Effects And Mechanisms Of METTL14 During Proliferation And Differentiation Of Ovine Skeletal Muscle Satellite Cell

The yield and quality of sheep meat depend on the development of skeletal muscle satellite cells,and it is important to explore the regulatory mechanism of skeletal muscle satellite cell proliferation and differentiation to improve economic traits such as lean meat percentage.m~6A modification is the most common type of RNA methylation modification,and m~6A modification plays an important role in regulating biological processes such as skeletal muscle growth and development,adipogenesis and osteogenic differentiation.However,there is a lack of studies on the mechanism of m~6A modification during proliferation and differentiation of sheep skeletal muscle satellite cells.In this study,we used sheep skeletal muscle satellite cells as the experimental material,and constructed transcriptomic profiles of proliferation and differentiation sheep skeletal muscle satellite cells based on RNA-seq method.Furthermore,the knockdown of METTL14 in proliferation of ovine skeletal muscle satellite cells,and then the m~6A expression profile was mapped using MeRIP-seq method to resolve the regulatory mechanism of METTL14 on the proliferation of sheep skeletal muscle satellite cells.The main results were as follows:(1)RNA-seq was performed on six cell samples from proliferation(D0)and differentiation(D7)of ovine skeletal muscle satellite cells,at least 6.9 Gb data volume was obtained for each sample,and the reference genome mapping rates were above 92%,the average Q20 and Q30 were above 96%and 91%,respectively.Using|log2Fold Change|≥2and q<0.01 as the screening standards for differentially expressed genes,a total of 1954differentially expressed genes(DEGs)were identified,of which 1288 DEGs were up-regulated and 666 DEGs were down-regulated.These DEGs were significantly enriched in AMPK,PI3K-Akt,Wnt and other signaling pathways.The PPI interaction network was constructed based on the top 50 up-regulated DEGs and top 50 down-regulated DEGs,and the hub genes such as MYBPC1,AREG and MYH1 were identified to be involved in the proliferation and differentiation of ovine skeletal muscle satellite cells.In addition,some immune factors related to skeletal muscle satellite cell development,including IL6 and IL11 were identified.(2)A total of 1479 alternative splicing events were identified during proliferation(D0)and differentiation(D7)of sheep skeletal muscle satellite cells,including 1050 SE,98 RI,128A3SS,100 A5SS and 103 MXE.SE accounted for 70.99%of the alternative splicing events,indicating that the variable m RNA of pre-m RNAs during proliferation and differentiation of sheep skeletal muscle satellite cells alternative splicing events were mainly dominated by SE.Among them,this alternative splicing occurred mainly enriched in MAPK,PI3K-Akt and other signaling pathways.In addition,a total of 253 transcription factors were annotated in this study,including MEF2C,NR4A2,FOXM1,SSRP1 and other transcription factors related to the proliferation and differentiation of skeletal muscle satellite cells.(3)METTL14 regulates the proliferation and differentiation of ovine skeletal muscle satellite cells.We found that the knockdown of METTL14 promoted proliferation and inhibited differentiation of ovine skeletal muscle satellite cells.(4)Six cell samples from the experimental(siMETTL14)groups and control(siNC)groups were sequenced by MeRIP-seq;and each sample more than 5 Gb of raw data with no less than 98%and 94%of Q20 and Q30 for each library,respectively.10405peaks and10122peaks,4420genes and 4232 genes were identified in the two groups,respectively.KEGG results showed that differential m~6A peaks and differential m~6A genes were enriched in Wnt,Hippo,and Notch signaling pathways.(5)RNA-seq and m~6A-seq data from the control(siNC)and experimental(siMETTL14)groups were integrated,and analysis identified that gene expression of TCF7,ALOX12,PANX3,CTF1 and ISLR were correlated with their m~6A modifications,and we hypothesized that these genes migth regulate sheep skeletal muscle satellite cell proliferation through METTL14-mediated m~6A modifications level.In summary,this study used sheep skeletal muscle satellite cells as the experimental material to construct the expression profile of cell proliferation and differentiation using RNA-seq sequencing method,and identified the key genes involved in cell proliferation and differentiation.In addition,this study was the first to construct the m~6A expression profile of ovine skeletal muscle satellite cells that knockdown of METTL14 during proliferation cell,which is important for elucidating the mechanism of m~6A modification involved in the regulation of skeletal muscle satellite cell proliferation.