Regulation Effect Of Lipid-derived Exosomes And Exosomal LncRNA-224 In Adipogenic Differentiation Of Muscle Satellite Cells In Yanbian Yellow Cattle

Intramuscular Fat(IMF)is the material basis for the formation of beef marbling.For years,research on IMF has been attracting much attention.Most researchers have focused on the single culture of adipocytes or muscle cells,and then explore the effect of functional molecules on the proliferation or differentiation of these cells to explain the cause of IMF formation.However,the genesis mechanism of IMF is complex,and single culture in cell experiments can not simulate the action environment in vivo very well.Studies have shown that almost all of the cells in the organism can transport their own genetic material to the target cells by secreting exosomes.There also exist studies shown that under the stimulate of certain conditions,muscle satellite cells can transdifferentiate into adipogenic direction,specifically reflected in up-regulated expression of adipogenic-related genes and increased triglyceride content.Based on the results of previous studies,this study assumes that the adipocytes in Yanbian yellow cattle secrete exosomes containing their own genetic substance,absorbed by muscle satellite cells via endocytosis effect,then the adipogenic genes and the triglyceride contentwere were both up-regulated.Finally,the phenomenon of adipogenic differentiation appeared.This study was conducted by establishing a co-culture system of adipocytes and muscle satellite cells.Molecular biology and cell biology related experiments were applied to verify this hypothesis,then the results of these experiments were analyzed and discussed deeply.Here are the results of this study as follows:1.In this study,pre-adipocytes and muscle satellite cells were isolated from Yanbian yellow cattle and identified,then the adipocytes and muscle satellite cells were seeded in to the transwell to constitute the co-culture system.After 5 days’ co-culture,the internal triglyceride content,m RNA and protein expression of PPARγ and CEBPA of muscle satellite cells were detected and analyzed.The results showed that the PPARγ(P <0.01),CEBPA(P<0.05)m RNA of the co-culture group are both higher than single culture group,and the protein expression of the PPARγ,CEBPA in co-culture group were both higher than the single culture group(P<0.001).Plus,a significant increase in triglyceride content were observed compared to the single culture group as well(P<0.001).Red oil O staining result indicated that lipid droplets were produced in muscle satellite cells after co-culture.These results showed that adipogenic transdifferentiation of muscle satellite cells occurred after co-culture with adipocytes.2.In order to explore the functional molecule related to the adipogenic differentiation of muscle satellite cells after co-culture deeply,we isolated and identified adipocyte-derived exosomes.On the one hand,the lnc RNA-seq was carried out with isolated exosomes and the sequencing results were verified by RT-q PCR.On the other hand,another part of the exosomes were cultured with muscle satellite cells together.The total protein was extracted to detect the PPARγ and CEBPA protein expression level.The results showed that the isolated extracellular vesicles had the physiochemical properties of exosomes in most of the studies,data of the lnc RNA-seq is reliable,and the protein expression of PPARγ and CEBPA levels are both increased in muscle satellite cells(P<0.01)after culturing with exosomes,this result indicates that the adipocyte-derived exosomes may have the ability to regulate the adipogenic differentiation of muscle satellite cells.3.In order to explore how the functional molecule of the adipogenic differentiation related adipose-derived exosomes act.Lnc RNA-seq was performed with adipocyte-derived exosomes in this study and the sequencing results were verified by RT-q PCR subsequently.According to the theory that lnc RNA competitively binds mi RNA and thus up-regulates target gene expression.Combining with the results of adipocytes-derived exosomal lnc RNA and the previous myotogenic mi RNA sequencing work of the research group,the adipogenic differentiation related lnc RNA-224 was screened for this study.The results of bioinformatic analysis showed that lnc RNA obeyed the rule of low conservation among species of non-coding RNA,and also has binding sites with target gene DGAT2 and myogenic bta-mi R-2455.Results of RT-q PCR shows that over-expression of lnc RNA-224 can down regulate the expression of bta-mi R-2455(P<0.01),and up-regulate the expression of DGAT2(P<0.05);Low-expression of lnc RNA-224 can up-regulate the expression of bta-mi R-2455(P<0.05),and down-regulate the expression of DGAT2(P<0.001).In summary,adipocytes may up-regulate the expression of adipogenic marker genes PPARγ and CEBPA by secreting exosomes,and the adipocyte-derived exosomal lnc RNA-224 can binding the bta-mi R-2455 competitively as ce RNA in muscle satellite cells,then up-regulate the adipogenetic related gene DGAT2,which is the target gene of lnc RNA-224.With the up-regulating of DGAT2,the content of triglyceride increased,which served as a phenomenon of lipogenic transdifferentiation in muscle satellite cells.These results may lay a relatively reliable theory basis for studying the molecular mechanism of the IMF deposition in beef cattle.