Dynamic Profiles Of MicroRNA In Giant Panda Milk Derived From Different Lactation

With these advantages that not only be feeding-convenient and rich nutrition, but also easy to digest, dairy products have become an indispensable nutrient in People’s daily life, with the improvement of living standard. Especially for human and other mammals, the breast milk is the best natural and ideal foods for babies to promote immune system development and function, which also further strengthen the whole body’s resistance to systemic disease. MicroRNA (miRNA) is a kind of endogenous non-coding small RNA which is about 22 nucleotide (nt), single chain and highly conserved. Detected widely in eukaryotic organisms, miRNA mainly target to the 3’untranslated regions of target gene’s mRNA, resulting in the translational inhibition or degradation of the target gene, which plays a very important role in the post-transcriptional regulation of target genes. A growing number of studies shown, numerous miRNAs are found being involved in the biosynthetic process of breast milk, and affect the immune regulation related functions. Nevertheless, the current research on giant panda(Ailuropoda melanoleuca) break milk is very few, and the potential mechanism during the generation of break milk. Thus, we selected high-throughput sequencing technology to analyze microRNAome expressed in giant panda breast milk collected from five different time point (0d,3d,7d,15d,30d), to explore the important role of miRNA in giant breast milk, and to conduct comparative analysis of their characteristics, to reveal the regulation mechanism of miRNA regulating secretion of breast milk at molecular level. The main results are shown as follow:(1) This study is the first research to build the dynamic profiles of microRNAome in lactation panda breast milk collected from five different time point (postnatal day 0d、3d、 7d、15d、30d), which filled the blank of poor available resource in current miRBase for panda miRNA information.(2) To assess the reliability of the experimental results and the operational stability, we have done correlation analysis to the results of parallel experiments and use Q-PCR to verify the high-throughput sequencing data. The results showed that:correlation coefficient between biological replicates was very high (r>0.95, P<0.05). This result indicates that biological individuals have very strong repeatability. There is also a good correlation between the results of high-throughput sequencing data and Q-PCR, indicating that the high-throughput sequencing results are accurate and reliable. In conclusion, the experimental operation have strong stability and reliability, and the results of subsequent analysis are credible and reliable.(3) The high-throughput sequencing analysis results indicated that the panda breast milk expressed a large number of miRNAs, and the majority sequencing reads can map to the panda genome, which can also map to many mammal precursor miRNA sequences (pre-miRNA) in miRBase database. Through a series of filter process, most clean reads are between 22-24nt in length, and the 22nt small RNA sequencing sequence is the most abundant reads (47.89%), followed by 21 (22.14%) and 23-nt (20.32%) small RNA sequences, which are typical sizes of Dicer-processed products, confirm the characteristics of small RNA in sequence structure.(4) Through the bioinformatics pipeline for miRNA discovery, seven sequencing libraries representing five different lactation stage (0d、3d-1、3d-2、3d-3、7d、15d、30d), in total, identified 320 panda mature miRNA corresponding to 226 pre-miRNA sequences.(5) The BLAST analysis result the miRNA sequence expressed by panda breast milk are extremely conserved among mammal species. In this study, the most sequence is conserved in 9 mammal species (18.75%), followed by 10 mammal species containing 51 miRNA sequences.Notably, there are 71 sequence is conserved in more than 10 mammal species (22.19%). In conclusion, the miRNA identified from this study are extremely conserved among mammal species, which more than 95% miRNA are conserved expressed at least in five mammal species.(6) Similar to previous miRNA expression profile researches on mammal species and tissues, the microRNAome in panda also share the rule that distinct pre-miRNAs and genomic loci that express identical mature sequences, which resulted in these 320 miRNAs sequences corresponding to 282 unique miRNAs sequences and indicated that more than 38 miRNA come from two or more distinct pre-miRNA sequence or genomic loci.(7) For the expression level, the miRNA profile expressed in all libraries shared the same pattern that only a few types of miRNA is extremely abundant highly expressed and comprised the majority of sequences, on the other hand, plenty of miRNA is is extremely poor expression.(8) Through the comparative analysis of miRNA profile expressed at different lactation stages, the result showed that the vast majority type of miRNAs are simultaneously identified at all lactation stages (154,54.61%). Further more, among these 154 miRNAs,39 kinds of miRNA sequences show a continuous high-abundant expression level (reads> 1000) during the whole lactation (from birth to postnatal day 30). Then, the results of pathway analysis for the target genes predicted from these 39 lactation-enriched miRNAs suggest that these lactation-enriched miRNAs exert an important regulation role in the development of breast ductal system and maintaining the secretion of breast milk in panda. Compared with the lactation-enriched miRNAs, although these stage-specific miRNAs is relatively few type of miRNAs (57,20.21%) with relatively low abundance (reads< 1000), but these stage-specific miRNAs contributed to the appropriate demand of different lactation stage, especially in colostrum stage, which improve the quality and secretion of breast milk through immune-related pathway and regulation elements.(9) We adopted STEM (short time series method) analysis for the expression data of miRNAs simultaneously expressed at whole lactation, to further explore the expression patterns among different lactation stages. Results revealed that two types of miRNA expression patterns were significantly enriched by these lactation expressed miRNAs. Furthermore, the colostrum enriched miRNAs were significantly enriched in secretion of milk and immune-related pathway, and the ordinary milk enriched miRNAs were significantly enriched in milk product.(10) Through hierarchical cluster analysis, we found that the microRNAome show typical lactation-specific pattern, which reveal obvious differences between colostrum and ordinary milk, In detail, the results of clustering support that the Od and 3d breast milk was the first cluster and the 7d,15d and 30 breast milk were grouped into another cluster.(11) Both high-throughput sequencing (submit the sequencing data to the genome of bamboo, and map sequences to miRBase, to identify these miRNA expressed in bamboo) and the real-time PCR results supported that bamboo-origined miRNAs appear at panda breast milk with good stability, which implied that that these bamboo-origined miRNAs may be transferred into the panda body directly via the digestive tract and exert its regulation function, potentially affecting the development of breast ductal system and secretion of milk in giant panda.In conclusion, this study is the first research to build the basic microRNAome in giant panda. We not only comparatively analysis the characteristics of miRNA profile expressed at different lactation panda breast milk, identified out numerous lactation-enriched or stage-specific miRNAs, but also predicted its biological regulation role during secretion of breast milk and explore to reveal the effect of the plant miRNA on breast milk secretion in giant panda. Our results facilitated to further explain the regulation role of miRNA in giant panda lactation and its well-developed immune systems.