The microRNA-144/451 cluster in plasma-derived microvesicles and erythrocytes in patients with history of pulmonary embolism

Chronic thromboembolic disease (CTED) and chronic thromboembolic pulmonary hypertension (CTEPH) are the complications that comprise a serious problem for patients with history of pulmonary embolism (PE). Erythrocytes, extracellular microvesicles (EMVs) and miRNAs play a substantial role in the procoagulant states. The aim. To analyze the levels of miR-144-3р, miR-451a, and miR-451b in blood plasma-derived EMVs and erythrocytes in patients with history of PE and in the control group. Materials and Methods. 18 patients with history of PE (13 CTEPH, 5 CTED) and 8 controls were enrolled into the study. All the participants had undergone clinical and biochemical blood tests as well as the coagulogram. We used flow cytometry to assess plasma-derived EMVs (CD9, CD41, CD45, CD235a, CD105). We measured the expression of miR-144-3р, miR-451a, miR-451b by real-time PCR with endogenous control (miR-152-3p) and five exogenous quality controls. Results. The levels of miR-144-3р and miR-451a in patients were lower than in controls, both in EMVs (р = 0.030; р = 0.065) and in erythrocytes (р = 0.023;р = 0.086). In female patients, the levels of miR-144-3р and miR-451a in CTEPH were lower than in CTED (р = 0.087; р = 0.031). Mir-451b in EMVs has not been detected, while in erythrocytes its levels have not differed between the groups. In patients, the levels of miR-144-3р and miR-451a directly correlated with each other both in EMVs (р = 0.004) and in erythrocytes (р = 0.042). In all the participants, the levels of miR-144-3р and miR-451a in EMVs directly correlated with those in erythrocytes (р = 0.002; р = 0.078). The number of erythrocyte-derived EMVs correlated with miR-451a levels both in EMVs (R = 0.472; p = 0.065) and in erythrocytes (R = –0.829; p = 0.011). The level of miR-451a in EMVs correlated with blood plasma levels of factor VIII and fibrinogen (R = 0.584; p = 0.022 and R= –0.489; p = 0.047), and with the International Normalized Ratio (R = 0.894; p = 0.041). Conclusion. The microRNA-144/451 cluster may influence both the hemostasis system and the risk of post-thromboembolic complications development. In the present study, miR-144-3р and miR-451a showed themselves as protective factors in relation to both the development of PE and severity of post-thromboembolic complications.

1. Pastori D, Cormaci VM, Marucci S, et al. A Comprehensive Review of Risk Factors for Venous Thromboembolism: From Epidemiology to Pathophysiology. Int J Mol Sci. 2023 Feb 5;24(4):3169. DOI: 10.3390/ijms24043169.

2. Валиева З.С., Мартынюк Т.В. Хроническая тромбоэмболическая легочная гипертензия: от патогенеза к выбору тактики лечения. Терапевтический архив. 2022;94(7):791–796. [Valieva ZS, Martynyuk TV. Chronic thromboembolic pulmonary hypertension: from pathogenesis to the choice of treatment tactics. Terapevticheskii Arkhiv (Ter. Arkh.). 2022;94(7):791–796. (In Russ.)]. DOI: 10.26442/00403660.2022.07.201741.

3. Антонова О.А., Якушкин В.В., Мазуров А.В. Коагуляционная активность мембранных микрочастиц. Биологические мембраны. 2019;36(3):155–175. [Antonova OA, Yakushkin VV, Mazurov AV. Coagulation activity of membrane microparticles. Biologicheskiye membrany = Biological membranes. 2019;36(3):155–175. (In Russ.)]. DOI: 10.1134/S0233475519030034.

4. Золотова Е.А., Симакова М.А., Жиленкова Ю.И. и др. Роль микро-РНК в патогенезе венозных тромбоэмболических осложнений. Российский журнал персонализированной медицины. 2022;2(1):43–50. [Zolotova EA, Simakova MA, Zhilenkova YuI et al. The role of miRNAs in the pathogenesis of venous thromboembolic complications. Russian Journal for Personalized Medicine. 2022;2(1):43–50. (In Russ.)]. DOI: 10.18705/2782-3806-2022-2-1-43-50.

5. Alberro A, Iparraguirre L, Fernandes A, Otaegui D. Extracellular vesicles in blood: sources, effects, and applications. Int J Mol Sci. 2021;22(15):8163. DOI: 10.3390/ijms22158163.

6. He Y, Wucorresponding Q. The effect of extracellular vesicles on thrombosis. J Cardiovasc Transl Res. 2022 Nov 28:1–16. DOI: 10.1007/s12265-022-10342-w.

7. Thangaraju K, Neerukonda SN, Katneni U, Buehler PW. Extracellular vesicles from red blood cells and their evolving roles in health, coagulopathy and therapy. Int J Mol Sci. 2021;22(1):153. DOI: 10.3390/ijms22010153.

8. Lander ES, Linton LM, Birren B, et al. Initial sequencing and analysis of the human genome. Nature. 2001;409(6822):860–921. DOI: 10.1038/35057062.

9. Xue Y, Chen R, Qu L, Cao X. Noncoding RNA: from dark matter to bright star. Sci China Life Sci. 2020;63(4):463–468. DOI: 10.1007/s11427-020-1676-5.

10. Alles J, Fehlmann T, Fischer U, et al. An estimate of the total number of true human miRNAs. Nucleic Acids Res. 2019;47(7):3353–3364. DOI: 10.1093/nar/gkz097.

11. Matsuyama H, Suzuki HI. Systems and synthetic microRNA biology: from biogenesis to disease pathogenesis. Int J Mol Sci. 2019;21(1):132. DOI: 10.3390/ijms21010132.

12. Saliminejad K, Khorram Khorshid HR, et al. An overview of microRNAs: biology, functions, therapeutics, and analysis methods. J Cell Physiol. 2019;234(5):5451–5465. DOI: 10.1002/jcp.27486.

13. Zhang X, Wang X, Zhu H, et al. Synergistic effects of the GATA-4-mediated miR-144/451 cluster in protection against simulated ischemia/reperfusion-induced cardiomyocyte death. J Mol Cell Cardiol. 2010;49(5): 841–50. DOI: 10.1016/j.yjmcc.2010.08.007.

14. Rasmussen KD, Simmini S, Abreu-Goodger C, et al. The miR-144/451 locus is required for erythroid homeostasis. J Exp Med. 2010 Jul 5; 207(7):1351–8. DOI: 10.1084/jem.20100458.

15. Wang X, Hong Y, Wu L et al. Deletion of microRNA-144/451 cluster aggravated brain injury in intracerebral hemorrhage mice by targeting 14-3-3ζ. Front Neurol. 2021;11:551411. DOI: 10.3389/fneur.2020.551411.

16. He Q, Wang F, Honda T, et al. Ablation of miR-144 increases vimentin expression and atherosclerotic plaque formation. Sci Rep. 2020;10(1):6127. DOI: 10.1038/s41598-020-63335-7.

17. Wang X, Zhu H, Zhang X, et al. Loss of the miR-144/451 cluster impairs ischaemic preconditioning-mediated cardioprotection by targeting Rac-1. Cardiovasc Res. 2012;94(2):379–390. DOI: 10.1093/cvr/cvs096.

18. Tao L, Yang L, Huang X, et al. Reconstruction and aof the lncRNAmiRNA-mRNA network based on competitive endogenous RNA reveal functional lncRNAs in dilated cardiomyopathy. Front Genet. 2019;10:1149. DOI: 10.3389/fgene.2019.01149.

19. Turczyńska KM, Bhattachariya A, Säll J, et al. Stretch-sensitive down-regulation of the miR-144/451 cluster in vascular smooth muscle and its role in AMP-activated protein kinase signaling. PLoS One. 2013;8(5): e65135. DOI: 10.1371/journal.pone.0065135.

20. Сироткина О.В., Ермаков А.И., Гайковая Л.Б. и др. Микрочастицы клеток крови у больных COVID-19 как маркер активации системы гемостаза. Тромбоз, гемостаз и реология. 2020;(4):35–40. [Sirotkina OV, Ermakov AI, Gaykovaya LB, et al. Microparticles of blood cells in patients with COVID-19 as a marker of hemostasis activation. Tromboz, gemostazireologija = Thrombosis, hemostasis and rheology. 2020;(4):35–40. (In Russ.)]. DOI: 10.25555/THR.2020.4.0943.

21. Kabanova S, Kleinbongard P, Volkmer J, et al. Gene expression analysis of human red blood cells. Int J Med Sci. 2009;6(4):156–159. DOI: 10.7150/ijms.6.156.

22. Groen K, Maltby VE, Lea RA, et al. Erythrocyte microRNA sequencing reveals differential expression in relapsing-remitting multiple sclerosis. BMC Med Genomics. 2018;11(1):48. DOI: 10.1186/s12920-018-0365-7.

23. Chen SY, Wang Y, Telen MJ, Chi JT. The genomic analysis of erythrocyte microRNA expression in sickle cell diseases. PLoS One. 2008;3(6):e2360. DOI: 10.1371/journal.pone.0002360.

24. Lamba V, Ghodke-Puranik Y, Guan W, Lamba JK. Identification of suitable reference genes for hepatic microRNA quantitation. BMC Res Notes. 2014;7:129. DOI: 10.1186/1756-0500-7-129.

25. Shen J, Wang Q, Gurvich I, et al. Evaluating normalization approaches for the better identification of aberrant microRNAs associated with hepatocellular carcinoma. Hepatoma Res. 2016;2:305–315. DOI: 10.20517/2394-5079.2016.28.

26. Wagner GM, Chiu DT, Yee MC, Lubin BH. Red cell vesiculation – a common membrane physiologic event. J Lab Clin Med. 1986;108(4):315–24.

27. Van Der Meijden PE, Van Schilfgaarde M, Van Oerle R, et al. Platelet- and erythrocyte-derived microparticles trigger thrombin generation via factor XIIa. J Thromb Haemost. 2012;10(7):1355–62. doi: 10.1111/j.1538-7836.2012.04758.x.

28. Koshiar RL, Somajo S, Norström E, Dahlbäck B. Erythrocyte-derived microparticles supporting activated protein C-mediated regulation of blood coagulation. PLoS One. 2014;9(8):e104200. DOI: 10.1371/journal.pone.0104200.

29. Papapetrou EP, Korkola JE, Sadelain M. A genetic strategy for single and combinatorial analysis of miRNA function in mammalian hematopoietic stem cells. Stem Cells. 2010;28(2):287–96. DOI: 10.1002/stem.257.

30. Fang X, Shen F, Lechauve C, et al. miR-144/451 represses the LKB1/AMPK/mTOR pathway to promote red cell precursor survival during recovery from acute anemia. Haematologica. 2018 Mar;103(3):406–416. DOI: 10.3324/haematol.2017.177394.

31. Yu D, dos Santos CO, Zhao G, et al. miR-451 protects against erythroid oxidant stress by repressing 14-3-3zeta. Genes Dev. 2010; 24(15):1620–1633. DOI: 10.1101/gad.1942110.

32. Feng L, Yang X, Liang S, et al. Silica nanoparticles trigger the vascular endothelial dysfunction and prethrombotic state via miR-451 directly regulating the IL6R signaling pathway. Part Fibre Toxicol. 2019;16(1):16. DOI: 10.1186/s12989-019-0300-x.

33. Oto J, Plana E, Solmoirago MJ, et al. microRNAs and markers of neutrophil activation as predictors of early incidental post-surgical pulmonary embolism in patients with intracranial tumors. Cancers (Basel). 2020;12(6):1536. DOI: 10.3390/cancers12061536.

34. Morelli VM, Brækkan SK, Hansen JB. Role of microRNAs in venous thromboembolism. Int J Mol Sci. 2020;21(7):2602. DOI: 10.3390/ijms21072602.

35. He F, Ni N, Wang H et al. OUHP: an optimized universal hairpin primer system for cost-effective and high-throughput RT-qPCR-based quantification of microRNA (miRNA) expression. Nucleic Acids Res. 2022;50(4):e22. DOI: 10.1093/nar/gkab1153.

36. Forero DA, González-Giraldo Y, Castro-Vega LJ, Barreto GE. qPCR-based methods for expression analysis of miRNAs. Biotechniques. 2019;67(4):192–199. DOI: 10.2144/btn-2019-0065.

37. Busk PK. A tool for design of primers for microRNA-specific quantitative RT-qPCR. BMC Bioinformatics. 2014;15:29. DOI: 10.1186/1471-2105-15-29.

38. D’Agata R, Spoto G. Advanced methods for microRNA biosensing: a problem-solving perspective. Anal Bioanal Chem. 2019;411(19):4425–4444. DOI: 10.1007/s00216-019-01621-8.

39. Zárybnický T, Matoušková P, Ambrož M, et al. The selection and validation of reference genes for mRNA and microRNA expression studies in human liver slices using RT-qPCR. Genes (Basel). 2019;10(10):763. DOI: 10.3390/genes10100763.

40. Tafrihi M, Hasheminasab E. MiRNAs: biology, biogenesis, their web-based tools, and databases. Microrna. 2019;8(1):4–27. DOI: 10.2174/2211536607666180827111633.

41. Felekkis K, Papaneophytou C. Challenges in using circulating micro- RNAs as biomarkers for cardiovascular diseases. Int J Mol Sci. 2020;21(2):561. DOI: 10.3390/ijms21020561.

42. Rogula S, Pomirski B, Czyżak N, et al. Biomarker-based approach to determine etiology and severity of pulmonary hypertension: Focus on microRNA. Front Cardiovasc Med. 2022;9:980718. DOI: 10.3389/fcvm.2022.980718