Regulatory Mechanism Study Of PLAG1 Gene In Bovine Fetal Growth And Development

Most animals date from fertilized oocytes,and form embryos,fetuses and adults gradually through cell proliferation and differentiation.Animal growth and development are affected by both genetic and environment factors.The study of growth and development in domestic animals is contribute to improving the productivity of domestic animals and the efficiency of animal husbandry.In order to explore the growth and development mechanism of bovine fetal,PLAG1,a stature trait candidate gene was selected to perform serious of experiment in myoblasts and chondrocytes in vitro.siRNA interference,adenovirus overexpression,CCK assay and mRNA-seq were used to study the effects of PLAG1 on proliferation and relative genes expression of bovine fetal skeletal muscle derived myoblasts and limbs long bone cartilage derived chondrocytes.ChIP-qPCR and dual luciferase report system were applied to verify the target genes of PLAG1 and explore its mechanism.The main results are as follows:1.Myoblasts derived from 3 months bovine fetal skeletal muscle were isolated with collagenase,and transfected with siRNA to interfere PLAG1 expression during the cell proliferation.After 1 day of interference,the expression levels of GHR and INSR in siRNA group were significantly higher than those in negative control(NC)group(P < 0.05),while IGF1 R and IGF2 expression were significantly lower than those in NC group(P < 0.05).Two days after interference,IGF1 R and IGF2 expression in siRNA group were significantly lower than those of NC group(P < 0.01 for IGF1 R,P < 0.05 for IGF2).The cell proliferation process was suppressed after interfering with PLAG1 siRNA.2.PLAG1 was overexpressed using adenovirus infection during the proliferation phase of myoblasts.The expression level of IGF2 in the overexpression group was significantly higher than that of the NC group after one day(P < 0.05),and the cell proliferation process was promoted.3.Chondrocytes derived from long bone cartilage of 3 months bovine fetuses were isolated using trypsin and collagenase.During the proliferation period,siRNA was used to interfere PLAG1 expression.One day after interference,the expression of GHR and BMP4 were significantly higher than those in NC group(P < 0.05 for GHR,P < 0.01 for BMP4),while INSR,IGF1 R and IGF2 R expression levels were significantly lower than those in NC group(P < 0.01 for INSR and IGF1 R,P < 0.05 for IGF2R).After 2 days of interference,GHR expression level was significantly higher than that in NC group(P < 0.05),while INSR,IGF1 R and IGF2 R expression levels were still significantly lower than those in NC group(P < 0.01).The cell proliferation process was inhibited after interfering.4.PLAG1 was overexpressed during the chondrocyte proliferation period using adenovirus,the expression levels of IGF1 R,GHR,IGF2 and BMP4 in the overexpression group were significantly higher than those in NC group(P < 0.01),and the chondrocyte proliferation process was promoted.5.The result of RNA-Seq showed that silencing PLAG1 in chondrocytes influence the expression of genes involve in the cell cycle,metabolic processes,cancer-related proteins,RNA regulatory pathways,and Ras and MAPK signaling pathways.6.ChIP-qPCR result showed that PLAG1 could directly bind to IGF2-P3,IGF1R-P1,INSR-P1 and INSR-P2 promoters.7.It was preliminarily confirmed that IGF1 R,IGF2 and INSR were PLAG1 target genes by the dual luciferase report system.Therefore,we inferred that PLAG1 may work on regulating growth and development of bovine fetal by directly activating the expression of related genes in the IGF system because of INSR,IGF2,and IGF1 R all belong to it.