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      Long Noncoding RNA (lncRNA) CTTN-IT1 Elevates Skeletal Muscle Satellite Cell Proliferation and Differentiation by Acting as ceRNA for YAP1 Through Absorbing miR-29a in Hu Sheep


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          Characterizing the factors that regulate the growth and development of muscle is central to animal production. Skeletal muscle satellite cells (SMSCs) provide an important material for simulating the proliferation and differentiation of muscle cells. YAP1, which can promote muscle growth, is closely related to the proliferation of SMSCs in Hu sheep ( Ovis aries). In addition, some miRNAs, such as miR-541-3p, miR-142-5p, and miR-29a, can play critical roles in muscle growth by specifically binding with their target mRNAs. Meanwhile, lncRNA can competitively bind these miRNAs and reduce the regulatory effect of miRNAs on their target genes and thus play critical roles themselves in muscle growth. However, the regulatory molecular mechanism of miRNA and lncRNA on SMSC proliferation through YAP1 remains unclear. Here, we characterized the regulatory network among YAP1 and its targeted miRNAs and lncRNAs in Hu sheep SMSCs. The potential ncRNAs that regulate YAP1 (miR-29a and CTTN-IT1) were predicted through multilevel bioinformatics analysis. Dual-luciferase assays, RT-qPCR, and western blots revealed that miR-29a can significantly reduce the mRNA and protein expression level by binding to a specific 3′-UTR of YAP1 ( P < 0.05), while CTTN-IT1 can restore the expression of YAP1 through competitive binding to miR-29a. Furthermore, the mRNA and protein expression levels of MyoG, MyoD, and MyHC showed that miR-29a can inhibit the expression of genes related to the differentiation of SMSCs, and CTTN-IT1 can increase the expression of these same genes. Thus, miR-29a may inhibit the differentiation of SMSCs and CTTN-IT1 can restore this inhibition. The EdU staining assay indicated that excessive miR-29a can significantly reduce the proliferation ability of SMSCs ( P < 0.05), while overexpression of CTTN-IT1 can significantly increase the proliferation of SMSCs ( P < 0.01). CTTN-IT1 is a novel lncRNA that is a competing endogenous RNA (ceRNA) of miR-29a and can promote SMSC proliferation and differentiation by restoring the expression of YAP1 when it is inhibited by miR-29a in Hu sheep. Overall, our findings construct a CTTN-IT1-miR-29a- YAP1 regulatory network that will help contribute new insight into improving the muscle development of Hu sheep.

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          MicroRNAs (miRNAs) are small endogenous RNAs that regulate gene-expression posttranscriptionally. MiRNA research in allergy is expanding because miRNAs are crucial regulators of gene expression and promising candidates for biomarker development. MiRNA mimics and miRNA inhibitors currently in preclinical development have shown promise as novel therapeutic agents. Multiple technological platforms have been developed for miRNA isolation, miRNA quantitation, miRNA profiling, miRNA target detection, and modulating miRNA levels in vitro and in vivo. Here we will review the major technological platforms with consideration given for the advantages and disadvantages of each platform.
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            Repairing skeletal muscle: regenerative potential of skeletal muscle stem cells.

            Skeletal muscle damaged by injury or by degenerative diseases such as muscular dystrophy is able to regenerate new muscle fibers. Regeneration mainly depends upon satellite cells, myogenic progenitors localized between the basal lamina and the muscle fiber membrane. However, other cell types outside the basal lamina, such as pericytes, also have myogenic potency. Here, we discuss the main properties of satellite cells and other myogenic progenitors as well as recent efforts to obtain myogenic cells from pluripotent stem cells for patient-tailored cell therapy. Clinical trials utilizing these cells to treat muscular dystrophies, heart failure, and stress urinary incontinence are also briefly outlined.
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              Expression of Cd34 and Myf5 Defines the Majority of Quiescent Adult Skeletal Muscle Satellite Cells

              Skeletal muscle is one of a several adult post-mitotic tissues that retain the capacity to regenerate. This relies on a population of quiescent precursors, termed satellite cells. Here we describe two novel markers of quiescent satellite cells: CD34, an established marker of hematopoietic stem cells, and Myf5, the earliest marker of myogenic commitment. CD34+ve myoblasts can be detected in proliferating C2C12 cultures. In differentiating cultures, CD34+ve cells do not fuse into myotubes, nor express MyoD. Using isolated myofibers as a model of synchronous precursor cell activation, we show that quiescent satellite cells express CD34. An early feature of their activation is alternate splicing followed by complete transcriptional shutdown of CD34. This data implicates CD34 in the maintenance of satellite cell quiescence. In heterozygous Myf5 nlacZ/+ mice, all CD34+ve satellite cells also express β-galactosidase, a marker of activation of Myf5, showing that quiescent satellite cells are committed to myogenesis. All such cells are positive for the accepted satellite cell marker, M-cadherin. We also show that satellite cells can be identified on isolated myofibers of the myosin light chain 3F-nlacZ-2E mouse as those that do not express the transgene. The numbers of satellite cells detected in this way are significantly greater than those identified by the other three markers. We conclude that the expression of CD34, Myf5, and M-cadherin defines quiescent, committed precursors and speculate that the CD34−ve, Myf5−ve minority may be involved in maintaining the lineage-committed majority.

                Author and article information

                Front Genet
                Front Genet
                Front. Genet.
                Frontiers in Genetics
                Frontiers Media S.A.
                07 August 2020
                : 11
                : 843
                [1] 1College of Animal Science and Technology, Yangzhou University , Yangzhou, China
                [2] 2Joint International Research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education of China, Yangzhou University , Yangzhou, China
                [3] 3Jiangsu Co-innovation Center for Important Animal Infectious Diseases and Zoonoses, Yangzhou University , Yangzhou, China
                Author notes

                Edited by: Naoyuki Kataoka, The University of Tokyo, Japan

                Reviewed by: Zhenhui Li, South China Agricultural University, China; Tao Zhong, Sichuan Agricultural University, China; Siyuan Zhan, Sichuan Agricultural University, China

                *Correspondence: Wei Sun, dkxmsunwei@ 123456163.com

                These authors share first authorship

                This article was submitted to RNA, a section of the journal Frontiers in Genetics

                Copyright © 2020 Wu, Wang, Wang, Zhang, Chen, Lv, Li, Hussain and Sun.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                : 13 May 2020
                : 13 July 2020
                Page count
                Figures: 4, Tables: 3, Equations: 0, References: 63, Pages: 13, Words: 0
                Funded by: National Natural Science Foundation of China 10.13039/501100001809
                Funded by: Jiangsu Agricultural Science and Technology Innovation Fund 10.13039/100007540
                Original Research

                yap1,mir-29a,lncrna,skeletal muscle,satellite cell,hu sheep
                yap1, mir-29a, lncrna, skeletal muscle, satellite cell, hu sheep


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