Scientia Agricultura Sinica ›› 2020, Vol. 53 ›› Issue (3): 642-657.doi: 10.3864/j.issn.0578-1752.2020.03.015

• ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT • Previous Articles     Next Articles

Analysis and Identification of circRNAs of Skeletal Muscle at Different Stages of Sheep Embryos Based on Whole Transcriptome Sequencing

SHI TianPei,WANG XinYue,HOU HaoBin,ZHAO ZhiDa,SHANG MingYu,ZHANG Li()   

  1. Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193
  • Received:2019-03-01 Accepted:2019-05-30 Online:2020-02-01 Published:2020-02-13
  • Contact: Li ZHANG E-mail:zhangli07@caas.cn

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Abstract:

【Objective】The meat production of livestock, which is closely related to the development of skeletal muscle, is an important economic trait to measure the quality of livestock. For mammals, the skeletal muscle development depends on the growth and differentiation of embryonic myocyte, which has a significant impact on the subsequent growing potential. In this study, the developmental mode of skeletal muscle, the important transformation nodes, the formation of muscle fibers and the molecular regulation mechanism of transformation were mainly explored. 【Method】 Based on the previous research, the important nodes D85, D105 and D135 related to the myotube development were used in the experiment, and the longissimus dorsi muscles were sequenced by whole transcriptome sequencing. The differentially expressed (DE) circRNAs were screened by bioinformatics analysis and verified by quantitative real-time PCR (qRT-PCR). 【Result】 1 126 DE circRNAs were obtained by conditional screening (|log2| ≥1 and P≤0.05). The 3 groups were compared and many specific expressions of circRNA were found at each stage, but in the D85 vs D135 group, the amount was the most. 374 DE circRNAs were obtained, which contained 201 up-regulated and 173 down-regulated, and 44.7% of the DE genes were differentially expressed with a difference of more than 4 times. These DE circRNAs were subjected to run GO and KEGG functional analysis and targeted prediction, and they were enriched into some pathways, such as energy metabolism and signal transduction, which involved in muscle differentiation and muscle fiber development, including MAPK, PI3K-Akt, Ras, regulation of actin cytoskeleton and other signal transduction pathways. According to the results, it was confirmed that the DE circRNAs enriched during D85 to D105 were mostly associated with cell proliferation and survival, regulation of myocyte development and cell cycle, while D105 to D135 were mainly related to energy conversion, material transport, RNA transport, and DNA repair. By drawing co-expression visualization network with the targeted prediction results used by Cytoscape, the core regulatory transcripts, such as circRNA8239, circRNA19073, circRNA2765 and circRNA1616, were identified. In the D105 period, a key factor circRNA7527 that regulated the conversion of fast and slow muscle types was found, which targets the bta-miR-135a, bta-miR-615, and chi-miR-133a-5p to regulate the MEF2C gene. According to the differential expression and functional prediction in three comparison groups, 4 circRNAs related to muscle development and 4 target miRNA were selected for qRT-PCR, and the results showed that the gene expression trend was consistent with the sequencing data. 【Conclusion】 It was verified that the stabilization of the number of muscle fibers occurred between sheep embryos at D85 and D105, and muscle fiber hypertrophy happened during the D105 to D135 period, which lead to the conclusion that D105 was probably a key time point. In this study, we firstly constructed a circRNA map in sheep embryonic skeletal muscle development based on the whole transcriptome sequencing. The transcriptome differences at key stages were revealed, and multiple circRNAs and miRNAs targeting MEF2C that involved in the MAPK signaling pathway were found, which provided reference for livestock myofiber development research and other research on non-coding RNA.

Key words: sheep, embryo, whole transcriptome, skeletal muscle, growth and development, circRNA

Table 1

The list of gene primers of qRT-PCR"

RNA 类型
RNA Type		 RNA 名称
RNA ID		 上游引物序列
Upstream primer sequence		 下游引物序列
Downstream primer sequence
circRNA内参基因
CircRNA reference gene		 Actin-β CGTGCGTGACATCAAAGAGAA AACCGCTCGTTGCCAATAGT
circRNA circRNA17939 CTGGATACTGTGTTACTTAGAC CATTCTCAGAGGATTAACGATT
circRNA circRNA1037 GGATGACTTGCCCAAGGAGAA CTTGATGGCGACTCGGACTT
circRNA circRNA1042 GGAGATTGGTTTGGAGAGAAAGC TCCTGTCCTCATCATTATCGTCTG
circRNA circRNA5212 TGCCCTCCTACAGACTTGA CGCTGGGTTGGAGATGTT
miRNA内参基因
miRNA reference gene		 U6 CTCGCTTCGGCAGCACA AACGCTTCACGAATTTGCGT
miRNA oar-miR-150 ACGCGTCTCCCAACCCTT GTCGTATCCAGTGCAGGGTCCGAGGTATTC
GCACTGGATACGACCACTGGG
miRNA oar-miR-410-5p CGGCGGAGGTTGTCTGTG GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCGAACT
miRNA oar-miR-133a-5p GGCGGAGCTGGTAAAATGG GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACATTTGG
miRNA bta-miR-365-3p
 CGCGTAATGCCCCTAAAAAT GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACATAAGG
通用下游
Universal downstream primer		 CAGTGCAGGGTCCGAGGTAT

Fig. 1

Distribution of circRNAs based on FPRM in various samples"

Fig. 2

Distribution of circRNAs based on FPRM in various samples"

Fig. 3

KEGG pathway analysis of differentially expressed circRNAs A: D85 vs D105; B: D105 vs D135; C: D85 vs D135; Y-axis: Pathway name; X-axis: enrichment factor; Bubble size reflect the number of differential expressions enriched to a certain pathway/all quantities in the background; Color reflect the significance of differential expression in a certain process"

Fig. 4

PI3K-Akt Signal Pathway The white box is a generalized and detailed metabolic map based on the existing data; the greenbox is the unique gene or enzyme of the species with more detailed information; the orange box indicates that the gene differential expressed in here"

Fig. 5

The heat map of differentially expressed Horizontal axis: embryo age; Vertical axis: circRNA name; Blue: down; Red: up"

Fig. 6

miRNA-circRNA interaction network diagram Orange ellipses indicate differentially expressed miRNAs; Green arrows indicate differentially expressed circRNAs; and Edges indicate interactions between the two. The color from light to dark represent the degree of difference in expression, and the deeper the color, the more significant the difference, and vice versa"

Fig. 7

circRNA-miRNA-mRNA interaction network map Orange ellipses indicate differentially expressed mRNA; Blue arrows indicate differentially expressed circRNA; Red diamonds indicate differentially expressed miRNAs; and Edges indicate interactions between the two"

Fig. 8

GO analysis of circRNA-miRNA-mRNA Y-axis: biological process; X-axis: enrichment factor; Bubble size reflect the number of differential expressions enriched to a certain GO_Term/all quantities in the background; Color reflect the significance of differential expression in a certain process"

Fig. 9

The results of qRT-PCR The figure shows the quantitative results of RNA-seq and qRT-PCR; the line graph shows the FPKM value of the sequencing and the histogram shows the relative quantitative results; the significance test: the D105 histogram is marked with the D85 vs D105 comparison result; 2 test result in D135 histogram: left is D85 vs D135 and right is D105 vs D135; *P≤0.05, ** P≤0.01"

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