage in experimental acute ischemia. Clinics (Sao Paulo). 2009; 64:245–252. PMID: 19330252.

10. Salloum FN, Hoke NN, Seropian IM, et al. Parecoxib inhibits apoptosis in acute myocardial infarction due to permanent coronary ligation but not due to ischemia-reperfusion. J Cardiovasc Pharmacol. 2009; 53:495–498. PMID: 19455055.

11. Zhao J, Wang F, Zhang Y, et al. Sevoflurane preconditioning attenuates myocardial ischemia/reperfusion injury via caveolin-3-dependent cyclooxygenase-2 inhibition. Circulation. 2013; 128(Suppl 1):S121–S129. PMID: 24030395.

12. Ruan Z, Wang S, Yu W, Deng F. LncRNA MALAT1 aggravates inflammation response through regulating PTGS2 by targeting miR-26b in myocardial ischemia-reperfusion injury. Int J Cardiol. 2019; 288:122. PMID: 31101226.

13. Yang J, Huang X, Hu F, Fu X, Jiang Z, Chen K. LncRNA ANRIL knockdown relieves myocardial cell apoptosis in acute myocardial infarction by regulating IL-33/ST2. Cell Cycle. 2019; 18:3393–3403. PMID: 31674275.

14. Zhou M, Zou YG, Xue YZ, et al. Long non-coding RNA H19 protects acute myocardial infarction through activating autophagy in mice. Eur Rev Med Pharmacol Sci. 2018; 22:5647–5651. PMID: 30229841.

15. Zhang H, Wang J, Du A, Li Y. MiR-483-3p inhibition ameliorates myocardial ischemia/reperfusion injury by targeting the MDM4/p53 pathway. Mol Immunol. 2020; 125:9–14. PMID: 32619931.

16. Zhao J, Chen F, Ma W, Zhang P. Suppression of long noncoding RNA NEAT1 attenuates hypoxia-induced cardiomyocytes injury by targeting miR-378a-3p. Gene. 2020; 731:144324. PMID: 31904498.

17. Rong J, Pan H, He J, et al. Long non-coding RNA KCNQ1OT1/microRNA-204-5p/LGALS3 axis regulates myocardial ischemia/reperfusion injury in mice. Cell Signal. 2020; 66:109441. PMID: 31644943.

18. Lin Y, Dan H, Lu J. Overexpression of microRNA-136-3p alleviates myocardial injury in coronary artery disease via the Rho A/ROCK signaling pathway. Kidney Blood Press Res. 2020; 45:477–496. PMID: 32434208.

19. Yang K, Shi J, Hu Z, Hu X. The deficiency of miR-214-3p exacerbates cardiac fibrosis via miR-214-3p/NLRC5 axis. Clin Sci (Lond). 2019; 133:1845–1856. PMID: 31434695.

20. Bai M, Pan CL, Jiang GX, Zhang YM, Zhang Z. CircHIPK3 aggravates myocardial ischemia-reperfusion injury by binding to miRNA-124-3p. Eur Rev Med Pharmacol Sci. 2019; 23:10107–10114. PMID: 31799682.

21. Liu K, Wang F, Wang S, Li WN, Ye Q. Mangiferin attenuates myocardial ischemia-reperfusion injury via MAPK/Nrf-2/HO-1/NF-κB in vitro and in vivo. Oxid Med Cell Longev. 2019; 2019:7285434. PMID: 31249649.

22. Xing X, Guo S, Zhang G, et al. miR-26a-5p protects against myocardial ischemia/reperfusion injury by regulating the PTEN/PI3K/AKT signaling pathway. Braz J Med Biol Res. 2020; 53:e9106. PMID: 31994603.

23. Zhao B, Li G, Peng J, et al. CircMACF1 attenuates acute myocardial infarction through miR-500b-5p-EMP1 axis. J Cardiovasc Transl Res. 2021; 14:161–172. PMID: 32162171.

24. Hu X, Ma R, Cao J, Du X, Cai X, Fan Y. CircSAMD4A aggravates H/R-induced cardiomyocyte apoptosis and inflammatory response by sponging miR-138-5p. J Cell Mol Med. 2022; 26:1776–1784. PMID: 33219594.

25. Tong H, Zhao K, Wang J, Xu H, Xiao J. CircZNF609/miR-134-5p/BTG-2 axis regulates proliferation and migration of glioma cell. J Pharm Pharmacol. 2020; 72:68–75. PMID: 31721211.

26. Wang S, Xue X, Wang R, et al. CircZNF609 promotes breast cancer cell growth, migration, and invasion by elevating p70S6K1 via sponging miR-145-5p. Cancer Manag Res. 2018; 10:3881–3890. PMID: 30288120.

27. Wu W, Wei N, Shao G, Jiang C, Zhang S, Wang L. circZNF609 promotes the proliferation and migration of gastric cancer by sponging miR-483-3p and regulating CDK6. Onco Targets Ther. 2019; 12:8197–8205. PMID: 31632070.

28. Sun H, Cai J, Xu L, et al. miR-483-3p regulates acute myocardial infarction by transcriptionally repressing insulin growth factor 1 expression. Mol Med Rep. 2018; 17:4785–4790. PMID: 29363719.

29. Yuan M, Zhang L, You F, et al. MiR-145-5p regulates hypoxia-induced inflammatory response and apoptosis in cardiomyocytes by targeting CD40. Mol Cell Biochem. 2017; 431:123–131. PMID: 28281187.