Int J Stem Cells.  2023 May;16(2):145-155. 10.15283/ijsc22187.

The Essential Function of miR-5739 in Embryonic Muscle Development

• Lee JH
• Kim MS
• Lee Js
• Lee DH
• Park C
• Lee DH
• Kim EY
• Chung HM

• KMID: 2542828
• DOI: http://doi.org/10.15283/ijsc22187

Abstract

Background and Objectives
Embryologically, mesodermal development is closely related to the development of various organs such as muscles, blood vessels, and hearts, which are the main organs that make up the body. However, treatment for mesoderm developmental disorders caused by congenital or acquired factors has so far relied on surgery and drug treatment for symptom relief, and more fundamentally, treatment for mesoderm developmental disorders is needed.
Methods and Results
In our study, microRNA (miRNA), which plays an important role in the mesoderm development process, was identified and the developmental function was evaluated. miRNAs consist of small nucleotides, which act as transcription factors that bind to the 3’ untranslated region and suppressed target gene expression. We constructed the human embryonic stem cell (hESC) knockout cell line and analyzed the function and characteristics of miR-5739, which plays an important role in mesoderm lineage. miR-5739 acts as a transcription factor targeting SMA, Brachyury T, Hand1, which controls muscle proliferation and differentiation, and KDR gene, which regulates vessel formation in vitro. In vivo results suggest a role in regulating muscle proliferation and differentiation. Gene ontology analysis confirmed that the miR-5739 is closely related to genes that regulate muscle and vessel proliferation and differentiation. Importantly, abnormal expression of miR-5739 was detected in somatic cells derived from patients with congenital muscle disease.
Conclusions
Our study demonstrate that miR-5739 gene function significantly affects transcriptional circuits that regu-late muscle and vascular differentiation during embryonic development.

Keyword

Human embryonic stem cell; Mesoderm development; microRNA; Muscle; Vessel

Figure

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  Fig. 1 miR-5739 knockout cell line construction and analysis. (A) Schematic overview of the miR-5739 gene and the Cas9/gRNA target site. (B) miR-5739 specific sgRNA RNP-mediated mutations measured with the T7E1 assay. (C) Real-time PCR for confirming miR-5739 expression reduction in miR-5739 knockout cells.

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  Fig. 2 miR-5739 knockout cells undifferentiated in vitro characterization. (A) H9 and miR-5739 knockout cells colony, AP staining (Scale bar 200 μm). (B) Representative karyotype miR-5739 cell lines showing a normal 48 chromosomes. (C) Pluripotent gene expression profile miR-5739 knockout cell lines compared with the H9.

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  Fig. 3 miR-5739 knockout cells three-germ layer differentiated in vitro cha-racterization. (A) Immunofluoresce-nce was identified using differenti-ation markers in three-germ layer differentiated miR-5739 knockout cells. Top (Endoderm-SOX17 (Green), FOXA2 (Red)), middle (Mesoderm-VEGF (Gre-en), PECAM (Red)), bottom (Ec-toderm-Map2b (Green), Tuj1 (Red), DAPI (blue)), (Scale bar 200 μm). (B) Expression of markers for all three germ layers in 3D culture condition differentiation. GAPDH, Pluri-potent-Nanog, POU5F1, Endoderm-SOX17 (SRY-box transcription factor 17), Hnf4a (Hepatocyte nuclear factor 4 alpha), FOXA2 (Forkhead box protein A2), AFP (Alpha fetoprotein), Mesoderm (Collagen type)-ACAN (Aggrecan), Col1A1 (Collagen type 1 alpha 1 chain), ALP (Alkaline phosphatase), Mesoderm (Muscle type)-KDR (Kinase insert domain receptor), Hand1 (Heart and neural crest derivatives expressed 1), SMA (Smooth muscle actin), T (Brachyury), Ectoderm-Nestin, Tuj1 (Class III beta-tubulin), PAX6.

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  Fig. 4 The effect of miR-5739 knockout cells injection on development in nude mouse. Gut epithelium (Hematoxylin & Eosin staining, magnification ×10, scale bar 100 μm), Muscle (Masson’s trichrome staining, magnification ×10, scale bar 100 μm), Cartilage (Alcian blue staining, magnification ×10, scale bar 100 μm), secretory epithelium (PAS staining, magnification ×10, scale bar 100 μm).

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  Fig. 5 Clue GO analysis of regulated miR-5739 in human embryonic stem cells. The distribution of four clusters visualized on network. Overview chart with functional groups including specific terms for regulated genes.

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  Fig. 6 Muscle and vessel specific differentiated in vitro characterization. (A) Immunofluorescence identified cells differentiated into specific mesoderm at miR-5739 knockout cells. Endothelial cells-VEGF (Green), PECAM (Red), Cardiomyocytes-cTnT(Green), sarcomeric actin(Red), DAPI (blue), (Scale bar 200 μm). (B) FACS analysis of endothelial cells and cardio-myocytes. CD31+H9 cells (22.19%), miR-5739 #39 cells (5.09%), miR-5739 #65 cells (2.95%). cTnT+ H9 cells (62.89%), miR-5739 #39 (1.59%), miR-5739 #65 (1.06%).

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  Fig. 7 Verification of miR-5739 expression using congenital muscle and vascular disease cells. Real-time PCR for confirming miR-5739 expression reduction in duchenne muscular dystrophy (DMD) patient’s cell lines and hereditary hemorrhagic telangiectasia patient’s cell lines. DMD cell lines male (27.68%), DMD cell lines female (14.01%) and HHT cell lines male (21.94%), HHT cell lines female (27.51%).

Reference

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