The Role Of MicroRNA-378b-3p In Regulating Porcine Skeletal Muscle Fiber Type Conversion And Its Mechanism

Muscle fibers are the basic unit of skeletal muscle and have four isoforms including MyHC-I,MyHC-IIa,MyHC-IIx and MyHC-IIb.Different muscle fiber types have great differences in metabolic and physicochemical characteristics,and is closely related to meat quality.Many factors can affect muscle fiber type conversion,previous studies have demonstrated that miRNAs play important roles in muscle fiber development and fiber type conversion.Due to many types of miRNAs in muscle,it is necessary to further investigate their functions and mechanisms.Therefore,the present study firstly compared the develpomental patterns of muscle fiber type and the expression patterns of muscle fiber type related miRNAs between different pigs,and then foused on the role of microRNA-378b-3p on muscle fiber conversion and its mechanism and explored the relationship between its expression and nutrition regulation,which will provide the experimental basis for deep understanding muscle fiber development and principle of meat quality formation,and developing nutritional technology for the regulation of meat quality.There are five experiments in this study:Experiment 1.Developmental patterns of muscle fiber type between different pig breeds and screening the differential expression miRNATo verify the relationship between miRNA and muscle fiber type,forty newborn Chenghua pigs and forty newborn Large White pigs were selected and slaughtered to collect longissimus doris muscle（LDM）for the determination of muscle fiber type and miRNAs expression at day 0,7,14,21,63,105,185 and 220 after birth.Meanwhile,the other tissues from newborn piglets,including heart,liver,spleen,lung,kidney,intestine,soleus（SOL）,tibialis anterior（TA）and psoas major muscle（PMM）were also collected for the determination of miR-378b-3p expression.The results were presented as follows:（1）the percentage of slow-fibers gradually increased before weaning and then declined.However,the developmental pattern of fast-fiber percentage showed the opposite trend.In addition,the percentage of slow-fiber in Chenghua pigs’LDM was higher than that in Large White pigs.（2）In all detected miRNAs,the expression pattern of miR-378b-3p showed a similar trend with slow-fiber composition.（3）miR-378b-3p was high expression in skeletal muscle and its expression in porcine oxidative muscle（SOL）was significantly higher than that in glycolytic muscle（LDM）.Conclusion:miR-378b-3p is a muscle-specific miRNA,and may be involved in regulating muscle fiber type conversion and promoting slow-fibers formation.Experiment 2.Effects of dietary sodium butyrate supplementation on skeletal muscle fiber type and miR-378b-3p expression of finishing pigsTo investigate whether the formation of slow-or oxidative muscle fiber promoted by sodium butyrate（NaB）is related to miR-378b-3p expression.Thirty-two DLY growing pigs（64.99±5.71 kg）were randomly allotted to either control or NaB diets（0.3%NaB）with 8 duplicates（pen）of two pigs.The experiment lasted 8 weeks.At the end of the feeding experiment,one pigs with medium body weight per pen was selected to slaughter for skeletal muscle sampling.The results showed that dietary supplemented with NaB significantly improved pH_{24 h},a_{45 min},intramscular fat content（P<0.05）and marbling score and decreased L_{24 h} and shear force（P<0.05）in LDM.Meanwhile,dietary NaB supplementation significantly increased the expressions of MyHC-I,MyHC-IIa and miR-378b-3p（P<0.05）,and decreased MyHC-IIb expression（P<0.05）.Conclusion:dietary NaB supplementation promoted the formation of slow-or oxidative muscle fiber,which may be related to the increased miR-378b-3p expression.Experiment 3.Effects of different concentrations of sodium butyrate treatment on in vitro porcine skeletal muscle fiber type and miR-378b-3p expressionTo further explore whether NaB treatment could influence the expression of miR-378b-3p in differentiated porcine myoblast.The porcine muscle cells were separated and then identified by flow cytometry with Pax7 antibody.Then porcine myoblast were treated with different concentrations of NaB（0,1.0,2.5,5.0 mM）from the 5th day of differentiation.The results showed that 98.9%separated muscle cells were Pax7 positive cells.Medium and high（2.5 and 5.0 mM）concentrations of NaB treatment promoted the expression of MyHC-I,MyHC-IIa,PGC-1αand miR-378b-3p,and decreased MyHC-IIb expression.In addition,NaB promoted the expressions of MyHC-I,MyHC-IIa and miR-378b-3p in a dose-dependent manner.Conclusion:NaB treatment could promote the expressions of oxidative muscle fiber（MyHC-I and IIa）and miR-378b-3p,and their expression trends are consistent.Experiment 4.Prediction and identification of miR-378b-3p target geneMiRNAs function in gene silencing and translational suppression by binding to the3′-UTR of their target mRNAs.Based on previous miRNA and mRNA sequencing results of fetal longissimus doris muscle from Meishan and Large White pigs in our laboratory,we found that correlation coefficient was high between miR-378b-3p and HDAC3（-0.8881）.RNA22and Segal Lab of Computational Biology analysis results showed that HDAC3 3′-UTR contain two binding sites for miR-378b-3p.Then we constructed wild-type and mutant HDAC3/siCHECK-2 recombinant plasmids for luciferase reporter gene detection.Additionally,the expression of miR-378b-3p and HDAC3 in tissues and differentiated myoblasts were also detected.The results showed that miR-378b-3p remarkably decreased the activities of luciferase in HEK-293T cells transfected with wild-type HDAC3/siCHECK-2 recombinant plasmid,and the expression of miR-378b-3p and HDAC3 in tissues and differentiated myoblasts presented the opposite trend.Conclusion:HDAC3 is the target gene of miR-378b-3p,which could inhibit HDAC3expression.Experiment 5.Effects of miR-378b-3p on porcine skeletal muscle fiber type conversion and its signaling pathwayTo reveal miR-378b-3p regulates muscle fiber type conversion through target gene HDAC3 and its signaling pathway.In differentiated porcine myoblast,miR-378b-3p mimics and inhibitor transfection showed that miR-378b-3p inhibited the expression of HDAC3 and MyHC-IIb,while promoted the expressions of MyHC-I,MyHC-IIa and PGC-1α,and mitochondrial DNA copy number.However,pcDNA3.1（+）-HDAC3recombinant plasmid transfection or RGFP966 treatment revealed that HDAC3 has the opposite functions with miR-378b-3p.Meanwhile,it has been demonstrated that HDAC3could deacetylate MEF2D,thus inhibited MEF2D-dependnet transcriptional activation of PGC-1α.The results of immunoprecipitation showed that pcDNA3.1（+）-HDAC3recombinant plasmid transfection decreased the acetylation level of MEF2D,whereas RGFP966 treatment increased the acetylation level of MEF2D.Conclusion:miR-378b-3p promoted the formation of slow-or oxidative fibersmainly through HDAC3/MEF2D/PGC-1αsignaling pathway.In summary,the development pattern of slow-fiber in porcine skeletal muscle is in line with the expression pattern of miR-378b-3p and sodium butyrate can regulate miR-378b-3p expression.Meanwhile,miR-378b-3p inhibits HDAC3 expression,thus increases the acetylation level of MEF2D.The acetylation level of MEF2D not only promotes the formation of slow-or oxidative fibers,but also improves MEF2D-dependent transcriptional activation of PGC-1α,thus promotes slow-fiber formation and mitochondrial biogenesis.The present study indicates that miR-378b-3p regulates skeletal muscle fiber type conversion and muscle fiber conversion modulated by NaB is related to miR-378b-3p expression.