Abstract
Circular RNAs (circRNAs) are a new class of RNA that have a stable structure characterized by covalently closed circular molecules and are involved in invasive pituitary adenomas and recurrent clinically nonfunctioning pituitary adenomas. However, information on circRNAs in the normal pituitary, especially in rats, is limited. In this study, we identified 4123 circRNAs in the immature (D15) and mature (D120) rat anterior pituitary using the Illumina platform, and 32 differentially expressed circRNAs were found. A total of 150 Gene Ontology terms were significantly enriched, and 16 KEGG pathways were found to contain differentially expressed genes. Moreover, we randomly selected eight highly expressed circRNAs and detected their relative expression levels in the mature and immature rat pituitary by qPCR. In addition, we predicted 90 interactions between 53 circRNAs and 57 miRNAs using miRanda. Notably, circ_0000964 and circ_0001303 are potential miRNA sponges that may regulate the Fshb gene. The expression profile of circRNAs in the immature and mature rat anterior pituitary may provide more information about the roles of circRNAs in the development and reproduction in mammals.
Introduction
Circular RNAs (circRNAs), a new and unique class of RNA, are different from linear RNA and form a stable structure characterized by covalently closed circular molecules (Shang et al. 2016, Wu et al. 2017). Recently, thousands of strongly expressed and stable circRNAs were detected in humans and animals; these circRNAs exhibit tissue/developmental-stage-specific expression (Memczak et al. 2013, Guo et al. 2014). Since circRNAs usually do not have poly-A tails, they have higher stability and higher sequence conservation than normal linear RNA molecules, such as miRNAs and long noncoding RNAs, in mammalian cells (Jeck et al. 2013, Jeck & Sharpless 2014). Although the functions of circRNAs are largely unknown, an increasing number of reports have shown that circRNAs can act as ‘miRNA sponges’ by binding to miRNAs to regulate the expression of genes (Kulcheski et al. 2016). For instance, circRNA_100290 acts as a sponge for miR-29b family members to regulate CDK6 expression and may be a potential target for oral squamous cell carcinoma (OSCC) therapy (Chen et al. 2017). Another study reported that the circRNA Cdr1as, as a sponge for miR-7, affected insulin secretion, which may be a new target for improving the function of diabetic β cells (Xu et al. 2015). An increasing number of reports have shown that circRNAs play key roles in diverse biological processes (Wilusz 2015, 2018). In 2018, Fu et al. reported a comprehensive profile of circRNAs in bovine cumulus cells (Fu et al. 2018).
As an important class of experimental animals, rats play a crucial role in many types of functional studies, particularly in reproductive development (Han et al. 2017a ). The pituitary is an important functional organ in the animal endocrine system and performs important functions in numerous physiological processes (Hong et al. 2016). The pituitary secretes seven types of hormones, such as follicle-stimulating hormone (FSH), luteinizing hormone (LH) and growth hormone; these hormones are essential for reproduction, growth and metabolic homeostasis, and they regulate developmental and physiological processes (Lupu et al. 2001, Samarasinghe et al. 2014). Moreover, reproductive development covers a wide range of components and is affected by many factors, such as vitamin A, probiotics and hormones (Clagett-Dame & Knutson 2011, Carnevali et al. 2013, Goodman et al. 2013). A few studies have reported that circRNAs are involved in the regulation of reproduction. Some studies have reported circRNA screening and expression patterns in the ovary, testis and placenta in humans (Dong et al. 2016, Qian et al. 2016, Quan & Li 2018). However, information about the effects of circRNAs on reproductive hormones in rats is limited.
In this study, we systematically investigated the circRNA content in the immature (D15) and mature (D120) rat anterior pituitary using the Illumina platform. Our findings will provide a powerful resource for more in-depth investigations of the regulatory functions of circRNAs in rats and will contribute to a better understanding of reproduction and development in mammals.
Materials and methods
Ethics statement
The experiments were strictly performed according to the guidelines of the Guide for the Care and Use of Laboratory Animals of Jilin University. In addition, all experimental protocols were approved by the Institutional Animal Care and Use Committee of Jilin University (Permit Number: 201801005).
Collection of tissue samples
Sprague–Dawley rats were provided by the School of Medical Science of Jilin University. Anterior pituitary samples were obtained from each of the 15-day-old (D15) and 4-month-old (D120) rats, and all the samples were immediately snap-frozen in liquid nitrogen and stored at −80°C until RNA extraction.
RNA sequencing and quality control
Equal amounts of total RNA (3 μg) from four rat pituitary samples were pooled into a single sample to construct a library. Ribo-Zero Gold Kits were used to remove the rRNA from the sample, and different index tags for library construction were selected according to the instructions of the NEB Next Ultra Directional RNA Library Prep Kit for the Illumina platform (NEB, Ipswich, MA, USA). The specific steps used for library construction are as follows: the kit was first used to remove ribosomal rRNA (if it was necessary to remove linear RNA molecules, then RNase R was added); fragmentation buffer was added to the reaction system to fragment the RNA into short fragments; the fragmented RNA was then used as a template; the first strand of the cDNA was synthesized using six-base random primers (random hexamers), and the second strand of the cDNA was synthesized by adding buffers dNTPs, RNase H and DNA polymerase I; and the cDNA was purified by the QiaQuick PCR kit and washed with EB buffer. After the repair was complete, poly-A tails and sequencing joints were added, and the size of the target fragment was determined by agarose gel electrophoresis. After DNase digestion of both strands of the cDNA and PCR amplification, the final size of fragments was determined by agarose gel electrophoresis, which completed the library preparation. Then, the libraries were sequenced by Anoroad Technologies (Beijing, China) using the Illumina platform. The raw reads generated by Illumina sequencing contain sequencing linker sequences and low-mass sequences. To ensure the quality of the data obtained from the analysis, we filtered the original downstream data sequences to obtain high-quality clean reads. Analysis and follow-up analysis were based on clean reads. For double-ended sequencing, if one end of the reads did not satisfy any of the following conditions, the pair of reads was removed: (1) reads from contaminated joints (the base number of the joint contaminated in the reads was greater than 5 bp); (2) low-quality reads (bases with Q ≤ 19 in the reads account for more than 50% of the total bases); (3) reads containing more than 5% N (the Q value is an abbreviation of the Phred quality score; an N base refers to an unknown base) and (4) reads matched with rRNA.
circRNA identification
The current research focuses on exon circularized ecircRNAs, and there are two models of the ring formation mechanism: Lariat-driven cyclization and intron-driven cyclization. In either model, the splice donor (SD) in the downstream exon of the circular RNA is trans-spliced to the splice acceptor (SA) in the upstream exon, so the sequence cannot be directly aligned to the reference genome. This concept is also the main idea for identifying circular RNAs: sequence-separating alignments and searching for GT-AG signals beside the junction site. CIRI is widely used as a rapid and efficient circular RNA recognition tool (Gao et al. 2015). We used CIRI_v2.0.3 to identify the circRNAs. First, we used the BWA-MEM algorithm to perform the sequence resolution comparison. Then, we compared the results of the SAM file scan, and we found the paired chiastic clipping (PCC) and paired-end mapping sites and the GT-AG splicing signal. Finally, the sequence of the junction site was realigned with a dynamic programming algorithm to ensure the reliability of the circRNAs.
Analysis of differentially expressed circRNAs
The expression level of the circRNAs was normalized by spliced reads per billion mapping (SRPBM). Differential expression of the circRNAs was assessed using DEseq2 (Love et al. 2014). P < 0.05 and |log2 (fold change)| ≥ 1.5 were set as the threshold for significant differential gene expression.
Target site prediction and enrichment analysis
MiRanda (3.3a) (John et al. 2004) was used to analyze the interactions between the circRNAs and miRNAs. In view of the known reports and the extractability of the sequences, only classical and antisense circRNAs were selected for miRNA target prediction. Host genes associated with the circRNAs were submitted to R-project for Gene Ontology (GO) analysis. We used KOBAS software (Mao et al. 2005) to test the statistical enrichment of differential gene expression in the KEGG pathways. GO terms and KEGG pathways for which P < 0.05 were considered significantly enriched.
Primer design and RT-qPCR analysis
Primers were designed by RiboBio Biotech Co., Ltd. (Guangzhou, China; Supplementary Table 1, see section on supplementary data given at the end of this article). Total RNA was converted into cDNA using a FastQuant RT kit (with gDNase) (Tiangen, China), and quantitative RT-PCR was performed on a Mastercycler ep Realplex2 system (Eppendorf, Germany) with SuperReal PreMix Plus (SYBR Green) (Tiangen, China) according to the manufacturer’s instructions.
Statistical analysis
The data are presented as mean ± s.d. from three independent experiments. The data were analyzed using SPSS 19.0. The significance of the differences was determined through one-way ANOVA, and P < 0.05 was considered significant.
Results
Deep sequencing of circRNAs in rats
To determine the identity and abundance of circRNAs in the rat anterior pituitary at different developmental stages, we constructed four cDNA libraries, including the immature (D15) and mature (D120) pituitary, with two replicates per group. The libraries were sequenced using the Illumina NextSeq 550AR platform following a stringent filtering pipeline (Fig. 1A). We identified a total of 4123 circRNAs from the RNA-seq data for D15 and D120, including classic circRNAs (72.6%), alter exon circRNAs (6.1%), intron circRNAs (0.8%), overlap exon circRNAs (13.0%), antisense circRNAs (1.1%) and intergenic circRNAs (6.4%, Fig. 1B). Moreover, these circRNAs were distributed on 20 autosomes and the X and Y chromosomes (Fig. 1C and Supplementary Table 2).
Deep sequencing of circRNAs in rats. (A) Identification pipeline for circRNAs. (B) Classification of 4123 circRNAs, including classic, alter exon, intron, overlap exon, antisense and intergenic circRNAs. (C) Distribution of circRNAs on the rat chromosomes. A full color version of this figure is available at https://doi.org/10.1530/JOE-18-0540.
Citation: Journal of Endocrinology 240, 3; 10.1530/JOE-18-0540
Expression patterns of circRNAs in the mature and immature rat anterior pituitary
The size of the circRNA candidates ranged from 80 nt to over 2000 nt, and most of the candidates were distributed between 300 nt and 700 nt. Approximately 95.1% of the circRNAs had a predicted spliced length of less than 2000 nt, whereas circRNAs with a length of more than 2000 nt accounted for 4.9% of the circRNAs (Fig. 2A). The number of exons in the circRNAs was typically 2–5 (Fig. 2B). Moreover, when a circRNA consisted of one exon, the length of the exon was significantly longer than that of the exons in a circRNA consisting of multiple exons (Fig. 2C). We used SRPBM to estimate the expression levels of the circRNAs. We found that the SRPBM value of D15 was slightly higher than that of D120 (Fig. 2D).
Expression pattern of circRNAs in the mature and immature rat anterior pituitary. Distributions of the sequence length (A), exon number (B) and exon length (C) of the circRNAs. (D) Boxplots of the expression levels (SRPBM value) of circRNAs in D15 and D120 rats. A full color version of this figure is available at https://doi.org/10.1530/JOE-18-0540.
Citation: Journal of Endocrinology 240, 3; 10.1530/JOE-18-0540
Identification of differentially expressed circRNAs
We used DEseq2 for differential expression analysis of circRNAs. When comparing the data for D15 and D120, an expression ratio greater than 1.5 and P < 0.05 were selected as the criteria for significantly differentially expressed circRNAs, and this approach was used to obtain the number of upregulated and downregulated circRNAs. We identified 32 circRNAs that were differentially expressed in the immature and mature rat anterior pituitary (Supplementary Table 3). The differentially expressed circRNAs included 16 upregulated and 16 downregulated circRNAs (Fig. 3A and B). We measured the expression patterns of the differentially expressed circRNAs through systematic cluster analysis to explore the similarities and compare the relationships between D15 and D120 (Fig. 3C).
Identification of differentially expressed circRNAs. (A) Proportion of upregulated and downregulated circRNAs in the rat anterior pituitary at different developmental stages. (B) Volcano plot analysis of differentially expressed circRNAs between D15 and D120. Red dots indicate upregulated genes; green dots represent downregulated genes. (C) Analysis of the expression patterns of differentially expressed circRNAs. The highest to lowest fold changes are marked from red to blue, respectively. A full color version of this figure is available at https://doi.org/10.1530/JOE-18-0540.
Citation: Journal of Endocrinology 240, 3; 10.1530/JOE-18-0540
Enrichment of differentially expressed circRNAs
We used GO and pathway enrichment analyses to analyze the enrichment of genes for differentially expressed circRNAs. GO analysis showed that 150 GO terms were significantly enriched (P < 0.05, Supplementary Table 4), and these terms were mainly associated with biological regulation (GO: 0065007), cellular process (GO: 0009987), cell part (GO: 0044464), organelle (GO: 0043226) and binding (GO: 005488). Figure 4 showed the GO annotation of upregulated and downregulated mRNAs covers the fields of cellular components, biological processes and molecular functions. In addition, 16 KEGG pathways were found to contain differentially expressed genes, such as the insulin secretion pathway, nucleotide excision repair pathway and MAPK signaling pathway (Supplementary Table 5).
Enrichment of differentially expressed circRNAs. The GO annotation of upregulated and downregulated mRNAs covers the fields of cellular components, biological processes and molecular functions. Red columns indicate upregulated genes, and green columns indicate downregulated genes. A full color version of this figure is available at https://doi.org/10.1530/JOE-18-0540.
Citation: Journal of Endocrinology 240, 3; 10.1530/JOE-18-0540
Interactions between circRNAs and miRNAs
To further analyze the functions of the circRNAs, we predicted the interactions between circRNAs and miRNAs using miRanda. A total of 90 interactions between 53 circRNAs and 57 miRNAs were identified (Fig. 5 and Supplementary Table 6). Two of the 53 circRNAs, rno_circ_0001772 and rno_circ_0000432, were differentially expressed in the immature and mature anterior pituitary of rats. Rno_circ_0001772 acted as a sponge with miR-145-5p, and rno_circ_0000432 acted as a sponge with both miR-181b-5p and miR-181d-5p. Moreover, in our previous study, we identified 18 miRNAs that may target the 3′ untranslated region (UTR) of Fshb by a luciferase reporter assay (Han et al. 2017b ). Interestingly, in the interaction analysis, we found that circ_0000964 acted as a sponge for miR-880 and circ_0001303 acted as a sponge for miR-204. Notably, miR-204 and miR-880 were among the 18 miRNAs that potentially target the 3′UTR of Fshb.
Validation of highly expressed circRNAs and two key circRNAs. (A) Expression of upregulated and downregulated circRNAs in the mature and immature rat anterior pituitary. (B and C) Expression levels of circ_0000964 and circ_0001303 in the mature and immature rat anterior pituitary. All experiments were repeated at least three times. The data are shown as the mean ± s.d. Statistical significance was analyzed by one-way ANOVA, P < 0.05 was considered significant, and differences are marked with the letters a and b. A full color version of this figure is available at https://doi.org/10.1530/JOE-18-0540.
Citation: Journal of Endocrinology 240, 3; 10.1530/JOE-18-0540
Validation of highly expressed circRNAs and two key circRNAs
To confirm the circRNA data generated by RNA-seq analysis, we randomly selected eight highly expressed circRNAs and detected their relative expression levels in the mature and immature rat pituitary by qPCR. When comparing D120 with D15, the eight circRNAs included four upregulated circRNAs (rno_circ_0000905, rno_circ_0003214, rno_circ_0002680 and rno_circ_0002395) and four downregulated circRNAs (rno_circ_0001324, rno_circ_0001024, rno_circ_0002072 and rno_circ_0002888). The results were highly consistent with the RNA-seq results (Fig. 6A). Moreover, we detected the expression of circ_0000964 and circ_0001303 in the immature and mature rat pituitary, and the results indicated that both circRNAs were downregulated in the mature pituitary compared to the immature pituitary (Fig. 6B and C). Therefore, circ_0000964 and circ_0001303 may be potential miRNA sponges that regulate the Fshb gene and further affect the regulation of related factors in the rat pituitary.
Interactions between circRNAs and miRNAs. The network was based on the miRanda program. The triangles represent the circRNAs, and the V shape represents the miRNAs. The pink color indicates the components that are upregulated in the mature vs immature rat pituitary, the purple color indicates the components that are downregulated, and the gray color indicates the components with no change in either group. The size of the graphs represents the expression of miRNAs and circRNAs, and a larger graph indicates higher expression. A full color version of this figure is available at https://doi.org/10.1530/JOE-18-0540.
Citation: Journal of Endocrinology 240, 3; 10.1530/JOE-18-0540
Discussion
Recently, with the continued development of sequencing technology and bioinformatics technology, circRNAs have been found in the expanding world of RNA. circRNAs are highly abundant, conserved and dynamically expressed in the mammalian brain (Rybak-Wolf et al. 2015). Recent studies showed that circRNAs are involved in invasive pituitary adenomas and recurrent clinically nonfunctioning pituitary adenomas (Wang et al. 2018) and are also involved in the prenatal and postnatal pituitary in sheep (Li et al. 2017); however, studies on circRNAs in the normal pituitary, especially in rats, are limited. In this study, we identified 4123 circRNAs in the immature (D15) and mature (D120) rat anterior pituitary using the Illumina platform. The pituitary is the most important endocrine gland, and in the immature and mature pituitary, the hormones produced affect body growth and reproductive capacity, respectively. This study provides the first identification of circRNAs in the rat anterior pituitary at different developmental stages.
There are many types of circRNAs; the major types of circRNAs are the classical exon circRNAs (ecircRNAs), which are formed by exon backsplicing events, and natural circular viral RNAs (Sanger et al. 1976) and intron circular RNAs (ciRNAs), which are formed by the degradation of intron lasso structures (Zhang et al. 2013). Both types of circRNAs can perform a number of functions (Zhao & Shen 2017). However, the function of a large number of circular RNAs remains unknown. In this study, we subdivided the circRNAs based on the splice site information for the circular RNA and the relative position of the gene structure, and classical circRNAs accounted for the largest proportion. circRNAs, as a novel type of noncoding RNA, are generally presented as developing covalently closed loop structures lacking 5′–3′ polarities (Wang et al. 1986, Qu et al. 2015). In our previous study, we provided a catalog of lncRNAs in the anterior pituitary immature and mature rats (Han et al. 2017a ). The length distribution had a similar tendency between circRNAs and lncRNAs; both types of RNAs had high proportions of RNAs with lengths of 200–800 bp and greater than 2000 bp. With respect to exon numbers, circRNAs mostly had 2–3 exons, while lncRNAs had two exons. Eight highly expressed lncRNAs were randomly selected to validate their expression levels in the rat anterior pituitary at different developmental stages, and we obtained results that were consistent with the RNA-seq data. Therefore, the results confirmed the identified circRNAs.
circRNAs are a new class of noncoding RNAs with different biological functions (You et al. 2015, Yang et al. 2018), including roles in diverse developmental and physiological processes (Dong et al. 2017, Ebbesen et al. 2017). Here, we identified 32 circRNAs that were differentially expressed between the immature and mature pituitary gland. These circRNAs may have a specific biological role in regulating development or reproduction. Recent studies have indicated that circRNAs are involved in the development and reproduction. Several studies have reported that circRNAs may be involved in embryonic development and epigenetic regulation (Veno et al. 2015, Dang et al. 2016, Dong et al. 2017). Moreover, another study reported that circRNAs that are highly abundant and dynamically expressed in a spatiotemporal manner in the porcine fetal brain may perform important functions during mammalian brain development (Veno et al. 2015). In a previous study, a highly expressed circRNA in the human and mouse brain was identified (Hansen et al. 2011), and the well-known circular, testis-specific sex-determining region Y (Sry) RNA (Sry circRNA) was revealed to act as an miR-138 sponge (Hansen et al. 2013). Therefore, we predict that differentially expressed circRNAs are potential reproduction- and development-related regulators.
Many studies have reported pathways related to development and reproduction in the pituitary, including the BMP, WNT, NOTCH, GnRH and thyroid hormone signaling pathways (Ericson et al. 1998, Raetzman et al. 2004, Potok et al. 2008, Li et al. 2017). However, information on pituitary circRNAs remains relatively limited. In this study, we identified 150 significant GO terms and 16 significant KEGG pathways. GO enrichment analysis revealed that the genes were mainly involved in biological regulation, cellular process, cell part, organelle and binding. The KEGG pathways included insulin secretion, nucleotide excision repair and MAPK signaling pathways. Insulin was reported to play a role in regulating neural control of reproduction by affecting GnRH secretion (Sliwowska et al. 2014). Insulin also affected reproduction and juvenile hormone metabolism in Drosophila females (Karpova et al. 2013). Our results may improve our understanding of the role of circRNAs in the rat pituitary on development and reproduction.
Although the functions of a large number of circRNAs are not well known and require further exploration, it is clear that circRNAs are involved in transcriptional and posttranscriptional gene expression regulation (Hansen et al. 2013). Many studies have reported that endogenous circRNAs can work as miRNA sponges (Memczak et al. 2013, Cortes-Lopez & Miura 2016, Qu et al. 2018), which means that the circRNAs bind to miRNAs and potentially affect the miRNA target genes. In 2017, a study reported that the identification of differentially expressed lncRNAs indicate the potential ceRNA networks during periodontal ligament stem cell (PDLSC) osteogenic differentiation (Gu et al. 2017). Moreover, circRNA-CER, as an miR-136 sponge, regulates MMP13 expression and participates in the process of chondrocyte extracellular matrix (ECM) degradation (Liu et al. 2016). Another circRNA, Atp9b, which acts as a sponge for miR-138-5p, regulates osteoarthritis (OA) progression by modulating extracellular matrix (ECM) catabolism and inflammation in chondrocytes (Zhou et al. 2018). In this study, we predicted 90 interactions between circRNAs and miRNAs in the rat pituitary. Among these interactions, circ_0001772 and circ_0000432 were two differentially expressed circRNAs that acted as a sponge with miR-145-5p, and both miR-181b-5p and miR-181d-5p, respectively. The two circRNAs might be potential key regulators in the rat pituitary. Furthermore, circ_0000964 and circ_0001303 acted as sponges for miR-880 and miR-204, respectively. Notably, miR-204 and miR-880 were among 18 miRNAs that potentially target the Fshb 3′UTR. Therefore, circ_0000964 and circ_0001303 may be potential miRNA sponges that regulate the Fshb gene and further affect reproduction regulation in the rat pituitary. These results may provide a new regulatory mechanism for competing endogenous RNA (ceRNAs) in the context of FSH.
In summary, we provided a catalog of circRNAs in the rat pituitary and identified the circRNAs that were differentially expressed in the immature and mature rat pituitary gland. Furthermore, we predicted two circRNAs as miRNA sponges, which may potentially regulate the Fshb gene. Our study provides an important resource for understanding circRNA biology in the context of development and reproduction and sheds light on the function of circRNAs in the pituitary.
Supplementary data
This is linked to the online version of the paper at https://doi.org/10.1530/JOE-18-0540.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This study was supported by the National Natural Science Foundation of China (31872349).
Author contribution statement
B Y and J B Z were responsible for the main experimental concept and design; the experiments were performed by D X H, X L S, J B L and C J W; D X H, C Z C, H J and Y G performed the data analyses and contributed reagents; and the manuscript was written by D X H, B Y and J B Z. All the authors approved the final version.
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