| Dairy goat lactation behavior show certain regularity during entire lactation period, milkyield is low in early lactation, hereafter, milk yield increased gradually, reached a peak andmaintain a certain period of time. Therefore, understanding the dairy goat mammary glanddevelopment and lactation regularity, improving the dairy goat lactation genetic performancethrough molecular techniques, have an important significance of extending the dairy goatlactation or increasing milk yield. Currently, many candidate genes related to lactationperformance have been found through candidate gene approach, while their regulationmechanisms involved in lactation regularity remained unclear.MicroRNA, a class of non-protein coding RNA with length of22nt, which is highlyconserved in animal evolution and exhibit spatial and temporal expression patterns,extensively involved in many biological processed, such as cell proliferation, differentiationand apoptosis and organ development. The mammary gland development and lactation is acycle process of proliferation, differentiation and apoptosis in mammary gland epithelial cell.Studies showed miRNA exhibit differential expression patterns in different periods ofmammary gland development, which also is a important regulatory factor for mammary glanddevelopment and lactation.In this study, mammary gland tissues in different lactation periods (Early lactation,20dpostpartum; Peak lactation,90d postpartum; Late lactation,210d postpartum) of fiveLaoshan dairy goats were used respectively for sRNA libraries construction. Illumina/Solexahighthrought-sequencing technology was used to investigate the sequences distribution inthree libraries, and the sequencing data was statisticed and analyzed through quality control,length distribution, genome location, sRNA classification annotation, and selected outdifferentially expressed miRNAs. The differentially expressed miRNAs were further analyzedusing bioinformatics, including target genes prediction, GO annotation and KEGG pathwayanalysis. As a result, we found miR-143has high expression abundance and was differentiallyexpressed in different lactation periods, the bioinformatics analysises showed the target genesinvolved in mammary gland development, cell growth and other biological processed. Dualluciferase reporter gene assay and qRT-PCR were used to detect the target regulationrelationship between miR-143and IGFBP5, the effents of miR-143on goat mammary glandepithelial cells proliferation and apoptosis were also studied by MTT, FluorescentHoechst33342/PI double staining and Flow Cytometry. The main results are as follows:(1)18,031,615,19,044,002and7,385,833clean reads were obtained in Early lactation, Peak lactation and Late lactation library, respectively, accounted for97.88%、98.45%and73.87%of high-quality reads.(2) Statistical results of sRNA length distribution showed, sRNA sequences were mainlydistributed in the range of19-24nt, the22nt was the most and accounted for44.04%、41.48%and33.96%in three libraries, respectively, followed by20nt and23nt. The sRNA havingdifferent length exhibited differential distribution in three libraries.(3) The sRNA location analysis results in sheep genome showed, there were44,928unitsequences (9,093,530reads),69,244unit sequences (8,941,279reads),32,509unit sequences(3,916,179reads) were located in sheep genome, accounted for50.43%、46.95%and53.02%of total reads, respectively。(4) In three libraries,336known miRNA were identified, and189miRNA weredifferential expressed between different libraries.5miRNAs, let-7a, let-7b, miR-143,miR-378, miR-148a, were more than1,000,000in expression abundance.10miRNAs,miR-30e-5p, miR-30d, miR-30a-5p, miR-21, miR-200c, miR-146b, miR-126, miR-103, let-7f,let-7c, have expression abundance in the range of100,000-1,000,000.289miRNAs wereco-expressed in three libraries,26in two libraries,21existed only in one library, of which,7miRNAs were specifically expressed in early lactation,8in peak lactation,6in late lactation.(5) Bioinformatics analysis of differential expressed miRNAs showed,10,325targetgenes for189differentially expressed miRNAs were predicted by TargetScan online software,among them,9,341genes were annotated in GO database through Blast2GO and DAVIDanalysis, and1,229terms were significantly enriched (FDR <0.05),68pathway were alsosignificantly enriched (P <0.05), of which, MAPK signaling pathway、Wnt signalingpathway、Insulin signaling pathway、Jak-STAT signaling pathway may be related tomammary gland development and lactation.(6)50novel miRANs were identified in three libraries, and bioinformatics analysisshowed,13,298EST were predicted as target genes,125,846annotations were obtained,involved in237pathways. Metabolic pathways, ECM-receptor interaction, which may berelated to mammary gland and lactation were ehriched.(7) The expression levels of miR-143and IGFBP5were detected in three lactationperiods by qRT-PCR, results showed miR-143and IGFBP5have similar expression trend,low expression level in the peak lactation compared with early lactation and late lactation.pcDNA3.1(+)-pre-miR-143eukaryotic expression vector, pGL3-promotor-IGFBP5andpGL3-promotor-Mut-IGFBP5dual luciferase reporter gene vector were constructed and transfected primary goat mammary gland epithelial cells, we found miR-143targets IGFPB53′-UTR and positively regulates its expression.(8) The effects on miR-143on goat mammary gland epithelial cells proliferation andapoptosis were detected by MTT, Hoechst33342/PI and FCM, as a result, miR-143inhibitsmammary gland epithelial cell prolification, and promotes their apoptosis.(9) After the over-expression of miR-143, BAX, a critical apoptosis gene, wassignificantly up-regulation compared with control (P<0.01). While BCL2, a criticalanti-apoptosis gene, was down-regulation. |
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