Open Access

ARTICLE

LncRNA ZFAS1 regulates cardiomyocyte differentiation of human embryonic stem cells

by YANG CAO^1,#, YINING LIU^1,#, YANG YU¹, XIAOFEI GUO¹, XIUXIU WANG¹, WENYA MA¹, HANJING LI², ZHONGYU REN², XINLU GAO², SIJIA LI², HAOYU JI², HONGYANG CHEN², HONG YAN², YANAN TIAN², XIN WANG², BENZHI CAI^1,2,*

1 Department of Pharmacy, The Second Affiliated Hospital of Harbin Medical University (Institute of Clinical Pharmacy, The University Key Laboratory of Drug Research, Heilongjiang Higher Education Institutions), Harbin, 150086, China
2 Department of Pharmacology (State-Province Key Laboratories of Biomedicine-Pharmaceutics of China, Key Laboratory of Cardiovascular Medicine Research, Ministry of Education), College of Pharmacy, Harbin Medical University, Harbin, 150081, China

* Corresponding Author: BENZHI CAI. Email:
# The first two authors contributed equally to this work

BIOCELL 2023, 47(6), 1407-1416. https://doi.org/10.32604/biocell.2023.029080

Received 31 January 2023; Accepted 20 March 2023; Issue published 19 May 2023

Abstract

Background: Cardiomyocytes derived from human embryonic stem cells (hESCs) are regulated by complex and stringent gene networks during differentiation. Long non-coding RNAs (lncRNAs) exert critical epigenetic regulatory functions in multiple differentiation processes. However, the involvement of lncRNAs in the differentiation of hESCs into cardiomyocytes has not yet been fully elucidated. Here, we identified the key roles of ZFAS1 (lncRNA zinc finger antisense 1) in the differentiation of cardiomyocytes from hESCs. Methods: A model of cardiomyocyte differentiation from stem cells was established using the monolayer differentiation method, and the number of beating hESCs-derived cardiomyocytes was calculated. Gene expression was analyzed by quantitative real-time PCR (qRT-PCR). Immunofluorescence assays were performed to assess the expression of cardiac troponin T (cTnT) and α-actinin protein in cardiomyocytes. Results: qRT-PCR showed that ZFAS1 expression in the mesoderm was significantly higher than that in embryonic stem cells, cardiac progenitor cells, and cardiomyocytes. Knockdown of ZFAS1 inhibited cardiomyocyte differentiation from hESCs, which was characterized by reduced expression of the cardiac-specific markers cTnT, α-actinin, myosin heavy chain 6 (MYH6), and myosin heavy chain 7 (MYH7). In contrast, ZFAS1 overexpression remarkably increased the percentage of spontaneously beating cardiomyocytes. In terms of the mechanism, we found that ZFAS1 is an antisense lncRNA at the 5′ end of the protein-coding gene ZNFX1. Knockdown of ZFAS1 could increase the mRNA expression level of ZNFX1. Furthermore, qRT-PCR demonstrated that the silencing of ZNFX1 led to an increase in cardiac-specific markers that predicted the promotion of cardiomyocyte differentiation. Conclusion: Altogether, these data suggest that lncRNA-ZFAS1 is required for cardiac differentiation by functionally inhibiting the expression of ZNFX1, which may provide a reference for the treatment of heart disease to a certain extent.

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