Pier-Luc Dudemaine

Comparative Analysis of the miRNome of Bovine Milk Fat, Whey and Cells.

Abundant miRNAs have been identified in milk and mammary gland tissues of different species. Typically, RNA in milk can be extracted from different fractions including fat, whey and cells and the mRNA transcriptome of milk could serve as an indicator of the transcriptome of mammary gland tissue. However, it has not been adequately validated if the miRNA transcriptome of any milk fraction could be representative of that of mammary gland tissue. The objectives of this study were to (1) characterize the miRNA expression spectra from three milk fractions- fat, whey and cells; (2) compare miRNome profiles of milk fractions (fat, whey and cells) with mammary gland tissue miRNome, and (3) determine which milk fraction miRNome profile could be a better representative of the miRNome profile of mammary gland tissue. Milk from four healthy Canadian Holstein cows in mid lactation was collected and fractionated. Total RNA extracted from each fraction was used for library preparation followed by small RNA sequencing. In addition, miRNA transcripts of mammary gland tissues from twelve Holstein cows in our previous study were used to compare our data. We identified 210, 200 and 249 known miRNAs from milk fat, whey and cells, respectively, with 188 universally expressed in the three fractions. In addition, 33, 31 and 36 novel miRNAs from milk fat, whey and cells were identified, with 28 common in the three fractions. Among 20 most highly expressed miRNAs in each fraction, 14 were expressed in common and 11 were further shared with mammary gland tissue. The three milk fractions demonstrated a clear separation from each other using a hierarchical cluster analysis with milk fat and whey being most closely related. The miRNome correlation between milk fat and mammary gland tissue (rmean = 0.866) was significantly higher than the other two pairs (p < 0.01), whey/mammary gland tissue (rmean = 0.755) and milk cell/mammary gland tissue (rmean = 0.75), suggesting that milk fat could be an alternative non-invasive source of RNA in assessing miRNA activities in bovine mammary gland. Predicted target genes (1802) of 14 highly expressed miRNAs in milk fractions were enriched in fundamental cellular functions, infection, organ and tissue development. Furthermore, some miRNAs were highly enriched (FDR <0.05) in milk whey (3), cells (11) and mammary gland tissue (14) suggesting specific regulatory functions in the various fractions. In conclusion, we have obtained a comprehensive miRNA profile of the different milk fractions using high throughput sequencing. Our comparative analysis showed that miRNAs from milk fat accurately portrayed the miRNome of mammary gland tissue. Functional annotation of the top expressed miRNAs in milk confirmed their critical regulatory roles in mammary gland functions and potentially to milk recipients.

MicroRNA roles in signalling during lactation: an insight from differential expression, time course and pathway analyses of deep sequence data

The study examined microRNA (miRNA) expression and regulatory patterns during an entire bovine lactation cycle. Total RNA from milk fat samples collected at the lactogenesis (LAC, day1 [D1] and D7), galactopoiesis (GAL, D30, D70, D130, D170 and D230) and involution (INV, D290 and when milk production dropped to 5 kg/day) stages from 9 cows was used for miRNA sequencing. A total of 475 known and 238 novel miRNAs were identified. Fifteen abundantly expressed miRNAs across lactation stages play regulatory roles in basic metabolic, cellular and immunological functions. About 344, 366 and 209 miRNAs were significantly differentially expressed (DE) between GAL and LAC, INV and GAL, and INV and LAC stages, respectively. MiR-29b/miR-363 and miR-874/miR-6254 are important mediators for transition signals from LAC to GAL and from GAL to INV, respectively. Moreover, 58 miRNAs were dynamically DE in all lactation stages and 19 miRNAs were significantly time-dependently DE throughout lactation. Relevant signalling pathways for transition between lactation stages are involved in apoptosis (PTEN and SAPK/JNK), intracellular signalling (protein kinase A, TGF-β and ERK5), cell cycle regulation (STAT3), cytokines, hormones and growth factors (prolactin, growth hormone and glucocorticoid receptor). Overall, our data suggest diverse, temporal and physiological signal-dependent regulatory and mediator functions for miRNAs during lactation.

Co-Expression Network and Pathway Analyses Reveal Important Modules of miRNAs Regulating Milk Yield and Component Traits

Co-expression network analyses provide insights into the molecular interactions underlying complex traits and diseases. In this study, co-expression network analysis was performed to detect expression patterns (modules or clusters) of microRNAs (miRNAs) during lactation, and to identify miRNA regulatory mechanisms for milk yield and component traits (fat, protein, somatic cell count (SCC), lactose, and milk urea nitrogen (MUN)) via miRNA target gene enrichment analysis. miRNA expression (713 miRNAs), and milk yield and components (Fat%, Protein%, lactose, SCC, MUN) data of nine cows at each of six different time points (day 30 (D30), D70, D130, D170, D230 and D290) of an entire lactation curve were used. Four modules or clusters (GREEN, BLUE, RED and TURQUOISE) of miRNAs were identified as important for milk yield and component traits. The GREEN and BLUE modules were significantly correlated (|r| > 0.5) with milk yield and lactose, respectively. The RED and TURQUOISE modules were significantly correlated (|r| > 0.5) with both SCC and lactose. In the GREEN module, three abundantly expressed miRNAs (miR-148a, miR-186 and miR-200a) were most significantly correlated to milk yield, and are probably the most important miRNAs for this trait. DDR1 and DDHX1 are hub genes for miRNA regulatory networks controlling milk yield, while HHEX is an important transcription regulator for these networks. miR-18a, miR-221/222 cluster, and transcription factors HOXA7, and NOTCH 3 and 4, are important for the regulation of lactose. miR-142, miR-146a, and miR-EIA17-14144 (a novel miRNA), and transcription factors in the SMAD family and MYB, are important for the regulation of SCC. Important signaling pathways enriched for target genes of miRNAs of significant modules, included protein kinase A and PTEN signaling for milk yield, eNOS and Noth signaling for lactose, and TGF β, HIPPO, Wnt/β-catenin and cell cycle signaling for SCC. Relevant enriched gene ontology (GO)-terms related to milk and mammary gland traits included cell differentiation, G-protein coupled receptor activity, and intracellular signaling transduction. Overall, this study uncovered regulatory networks in which miRNAs interacted with each other to regulate lactation traits.

Integration of miRNA weighted gene co-expression network and miRNA-mRNA co-expression analyses reveals potential regulatory functions of miRNAs in calf rumen development

This study aimed to explore the roles of microRNAs (miRNAs) in calf rumen development during early life. Rumen tissues were collected from 16 calves (8 at pre-weaning and 8 at post-weaning) for miRNA-sequencing, differential expression (DE), miRNA weighted gene co-expression network (WGCNA) and miRNA-mRNA co-expression analyses. 295 miRNAs were identified. Bta-miR-143, miR-26a, miR-145 and miR-27b were the most abundantly expressed. 122 miRNAs were significantly DE between the pre- and post-weaning periods and the most up- and down-regulated miRNAs were bta-miR-29b and bta-miR-493, respectively. Enrichment analyses of the target genes of DE miRNAs revealed important roles for miRNA in rumen developmental processes, immune system development, protein digestion and processes related to the extracellular matrix. WGCNA indicated that bta-miR-145 and bta-miR-199a-3p are important hub miRNAs in the regulation of these processes. Therefore, bta-miR-143, miR-29b, miR-145, miR-493, miR-26a and miR-199 family members might be key regulators of calf rumen development during early life.

Integration of lncRNA and mRNA Transcriptome Analyses Reveals Genes and Pathways Potentially Involved in Calf Intestinal Growth and Development during the Early Weeks of Life

A better understanding of the factors that regulate growth and immune response of the gastrointestinal tract (GIT) of calves will promote informed management practices in calf rearing. This study aimed to explore genomics (messenger RNA (mRNA)) and epigenomics (long non-coding RNA (lncRNA)) mechanisms regulating the development of the rumen and ileum in calves. Thirty-two calves (≈5-days-old) were reared for 96 days following standard procedures. Sixteen calves were humanely euthanized on experiment day 33 (D33) (pre-weaning) and another 16 on D96 (post-weaning) for collection of ileum and rumen tissues. RNA from tissues was subjected to next generation sequencing and 3310 and 4217 mRNAs were differentially expressed (DE) between D33 and D96 in ileum and rumen tissues, respectively. Gene ontology and pathways enrichment of DE genes confirmed their roles in developmental processes, immunity and lipid metabolism. A total of 1568 (63 known and 1505 novel) and 4243 (88 known and 4155 novel) lncRNAs were detected in ileum and rumen tissues, respectively. Cis target gene analysis identified BMPR1A, an important gene for a GIT disease (juvenile polyposis syndrome) in humans, as a candidate cis target gene for lncRNAs in both tissues. LncRNA cis target gene enrichment suggested that lncRNAs might regulate growth and development in both tissues as well as posttranscriptional gene silencing by RNA or microRNA processing in rumen, or disease resistance mechanisms in ileum. This study provides a catalog of bovine lncRNAs and set a baseline for exploring their functions in calf GIT development.

Integration of miRNA and mRNA Co-Expression Reveals Potential Regulatory Roles of miRNAs in Developmental and Immunological Processes in Calf Ileum during Early Growth

This study aimed to investigate the potential regulatory roles of miRNAs in calf ileum developmental transition from the pre- to the post-weaning period. For this purpose, ileum tissues were collected from eight calves at the pre-weaning period and another eight calves at the post-weaning period and miRNA expression characterized by miRNA sequencing, followed by functional analyses. A total of 388 miRNAs, including 81 novel miRNAs, were identified. A total of 220 miRNAs were differentially expressed (DE) between the two periods. The potential functions of DE miRNAs in ileum development were supported by significant enrichment of their target genes in gene ontology terms related to metabolic processes and transcription factor activities or pathways related to metabolism (peroxisomes), vitamin digestion and absorption, lipid and protein metabolism, as well as intracellular signaling. Integration of DE miRNAs and DE mRNAs revealed several DE miRNA-mRNA pairs with crucial roles in ileum development (bta-miR-374a—FBXO18, bta-miR-374a—GTPBP3, bta-miR-374a—GNB2) and immune function (bta-miR-15b—IKBKB). This is the first integrated miRNA-mRNA analysis exploring the potential roles of miRNAs in calf ileum growth and development during early life.