| Muscles are critical tissues for mammals due to their close association with movement and physiology.Myogenesis involves proliferation,differentiation,and fusion of myoblasts.Besides,many well-known protein-coding genes,as well as linear non-codingRNAs such as microRNAs(miRNAs),are involved in myogenesis.Recently,circularRNAs(circRNAs)have attracted much attention since several circRNAs are known to play significant roles in muscle development and diseases through limited mechanisms,particularly through sponging miRNAs.Through advanced researches,increasing evidence suggests that Cerebellar DegenerationRelated Protein 1 antisense(CDR1as)is an important circRNA that regulates the levels of mRNA expression via competitively sponged miRNAs.Here,we screened for miRNAs and mRNA associated with CDR1 as from the Longissimus dorsi(LD)muscles of a newborn goat(3-day old female Nanjiang Brown goat)skeletal muscle satellite cells using an Illumina Hiseq2500 platform.We further characterized their regulatory functions during muscle differentiation.The potential CDR1as/miRNAs/mRNA pathway will provide the basis for further research on the function of CDR1 as in muscle development,and eventually extend the versatile roles of CDR1 as in mammals.The main results are as follows:(1)After knocked down of CDR1 as in SMSCs,the totalRNA from the samples were extracted and were used forRNA sequencing.An average of 59,821,102 and 58,552,463 mRNAs were generated from raw and clean reads,respectively.Furthermore,an average of15,350,087 raw reads and 14,614,375 clean reads of miRNAs were produced.A correlation coefficient of the si CDR1as-1,2,and 3 in SMSCs ranged from 0.97 to 0.99(an average of 0.98)were obtained,demonstrating that the samples replicated very well biologically.(2)After sequencing,a total of 789 mRNAs were differentially expressed,of which 401 mRNAs were downregulated and 388 mRNAs were upregulated.Moreover,some muscle-related genes such as ANGPT1,E2F2,CCN1,FGFR1,and MEF2 C were downregulated according to the sequencing data and q RT-PCR.Some KEGG signaling pathways,including the PI3K-AKT signaling pathway,the Rap1 signaling pathway,and the MAPK signaling pathway,were also linked with the downregulated mRNAs.These show that the downregulated mRNAs obtained after knocking down CDR1 as are associated with myogenic signaling pathways.(3)Also,a total of 43 miRNAs were differentially expressed,comprising 16 downregulated miRNAs(15 known and 1 novel)and 27 upregulated miRNAs(24 known and 3 novels)were obtained after sequencing the SMSCs.Furthermore,the expressions of five upregulated(miR-107-3p,miR-125b-5p,miR-140-5p,miR-29a-3p,and miR-27a-3p)and five downregulated(miR-143-3p,miR-378-5p,miR-140-3p,miR-184,and miR-26a-5p)miRNAs were randomly selected and confirmed by q RT-PCR to validate theRNA-sequenced data.Besides,through KEGG analysis some popular myogenic signaling pathways,such as MAPK signaling pathway,Focal adhesion,PI3K-AKT signaling pathway,Rap1 signaling pathway,Wnt signaling pathway,and Hippo signaling pathways,were indicated to be linked with the upregulated miRNAs.These demonstrate that the upregulated miRNAs are associated with numerous myogenic signaling pathways which are responsible for muscle development.(4)Further,through western blotting and immunofluorescence analysis we identified that the knockdown of CDR1 as prevent the expression of Myo D and myoblast differentiation,respectively.With the use ofRNAhybrid and Targetscan,we predicted the miR-27a-3p recognition sequence for goat CDR1 as.Luciferase assay also indicated that miR-27a-3p significantly decreased Rluc expression of p CK-CDR1as-WT.These show that CDR1 as has a significant impact on muscle development and can sponge miR-27a-3p during SMSCs differentiation.(5)In addition,western blotting analysis shows that miR-27a-3p inhibits the expression level of Myo D as compared to the NC.Moreover,we recognized that miR-27a-3p reduced the differentiation of myotubes through an immunofluorescence assay.Ed U assay shows that miR-27a-3p enhances the proliferation of SMSCs.These demonstrate that miR-27a-3p promotes proliferation but inhibits differentiation of SMSCs during myogenesis.(6)Through q RT-PCR analysis,we noticed that overexpression of ANGPT1 elevated theRNA expression level of Myo D.Also,western blotting analysis indicates that knockdown of ANGPT1 reduced the protein level of Myo D,while overexpression of ANGPT1 increased the protein level of Myo D.An immunofluorescence analysis also indicates that knockdown of ANGPT1 decreased the differentiation of myotubes in SMSCs.Despite this,knockdown of ANGPT1 increased the proliferation of SMSCs.These show that ANGPT1 positively regulates differentiation of SMSCs during myogenesis.(7)With the use ofRNAhybrid and Targetscan,we predicted the miR-27a-3p recognition sequence for goat ANGPT1.Through a luciferase assay,we found that miR-27a-3p is a target of psi-ANGPT1-3’ UTR WT and significantly reduces its expression.This result shows that miR-27a-3p negatively regulates the expression of ANGPT1 mRNA during myogenesis of SMSCs.(8)In conclusion,the findings indicate that knockdown of CDR1 as prevents muscle development by decreasing differentiation of SMSCs via enhancing miR-27a-3p to inhibit ANGPT1. |
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