MicroRNA And Transcriptome Sequencing Analysis Of Lactating Mammary Gland Of Jinhua And Yorkshire Pig

The production and quality of breast milk can influence the growth, health and survival rate of suckling piglets and may have an impact on their weaning weight and weaning age even later development performance. The lactation performance of sow depends on the growth and development of the mammary gland, for example, the number and vitality of epithelial cells. It is important to know about biological characteristics of the sow mammary gland to obtain better growth performance for piglets. The development of mammary gland goes through many stages, from embryolic period, puberty period, gestation period, lactation period to degradation period, making it unique organ which can develop more than once after born in mammal. The weight, DNA content and lactation yield of mammary gland come to reach the peak at day 21 of lactation period. It is reported that a lot of growth factors and hormones take part in the processes of development of mammary gland and in lactation. MicroRNA plays wide roles in many biological processes, such as development, differentiation, cell proliferation, and apoptosis. Studies also showed that miRNA is an important regulatory factor for mammary gland development and lactation. Even the research on mammary gland development and lacation started very early and there are many researches about human, mouse, cow and sheep, very little research about pig has been reported.In our study, three sows at 21 day of lactation for both Jinhua and Yorkshire were chosen as experiment animals, mammary gland samples were collected to extract total RNA. MiRNA and transcriptome RNA-Sequencing of Jinhua and Yorkshire mammary gland was performed using Illumia/Solexa technology to obtain useful information of their expression pattern between the two breeds. Differentially expressed miRNAs and genes were filtered and then bioinformatics methods were used to analyze their target genes and perform gene ontology (GO) and KEGG pathway analysis. Real-time PCR was performed to compare the expression level ofgenes which are related to mammary gland development and lactation between the two pig breeds. At last, Jinhua mammary gland epithelial cells were successfully cultured in vitro, mimics and inhibitor of miR-148a were also both successfully transfected in the cells.The results are as follows:(1) Small RNA sequencing results of mammary gland in Jinhua and Yorkshie pigAfter filtering low-quality reads and adaptor sequences,17,044,789 and 16,545,454 mappable reads were obtained from Jinhua and Yorkshire libraries, respectively. After filtering reads mapped to the mRNA, RFam, or repbase, there were 9,672,024 and 6,946,954 reads representing 44.7% and 29.7% of the total mappable reads in Jinhua and Yorkshire libraries were mapped to the porcine miRNAs in miRBase version 19.0, respectively. Length distribution analysis revealed that 20-24nt was the major length for both breeds, consistent with the typical size range of miRNA. The conservation of the identified miRNA with other species showed that the porcine miRNA population is most conserved in Homo sapiens, Bos taurus and Mus musculus. Characteristics of chromosomal locations of miRNAs in porcine mammary glands showed that chromosome 18, chromosome 2 and chromosome 6 carried the most miRNAs in both pig breeds. The identification of miRNAs that were differentially expressed between the two libraries was performed after their numbers were normalized to transcripts per million. The results show that 417 miRNAs corresponced to 228 pre-miRNA were differentially expressed (P<0.05) between the two libraries. GO and KEGG pathway analysis of target genes were performed to identify the pathways that were actively regulated by miRNAs in mammary gland. The results discovered the involved pathways including TGF-β、MAPK、PPAR signaling pathways, glutathione metabolism, biosynthesis of unsaturated fatty acids and so on which may play important roles in the regulation of mammaty gland growth and lactation.(2) Transcriptome sequencing results of mammary gland in Jinhua and Yorkshire pigWe investigate the transcriptome changes in the mammary gland between Jinhua and Yorkshire pig using high-throughput deep sequencing technology, a total of 2.27Gb and 2.34Gb reads were obtained with 21467 and 29572 transcripts were calculated in Jinhua and Yorkshire libraries, respectively. Chromosome 2 carries the most number of transcripts, chromosomeMT carries the least in both libraries. We identified 3032 differentially expressed genes between two libraries and the fold-change of their expression level ranging from -20.0722 to 17.3565. Among the differentially expressed genes,1755 were up-regulated and 1247 were down-regulated in Yorkshire related to Jinhua. GO and KEGG Pathway were also analyzed,156 functional categories were involved, especially in amino acid metabolism, fatty acid biosynthesis, insulin signaling pathway, TGF-β signaling pathway, oxidative phosphorylation, glycerophospholipid metabolism, ubiquitin mediated proteolysis, protein export and other pathways.(3)RT-PCR was performed to compare the expression level of GHR、IGF-1R、INSR and PRLR in Jinhua and Yorkshire. The results showed that the expression level of these genes were all significantly higher in Yorkshire than Jinhua (P<0.01). The expression level of β-case in was higher in Jinhua than Yorkshire (P<0.01), however, for LALBA gene, there was no different between two breeds (P>0.05). The expression level of microRNA was also tested, six miRNAs were chosen, miR-138 was more expressed in Yorkshire than Jinhua while miR-26a、miR-186、miR-126、 miR-92a and miR-let-7g were all less expressed in Yorkshire than Jinhua(P<0.01)(4) Mammary gland epithelial cells were successfully cultured and showed the typical epithelial cells morphological characteristics. After transfection of miR-148a mimics or inhibitor, the expression level of miR-148a was significantlyup-regulated or down-regulated (P<0.01)。...