| Miniature pigs have many similarities with human beings in anatomy,morphology,physiology,and have irreplaceable advantages as a biomedical animal model.Understanding the genetic background of miniature pigs and the mechanism of dwarfism is a prerequisite for scientific research using miniature pig models,but it is a lack of systematic research on the formation mechanism of short size in miniature pigs.In addition,animal body bone size(bone mass,volume),bone growth,muscle growth and development are considered to be important indicators of mammalian body size and influenced by growth factors and hormone-controlled cell signaling pathways.The large and miniature pigs have accumulated a large amount of heritable variation under growth axis of GH-IGF-1.The extracellular domain(ECD)of insulin-like growth factor 1 receptor(IGF-1R)plays a critical role in regulating growth,bone matrix mineralization,and muscle development by binding to IGF-1 to receive signals and then activating downstream signaling pathways.Therefore,elucidating the mechanism of Igf1 r gene variation can provide theoretical support for exploring the formation mechanism of short size in miniature pigs.Nine strongly linked synonyms mutations were screened between Igf1 r coding region sequences(CDS)of large and miniature pigs in the previous study,of which four located in the ECD coding region and five located in the ICD coding region.Furthermore,Igf1 r synonymous mutations in the intracellular domain affect cell proliferation and alter kinase activity.On this basis,this study intended to elucidate the effect and mechanism of two haplotypes formed by four synonym mutations in the Igf1 r ECD coding region on osteoblasts and skeletal muscle cells differentiation.First,CRISPR/Cas9 technology was used to construct Igf1 r knockout cell line,named MC3T3-KO cells(KO group),to avoid interference of intracellular IGF-1R.Two pairs of expression vectors containing different haplotypes of Igf1 r in large pigs(LP)and miniature pigs(BM)were constructed,which were named p B513B-LP,p B513B-BM,pc DNA.3.1-LP and pc DNA.3.1-LP,respectively.Then,using piggy Bac transposable system,vectors containing two haplotypes of IGF-1R(PB513B-LP and PB513B-BM)were transferred into MC3T3-KO cells,respectively.The expression of the two haplotypes of Igf1 r were used verified by immunofluorescence and Western Blot?The results showed that two haplotypes of Igf1 r were stable expressed in MC3T3-KO cells,and were named MC3T3-LP cells(LP group)and MC3T3-BM cells(BM group),respectively.The expression levels of two haplotypes of Igf1 r were detected by q RT-PCR and Western Blot,and the results showed that the expression levels of IGF-1R in BM group were significantly lower than that in LP group at the transcriptional and the translation level(P<0.05).Secondly,pc DNA.3.1-LP and pc DNA.3.1-BM were transfected into porcine skeletal muscle satellite cells and myoblast cells,respectively.Western Blot showed that the expression of IGF-1R in BM group was significantly lower than that in LP group at the translation level(P<0.05).In order to further explore the molecular mechanism by which four synonym mutations of Igf1 r ECD affect gene expression,software prediction showed that two haplotypes of Igf1 r changed the m RNA secondary structure of Igf1 r coding genes.The effects of two haplotypes of Igf1 r on the m RNA and protein stability of Igf1 r were further investigated by Act D and CHX treatment experiments.The results showed that the m RNA and protein stability of Igf1 r in BM group were significantly higher than that in LP group(P<0.05).Because the amino acid encoded by synonym mutation was nearly located the IGF-1 binding region,the influence of two haplotypes of Igf1 r on the IGF-1 binding efficiency was determined by Co-IP technique.The results showed that the binding rate of IGF-1R and ligand IGF-1 in BM group was significantly lower than that in LP group(P<0.05).Because receptors bound to ligand in the cell membrane,the expression level of IGF-1R on the cell membrane surface of the two groups was detected by flow cytometry.The results showed that the relative membrane expression rate of IGF-1R in BM group was lower than that in LP group(P<0.05).To further explore the reasons that may lead to the binding difference between IGF-1R and IGF-1,we used the conformation-sensitive IGF-1R antibody to detect the influence of IGF-1R haplotype on the protein conformation.The results showed that two haplotypes produced by four synonym mutations of Igf1 r affected the protein conformation of IGF-1R.CCK-8 assay was used to investigate the effect of two haplotypes produced by four synonym mutations of Igf1 r on osteoblasts proliferation.The results showed that among the three groups,the KO group had the lowest proliferation ability,and the LP group showed better cell proliferation than BM group(P<0.05).Further,q RT-PCR and alizarin red staining were used to detect osteoblast differentiation indicator.The results showed that the ALP activity of osteoblasts in the LP and BM groups was significantly higher than that in the KO group.The early stage osteogenic differentiation was significantly promoted in the BM group,while the last stage osteogenic differentiation was promoted in the LP group(P<0.05).Quantitative analysis of key osteogenic differentiation genes(Col-1,Alp,Opn,Ocn,Runx2,and Osx)also supported the results(P<0.05);Osteogenic mineralization was detected as the last step,and late-stage osteogenic mineralization was obvious in the LP group(P<0.05).Further,Western Blot detection of related pathway factors showed that the phosphorylation level of AMPK-T172 in BM group was significantly higher than that in LP group(P<0.05);From 3 to 9 days,the phosphorylation level of AMPK in BM group was significantly lower than that in LP group(P<0.05);From 3 to 21 days,the phosphorylation level of AKT in BM group was consistently lower than that in LP group(P<0.05).CCK-8 assay was used to investigate the effect of two haplotypes of four synonym mutations of Igf1 r on proliferation of skeletal muscle cells.The results showed that compared with BM group,cells in LP group showed better proliferation ability(P<0.05).Furthermore,the expression of proliferation-related factors and key factors of downstream signaling pathway was detected by Western Blot to elucidate the mechanism.The results showed that the content of Cyclin D1 in BM group was significantly lower than that in LP group(P<0.05).Pathway detection results showed that LP group promoted the PI3K/ Akt signaling pathway more significantly than BM group(P<0.05).To explore the effect of two haplotypes of IGF1 R on muscle differentiation,Western Blot was used to detect the expression of differentiation-related factors and key factors of downstream signaling pathway.The results showed that compared with BM group,LP group showed better cell differentiation ability(P<0.05).Further detection of differentiation-related factors showed that the expression of Myo D differentiation factor in BM group was significantly lower than that in LP group(P<0.05);The pathway detection also supported this result,the LP group promoted the PI3K/AKT signaling pathway more significantly than BM(P<0.05).The above results indicate that the four synonymous mutations in the coding region of the extracellular domain of IGF-1R affect gene transcription,translation the different IGF-1R protein conformations and their binding to IGF-1,which affect the differential activation of the downstream pathways of IGF-1R,leading to differences in osteoblasts and myocytes differentiation,to provide theoretical data for the formation mechanism of dwarf in miniature pigs. |
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