| In order to study the mechanism of circular RNA on muscle development of Longlin goat,In this study,we selected Longlin goats aged 1 month and 10 months as sequencing materials.The differentially expressed circ RNAs were screened by RNA-seq technology,and the source genes of differentially expressed circ RNAs were analyzed by bioinformatics analysis method;At the same time,we used skeletal muscle satellite cells of Longlin goat as materials,Overexpression and interference technology,EDU,CCK8,Immunofluorescence,RT-q PCR and other experimental methods were used to verify the function of differentially expressed circ RNA that circ LOC102191280 screened from longissimus dorsi muscle at different stages,the main results are as follows.1.In this experiment,six circ RNA sequencing libraries were successfully constructed.After data filtering,a total of 356167236 clean reads were obtained,of which 82.67% ～ 90.12% clean reads can be compared to the goat genome,and 56.67% ～ 68.6% of the sequences have a single gene position.A total of26212 circ RNAs were identified in the dorsal muscle of goats aged 1 month and10 months.531 circ RNAs were differentially expressed in two different stages,of which 274 were up-regulated and 257 were down regulated.The source genes of different circ RNAs were analyzed by functional enrichment of GO and KEGG.GO enrichment showed that they were involved in cell components,enzyme activity changes,biogenesis and metabolism etc.KEGG enrichment showed that the source genes of circ RNAs were involved in c GMP-PKG,Oxytocin signal pathway,fatty acid elongation,Calcium ion signal pathway,adhesion junction and other important pathways.2.PCR,Sanger sequencing and RT-q PCR were used to verify the cyclization sites and expression trend of the six circ RNAs.The results showed that the six circ RNAs had a cyclization structure.The circ RNA expression trend detected by RT-q PCR is consistent with the results provided by the sequencing company.3.circ-0029745 is derived from exons 4-6 of LOC120191280 host gene,with a length of 532 bp.Therefore,it is named circ LOC102191280.First generation sequencing and RNase digestion experiments confirmed that circ LOC102191280 was real,RRT-q PCR results showed that after overexpression of circ LOC102191280,the expression of related proliferation genes PCNA and CDK2 increased significantly(P<0.05),and the expression of related differentiation genes Myo D,Myo G,Myf5 and My HC decreased significantly(P<0.05);It interfered with circ LOC102191280,on the contrary.It shows that circ LOC102191280 has a certain effect on the proliferation and differentiation of skeletal muscle satellite cells in Longlin goat,Promote cell proliferation and inhibit differentiation.4.The location of circ LOC102191280 in the cell was located by nucleocytoplasmic separation experiment.The results showed that circLOC102191280 was distributed in the cytoplasm and nucleus,with the proportion of cytoplasm 51% and nucleus 49%,indicating that circLOC102191280 can play the role of competitive binding miRNA.Rnahybrid prediction results showed that circ LOC102191280 had potential binding sites with miR-214-5p.Double Luciferase Report experiment confirmed that circ LOC102191280 had binding sites with miR-214-5p.Using RT-qPCR,CCK8,EDU and Immunofluorescence experiments,it was found that after overexpression of mi R-214-5p,the expression of related proliferation genes PCNA and CDK2 decreased significantly(P<0.05),and the expression of related diffe rentiation genes Myo D,Myo G,Myf5 and My HC increased significantly(P<0.05).When circ LOC102191280 was overexpressed,this effect could be reversed.It shows that there is a ce RNA mechanism between circ LOC102191280 and mi R-214-5p,which jointly regulates the proliferation and differentiation of goat skeletal muscle satellite cells.5.RNAhybrid predicted that mi R-214-5p and IGFBP5 had potential binding sites,and double Luciferase Report experiment confirmed the interaction between them.In the proliferative stage,the expression of IGFBP5 decreased significantly when mi R-214-5p was overexpressed(P<0.01),while the expression of IGFBP5 increased significantly when circ LOC102191280 was overexpressed(P<0.05).During the differentiation stage,the expression of IGFBP5 increased significantly when mi R-214-5p was overexpresse d(P<0.01),while the expression of IGFBP5 decreased significantly when circ LOC102191280 was overexpressed(P<0.05),It shows that circ LOC102191280 regulates the expression of IGFBP5 by Sponging mi R-214-5p.In conclusion,this study successfully screened circ LOC102191280 related to muscle growth and development through 1-month-old and 10-month-old Longlin goat Longissimus dorsi muscle sequencing and bioinformatics analysis,and verified the new ce RNA mechanism that circ LOC102191280 can regulate the expression of IGFBP5 through competitive binding to mi R-214-5p,It is involved in the regulation of proliferation and differentiation of skeletal muscle satellite cells in Longlin goat,it will provide a theoretical reference for exploring the molecular mechanism of circ RNAs in goat muscle development. |
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