Research resources: comparative microRNA profiles in human corona radiata cells and cumulus oophorus cells detected by next-generation small RNA sequencing

Abstract

During folliculogenesis, cumulus cells surrounding the oocyte differentiate into corona radiata cells (CRCs) and cumulus oophorus cells (COCs), which are involved in gonadal steroidogenesis and the development of germ cells. Several studies suggested that microRNAs (miRNAs) play an important regulatory role at the post-transcriptional level in cumulus cells. However, comparative miRNA profiles and associated processes in human CRCs and COCs have not been reported before. In this study, miRNA profiles were obtained from CRCs and COCs using next generation sequencing in women undergoing controlled ovarian stimulation for IVF. A total of 785 and 799 annotated miRNAs were identified in CRCs and COCs, while high expression levels of six novel miRNAs were detected both in CRCs and in COCs. In addition, different expression patterns in CRCs and COCs were detected in 72 annotated miRNAs. To confirm the miRNA profile in COCs and CRCs, quantitative real-time PCR was used to validate the expression of annotated miRNAs, differentially expressed miRNAs, and novel miRNAs. The miRNAs in the let-7 family were found to be involved in the regulation of a broad range of biological processes in both cumulus cell populations, which was accompanied by a large amount of miRNA editing. Bioinformatics analysis showed that amino acid and energy metabolism were targeted significantly by miRNAs that were differentially expressed between CRCs and COCs. Our work extends the current knowledge of the regulatory role of miRNAs and their targeted pathways in folliculogenesis, and provides novel candidates for molecular biomarkers in the research of female infertility.

Figure 1. The miRNA expression profiles in CRCs and COCs are presented by volcano plots.

The vertical lines correspond to 2-fold up- and down-regulation, respectively, and the horizontal line represents a p-value of 0.05. The red point in the plot represents the similarly expressed miRNAs without statistical significance, while the blue point in the plot represents the differentially expressed miRNAs with statistical significance.

Figure 2. Confirmation of miRNA expression detected by NGS.

Validation of the expression of ten miRNAs at different expression levels in CRCs (A) and COCs (B). In CRCs, the expression of miR-26b-5p was set as 1 and the expression levels of other miRNAs were compared with that of miR-26b-5p. In COCs, the expression of miR-23b-3p was set as 1 and the expression levels of other miRNAs were compared with that of miR-26b-5p.

Figure 3. Confirmation of the expression of the most abundant miRNAs in both COCs and CRCs.

Validation of the expression of ten miRNAs by NGS (A) and quantitative real-time PCR (B), demonstrating a similar expression pattern in COCs and CRCs.

Figure 4. Confirmation of the differentially expressed miRNAs between COCs and CRCs.

Validation of the expression of ten miRNAs by NGS (A) and quantitative real-time PCR (B), showing differentially expressed miRNAs in COCs and CRCs.

Figure 5. Confirmation of novel miRNA expression detected by NGS.

Validation of the expression of ten novel miRNAs at different expression levels in CRCs (A) and COCs (B). In CRCs, the expression of novel miR-m0042 was set as 1 and the expression levels of other miRNAs were compared with that of novel miR-m0042. In COCs, the expression of novel miR-m0036 was set as 1 and the expression levels of other miRNAs were compared with that of novel miR-m0036.