Identification Of Differentially Expressed Proteins In Skeletal Muscle Between The High-and Low-feed Efficiency Pigs And The Function Study Of Muscle-specific MiR-208b

Feed accounts for more than 60% of the total costs of pig production.Thus,improving feed efficiency(FE)is an important approach for reducing pig production expenses.It is well known that the skeletal muscle is the largest tissue in pigs and accounts for 40-50% of the total body weight.Furthermore,muscle contraction and basal metabolism consume large amounts of energy.Therefore,we systematically analyzed the differentially expressed proteins(DEPs)in the skeletal muscle tissues of highand low-FE pigs using the i TRAQ approach.Meanwhile,we investigated whether these differentially expressed proteins in the skeletal muscle proteome of high-FE and low-FE pigs were regulated by miRNA.Interestingly,we found miR-208 b could play an important role in skeletal muslce development.We explored the mechanism of the miR-208 b during skeletal muscle growth,development,and energy metabolism.The results are as follows:(1)The skeletal muscle proteome of high-FE and low-FE pigs were investigated by the i TRAQ approach.A total of 1780 proteins were identified,among which 124 proteins were differentially expressed between the high-and low-FE pigs,with 74up-regulated and 50 down-regulated in the high-FE pigs.(2)Gene ontology(GO)analysis showed that all the 25 DEPs located in mitochondria were down-regulated in the high-FE pigs.Furthermore,the glucose-pyruvate-tricarboxylic acid(TCA)-oxidative phosphorylation energy metabolism signaling pathway was found to be differentially expressed between highand low-FE pigs.Mitochondrial energy metabolism in skeletal muscle tissue is negatively correlated with feed efficiency in pigs.(3)The function of miR-208 b on skeletal muscle proliferation and differentiation was verified.In vitro,miR-208 b promoted C2C12 myoblasts proliferation with cell cycle phase,the cell growth dynamics monitored by real-time x CELLigence system,and the proliferating cells stained by Ed U in C2C12 cells transfected with miR-208 b.In C2C12 differentiation,miR-208 b repressed C2C12 myoblasts differentiating into myotubes by immunofluorescence,Western blot,Q-PCR,and the analysis of differentially expressed skeletal muscle differentiation genes in the transcriptome of overexpressing miR-208 b C2C12 cells.In vivo,overexpressing miR-208 b transgenic mices showed less body weights at postnatal day 28,and had smaller myofibers than those WT mices.The m RNA and protein expression of skeletal muscle marker genes were down-regulated by Western blot and Q-PCR,respectively.Interestingly,miR-208 b overexpressed transgenic mice increased the number of Pax7 positive cells in muscles by Pax7 immunostaining.(4)miR-208 b regulates skeletal muscle growth and development by TCF12.Firstly,TCF12 was verified to be a target gene of miR-208 b by targetscan predicting,Western blot,and Dual-Luciferase reportor system.TCF12 repressed C2C12 myoblasts proliferation according to the results of cell cycle phase and the cell growth dynamics monitored by real-time x CELLigence system.The results of immunofluorescence,Western blot,and Q-PCR showed that TCF12 promoted C2C12 myoblasts differentiating into myotubes,which also supported by differentially expressed skeletal muscle marker genes in the transcriptome of inhibition of TCF12 C2C12 cells.(5)In addition,miR-208 b increases the number of slow muscle fibers and positively regulates energy metabolism.After C2C12 cells were transfected with miR-208 b,miR-208 b increases the number of mitochondria by Mito-Tracker Green labeled mitochondria method,and induced ATP production.The results of Q-PCR showed that differentially expressed genes related to mitochondria were up-regulated.The differentially expressed genes related to oxidative phosphorylation also up-regulated in the transcriptome of overexpressing miR-208 b C2C12 cells.After C2C12 myotubes were transfected with miR-208 b or NC,miR-208 b increased the number of slow muscle fibers by immunofluorescence,Western blot,and Q-PCR.According to the results of immunohistochemical and Western blot,overexpressing miR-208 b transgenic mice muscles also increased the number of slow skeletal muscle fibers.(6)miR-208 b regulates the conversion of myofiber types and energy metabolism by FNIP1.Based on the results of targetscan predicting,Western blot,and Dual-Luciferase reportor system,FNIP1 was firstly proved to a target of miR-208 b that regulates the conversion of myofiber types and energy metabolism.si-FNIP1 could increase the number of slow skeletal muscle fibers and positively regulates energy metabolism with immunofluorescence,Western blot,and Q-PCR experiment.(7)miR-208 b directly targets FNIP1,an AMPK interacting protein that negatively regulates AMPK-PGC-1a signaling and mitochondrial energy metabolism.Inhibition of FNIP1 reactivated p-AMPK,and promoted the protein expression of PGC-1a and CYC1 in C2C12 cells.Meanwhile,After C2C12 myotubes were transfected with miR-208 b or NC,overexpressing miR-208 b reactivated AMPK-PGC1 a signaling,and promoted the expression of CYC1 protein.Furthermore,compared with WT mice,the up-regulated expression of miR-208 b also activated AMPK-PGC1 a signaling and promoted mitochondrial energy metabolism in leg muscles from overexpressing miR-208 b transgenic mice.We concluded that the glucose-pyruvate-tricarboxylic acid(TCA)-oxidative phosphorylation is an important pathway for the improvement of FE in pigs.Mitochondrial energy metabolism in skeletal muscle tissue is negatively correlated with feed efficiency in pigs.Meanwhile,our study concluded that targeting TCF12 is a key pathway for miR-208 b regulates skeletal muscle growth and development.Besides,we found that miR-208 b directly targets FNIP1 that negatively regulates AMPK pathway and mitochondrial energy metabolism.