IGF2 Gene-derived IGF2 AS And MiR-483 Regulate Proliferation And Differentiation Of Bovine Skeletal Muscle Cells

Skeletal muscle is an important part of livestock and poultry meat,and its muscle fiber type composition is a vital factor for meat quality.Therefore,studying skeletal muscle development has certain guiding significance for how to improve meat production and quality.In the embryonic stage,Pax3⁺/Pax7⁺myogenic progenitor cells migrate from the mesoderm.The myogenic regulatory factors Myf5,MyoD and MRF4 are involved in the regulation of myogenic determination.The myogenic cells are further proliferated and differentiated to form muscle fibers.After birth,the number of muscle fibers is basically determined,and the increase in muscle mass mainly depends on the growth and thickening of muscle fibers.When the skeletal muscle injuried or excercised,the skeletal muscle has the ability to regenerate and repair,which mainly depends on the activation of the myogenic stem cells?satellite cells,Pax7⁺/Myf5^-?,and the activated satellite cells?Pax7⁺/Myf5⁺?can differentiate into myoblasts and fuse to form new muscle fibers for the purpose of repairing injured muscle fibers.Whether the skeletal muscle formation in the embryonic stage or the regeneration of skeletal muscle after birth,besides the regulation of Pax3/Pax7 and MRFs,which is also affected by MEF2 family,Wnt,IGFs and non-coding RNA.In recent years,research on the regulation of skeletal muscle development by non-coding RNA has received much attention,and its importance cannot be ignored.In this study,high-throughput RNA sequencing technology was used to screen the difference expressed non-coding RNAs?lncRNAs and miRNAs?in skeletal muscle during different developmental stages?embryo,birth and adult?of Qinchuan cattle.The experimental techniques of 5'RACE and 3'RACE,RT-qPCR,FISH,RIP,RNA pull down,flow cytometry were used to explore the function and mechanism of IGF2 AS and miR-483 from the intron region of IGF2 in bovine skeletal muscle development.The main findings are as follows:1.Screening,identification and expression analysis of lncRNAs in muscle tissueBased on the high-throughput lncRNA sequencing of skeletal muscle in different developmental stages?embryonic,postnatal and adult stages?of Qinchuan cattle,this study further explored the identification and analysis of lncRNAs from these 401 differentially expressed lncRNAs.An antisense transcript was found in the intron region of IGF2 gene,which was named IGF2 antisense transcript?IGF2 AS?.By rapid amplification of cDNA ends?RACE?,this transcript consisted of 698 nucleotides including a single exon.Through the protein coding potential software CPC predictive analysis,the protein coding ability of IGF2 AS was weak that is a non-coding RNA.Both high-throughput sequencing and RT-qPCR validation confirmed that IGF2 AS was mainly expressed in skeletal muscle at the embryonic stage.In the growth stage of bovine skeletal muscle cells,the expression of IGF2AS was relatively high.During the differentiation stage of bovine skeletal muscle cells,the expression of IGF2 AS showed an increasing trend.2.Function of IGF2 AS regulating proliferation,differentiation and apoptosis of bovine skeletal muscle cellsRT-qPCR,Western blot,Flow cytometry,EdU,CCK8,RNA-seq,and cell immunofluorescence were used to valicate the function of IGF2 AS during bovine skeletal muscle development.During the growth stage of bovine skeletal muslce cells,knockdown of IGF2 AS inhibited the expression of cell proliferation rlative genes,leading to G₁ phase cells population increase and S phase cells population decrease,and the cell proliferation activity reduced,while the overexpression of IGF2 AS resulted in the opposite phenomenon.In the stage of bovine skeletal muscle cells differentiation,knockdown of IGF2 AS inhibited the expression of myogenic regulatory factors,and the number of MyHC positive myotubes decreased.In contrast,overexpression of IGF2 AS resulted in the opposite effect.In addition,knockdown of IGF2 AS resulted in an increase in the number of apoptotic cells in the growth phase of bovine skeletal muscle cells,whereas overexpression of IGF2 AS inhibited apoptosis in bovine skeletal muscle cells.3.Mechanism of IGF2 AS regulating proliferation,differentiation and apoptosis of bovine skeletal muscle cellsThe results of RNA nuclear and cytoplasmic separation and fluorescence in situ hybridization?FISH?showed that IGF2 AS was both expressed in the cytoplasm and nucleus of bovine skeletal muscle cells.After treatment of total RNA by DNase I and RNase A,PCR results showed that IGF2 AS and the precursor sequences of IGF2 mRNA could form a complementary double-stranded structure.Actinomycin D treatment of bovine skeletal muscle cells at the growth stage revealed that expression of IGF2 AS stabilized the expression of different transcripts of IGF2.The results of RIP analysis indicated that Ago-2protein could bind to IGF2 AS,suggesting that IGF2 AS has the potential of competitive endogenous RNA.The dual luciferase reporter assay showed that miR-221/miR-222 has a competitive binding relationship with IGF2 AS.Further functional verification result indicated that overexpression of IGF2 AS restored the expression of miR-221/miR-222 target gene MyoD.RNA pull down,mass spectrometry and RIP assays showed that ILF3 protein could bind to IGF2 AS.IGF2 AS positively regulated the expression of ILF3,knockdown of ILF3 inhibited the proliferation and differentiation of bovine skeletal muscle cells,and promoted the apoptosis of bovine skeletal muscle cells.4.miR-483 regulates proliferation and differentiation of bovine skeletal muscle cells via IGF1/PI3K/Akt signaling pathwayHigh-throughput sequencing of miRNAs analysis showed that miR-483,a miRNA located in the intron region of the IGF2 gene,which was highly expressed in embryonic stage skeletal muscle.Functional studies showed that overexpression of miR-483 inhibited the proliferation and differentiation of bovine skeletal muscle cells,and interfered with miR-483 inhibitor promoted the proliferation and differentiation of bovine skeletal muscle cells.Mechanistic studies indicated that IGF1 was a target gene of miR-483,and that miR-483 could influence the expression of key proteins in PI3K/Akt signaling pathway by downregulation the expression of IGF1.In this study,high-throughput sequencing technology was used to screen differentially expressed lncRNA and microRNA.The purpose of our research was to elucidate the function and mechanism of lncRNA and microRNA in bovine skeletal muscle development,and provided a theoretical basis for studying non-coding RNA in regulation of bovine skeletal muscle development.