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.

IF 1.6 Q4 BIOCHEMISTRY & MOLECULAR BIOLOGY Reports of Biochemistry and Molecular Biology Pub Date : 2023-04-01 DOI:10.52547/rbmb.12.1.136
Behnaz Maleki, Mahdi Noureddini, Somayeh Saadat, Javad Verdi, Alireza Farrokhian, Hossein Ghanbarian, Ebrahim Cheraghi, Behrang Alani
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Abstract

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.

Conclusion: 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.

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miR-18a-5p、miR-19a-3p和miR-20a-5p通过调节Smad4表达对人子宫内膜组织衍生干细胞体外心肌细胞分化的影响。
背景:Smad4调节心脏稳态所需基因的表达。关于微小RNA在心脏生物学中的核心作用,我们研究了三种Smad4靶向miRNA,即miR-18a-5p、miR-19a-3p和miR-20a-5p,以及Smad4在人子宫内膜来源的间充质干细胞(hEMSCs)分化为心肌细胞(CMs)过程中的表达,分别通过流式细胞术进行免疫表型分析和分化为成骨细胞和脂肪细胞。为了转分化为CMs,将hEMSC暴露于由5-aza和bFGF组成的心肌原性培养基中30天。每6天通过qRT-PCR比较Nkx2-5、GATA4、Smad4、TNNT2、TBX5、miR-18a-5p、miR-19a-3p和miR-20a-5p的转录表达水平,以及通过免疫荧光染色比较Nkx2-5、Smad四和cTnT的蛋白水平。结果:在体外,证实了hEMSCs的间充质干细胞表型及其分化为其他MSCs的能力。分化的hEMSC具有CM的形态学特征。Nkx2-5、Smad4和cTnT蛋白阳性细胞的百分比在诱导后增加,并在第24天达到峰值。此外,Nkx2-5、GATA4、Smad4、TNNT2和TBX5的mRNA水平也表现出相同的趋势。所研究的miRNA的表达依次显著降低。观察到Smad4的表达与所研究的miRNA之间存在显著的负相关。结论:我们的研究结果表明,miR-18a-5p、miR-19a-3p和miR-20a-5p可能通过调节Smad水平参与hEMSC的心脏分化倾向。尽管如此,还需要更多的机械实验来证实这一观点。
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来源期刊
Reports of Biochemistry and Molecular Biology
Reports of Biochemistry and Molecular Biology BIOCHEMISTRY & MOLECULAR BIOLOGY-
CiteScore
2.80
自引率
23.50%
发文量
60
审稿时长
10 weeks
期刊介绍: The Reports of Biochemistry & Molecular Biology (RBMB) is the official journal of the Varastegan Institute for Medical Sciences and is dedicated to furthering international exchange of medical and biomedical science experience and opinion and a platform for worldwide dissemination. The RBMB is a medical journal that gives special emphasis to biochemical research and molecular biology studies. The Journal invites original and review articles, short communications, reports on experiments and clinical cases, and case reports containing new insights into any aspect of biochemistry and molecular biology that are not published or being considered for publication elsewhere. Publications are accepted in the form of reports of original research, brief communications, case reports, structured reviews, editorials, commentaries, views and perspectives, letters to authors, book reviews, resources, news, and event agenda.
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