Study On The Role Of IDE Gene In Porcine Skeletal Muscle Myoblasts

Insulin-degrading enzyme(IDE)is a multifunctional thiol-rich Zn metalloproteinase that is highly conserved among species.It has been reported that IDE can specifically degrade insulin and β-amyloid,and therefore is related to type 2 diabetes and Alzheimer’s disease.IDE was considered as the bridge between type 2 diabetes and Alzheimer’s disease.Skeletal muscle tissue is an important insulinregulating organ in the body.Recently,our colleagues found that IDE may be involved in the difficulty standing resulted from poor muscle development in pigs.Therefore,we studied the role of IDE gene in porcine skeletal muscle myoblasts,aiming to learn the possible mechanism of IDE influencing muscle development.Firstly,we used the mouse C2C12 myoblast cell line to understand the role of Ide in myoblast proliferation and differentiation as well as the possible molecular mechanism.After transfection of Ide-siRNA into C2C12,qRT-PCR and Western-blot results showed that Ide expression decreased significantly.Compared with the control group,after 48 h of Ide-siRNA transfection,CCK-8 analysis showed a significant increase in cell viability,and the expression of cell cycle-related factors Pcna,CyclinB1,and CyclinA2 was significantly increased.Accordingly,the expression of MyoD and MyoG was significantly decreased.These results indicated that Ide knockdown promoted C2C12 proliferation.Meanwhile,we found that as Ide expression decreased,Bcl2 expression was increased,and Bax expression was decreased significantly,indicating that Ide knockdown inhibited C2C12 apoptosis.In addition,qRT-PCR analysis revealed that the Ide expression at day 1(D1)and day 5(D5)of C2C12 differentiation showed an increasing trend,suggesting that Ide might also play a role in C2C12 differentiation.At day 0(D0)and day 3(D3)of differentiation,Ide-siRNA was transfected twice.As a result,compared with the control group,the expression of MyoD and MyoG was significantly decreased at D5 of differentiation in the Ide-siRNA transfected group.The expression of differentiation marker gene Myhc was significantly declined,and the expression Akt2 as well as phosphorylated AKT(P-AKT)was also declined.These results indicated that knockdown of Ide expression might inhibit C2C12 differentiation through the Akt2 / P-AKT / MyoG pathway.We further studied the expression and role of IDE gene using large white pig tissues and myoblasts.qRT-PCR and Western-blot results showed that IDE was widely expressed in large white pig tissues(including kidney,lung,spleen,liver,heart,gastrocnemius muscle,dorsal longest muscle and biceps femoris).After interfering IDE expression in porcine myoblasts,CCK-8 anaysis showed that the cell viability was significantly increased after 24 h,48 h and 72 h of IDE-siRNA transfection,and accordingly,the cell counting results showed a significant increase in cell number.After 48 h of IDE-siRNA interference,the expression of MYOD and MYOG was significantly down-regulated,and the expression of cell cyclerelated factors PCNA,CCNA2,CCNB1,CCND1,and CCNE2 was significantly increased,indicating knockdown of IDE expression also promoted the proliferation of large white pig myoblasts.However,with the decrease of IDE expression,the expression of BCL2 and BAX did not change significantly,indicating that knockdown of of IDE expression did not lead to apoptosis in large white pig myoblasts.Transcriptome analysis of large white pig myoblasts transfected with IDE-siRNA and NC-siRNA(negative control-siRNA)for 48 h identified 5194 differentially expressed genes,including 2658 upregulated genes and 2536 down-regulated genes.GO functional enrichment analysis found that 17 GO terms were significantly enriched in cellular component.KEGG pathway enrichment analysis showed 10 significantly enriched pathways,including DNA replication,cell cycle,and cell senescence and so on.Taken together,our results indicate that IDE negatively regulates the proliferation and positively regulates the differenation of mouse myoblasts.IDE is widely expressed in many tissues of large white pigs and negatively regulates the proliferation of large white pig myoblasts as well.Transcriptome analysis revealed differentially expressed genes and pathways regulated by IDE,which provides information for subsequent molecular mechanism-related researches.Therefore,this study analyzed the regulation role and possible molecular mechanism of IDE in skeletal muscle myoblasts,and provides reference information for the basic researches related to pig muscle development and genetic improvement of meat production performance.It also provides a candidate molecular marker for pig breeding.