Analysis Of MiRNAs Regulated By CDR1as In The Differentiation Of Goat Skeletal Muscle Satellite Cells

The molecular regulation mechanism of muscle growth and development has been regarded as the key point of molecular breeding improvement of livestock and poultry.Previously our group found out that CDR1 as,the antisense transcribed circularRNA of the cerebellar degeneration-related protein 1(CDR1)gene,can promote the differentiation of goat skeletal muscle satellite cells(SMSCs)through sponging miR-7 and consequently releasing its degradation of the target gene IGF1R(Insulin Like Growth Factor 1 Receptor),but whether there are other downstream miRNAs regulated by CDR1 as remains unclear.Therefore,in order to comprehensively screen out the miRNAs that closely related with CDR1 as may adsorb during the differentiation of SMSCs,we knocked down levels of CDR1 as by small interferingRNA in SMSCs.extract total cellularRNA and constructed miRNA library to perform high-throughput sequencing.The differentially expressed miRNAs(DE miRNAs)were identified and verified by q PCR,and further we also employed functional enrichment analysis for downstream genes regulated by these DE miRNAs.The main results were as follows:(1)Through smallRNA sequencing,the total number of clean reads obtained was92,362,973.The Q20 and Q30 of the sequencing data were above 98.31% and 94.9%,respectively.The GC content was ranged between 48.88% and 49.01%.In addition,more than 97.21% of the clean reads could be mapped to the goat genome(GCF＿001704415.1).(2)The content of detected smallRNA were mainly classified into known miRNA(above 63.91%),with about 0.07% novel miRNA and less than 1.5% rRNA.A total of 529 miRNAs were identified in these 6 samples,including 400 known miRNA mature bodies and 260 precursors;129 predicted novel miRNA mature bodies and 137 precursors.The first base was profered to be U of the known miRNA detected here.(3)The miRNA expression of the CDRas group and the control group was analyzed and compared.A total of 43 miRNAs were differentially expressed(DE miRNAs),of which 27 were significantly up-regulated and 16 were significantly down-regulated.(4)Furthurmore,we performed GO enrichment analysis by using predicted target genes of DE miRNAs.The main GO terms enriched were included positive regulation of cellular metabolic process,positive regulation of macromolecule metabolic process,and molecular function.KEGG analysis showed that these candidate target genes were mainly enriched in signaling pathways related to muscle development such as metabolic pathway,MAPK signaling pathway,and Hippo signaling pathway.(5)To validate the expression from high-throughput sequencing data,a total of ten DE miRNAs(5 significantly up-regulated and 5 down-regulated)were randomly selected and quantified theirRNA levels by using q-PCR.The miRNA expression trends were generally in line with the sequencing data.(6)Based on the expression and base-pairing prediction,seven miRNAs(miR-199a-3p,miR-30e-5p,mir-20a-5p,miR-29 a,miR-125 b,miR-374 b and miR-146)may be likely regulated by CDR1 as in the differentiation of SMSCs.Taken together,it is possible that except for the well-known miR-7 sponged by CDR1 as,other downstream miRNAs may also be regulated by CDR1 as and mediate its function on the muscle growth and development.