Reports of Biochemistry
and Molecular Biology
Tue, May 7, 2024
[Archive]
Volume 12, Issue 1 (Vol.12 No.1 Apr 2023)
rbmb.net 2023, 12(1): 136-146 |
Back to browse issues page
Download citation:
BibTeX | RIS | EndNote | Medlars | ProCite | Reference Manager | RefWorks
Send citation to:
BibTeX | RIS | EndNote | Medlars | ProCite | Reference Manager | RefWorks
Send citation to:
Maleki B, Noureddini M, Saadat S, Verdi J, Farrokhian A, Ghanbarian H, et al . Effect of miR-18a-5p, miR-19a-3p, and miR-20a-5p on In Vitro Cardiomyocyte Differentiation of Human Endometrium Tissue-Derived Stem Cells Through Regulation of Smad4 Expression. rbmb.net 2023; 12 (1) :136-146
URL: http://rbmb.net/article-1-1115-en.html
URL: http://rbmb.net/article-1-1115-en.html
Behnaz Maleki 
, Mahdi Noureddini , Somayeh Saadat , Javad Verdi , Alireza Farrokhian , Hossein Ghanbarian , Ebrahim Cheraghi , Behrang Alani *
, Mahdi Noureddini , Somayeh Saadat , Javad Verdi , Alireza Farrokhian , Hossein Ghanbarian , Ebrahim Cheraghi , Behrang Alani *
Department of Applied Cell Sciences, Faculty of Medicine, Kashan University of Medical Sciences, Kashan, Iran.
Abstract: (995 Views)
Background: Smad4 regulates the expression of the genes required for heart homeostasis. Regarding the central role of microRNAs in cardiac biology, we investigated the expression of the three Smad4-targeting miRNAs, namely miR-18a-5p, miR-19a-3p, and miR-20a-5p, as well as Smad4 during differentiation of human endometrium-derived mesenchymal stem cells (hEMSCs) into cardiomyocytes (CMs).
Methods: To evaluate mesenchymal phenotype and multi-lineage differentiation ability of hEMSCs, immunophenotyping by flow cytometry and differentiation into osteoblasts and adipocytes were performed, respectively. For transdifferentiation into CMs, hEMSCs were exposed to a cardiomyogenic medium composed of 5-aza and bFGF for 30 days. The comparison between transcriptional expression levels of Nkx2-5, GATA4, Smad4, TNNT2, TBX5, miR-18a-5p, miR-19a-3p, and miR-20a-5p by qRT-PCR, as well as protein levels of Nkx2-5, Smad4, and cTnT by immunofluorescence staining, was conducted in every 6 days.
Results: In vitro, the mesenchymal stem cell phenotype of hEMSCs and their potency for differentiation into other MSCs were confirmed. Differentiated hEMSCs had morphological characteristics of CMs. The percentage of positive cells for Nkx2-5, Smad4, and cTnT proteins was increased following induction and culminated on the 24th day. Also, mRNA levels of Nkx2-5, GATA4, Smad4, TNNT2, and TBX5 exhibited the same trend. The expression of investigated miRNAs was significantly decreased sequentially. A significant negative correlation between expressions of Smad4 and investigated miRNAs was observed.
Conclusions: Our results indicate that miR-18a-5p, miR-19a-3p, and miR-20a-5p are involved in the cardiac differentiation propensity of hEMSCs potentially by regulation of Smad levels. Although, more mechanistic experiments are required to confirm this idea.
Methods: To evaluate mesenchymal phenotype and multi-lineage differentiation ability of hEMSCs, immunophenotyping by flow cytometry and differentiation into osteoblasts and adipocytes were performed, respectively. For transdifferentiation into CMs, hEMSCs were exposed to a cardiomyogenic medium composed of 5-aza and bFGF for 30 days. The comparison between transcriptional expression levels of Nkx2-5, GATA4, Smad4, TNNT2, TBX5, miR-18a-5p, miR-19a-3p, and miR-20a-5p by qRT-PCR, as well as protein levels of Nkx2-5, Smad4, and cTnT by immunofluorescence staining, was conducted in every 6 days.
Results: In vitro, the mesenchymal stem cell phenotype of hEMSCs and their potency for differentiation into other MSCs were confirmed. Differentiated hEMSCs had morphological characteristics of CMs. The percentage of positive cells for Nkx2-5, Smad4, and cTnT proteins was increased following induction and culminated on the 24th day. Also, mRNA levels of Nkx2-5, GATA4, Smad4, TNNT2, and TBX5 exhibited the same trend. The expression of investigated miRNAs was significantly decreased sequentially. A significant negative correlation between expressions of Smad4 and investigated miRNAs was observed.
Conclusions: Our results indicate that miR-18a-5p, miR-19a-3p, and miR-20a-5p are involved in the cardiac differentiation propensity of hEMSCs potentially by regulation of Smad levels. Although, more mechanistic experiments are required to confirm this idea.
Keywords: Endometrium-derived mesenchymal stem cells (EMSCs), miR-18a-5p, miR-19a-3p, miR-20a-5p, Smad4.
Type of Article: Original Article |
Subject:
Cell Biology
Received: 2022/12/21 | Accepted: 2023/01/22 | Published: 2023/08/15
Received: 2022/12/21 | Accepted: 2023/01/22 | Published: 2023/08/15
References
1. Sadabadi F, Gholoobi A, Heidari-Bakavol A, Mouhebati M, Javandoost A, Asadi Z, et al. Decreased Threshold of Fasting Serum Glucose for Cardiovascular Events: MASHAD Cohort Study. Rep Biochem Mol Biol. 2020;9(1):64-70. [DOI:10.29252/rbmb.9.1.64] [PMID] [PMCID]
2. Leong DP, Joseph PG, McKee M, Anand SS, Teo KK, Schwalm JD, et al. Reducing the Global Burden of Cardiovascular Disease, Part 2: Prevention and Treatment of Cardiovascular Disease. Circ Res. 2017;121(6):695-710. [DOI:10.1161/CIRCRESAHA.117.311849] [PMID]
3. Müller P, Lemcke H, David R. Stem Cell Therapy in Heart Diseases - Cell Types, Mechanisms and Improvement Strategies. Cell Physiol Biochem. 2018;48(6):2607-55. [DOI:10.1159/000492704] [PMID]
4. Pittenger MF, Discher DE, Péault BM, Phinney DG, Hare JM, Caplan AI. Mesenchymal stem cell perspective: cell biology to clinical progress. NPJ Regen Med. 2019;4(1):22. [DOI:10.1038/s41536-019-0083-6] [PMID] [PMCID]
5. Bartolucci J, Verdugo FJ, González PL, Larrea RE, Abarzua E, Goset C, et al. Safety and Efficacy of the Intravenous Infusion of Umbilical Cord Mesenchymal Stem Cells in Patients With Heart Failure: A Phase 1/2 Randomized Controlled Trial (RIMECARD Trial [Randomized Clinical Trial of Intravenous Infusion Umbilical Cord Mesenchymal Stem Cells on Cardiopathy]). Circ Res. 2017;121(10):1192-204. [DOI:10.1161/CIRCRESAHA.117.310712] [PMID] [PMCID]
6. Liu Y, Liu T, Han J, Yang Z, Xue X, Jiang H, et al. Advanced age impairs cardioprotective function of mesenchymal stem cell transplantation from patients to myocardially infarcted rats. Cardiology. 2014;128(2):209-19. [DOI:10.1159/000360393] [PMID]
7. Fan X, He S, Song H, Yin W, Zhang J, Peng Z, et al. Human endometrium-derived stem cell improves cardiac function after myocardial ischemic injury by enhancing angiogenesis and myocardial metabolism. Stem Cell Res Ther. 2021;12(1):344. [DOI:10.1186/s13287-021-02423-5] [PMID] [PMCID]
8. Ehtesham N, Mosallaei M, Karimzadeh MR, Moradikazerouni H, Sharifi M. microRNAs: key modulators of disease-modifying therapies in multiple sclerosis. Int Rev Immunol. 2020;39(6):264-79. [DOI:10.1080/08830185.2020.1779712] [PMID]
9. Maleki B, Alani B, Tamehri Zadeh SS, Saadat S, Rajabi A, Ayoubzadeh SMJ, et al. MicroRNAs and exosomes: Cardiac stem cells in heart diseases. Pathol Res Pract. 2022;229:153701. [DOI:10.1016/j.prp.2021.153701] [PMID]
10. Ventura A, Young AG, Winslow MM, Lintault L, Meissner A, Erkeland SJ, et al. Targeted deletion reveals essential and overlapping functions of the miR-17 through 92 family of miRNA clusters. Cell. 2008;132(5):875-86. [DOI:10.1016/j.cell.2008.02.019] [PMID] [PMCID]
11. Chen J, Huang ZP, Seok HY, Ding J, Kataoka M, Zhang Z, et al. mir-17-92 cluster is required for and sufficient to induce cardiomyocyte proliferation in postnatal and adult hearts. Circ Res. 2013;112(12):1557-66. [DOI:10.1161/CIRCRESAHA.112.300658] [PMID] [PMCID]
12. Mahmoudi Rad M, Mahmoudi Rad N, Mirdamadi Y. Expression of TGF-β3 in Isolated Fibroblasts from Foreskin. Rep Biochem and Mol Biol. 2015;3(2):76-81.
13. Hanna A, Frangogiannis NG. The Role of the TGF-β Superfamily in Myocardial Infarction. Front Cardiovasc Med. 2019;6:140. [DOI:10.3389/fcvm.2019.00140] [PMID] [PMCID]
14. Saadat S, Noureddini M, Mahjoubin-Tehran M, Nazemi S, Shojaie L, Aschner M, et al. Pivotal Role of TGF-β/Smad Signaling in Cardiac Fibrosis: Non-coding RNAs as Effectual Players. Front Cardiovasc Med. 2020;7:588347. [DOI:10.3389/fcvm.2020.588347] [PMID] [PMCID]
15. Umbarkar P, Singh AP, Gupte M, Verma VK, Galindo CL, Guo Y, et al. Cardiomyocyte SMAD4-Dependent TGF-β Signaling is Essential to Maintain Adult Heart Homeostasis. JACC Basic Transl Sci. 2019;4(1):41-53. [DOI:10.1016/j.jacbts.2018.10.003] [PMID] [PMCID]
16. Hu W, Dong A, Karasaki K, Sogabe S, Okamoto D, Saigo M, et al. Smad4 regulates the nuclear translocation of Nkx2-5 in cardiac differentiation. Sci Rep. 2021;11(1):3588. [DOI:10.1038/s41598-021-82954-2] [PMID] [PMCID]
17. Qi X, Yang G, Yang L, Lan Y, Weng T, Wang J, et al. Essential role of Smad4 in maintaining cardiomyocyte proliferation during murine embryonic heart development. Dev Biol. 2007;311(1):136-46. [DOI:10.1016/j.ydbio.2007.08.022] [PMID]
18. Huang H-Y, Lin Y-C-D, Cui S, Huang Y, Tang Y, Xu J, et al. miRTarBase update 2022: an informative resource for experimentally validated miRNA-target interactions. Nucleic Acids Res. 2021;50(D1):D222-D30. [DOI:10.1093/nar/gkab1079] [PMID] [PMCID]
19. Rossmeislová L, McNeil M, Murrell A, Mynatt R, Smith S. Human Mesenchymal Stem Cells as an in Vitro Model for Human Adipogenesis. Obes Res. 2003;11:65-74. [DOI:10.1038/oby.2003.11] [PMID]
20. Song I-H, Caplan A, Dennis J. In Vitro Dexamethasone Pretreatment Enhances Bone Formation of Human Mesenchymal Stem Cells In Vivo. J Orthop Res. 2009;27:916-21. [DOI:10.1002/jor.20838] [PMID]
21. Xu W, Zhang X, Qian H, Zhu W, Sun X, Hu J, et al. Mesenchymal stem cells from adult human bone marrow differentiate into a cardiomyocyte phenotype in vitro. Exp Biol Med (Maywood). 2004;229(7):623-31. [DOI:10.1177/153537020422900706] [PMID]
22. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25(4):402-8. [DOI:10.1006/meth.2001.1262] [PMID]
23. Jiang S, Zhang S. Differentiation of cardiomyocytes from amniotic fluid‑derived mesenchymal stem cells by combined induction with transforming growth factor β1 and 5‑azacytidine. Mol Med Rep. 2017;16(5):5887-93. [DOI:10.3892/mmr.2017.7373] [PMID] [PMCID]
24. Shi S, Wu X, Wang X, Hao W, Miao H, Zhen L, et al. Differentiation of Bone Marrow Mesenchymal Stem Cells to Cardiomyocyte-Like Cells Is Regulated by the Combined Low Dose Treatment of Transforming Growth Factor-β1 and 5-Azacytidine. Stem Cells Int. 2016;2016:3816256. [DOI:10.1155/2016/3816256] [PMID] [PMCID]
25. Xing Y, Lv A, Wang L, Yan X. The combination of angiotensin II and 5-azacytidine promotes cardiomyocyte differentiation of rat bone marrow mesenchymal stem cells. Mol Cell Biochem. 2012;360(1-2):279-87. [DOI:10.1007/s11010-011-1067-z] [PMID]
26. Shen X, Pan B, Zhou H, Liu L, Lv T, Zhu J, et al. Differentiation of mesenchymal stem cells into cardiomyocytes is regulated by miRNA-1-2 via WNT signaling pathway. J Biomed Sci. 2017;24(1):29. [DOI:10.1186/s12929-017-0337-9] [PMID] [PMCID]
27. Zhang JF, Fu WM, He ML, Xie WD, Lv Q, Wan G, et al. MiRNA-20a promotes osteogenic differentiation of human mesenchymal stem cells by co-regulating BMP signaling. RNA Biol. 2011;8(5):829-38. [DOI:10.4161/rna.8.5.16043] [PMID]
28. Luo T, Yang X, Sun Y, Huang X, Zou L, Liu J. Effect of MicroRNA-20a on Osteogenic Differentiation of Human Adipose Tissue-Derived Stem Cells. Cells Tissues Organs. 2019;208(3-4):148-57. [DOI:10.1159/000506304] [PMID]
29. Ai F, Zhang Y, Peng B. miR-20a regulates proliferation, differentiation and apoptosis in P19 cell model of cardiac differentiation by targeting Smoothened. Biol Open. 2016;5(9):1260-5. [DOI:10.1242/bio.019182] [PMID] [PMCID]
30. Ebrahimi-Barough S, Massumi M, Kouchesfahani HM, Ai J. Derivation of pre-oligodendrocytes from human endometrial stromal cells by using overexpression of microRNA 338. J Mol Neurosci. 2013;51(2):337-43. [DOI:10.1007/s12031-013-0101-x] [PMID]
31. Ebrahimi-Barough S, Kouchesfehani HM, Ai J, Mahmoodinia M, Tavakol S, Massumi M. Programming of human endometrial-derived stromal cells (EnSCs) into pre-oligodendrocyte cells by overexpression of miR-219. Neurosci Lett. 2013;537:65-70. [DOI:10.1016/j.neulet.2013.01.022] [PMID]
32. Hasani Lialestani S, Javeri A, Asadi A, Fakhr Taha M. Improved cardiac differentiation of human adipose tissue-derived stem cells using a combination of bFGF and BMP4. Research in Medicine. 2018;42(1):21-7.
33. Markmee R, Aungsuchawan S, Narakornsak S, Tancharoen W, Bumrungkit K, Pangchaidee N, et al. Differentiation of mesenchymal stem cells from human amniotic fluid to cardiomyocyte‑like cells. Mol Med Rep. 2017;16(5):6068-76. [DOI:10.3892/mmr.2017.7333] [PMID] [PMCID]
34. Rahimi M, Zarnani AH, Mohseni-Kouchesfehani H, Soltanghoraei H, Akhondi MM, Kazemnejad S. Comparative evaluation of cardiac markers in differentiated cells from menstrual blood and bone marrow-derived stem cells in vitro. Mol Biotechnol. 2014;56(12):1151-62. [DOI:10.1007/s12033-014-9795-4] [PMID]
35. Zhang Y, Chu Y, Shen W, Dou Z. Effect of 5-azacytidine induction duration on differentiation of human first-trimester fetal mesenchymal stem cells towards cardiomyocyte-like cells. Interact Cardiovasc Thorac Surg. 2009;9(6):943-6. [DOI:10.1510/icvts.2009.211490] [PMID]
36. Subbarao RB, Ullah I, Kim EJ, Jang SJ, Lee WJ, Jeon RH, et al. Characterization and Evaluation of Neuronal Trans-Differentiation with Electrophysiological Properties of Mesenchymal Stem Cells Isolated from Porcine Endometrium. Int J Mol Sci. 2015;16(5):10934-51. [DOI:10.3390/ijms160510934] [PMID] [PMCID]
37. Hida N, Nishiyama N, Miyoshi S, Kira S, Segawa K, Uyama T, et al. Novel cardiac precursor-like cells from human menstrual blood-derived mesenchymal cells. Stem Cells. 2008;26(7):1695-704. [DOI:10.1634/stemcells.2007-0826] [PMID]
38. Meng X, Ichim TE, Zhong J, Rogers A, Yin Z, Jackson J, et al. Endometrial regenerative cells: a novel stem cell population. J Transl Med. 2007;5:57. [DOI:10.1186/1479-5876-5-57] [PMID] [PMCID]
39. Iijima Y, Nagai T, Mizukami M, Matsuura K, Ogura T, Wada H, et al. Beating is necessary for transdifferentiation of skeletal muscle-derived cells into cardiomyocytes. FASEB J. 2003;17(10):1361-3. [DOI:10.1096/fj.02-1048fje] [PMID]
40. Huang Y, Qi Y, Du JQ, Zhang DF. MicroRNA-34a regulates cardiac fibrosis after myocardial infarction by targeting Smad4. Expert Opin Ther Targets. 2014;18(12):1355-65. [DOI:10.1517/14728222.2014.961424] [PMID]
41. Di YF, Li DC, Shen YQ, Wang CL, Zhang DY, Shang AQ, et al. MiR-146b protects cardiomyocytes injury in myocardial ischemia/reperfusion by targeting Smad4. Am J Transl Res. 2017;9(2):656-63.
42. Li Y, Du Y, Cao J, Gao Q, Li H, Chen Y, et al. MiR-130a inhibition protects rat cardiac myocytes from hypoxia-triggered apoptosis by targeting Smad4. Kardiol Pol. 2018;76(6):993-1001. [DOI:10.5603/KP.a2018.0040] [PMID]
Send email to the article author
| Rights and permissions | |
 |
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. |
