Identification Of Lactation-Related Mirna From Mammary Gland In Dairy Goat And Analysis On The Regulation Roles Of MIR-2478

It is well reported that the miRNA (also known as microRNA) plays an important role in growth and development, hormone secretion and milking in animals by taking part in post-transcription regulation. Mammary gland is the most important lacation organ of animals.Milk traits are the most important quantitative economic traits in dairy goat, and significantly link to mammary gland. Thus, next-generation sequencing technology, quantitative PCR, gene clone, bioinformatics and cell culture were used to chosen lactation-related miRNA and reveal the regulatory mechanism. The research contents includes miRNA libraries of mammary gland will be constructed by next generation sequencing and thecandidate differential expressed miRNAs will also be chosen to confirm their expression profiles; verify target genes of the miR-2478and its regulatory mechanism. Moreover, we carried out the genetic variation analysis of miRNA and PrRP gene in goat. The main results are as follow:1. Identification of miRNAs and mammary gland small RNA by the Solexa deep sequencing techniqueTwo small RNA libraries from dry period (D) and peak lactation (P) were constructed using Solexa high-throughput technology. The clean reads of less than18nt was a total of15,712,891and14,851,375. In the dry period library,70%of small RNAs are21-23nt long, while about40%of the total reads in the peak lactation library are21-23nt. A total of346conserved miRNAs and95novel miRNAs were identified, and294of these were present in both libraries. A total of303miRNAs and54novle miRNAs were detected in the P library,337miRNAs and76novle miRNAs were detected in the D library. There are significantly differential expression between the dry period and peak lactation in the mammary gland. This difference may be due to the fact that samples were obtained from different developmental stages, suggesting that some of the miRNAs were expressed in specific temporal patterns and dynamic.2. Experimental validation of caprine miRNAs from mammary glandBecause the identification of miRNAs and prediction of novel miRNAs were based on the bovine genome sequences, these sequences may have few sequence differences in the caprine genome. A total of20conserved miRNAs were randomly selected and6with high expression and3specific miRNAs were selected for experimental validation by PCR and stem-loop RT-PCR. The corresponding caprine genomic regions were sequenced in an attempt to predict possible precursor sequences. These results reliably indicate that that the cloned caprine sequences were conserved miRNAs or novel miRNAs.3. The analysis of screening, expression profiles and function lactation-related miRNAThe 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 a total of169miRNAs were differentially expressed between the P and D libraries, of which161were down-regulated and8were up-regulated in the mammary gland during peak lactation compared to the dry period. We selected the above-mentioned differentially expressed miRNAs, including the3miRNAs that were up-regulated during peak lactation, the10miRNAs that were down-regulated during peak lactation, and6with high expression and3specific miRNAs, that were examined by the stem loop RT-PCR. Conserved miRNAs are not tissue specific, and are highly expressed in liver and muscle. A comparison of the expression profiles of the novel miRNAs among the different tissues revealed that the expression of novel_miR-12is tissue specific. The let-7family is highly expressed and conserved across animal species, including mammals, flies, worms and plants. We found that6miRNAs, including miR-21,-30a,-101,-103/107and-148a, were among the top20most abundant miRNAs in the other tissues or organs. Comparing the miRNAs from mammary gland with miRNAs from milk, there are a half of same miRNAs in both mammary gland and milk. Thus, our results, together with those of others, suggest that the immune-related miRNAs in the milk may be actively secreted by the mammary gland and transferred into the infant’s body via the digestive tract, where they could play a key role in the development of the immune system in infants. GO enrichment analysis and KEGG pathway analysis of target genes showed that most of target genes were involved in Insulin signaling pathway, Jak-STAT signaling pathway, TGFβ signaling pathway and MAPK signaling pathway and so on. These results indicated that some miRNAs might be involved in mammary gland lactation and physiology, and function in mammary gland cell proliferation, apoptosis, and differentiation.4. To predict and verify target genes of the miR-2478We predict target genes of the miR-2478using bioinformatics method and select SRA1、 FBXO11、NCOR1、TGFβ1、ING4、ING2and INSR target gene. The corresponding gene were cloned in goat and the FBX011、TGFβ1、ING4and INSR gene were selected by homology. Moreover, the predicted target gene of the miR-2478were verified using dual fluorescence report assay experiment. The results indicated that TGFβ1gene is a target of miR-2478.5. Primary analysis on the regulation mechanism of miR-2478We had carried out the experiment to identify the transcriptional activity region of TGFβ1gene. The results showed that TGFβ1gene had two transcriptional activity regions, locating the-904to-690bp and-79to+197bp from the transcriptional start site. Futhermore, there were many binding site for transcriptional factors and a CpG island with850bp length in two transcriptional activity regions. miR-2478maybe regulate the downstream promoter element of TGFβ1gene. There is a binding site of transcriptional factor, named RBPJ, in recognition sequence prediction of miR-2478and TGFβ1gene. These results indicated that miR-2478regulate TGFβ1gene by targeting the gene promoter.6. The genetic variation analysis of lactation-related miRNA and target geneWe detected the genetic variation of20miRNAs and PrRP gene by DNA pooling sequencing in Xinong Saanen dairy goat. The results showed that3mutations (-217C>T in miR-431,+107C>T in miR-196a and a indel-UC in miR-2478) were detected in goat. In the third lactation, the animals with TT genotype owned significantly higher milk yield than the ones with TC and CC genotype at miR-431polymorphism locus (P<0.05). In the second and third lactation, the animals with CT genotype owned significantly higher milk yield than the ones with TT genotype at miR-196a polymorphism locus (P<0.05). The ablity of binding target gene of miR-2478with "UC" deletion significantly reduced by dual fluorescence report assay experiment. However, the miR-2478with "UC" deletion significantly reduced the luciferase activity of the wild-type TGFβ1reporter compared to the control. Moreover, we detected four sequence variations of PrRP gene (1150G>A,1242T>C,1413A>G and1677indel22bp). The mutations1242T>C and1413A>G under completely linkage. The results revealed that haplotype1had the highest haplotype frequencies in the dairy populations and was highly significantly associated with body length (P<0.01), chest circumference (P<0.01) and milk yield (P<0.05) in the analyzed populations, respectively. Our results provide evidence that polymorphisms in the PrRP gene are associated with production traits, and may be as a molecular marker in goat breeding practices.