The Molecular Mechanism Of LNCRNA-TCONS₀0153891-miRNA29a-YAP1 Regulating The Proliferation And Of Muscle Cells In Hu Sheep

Hu sheep,one of the most famous sheep breeds all around the world,not only has the characteristics of high reproduction rate,but also has tender and juicy meat with high ratio between meat and bone,which is deeply popular with people.Therefore,it has a great significance to study the regulatory mechanism of mutton traits in Hu sheep scientifically.The main findings are as follows:Our previous experimental study has found that YAP1 gene,as the major gene in the Hippo channel,plays an important role in the growth and development of Hu sheep muscle.On this basis,the study obtained four miRNAs(miRNA-200,miRNA-181a,miRNA-29a and miRNA-541-3p)through a serious of bioinformatics analysis on a variety of online sites,which may have a targeted binding site with YAP1 gene in Hu sheep;The relative expression of miRNA-181a,miRNA-29a and miRNA-541-3p at the age of 6 months old in the longissimus dorsi muscle tissue was significantly higher than the relative expression at the age of 1 months old by RT-qPCR,which means these three miRNAs may negatively regulate the proliferation and of muscle cells;According to the literature reviewed,only miRNA-29a has been studied on the proliferation and of skeletal muscle cells,therefore,miRNA-29a was selected as a further research object.In the targeted validation of miRNA-29a and YAP1,the optimum amount of miRNA-29a mimic in a 6-well plate was 4 μL,the optimum amount of miRNA?29a inhibitor was 8 μL;The wild vector and mutant vector of YAP1 were also successfully constructed;The results of the dual luciferase assay showed that the luciferase relative activity with co-transfecting miRNA-29a mimic and the wild-type vector of wild-YAP1 was significantly lower than that of the other group,indicating that miRNA-29a and YAP1 may have a targeted binding site;Through RT-qPCR we found that the relative expression levels of YAP1,MyoG,MyoD and MyHC were extremely significantly lower than those of the control group under the overexpression of miRNA-29a.The relative expression of YAP1 and MyoG under the inhibition of miRNA-29a were extremely significantly higher than the control group,while the relative expression of MyHC was significantly higher than that of the control group.The difference between MyoD and the control group was not significant;The results of CCK-8 assay showed that the overexpression of miRNA-29a significantly decreased the proliferation of Hu sheep muscle satellite cells,and the decrease of miRNA-29a significantly increased the cell proliferation.Basing on the result of bioinformatics prediction,we found three binding sites between LNCRNA-TCONS 00153891 and miRNA-29a;The relative expression of miRNA-29a in the group over-expressed LNCRNA-TCONS 00153891 had a great lower significance than the group NC,and the relative expression of miRNA-29a in the group siRNA-1 and group siRNA-3 was extremely significantly higher than that in the group NC.After overexpression of LNCRNA-TCONS 00153891,the relative expression levels of YAP1,MyoG,MyoD and MyHC were extremely significantly higher than group control.The relative expression levels of YAP1,MyoG and MyoD genes were significantly lower than group control under interference of siRNA-3.The proliferation of Hu sheep muscle satellite cells in the group of LNCRNA-TCONS 00153891+miRNA-29a inhibitor was significantly higher than that in the other three groups by CCK-8 technology,and only in the group of siRNA-3,the proliferation of the lake sheep muscle satellite cells in the group of siRNA-3-NC+miRNA-29a inhibitor was significantly higher than the other three groups.Under the condition of overexpression and inhibition of miRNA-29a in Hu sheep muscle satellite cells,the protein expression levels of YAP1 and muscle-related genes were lower and higher the group NC,respectively;Under the condition of over-expressed LNCRNA-TCONS 00153891,the protein expression levels of YAP1 and muscle-related genes were higher than NC.The protein levels of YAP1 and muscle-related genes were significantly higher in the group miRNA-29a inhibitor than the group miRNA-29a mimic.The results indicated that the interference site of siRNA-3 was the binding site between LNCRNA-TCONS 00153891 and miRNA-29a5 which inhibited the silencing of YAP1 by miRNA-29a,and played a major role in promoting the proliferation and of Hu sheep muscle cells.