Screening Of Differential MiRNA In Ningxiang Pig Adipose Tissue And Verification Of Circular RNA

As one of the well-known breeds in China,Ningxiang pig has rich and even intramuscular fat（IMF）content,and the percentage of unsaturated fatty acids in intramuscular fatty acids is as high as 59.6%,which is significantly higher than some of foreign breeds.Studies have proved that micro RNAs in adipose tissue plays a regulatory role in the process of growth and development,but the miRNA research in Ningxiang pig is relatively small,and the circular RNA as upstream of the miRNA target genes,the sponge adsorption effects of micro RNAs,so when the miRNA to carry on the regulation of the Ningxiang pig fat tissue and circular RNA will also play a role of its sponges adsorption,indirect control of adipose tissue.In this experiment,genome sequencing technology was used to screen candidate miRNAs that affected the growth and development of Ningxiang pig adipose tissue.At the same time,target genes were predicted for these candidate miRNAs.Using Sanger sequencing technology,gel electrophoresis PCR technology,real-time fluorescence quantitative PCR technology（q RT-PCR）,RNase R tolerance experiment,fluorescence in situ hybridization experiment（FISH）,etc.,the circular RNA obtained by the deep sequencing in the previous stage of the research group:Circ＿22931 and Circ＿7903 were tested for loop formation and cell distribution.The specific research results are as:1.Using sequencing methods,a total of 16855381 raw reads in Ningxiang pigs’30-day adipose tissue were obtained,of which 16750418 were clean reads;a total of 26308855 raw reads were in 210-day adipose tissues,of which 1130236 were clean reads.2.By comparing and analyzing the 30-day and 210-day adipose tissue sequencing data,383 differentially expressed known miRNAs were obtained,among which 97 were significantly differentially expressed miRNAs,31 of these 97 miRNAs were up-regulated and66 were down-regulated.At the same time,495 newly predicted miRNAs with significant differences were screened,of which 393 were down-regulated and 102 were up-regulated.10miRNAs with significant differences were selected for expression analysis and target gene prediction.It was found that these 20 miRNAs could predict fat-related genes such as:SLC27A1-204,FAR1-201,ELOVL5-201,FAR2-201 etc.3.Based on the previous study of Ningxiang pig circular RNA in this research group,we selected the circular RNA that may be related to Ningxiang pig fat growth:Circ＿7903 and Circ＿22931,by designing primers to perform sequence amplification on the two circular RNAs,and at the same time The amplified sequence was subjected to Sanger sequencing,and the sequence obtained was compared with the original sequence to verify the reliability of the primers,and the loop formation of circular RNA was verified by gel electrophoresis PCR technology.The miRanda software was used to analyze the interaction between the 10 known miRNAs selected earlier and Circ＿7903 and Circ＿22931.It was found that Circ＿7903interacted with ssc-miR-31 and ssc-miR-9843-3p,while ssc-miR-31 ssc-miR-9843-3p also predicted target genes related to fat synthesis,thus establishing a regulatory network of circular RNA-miRNA-target genes-adipose tissue.4.The real-time fluorescence quantitative PCR technique and the 2^-ΔΔCt method were used to construct the time-expression profiles of Circ＿7903 and Circ＿22931 in Ningxiang pig adipose tissue at 30 days,90 days,150 days and 210 days.That is,the deposition of fat grows.At the same time,the distribution of circ＿7903 and circ＿22931 in Ningxiang porcine adipocytes was detected by fluorescence in situ hybridization（FISH）.This study revealed the reason for the relatively high fat content of Ningxiang pigs from the transcriptome level,and screened the candidate miRNAs related to Ningxiang pig fat development and growth,and at the same time carried out the circular RNAs related to Ningxiang pig fat deposition.Preliminary verification and research have established a regulatory network of circular RNA-miRNA-target genes,which provides a solid foundation for the study of Ningxiang pig intramuscular fat,a high-quality meat quality trait.