Screening Of Porcine LncRNA-MEG3 SNPs And The Effects On The Proliferation And Differentiation Of Porcine Skeletal Muscle Satellite Cells

Meat quality has always been an important economic indicator in Chinese pig industry.The muscle fiber formed by the differentiation and fusion of muscle cells is the basic unit of muscle,which is closely related to meat quality.Therefore,it is of great significance to study the molecular development mechanism of pig muscle cells to improve the meat quality and production of pigs.Bama Xiang pig(BM)is a unique local pig breed in China,which presented short stature,a low growth rate,high-class meat quality,low lean meat ratio and high back fat thickness,compared with Large White pigs,Duroc pigs,Landrace pigs and Pietrain pigs introduced from abroad.Therefore,Bama Xiang pig and foreign introduced pig breeds are widely used as the model to study muscle development of different types of pigs.The growth and development of skeletal muscle is regulated by long non-coding RNA(lncRNAs),in which lncRNA-MEG3 plays an important role in this progress,while the effect and mechanism of lncRNA-MEG3 polymorphism on the growth and development of porcine skeletal muscle satellite cells(SCs)are largely unknown.In this study,SNPs were screened from the lncRNA-MEG3 exons of typical Lean-type pigs(Duroc,Large White,Landrace,Pietrain)and fat-type pigs(Bama Xiang pig),meawhile analyze the functions of these SNPs.Eight SNPs were detected in the lncRNA-MEG3 exons,among which seven SNPs have significant correlation with the economic type(P< 0.001),but only four of them were in accordance with Hardy-Weinberg equilibrium,they were g.3087 a > G,g.3108 a > G,g.3398 a > G and g.3971 t > G,respectively.In addition,based on the different economic type of pigs,there were linkage disequilibrium effects in these four SNPs,the TTCC was the advantage haplotype of lean-type pigs,while the CCCA was the dominant haplotype in fat-typepigs,and the haplotype frequency was significantly different between the two meat-type(P < 0.001).Two dominant haplotypes of pcDNA3.1-MEG3-TTCC(abbreviation for p-M-T)and pcDNA3.1-MEG3-CCCA(abbreviation for p-M-C)were constructed and transiently transfected into porcine skeletal muscle satellite cells to detect the relative expression of lncRNA-MEG3.The results showed that relative expression of lncRNA-MEG3 in p-M-T group was significantly higher than p-M-C group(P< 0.01).Futhermore,actinomycin D was used to interfere with the synthesis of lncRNA to test the RNA stability.The results showed that lncRNA-MEG3 expressed by p-M-C was more stable than p-M-T(P< 0.05).At the same time,the RNA secondary structure and the minimum free energy of different haplotypes were analyzed by Mfold web.The results showed that the two haplotypes presented completely different secondary structures.The minimum free energy of MEG33-CCCA haplotype and MEG3-TTCC haplotype was-542.01 kcal/mol and-530.92 kcal/mol respectively,all these evidences proved that the lncRNA secondary structure of MEG33-CCCA haplotype is more stable than that of MEG3-TTCC haplotype.Through overexpression of p-M-C and p-M-T vector,we detected the effects of two haplotypes on the proliferation and differentiation of skeletal muscle satellite cells by using CCK-8,qRT-PCR and Western blot.The results suggested that overexpression of p-M-C and p-M-T could inhibit the proliferation of porcine skeletal muscle satellite cells.Compared with overexpression of p-M-C group,overexpression of p-M-T group had a stronger inhibitory effect on the proliferation of porcine skeletal muscle satellite cells(P< 0.05).Meanwhile,Western blot results showed that overexpression of p-M-T significantly inhibited PI3K/Akt and MAPK/ERK1/2signaling pathways(P< 0.05).On the contrary,overexpression of p-M-C and p-M-T promoted the genes and signal pathways in the differentiation process of pig skeletal muscle satellite cells.Compared with overexpression of p-M-C group,overexpression of p-M-T strongly promoted the differentiation process of pig skeletal muscle satellite cells(P< 0.05),and had a more significant stimulation on JAK2/STAT3 signal pathway(P< 0.05).The above results suggested that SNPs on the lncRNA-MEG3 exons can affect the transcription,stability,secondary structure and function of lncRNA-MEG3,which provides a theoretical basis for the study of lncRNAs polymorphism.