The Effect And Mechanism Of MiR-432 On Myoblast Proliferation And Differentiation

Skeletal muscle is the dominant executant in locomotion and regulator in energy metabolism.And the growth and development of skeletal muscle are controlled by a cascade of transcription factors and epigenetic regulatory mechanisms.MicroRNA,a family of non-coding RNA of 18-24 nucleotides in length,post-transcriptionally regulates expression of mRNA by pairing the seed sequence to 3'UTR of target mRNA.Previous studies have indicated that miRNAs are important regulators in diverse myogenic processes.By high-throughput sequencing,the profiling of miRNA expression revealed that miR-432-5p is more enriched in the longissimus dorsi of 35-day-old piglets than that of adult pigs and difference is as high as 7 times.In addition,miR-432 highly conserved in seed sequence between mice,human and pig,which means that miR-432 displays a similar role in myogenesis between mice and pig.Hence,we explore the effect and mechanism of miR-432 on myoblast proliferation and differentiation in C2C12 myoblast cell line.In our study,we investigated the function of miR-432 on myoblast proliferation and differentiation by transfecting miR-432 mimics or inhibitor,respectively.Mechanically,we used bioinformatic analysis and dual luciferase report assays to predict and verify the target genes of miR-432.Moreover,we discovered the molecular mechanism underlying the effect of miR-432 on proliferation and differentiation of myoblast.The main results of this study are shown as follows:1.miR-432 inhibits myoblast proliferation.Overexpression of miR-432 suppressed cell cycle-related genes(cyclin E,cdk2 and PCNA)expression,both in level of mRNA and protein.At the same time,the percentage of S-phage cells(P < 0.01)and total cell number was reduced in miR-432 mimics-treated group compared with NC.However,myoblast proliferation was promoted when we transfected with miR-432 inhibitor into myoblast.2.miR-432 suppresses myogenic differentiation.Overexpression of miR-432 significantly inhibited the expression of MyoG,a key myogenic transcription factor,and MyHC,both in mRNA and protein level.Moreover,immunofluorescence staining technique was used to demonstrated that miR-432 inhibited the formation of myotube and down-regulated the differentiation index.3.miR-432 targets E2F3 and P55 PIK directly.The relative luciferase activities of the psiCHECK-2-E2F3-3'UTR reporter(P < 0.05)and psiCHECK-2-P55PIK-3'UTR reporter(P < 0.05)were significantly inhibited when responded to miR-432 mimics.However,this change was not observed in mutated reporter vector.Also,we observed that mRNA expression of E2F3 and P55 PIK reduced in a concentration-dependent manner.4.miR-432 regulates myogenic differentiation through the PI3K/AKT/mTOR signaling pathway.Overexpression of miR-432 down-regulated the ratios of p-AKT/AKT and p-mTOR/mTOR during myogenic differentiation,but not proliferation stage.The inhibitory effect of miR-432 on myogenic differentiation was attenuated and even eliminated when treated with 10 nM and 100 nM insulin,respectively.Based on miRNAs sequencing,our study analyzed the characteristics in muscle development of Rongchang pigs,which have good meat quality in western region of our country.We found that miR-432 is more enriched in the longissimus dorsi of 35-day-old piglets than that of 287-day-old,and further research showed that the miRNA targeting E2F3 and P55 PIK inhibits myogenesis through PI3K/AKT/mTOR signaling pathway.This study provides a new candidate genes to explore the non-coding RNA regulation on meat characteristics of local pig breeds in China,as well as offers a scientific basis for selection of fine meat quality traits in breeding pigs.