| In livestock, all muscle fibers are formed during the prenatal stage, postnatal development of muscle cells is characterised by an increase in the size of myofibres. The postnatal growth and the regenerative ability of adult skeletal muscle are dependent on adult muscle stem cells known as satellite cells which reside beneath the basal lamina of the mature fibers. Activated satellite cells give rise to myoblast cells that fuse with myofibres, thus affect the development of muscle fibers.It can effectively improve livestock meat quality by controlling the differentiation of muscle satellite cells. Myogenin(Myo G) is one of the Myogenic regulatory factors(MRFs). As a differentiation factor, Myo G is implicated in the subsequent activation of muscle-specific genes during myogenic differentiation, so therefore Myo G is essential for the regulation of the differentiation. In this study, we identified differentially expressed mi RNAs and m RNA in bovine skeletal muscle-derived satellite cells(MDSCs) during different differentiation stages by deep sequencing technology, and predicted the mi RNAs and transcription factors whose could regulate the target Myo G gene expression by the method of bioinformatics. mi R-2400 and transcription factors EGR1 were screened for the candidate factors to regulate the expression of Myo G gene. To confirm this, the regulation of mi R-2400 on the MyoG gene expression was examined by the methods of luciferase reporter assay, overexpression and inhibition of mi R-2400,q RT-PCR, Western blot, Ed U and Flow cytometry. Also, the regulation of transcription factors EGR1 on the Myo G gene expression was examined by the methods of cris PRi, virus expression vector, qRT-PCR, Western blot and Ch IP. The results showed that in the process of MDSCs differentiation, mi R-2400 and EGR1 could regulate the expression of Myo G gene for the first time.The results contribute to further understanding the molecular mechanism for regulation of Myo G gene, as well as the mechanism of action of the muscle differentiation. This may help to livestock genetic breeding and improve the meat production.The main results were as follows:1. This study performed a comprehensive expression profiling of mi RNAs expressed in MDSCs during the differentiation process by RNA sequencing. Total 564 known mi RNAs and 53 novel miRNAs in MDSCs during the differentiation stages(0h, MDSC-P; 24 h, MDSC-D1; and72 h, MDSC-D3) were identified. Compared to MDSC-P, there were 9 up-expressed, 165down-expressed mi RNAs in MDSC-D1, and 15 up-expressed, 145 down-expressed in MDSC-D3,respectively. Furthermore, there were 17 up-expressed, 55 down-expressed in MDSC-D3,compared to those in MDSC-D1. There were 65 mi RNA which could regulate Myo G gene by bioinformatics prediction, of which the fold Change of 12 mi RNA were greater than 2. They were bta-mi R-122, bta-mi R-129-3p, bta-mi R-2450 a, bta-mi R-331, bta-miR-2398, bta-miR-376 b,bta-miR-196 a, bta-mi R-2400, bta-mi R-29 a, bta-mi R-155, bta-mi R-28 and bta-mi R-449 a. The mi R-2400 was a novel and unique mi RNA from bovine.2. This study performed a comprehensive expression profiling of m RNAs expressed in MDSCs during the differentiation process mentioned above by RNA sequencing. Compared to MDSC-P, there were 2,026 up-expressed, 2,125 down-expressed m RNAs in MDSC-D1, and 2,508up-expressed, 2,381 down-expressed in MDSC-D3, respectively. Furthermore, there were 1,243up-expressed, 863 down-expressed in MDSC-D3, compared to those in MDSC-D1. Additionally,there were six transcription factors were candidates for regulation of Myo G gene, they were Myo D,EGR1, MEF2, MEF3, SRF and MEF-1. Furthermore, compared to MDSC-P, transcription factors EGR1 gene was up-regulated up to 10.49-fold in MDSC-D3, therefore, EGR1 was regarded as an candidate transcription factors to regulate Myo G gene in our further study.3. The expression levels of mi R-2400 and Myo G gene in MDSCs during differentiation were determined by stem-loop RT-PCR. The results showed that the expression levels of mi R-2400 were down-regulated and MyoG were up-regulated during MDSCs differentiation. The bioinformatics prediction and dual-luciferase reporter analysis confirmed that the 3′UTR of the Myo G mRNA contained a conserved binding site(position of 590-597) for the seed sequence of mi R-2400, this indicated mi R-2400 may negatively regulate MYOG gene. Furthermore, the expression of Myo G gene was examined through q RT-PCR and Western blot after transfected with pc DNA3.1(+)-miR-2400 and mi R-2400-I. The results showed that over-expression of mi R-2400 in MDSCs derepressed the expression of Myo G gene and increased MDSCs proliferation, whereas inhibition of mi R-2400 caused increased the expression of Myo G gene and inhibited MDSCs proliferation. Mi R-2400 located at BTA27 and within the 8th introns of WHSC1L1 gene. The CRISPR interference analysis and dual-luciferase reporter assay confirmed that miR-2400 was processed from the WHSC1L1 intron rather than being transcribed as a separate RNA. These results confirmed that mi R-2400 was a regulatory factor which could regulate the expression of Myo G gene.4. The expression levels of EGR1 and Myo G gene in MDSCs during differentiation were determined by RT-PCR, werstern blot and immunofluorescence. The results showed that the expression level of EGR1 was up-regulated prior to the expression of Myo G and MHC genes,which were also up-regulated. During MDSCs differentiation, transcription factor EGR1 wasobserved to transfer into the nucleus through fluorescence microscope and confocal microscopy.Further study showed that over-expression of EGR1 in MDSCs increased the expression levels of Myo G and MHC genes, and inhibition of EGR1 derepressed Myo G and MHC expression. These results indicated that the transcription factor EGR1 had a positive regulation role on the expression of Myo G gene. The MDSCs proliferation was significantly reduced after overexpression of EGR1 in MDSCs, with an increase in G1/G0 phase and reductione in S phase cell. Meanwhile the expression of CCND2 and CCNB1 genes were reduceddecreased too. Whereas inhibition of EGR1 reduced G1/G0 cell proportion, increased G2/M phase cells proportion, and the expression of CCND2 and CCNB1 genes were upregulated. Additioanlly, EGR1 was observed to bind to the promoter of the EGR1 binds site in MyoG gene by the method of Ch IP, we confirmed for the first time that EGR1 could bind to the promoter of the EGR1 binds site in Myo G gene. These results confirmed that EGR1 as a transcription factor could bind to the promoter of Myo G gene,hence positively regulating it’s expressionIn conclusion, for the first time we confirmed that mi R-2400 could negatively regulate the expression of Myo G gene, and transcription factor EGR1 could positively regulated it’s expression.This may be a potential mechanism for the regulation of MDSCs differentiation. |
PDF Full Text Request