Identification and Profiling of microRNAs in Goat Endometrium during Embryo Implantation

<div><p>Background</p><p>MicroRNAs (miRNAs) are short, highly conserved small noncoding RNAs that had fundamental roles in post-transcriptional gene expression, and they are crucial for proper control of biological processes and known to participate in embryo implantation. However, miRNA expression profiles in the pre-receptive and receptive phases of the goat endometrium during embryo implantation are unknown.</p><p>Results</p><p>A total of 1,069 and 847 miRNAs were expressed in receptive (R) and pre-receptive (P) goat endometrium, and 632 miRNAs were co-expressed in both phases. We identified 545 (50.98%) known miRNAs in the R library and 522 (61.63%) in the P library. There were 110 up-expressed miRNAs and 33 down-expressed miRNAs in receptive endometrium compared with the pre-receptive endometrium meeting the criteria of <i>P</i>-values< 0.05. Moreover, GO and KEGG analysis of the target genes of the differentially expressed miRNAs revealed some candidate miRNAs, genes and pathways that may involve in the formation of the receptive endometrium. Based on stem-loop RT-qPCR, 15 miRNAs were detected and the results suggested that the majority of the miRNA expression data measured by Solexa deep sequencing could represent actual miRNA expression levels.</p><p>Conclusions</p><p>Our data revealed the first miRNA profile related to the biology of the goat receptive endometrium during embryo implantation, and the results suggested that a subset of miRNAs might play important roles in the formation of endometrial receptivity. Thus, elucidating the physiological roles of endometrial miRNAs will help us better understand the genetic control of embryo implantation in goats.</p></div

The size distribution of the small RNAs in R and P libraries.

<p>The size distribution of the small RNAs in R and P libraries.</p

miRNA-gene network of induction of apoptosis by extracellular signals (GO: 0008624).

<p>Note: White circular nodes represent genes, and red rectangular and pink rounded rectangle nodes represent miRNAs. The size of the nodes represents the power of the interrelation among the nodes, and edges between two nodes represent interactions between genes. The more edges a gene has, the more miRNAs that interact with it, and the more central a role it had within the network. The top five key miRNAs in the network were miR-449a, miR-182, PC-5p-6424_145, PC-5p-2673_284, and miR-138-5p. The top three key mRNAs’ serial numbers were JR128745.1 (Caspase 8, apoptosis-related cysteine peptidase (CASP8)), JR133467.1 (Y-linked ubiquitin-specific protease 9 (USP9Y)), and JR132030.1 (CD5 molecule (CD5)) in NCBI (National Center for Biotechnology Information).</p

Venn diagrams of detected miRNAs.

<p>Venn diagrams of detected miRNAs.</p

Stem-loop structures of 7 selected novel miRNAs in goat.

<p>Note: The secondary structures were generated from the goat genome and EST sequences. The uppercase letters refer to mature sequences and were indicated in yellow. A: PC-3p-22067_41, B: PC-3p-61234_13, C: PC-5p-21385_42, D: PC-5p-21767_42, E: PC-5p-32617_26, F: PC-5p-63323_12, G: PC-5p-104298_5.</p

The expression levels of 15 miRNAs were determined by Stem-loop RT-qPCR.

<p>Note: <i>18S</i> was used as internal control gene for normalization in our experiments. Values were “means ± SD” in receptive endometrium (n = 9) that were relative to pre-receptive endometrium (n = 9), whose values all were 1 in this manuscript because of the method of 2^-ΔΔCt, ΔΔ Ct = (CT _miRNA − CT _18s) _R − (CT _miRNA − CT _18s) _P.</p

Some predicted novel miRNA.

<p>Note: Len represented the bases number of miRNA. SS represented secondary structure of predicted hairpin RNA (pre-miRNA) structures that were predicted with RNAfold software (<a href="http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi" target="_blank">http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi</a>). R-NE represented the normalized expression level of miRNAs in the small RNA library generated from receptive endometrium of Xinong Saanen dairy goats. P-NE represented the normalized expression level of miRNAs in the small RNA library generated from pre-receptive endometrium of Xinong Saanen dairy goats.</p><p>Some predicted novel miRNA.</p

Some differential expressed miRNAs (|log2 (fold change)|> 3) and their targets.

<p>Note: R-NE represented the normalized expression level of miRNAs in the small RNA library generated from receptive endometrium of Xinong Saanen dairy goats. P-NE represented the normalized expression level of miRNAs in the small RNA library generated from pre-receptive endometrium of Xinong Saanen dairy goats. Fold (R /P) and log2 (R/P) indicated the fold change of the miRNAs between samples. <i>P</i>-Value manifested the significance of miRNAs differential expression between two samples. Number of Targets represented putative target genes for a certain miRNA predicted by two software programs (Target Scan v.50 and Mireap v.3.3a).</p><p>Some differential expressed miRNAs (|log2 (fold change)|> 3) and their targets.</p

Some known miRNA of which read number was more than 100,000 in this study.

<p>Note: rep_miR ID was the representative known miRNA ID. R-NE represented the normalized expression level of miRNAs in the small RNA library generated from receptive endometrium of Xinong Saanen dairy goats. P-NE represented the normalized expression level of miRNAs in the small RNA library generated from pre-receptive endometrium of Xinong Saanen dairy goats.</p><p>Some known miRNA of which read number was more than 100,000 in this study.</p

Differential expression levels of miRNAs between R and P libraries.

<p>Note: R-NE represented the normalized expression level of miRNAs in the small RNA library generated from receptive endometrium of Xinong Saanen dairy goats. P-NE represented the normalized expression level of miRNAs in the small RNA library generated from pre-receptive endometrium of Xinong Saanen dairy goats. Fold (R/P) and log2 (R/P) indicated the fold change of the miRNAs between samples. P-Value manifested the significance of miRNAs differential expression between two samples.</p><p>Differential expression levels of miRNAs between R and P libraries.</p