Regulation Of MiRNA Y-56 On Proliferation Of Porcine Skeletal Muscle Satellite Cells By Targeting IGF-1R And Mechanism Exploration

Pig is a kind of domestic mammal,which has significant similarities with human beings in anatomy,immunity,physiology,metabolism,dietary habits and evolution.Pigs have become indispensable livestock in people's daily life and excellent medical model.Bama Xiang pigs have the characteristics of short stature,slow growth,low lean meat rate and good meat quality,while Landrace pigs have distinct skeletal muscle phenotypes.Elucidating the different mechanisms of skeletal muscle formation of two kinds of pigs will provide theoretical basis for improving the production performance of pigs and further using pigs as medical models.As key post-transcriptional regulators,mi RNAs play an indispensable role in skeletal muscle development.Skeletal muscle satellite cells are the source of DNA that promotes muscle fiber growth after birth.Mi RNA Y-56 is a novel mi RNA detected in pigs by Rui-Song Ye et al.In our previous study,we found that mi RNA Y-56 was highly expressed in muscle,but there is no relevant study on it at present.The mi RNAs sequencing data published by Rui-Song Ye et al were analyzed by bioinformation related software,and mi RNA Y-56 was selected as the candidate mi RNA according to the expression level of mi RNAs in BM and the expression difference between BM and LP.Then the expression levels of differential mi RNAs in different tissues of pigs were detected by q RT-PCR.The results showed that a novel mi RNA Y-56 was highly expressed in porcine muscle,and the expression level of mi RNA Y-56 was significantly different in the muscle of Bama Xiang pigs and Landrace pigs.Subsequently,PSCs were isolated and cultured from the legs of pigs,then the expression of Pax7 was detected by immunofluorescence and the cell purity was over 95% under microscope.PSCs were transfected with Y-56 mimics and Y-56 inhibitor by liposome,then the cell proliferation was detected by Ed U assay and CCK-8,and the cell cycle progression was detected by flow cytometry.What's more,expression levels of cyclock-related factors were detected by q RT-PCR and Western blotting.The results showed that overexpression of Y-56 inhibited the cell proliferation,cell cycle progression and the cycle-related factors of PSCs compared with the mimics-NC group(P < 0.05).While Y-56 inhibitor group significantly promoted the cell proliferation and the cell cycle progression of PSCs compared with the inhibitor-NC group.And Y-56 inhibitor promoted the expression of period-related factors(P < 0.05).Through sequence alignment,it was found that the seed sequence of Y-56 targeted the 3'UTR of IGF-1R,and the results of RNA secondary structure prediction showed that the Mfe was-27.4kcal/mol.q RT-PCR was used to detect the expression level of IGF-1R in different tissues of pigs.The results showed that the expression level of IGF-1R in muscle was lower than that of other tissues,and the expression trend of IGF-1R was opposite to that of Y-56 in different tissues of pigs.In order to further verify the targeting relationship between IGF-1R and Y-56,a double luciferase reporter vector was constructed,and the target direction relationship between Y-56 and IGF-1R was confirmed by the double luciferase reporter.After transient transfection with Y-56 mimics into the PSCs,q RT-PCR and Western blotting experiments showed that overexpression of Y-56 significantly reduced the m RNA and protein expression levels of IGF-1R compared with mimics-NC group.To confirm that Y-56 inhibits the proliferation and cycle progression of PSCs by targeting IGF-1R,we constructed an IGF-1R overexpression vector and synthesized IGF-1R si RNA,which was transiently transfected into PSCs.The effects of IGF-1R on the cell proliferation and the cell cycle of PSCs were verified by Ed U,CCK-8,flow cytometry,q RT-PCR and Western blotting.The results showed that compared with the control group,overexpression of IGF-1R significantly promoted the cell proliferation and the cell cycle progression of PSCs(P < 0.05),while si-IGF-1R group significantly inhibited the cell proliferation and the cell cycle progression of PSCs(P < 0.05).To further confirm that Y-56 plays a role by targeting IGF-1R,Y-56 mimics and IGF-1R overexpression vector were co-transfected into PSCs.The cell proliferation and cell cycle progression of PSCs cells and expression levels of related factors were observed by Ed U,CCK-8,flow cytometry,q RT-PCR and Western blotting experiments.The results showed that compared with the control group,overexpression of IGF-1R partially restored the inhibitory effect of overexpression of Y-56 on PSCs proliferation(P < 0.05),and partially restored the inhibitory effect of overexpression of Y-56 on cell cycle progression(P < 0.05).Moreover,overexpression of IGF-1R partially recovered the inhibitory effect of Y-56 overexpression on cyclin expression level(P < 0.05).In order to further explore the relevant signaling pathways,Western blotting was used to detect the expression levels of key signaling factors in the downstream classical signaling pathways of IGF-1R.The results showed that compared with the mimics-NC group,the phosphorylation of AKT and ERK was significantly inhibited in Y-56 mimics group(P < 0.05),while the phosphorylation of AKT and ERK was significantly increased in Y-56 mimics group compared with inhibitor-NC group(P < 0.05).In addition,reply experimental results showed,overexpression of IGF-1R partially restored the inhibitory effect of overexpression of Y-56 on the activation of AKT and ERK signaling pathways(P <0.05).These results suggest that mi RNA Y-56 inhibits the expression of IGF-1R and the activation of AKT and ERK signaling pathways by targeting the 3'UTR of IGF-1R,and ultimately inhibits the cell proliferation and the cell cycle progression of PSCs.These results provide a theoretical basis for elucidating the different mechanisms of skeletal muscle formation in Bama Xiang pigs and Landrace pigs.