circAkap17b acts as a miR-7 family molecular sponge to regulate FSH secretion in rat pituitary cells

in Journal of Molecular Endocrinology
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Chang-Jiang WangDepartment of Laboratory Animals, Jilin Provincial Key Laboratory of Animal Model, Jilin University, Changchun, Jilin, China

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Fei GaoDepartment of Laboratory Animals, Jilin Provincial Key Laboratory of Animal Model, Jilin University, Changchun, Jilin, China

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Yi-Jie HuangDepartment of Laboratory Animals, Jilin Provincial Key Laboratory of Animal Model, Jilin University, Changchun, Jilin, China

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Dong-Xu HanDepartment of Laboratory Animals, Jilin Provincial Key Laboratory of Animal Model, Jilin University, Changchun, Jilin, China

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Yi ZhengDepartment of Laboratory Animals, Jilin Provincial Key Laboratory of Animal Model, Jilin University, Changchun, Jilin, China

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Wen-Hua WangDepartment of Laboratory Animals, Jilin Provincial Key Laboratory of Animal Model, Jilin University, Changchun, Jilin, China

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Hao JiangDepartment of Laboratory Animals, Jilin Provincial Key Laboratory of Animal Model, Jilin University, Changchun, Jilin, China

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Yan GaoDepartment of Laboratory Animals, Jilin Provincial Key Laboratory of Animal Model, Jilin University, Changchun, Jilin, China

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Bao YuanDepartment of Laboratory Animals, Jilin Provincial Key Laboratory of Animal Model, Jilin University, Changchun, Jilin, China

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https://orcid.org/0000-0003-3490-0755
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Jia-Bao ZhangDepartment of Laboratory Animals, Jilin Provincial Key Laboratory of Animal Model, Jilin University, Changchun, Jilin, China

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Correspondence should be addressed to B Yuan or J-B Zhang: yuan_bao@jlu.edu.cn or zjb@jlu.edu.com

*(C-J Wang and F Gao contributed equally to this work)

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The pituitary gland functions as a prominent regulator of diverse physiologic processes by secreting multiple hormones. Circular RNAs (circRNAs) are an emerging novel type of endogenous noncoding RNA that have recently been recognized as powerful regulators participating in various biological processes. However, the physiological roles and molecular mechanisms of circRNAs in pituitary remain largely unclear. Herein, we concentrated on expounding the biological function and molecular mechanism of circRNA in rat pituitary. In this study, we identified a novel circRNA in pituitary tissue, circAkap17b, which was pituitary- and stage-specific. Then, we designed circAkap17b siRNA and constructed an overexpression plasmid to evaluate the effect of loss- and gain-of-circAkap17b function on FSH secretion. Interestingly, silencing circAkakp17b significantly inhibited FSH expression and secretion, while overexpression of circAkap17b enhanced FSH expression and secretion. Furthermore, dual luciferase reporter and RNA immunoprecipitation (RIP) assays confirmed that circAkap17b could serve as miR-7 sponge to regulate target genes. Additionally, miR-7b suppressed FSH expression and secretion by directly targeting Fshb through the dual luciferase reporter and RT-qPCR analysis. Additionally, rescue experiments showed that circAkap17b could regulate FSH secretion in pituitary cells through a circAkap17b-miR-7-Fshb axis. Collectively, we demonstrated that circAkap17b could act as a molecular sponge of miR-7 to upregulate expression of the target gene Fshb and facilitate FSH secretion. These findings provide evidence for a novel regulatory role of circRNAs in pituitary.

Abstract

The pituitary gland functions as a prominent regulator of diverse physiologic processes by secreting multiple hormones. Circular RNAs (circRNAs) are an emerging novel type of endogenous noncoding RNA that have recently been recognized as powerful regulators participating in various biological processes. However, the physiological roles and molecular mechanisms of circRNAs in pituitary remain largely unclear. Herein, we concentrated on expounding the biological function and molecular mechanism of circRNA in rat pituitary. In this study, we identified a novel circRNA in pituitary tissue, circAkap17b, which was pituitary- and stage-specific. Then, we designed circAkap17b siRNA and constructed an overexpression plasmid to evaluate the effect of loss- and gain-of-circAkap17b function on FSH secretion. Interestingly, silencing circAkakp17b significantly inhibited FSH expression and secretion, while overexpression of circAkap17b enhanced FSH expression and secretion. Furthermore, dual luciferase reporter and RNA immunoprecipitation (RIP) assays confirmed that circAkap17b could serve as miR-7 sponge to regulate target genes. Additionally, miR-7b suppressed FSH expression and secretion by directly targeting Fshb through the dual luciferase reporter and RT-qPCR analysis. Additionally, rescue experiments showed that circAkap17b could regulate FSH secretion in pituitary cells through a circAkap17b-miR-7-Fshb axis. Collectively, we demonstrated that circAkap17b could act as a molecular sponge of miR-7 to upregulate expression of the target gene Fshb and facilitate FSH secretion. These findings provide evidence for a novel regulatory role of circRNAs in pituitary.

Introduction

The pituitary gland, as the most important endocrine organ secreting hormones essential for homeostasis, is composed of the adenohypophysis and neurohypophysis and plays a critical role in regulating diverse physiological progression (Fauquier et al. 2002, Hong et al. 2016). Follicle-stimulating hormone (FSH) is synthesized and secreted by the adenohypophyseal portion (Sheng et al. 2018), which contributes to modulating reproductive and development. In females, FSH induces follicular growth and maturation and contributes to LH-triggered ovulation and luteinization (Howles 2000, McGee & Hsueh 2000). In males, FSH stimulates proliferation, development, and maturation of Sertoli cells and accelerates the secretion of androgen-binding protein, inducing and maintaining normal sperm production (Simoni et al. 1999). Furthermore, FSH can directly regulate bone mass, and blocking FSH can not only prevent bone loss, but also reduce body fat accumulation and maintain energy homeostasis (Sun et al. 2006, Liu et al. 2017). Consequently, it is crucial to further address the molecular mechanisms involved in FSH regulation.

Circular RNAs (circRNAs), a novel class of noncoding RNAs, have attracted great research interest and become an important research field in recent years (Qu et al. 2015). However, circRNAs were once thought to be by-products of abnormal RNA splicing errors in the process of gene transcription (Cocquerelle et al. 1993, Hentze & Preiss 2013) until the rapid development of next-generation sequencing technology and bioinformatics analysis, which have transformed circRNAs from waste to treasure (Dong et al. 2017, Guo et al. 2017). In contrast to linear mRNA, circRNAs are characterized by a special covalently closed continuous loop structure with neither a 5′ cap nor a 3′ polyadenylated tail (Chen & Yang 2015, Qu et al. 2015). Due to their cyclization specificity, circRNAs are highly stable, conserved across species, preferentially distributed in the cytoplasm, and show a characteristic tissue or developmental stage-specific pattern (Jeck et al. 2013, Chen 2016). There is accumulating evidence that circRNAs are involved in various physiological processes such as myogenesis and differentiation (Legnini et al. 2017, Li et al. 2019b, c), mammary development and milk synthesis (Liu et al. 2019d), proliferation (Zhou et al. 2019), apoptosis (Guo et al. 2019b), cardiac fibrosis (Zhu et al. 2019), oxidative stress, and inflammation (Chen et al. 2019a). Furthermore, the occurrence of disease is closely associate with the aberrant expression of some circRNAs (Dube et al. 2019, Garikipati et al. 2019, Guo et al. 2019a, Huang et al. 2019a, Liu et al. 2019a Tian et al. 2019, Wu et al. 2019a). Notably in cancers, circRNAs can act as the sponge of miRNAs to affect tumor pathological progression by inhibiting cancer cell proliferation, metastasis, invasion, and epithelial-mesenchymal transition (EMT) (Chen et al. 2018, Huang et al. 2019b, Su et al. 2019, Xue et al. 2019, Yuan et al. 2019, Zhang et al. 2019a,b, Zhen et al. 2019). In addition to a competing endogenous RNA (ceRNA) mechanism, circRNAs can interact with RNA-binding proteins to regulate gene expression, and some circRNAs can encode extraordinary proteins (Legnini et al. 2017, Yang et al. 2017b,c, Wu et al. 2018).

miR-7, with a high degree of pituitary specificity (Amar et al. 2012, He et al. 2018, Wang et al. 2019a), plays a vital role in regulating diverse biological processes such as insulin signaling (Fernandez-de Frutos et al. 2019), neuronal processes (Hu et al. 2019), diet-induced obesity (Gao et al. 2019), EMT, trophoblast invasion (Shih et al. 2019), hormone secretion, and hypogonadism (Ahmed et al. 2017, Zhang et al. 2017a, Wang et al. 2019a). In our previous study, we analyzed the expression profiles of circRNA between mature and homologous immature rat pituitary tissue samples (Han et al. 2019). Based on next-generation sequencing with circRNA and bioinformatics, we constructded circRNA-miRNA-mRNA networks in the rat anterior pituitary. We further predicted and screened differentially expressed rno_circ_0004036 that may act as potential miRNA molecules to regulate rat Fshb expression. However, the role and mechanism of circRNAs in the rat pituitary have not been comprehensively defined.

In this study, rno_circ_004036, designated circAkap17b, was initially identified based on a previous study and bioinformatics. Subsequently, we explored whether circAkap17b could regulate FSH secretion by sponging miR-7 through a series of RT-qPCR, RIP, FISH, and luciferase reporter functional assays. Our results were expected to elucidate potential biological functions of circRNA in the rat pituitary gland and enrich research on the regulation of hormones by ncRNA.

Materials and methods

Ethics statement

The experiment was carried out in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of Jilin University. All rats were fed in the Jilin Provincial Key Laboratory of Animal Model. Animals had free access to food and water that had been disinfected and sterilized. They were housed in a capacious and comfortable room with a 12 h light:12 h darkness cycle. Animals were euthanized through inhalation of superfluous carbon dioxide without pain and suffering. Animal carcasses were treated harmlessly after completion of the experiments.

This study was approved by the Institutional Animal Care and Use Committee of Jilin University (Permit Number: SY201901010).

Animals and cell lines and culture

Sprague–Dawley (SD) rats were purchased from ChangSheng (Liaoning) with a production license and quality certificate. The 6-month-old pituitaries of male rat were used for primary cell culture. The pituitary adenomas cell lines (MMQ, GH3) were obtained from the National Infrastructure of Cell Line Resource (Resource number: 3111C0001CCC000008, 3111C0001CCC000081). In addition, the cultured H9C2 cell line (rat) was used in the RIP experiment. All primary cell and adenomas cell lines were cultured at 37°C in a 5% CO2 atmosphere.

The culture method for rat primary pituitary cells has been clearly described in our previous study (Jeck et al. 2013).

Total RNA and gDNA extraction

Total RNA was isolated using TRIzol reagent (Invitrogen), and gDNA was extracted using the Genomic DNA Isolation Kit (Sangon Biotech, Changchun, China) according to the manufacturer’s recommended protocol.

Nucleic acid preparation and qRT-PCR

cDNA was synthesized using the FastQuant RT Reagent Kit with a gDNA wiper (Tiangen Beijing, China). For PCR, we used PCR Master Mix (2×) (Thermo Fisher Scientific), and the cDNA and gDNA PCR amplification products were examined using 2% agarose gel electrophoresis. The DNA marker used was DL2000 (Vazyme Biotech Co, Ltd). The bands were observed by UV irradiation. Real-time PCR analyses were performed using a SuperReal PreMix Plus Kit (SYBR Green). GAPDH was used in the circRNA and mRNA RT-qPCR analyses as an internal control, while small nuclear U6 served as the internal control in the miRNA quantitation. The relative expression levels were calculated by the 2−ΔΔCt method compared with the Ct values. The sequence information for the primers is listed in additional file (see section on supplementary materials given at the end of this article).

RNase R treatment

For RNase R digestion, the original total RNAs were divided into two equal parts, respectively, one for RNase R treatment (RNase R) and the other for non-treatment (mock). Approximately 2 μg of total RNA was incubated with or without 3 U/μg RNase R (Jisai Biotech, Guangzhou, China) for 30 min at 37°C. Next, the digested RNA was purified according to the RNase R instructions and subsequently subjected to electrophoresis detection or RT-qPCR analysis.

Vector construction and cell transfection

The full-length sequence of circAkap17b was cloned into the original pCD2.1-ciR (7955 bp) plasmid to construct an overexpression plasmid of circAkap17b (pCD2.1-circAkap17b). We selected approximately 200 sequences upstream and downstream of the circRNA binding site of miR-7b to construct the pmirGLO vector, obtaining the pmirGLO-circAkap17b-WT plasmid. The target sites of circAkap17b and miR-7b were then mutated to obtain the pmirGLO-circAkap17b-MUT plasmid. Similarly, a Luciferase reporter vector with the WT or mutant sequence of the 3’ UTR of Fshb was constructed. All construct products were confirmed by sequencing (JinKairui Biotech Co. Ltd., Guangzhou, China). The detailed plasmid information is provided in the Supplementary materials. Overexpression circAkap17b plasmid was transfected into cells using Liposomal 2000 Reagent (Invitrogen by Thermo Fisher Scientific) according to the manufacturer’s instruction. miR-7 miRNA mimics and inhibitors were synthesized and provided by Guangzhou RiboBio Biotech Co. Ltd, and riboFECTTM CP transfection reagent was used for oligo nucleic acid transfection (e.g. siRNA, mimic, inhibitor). Cells were cultured at a density of 70–80% per well in a cell culture plate. The final concentrations of the miRNA mimics, inhibitors, and negative controls were 100 nM. The 100 nM of miRNA mimics, inhibitors, and negative controls were used as reference concentrations.

Fluorescence in situ hybridization (FISH)

circAkap17b probes labeled with Cy3 and Fam-labeled miR-7 probe were designed and synthesized by RiboBio (Guangzhou, China). A Fluorescent In Situ Hybridization Kit (Ribobio Biotech) was used to determine the position of circRNA and miRNA. The hybridization operating procedures and methods were performed according to the manufacturer’s instruction. A fluorescence microscope (Nikon) was used to capture images.

RIP RNA immunoprecipitation assay

RNA immunoprecipitation (RIP) experiments were performed using the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore) according to the manufacturer’s instructions. CST-Argonaute2 (C34C6) Rabbit mAb was purchased for RIP. Approximately 1 x 107 cells were collected and resuspended in RIP lysis buffer with protease and RNase inhibitor. Cell lysates were incubated with 5 mg control mouse IgG or anti-AGO2 antibody-coated beads, respectively, at 4°C overnight. After treatment with proteinase K, immunoprecipitated RNA was extracted by phenol–chloroform extraction.

FSH detection

To avoid the effects of hormones in the serum on the accuracy of the experiment, we culture pituitary cells with serum-free medium instead of DMEM-F12 (15%FBS) 24 h after transfection of plasmid or small RNA. Next, we collected serum-free medium to detect the FSH concentration after approximately 24 h. The Rat FSH ELISA Kit (Melian Biotech Co. Ltd., Shanghai, China) was used to measure the level of FSH secreted into the culture medium.

Luciferase activity assays

We seeded 293T cells in 24-well plates with DMEM containing 10% FBS at a density of 70–80% per well for 24 h before transfection. Approximately 2 μL of lipo2000 transfection reagent was diluted into 50 μL of serum-free DMEM, and the plasmid and mimics were diluted into 50 μL of serum-free DMEM medium (2 μg of total plasmid) and then mixed and incubated at room temperature for 20 min, supplemented with 100 μL of serum-free DMEM. The cells were cotransfected with a mixture in a 37°C incubator. After 5 h, the original medium was discarded and 15% FBS DMEM added to the cell culture. After 48 h of incubation, luciferase activity was assessed using the dual luciferase reporter assay system (Infinite M200 Pro, Tecan) according to the manufacturer’s instructions. The luciferase values were normalized to the corresponding Renilla luciferase values, and then the fold changes were calculated.

Statistical analysis

All statistical analyses were performed using SPSS 19.0 software (IBM, SPSS). The unpaired t-test was used for two-group comparisons, and all data are expressed as the mean ± s.e.m. of three independent experiments. Comparisons among three or multiple groups were performed by one-way ANOVA. Statistical significance was indicated by p values less than 0.05. *P < 0.05, **P < 0.01, ***P < 0.001.

Results

Identification and characterization of circAkap17b in rat pituitary cells

To verify the accuracy of the circRNA sequencing, we respectively detected the expression of rno_circ_0004036 in the pituitary of rats at 15 and 60 days by quantitative RT PCR (qRT-PCR), and we found that the expression of rno_circ_0004036 was significantly lower in mature rat anterior pituitary tissues (60D), which was consistent with the sequencing results (Supplementary material S1). Next, we affirmed the location information of rno_circ_0004036 on the chromosome via sequence analysis and blat of circ_004036. It was derived from Exon 3 to Exon 6 of the A kinase (PRKA) anchor protein 17B (Akap17b) gene (Rat genome (Rnor_6.0), ChrX:123,217,326-123,254,557), herein termed circAkap17b (1204 nt) and initially identified (Fig. 1A). We subsequently designed divergent primers to amplify the back-spliced junction of circAkap17b and analyzed it by agarose gel electrophoresis and Sanger sequencing (Fig. 1B).

Figure 1
Figure 1

Identification and characterization of circAkap17b in rat pituitary cells. (A) Schematic illustration demonstrating the formation of circAkap17b. circAkap17b is produced by the Akap17b gene of exons 3–6. (B) The presence of circAkap17b was validated by RT-PCR and agarose gel electrophoresis, followed by Sanger sequencing. The back-splice junction of circAkap17b is indicated by the black arrow. (C) RT-PCR analysis of the presence of circAkap17b in the pituitary. circAkap17b was amplified by divergent primers from cDNA but not gDNA. (D) The relative circAkap17b levels were analyzed by RT-qPCR after reverse transcription with random hexamer or oligo (dT)18 primers. (E) RT-qPCR analysis of circAkap17b and Akap17b mRNA relative expression after treatment with RNase R. (F) RNA FISH for circAkap17b. circAkap17b probes were labeled with Cy3. Nuclei were stained with DAPI. At least three replicates of each experiment were performed. Mean values and standard deviations (SDs) of the data are shown. One-way ANOVA and the Chi-square test were applied to analyze statistical significance. P < 0.05 was considered significant. *P < 0.05. A full color version of this figure is available at https://doi.org/10.1530/JME-20-0036.

Citation: Journal of Molecular Endocrinology 65, 4; 10.1530/JME-20-0036

To confirm the circular characteristics of circAkap17b, divergent primers and convergent primers were designed to detect circAkap17b and linear Akap17b mRNA in reverse-transcribed RNA (cDNA) and genomic DNA (gDNA). The results demonstrated that the divergent primers could amplify circAkap17b products from cDNA, while no amplification products were observed using gDNA (Fig. 1C). Furthermore, due to the circular structure of circRNA, it lacks a 3' poly(A) tail and cannot be amplified by RT using oligo dT, while random hexamers can reverse transcribe and amplify circRNA. Therefore, this experiment can be used as a method to verify the properties of circRNA. We used random hexamer or oligo (dT)18 primers to carry out RT. We found that the relative expression of circAkap17b was barely detected using oligo (dT)18 primers compared with random hexamer primers (Fig. 1D). To validate the stability of circAkap17b, we detected the expression of circAkap17b after RNase R treatment. As shown in Fig. 1E, circAkap17b was highly resistant to RNase R due to it closed loop structure, while linear Akap17b mRNA was completely digested by RNase R (Fig. 1E). Fluorescence in situ hybridization (FISH) demonstrated that circAkap17b was preferentially expressed in the cytoplasm (Fig. 1F).

Taken together, these results suggest that circAkap17b is indeed a stable cytoplasmic circular molecular.

circAkap17b expression pattern in rat anterior pituitary

To determine the biological implications of circAkap17b in rat pituitary, we measured the expression of circAkap17b in six groups of rat anterior pituitary tissues: 0, 2, 3, 4, 6, and 8 weeks. The results showed that the expression level of circAkap17b was significantly reduced whereas that of circAkap17b as the same after 2 weeks (Fig. 2A). Next, we examined the circAkap17b expression level in different rat tissues. We observed lower circAkap17 expression in the heart compared with other rat tissues. In addition, the pituitary gland appeared to present the same circAkap17b expression level in the other rat tissues (Fig. 2B). We analyzed the expression pattern of circAkap17b in MMQ, GH3 cell lines and primary cells. The results showed that circAkap17b was significantly reduced in hypophysomal cells (Fig. 2C). Subsequently, we investigated the expression pattern of related Akap17b mRNAs in the pituitary. We found that Akap17b expression patterns were roughly consistent with circAkap17b. Akap17b mRNA expression was downregulated in mature rat pituitary (Fig. 2D) and hypophysomal cell lines (GH3, MMQ) (Fig. 2F). Moreover, Akap17b mRNA was also preferentially expressed in the pituitary and spleen (Fig. 2E).

Figure 2
Figure 2

circAkap17b expression pattern in rat anterior pituitary. (A and D) RT-qPCR analysis of circAkap17b and Akap17b mRNA relative expression in different stages. (B and E) The relative expression level of circAkap17b and mAkap17b in mature rat different tissues. (C and F) circAkap17b and mAkap17b expression in pituitary primary cells and GH3, MMQ cell lines. At least three replicates of each experiment were performed. Mean values and s.d. of the data are shown. One-way ANOVA and the Chi-square test were applied to analyze statistical significance. P < 0.05 was considered significant. Different letters (a, b and c) indicate significant differences between groups (P < 0.05).

Citation: Journal of Molecular Endocrinology 65, 4; 10.1530/JME-20-0036

Effects of circAkap17b overexpression/blockade on Fshb transcription

To investigate the regulatory function of circAkap17b on FSH secretion in the pituitary, we designed three short interfering RNAs (si-circAkap17b) that specifically target the back-spliced junction point of circAkap17b (Fig. 3A). A transient transfection method was used to transfect circAkap17b siRNAs into pituitary primary cells, and the siRNA knockdown efficiency was detected. We found that circAkap17b siRNA-1,2 could successfully knock down circAkap17b expression and did not influence the expression of Akap17b linear mRNA (Fig. 3B). Considering that siRNA-1 exhibited a higher knockdown efficiency, we selected circAkap17b siRNA-1 for the following qRT-PCR analysis, which showed that circAkap17b silencing significantly suppressed Fshb expression and FSH secretion (Fig. 3C and D). Next, we constructed an overexpression plasmid of circAkap17b based on the circularization mechanism of circRNA (Fig. 3E). In contrast, stably overexpressing circAkap17b (Fig. 3F) markedly promoted Fshb expression and FSH secretion, as shown in Fig. 3G and H. Together, these results demonstrated that circAkap17b might a potential regulatory role in FSH secretion.

Figure 3
Figure 3

Effects of circAkap17b overexpression/blockade on Fshb transcription. (A) Schematic illustration showing three siRNAs at the back-splicing junction site of circAkap17b. Schematic illustration showing siRNAs and circPRKCI expression vectors. (B) The expressions of circRNA-MYLK were determined by RT-qPCR using pituitary cells were transfected with siRNAs. RT-qPCR (C) and ELISA experiment (D) revealed Fshb expression and FSH secretion after transfection with circAkap17b siRNA-1. (E) Schematic illustration showing the circAkap17b vector structure. (F and H) The Fshb expression and FSH concentration analysis after transfection of the circAkap17b overexpression vector. At least three replicates of each experiment were performed. Mean values and s.d. of the data are shown. One-way ANOVA and the Chi-square test were applied to analyze statistical significance. P < 0.05 was considered significant. *P < 0.05. A full color version of this figure is available at https://doi.org/10.1530/JME-20-0036.

Citation: Journal of Molecular Endocrinology 65, 4; 10.1530/JME-20-0036

circAkap17b may function as a sponge for miR-7

To explore the mechanism of ceRNA in the rat pituitary, we predicted potential target miRNAs of circAkap17b using different public databases including RegRNA2.0 (http://regrna2.mbc.nctu.edu.tw/detection.html), RNA22 (https://cm.jefferson.edu/rna22/Interactive/) and RNAhybrid (https://bibiserv.cebitec.uni-bielefeld.de/rnahybrid/). Filtering restrictions were as follows: (i) total score ≥150; (ii) free energy <−25 kcal/mol; and (iii) number of estimated binding sites ≥ 1. We identified eight candidate miRNAs that could be potential binding targets of circAkap17b and selected miR-7b based on the ranking and Targetscan database (Fig. 4A) (Supplementary material 3A). Subsequently, we found binding sites between miR-7 and circAkap17b using the RNAhybrid program (Fig. 4B). To validate whether miR-7b could interact with circAkap17b, we investigated the correlation between circAkap17b and miR-7 in pituitary cells. The results showed a negative correlation between miRNA and circRNA expression. Silencing/overexpressing circAkap17b could significantly suppress/promote miR-7 expression (Fig. 4C and D). Moreover, circAkap17b expression levels were significantly decreased after transfection of miR-7 mimic but increased after transfection of miR-7 inhibitor (Fig. 4E and F). To further verify the direct binding between circAkap17b and miR-7, AGO2 immunoprecipitation and the dual luciferase reporter assay were performed. As shown in Fig. 4G, miR-7b and circAkap17b were specifically enriched in miR-7b-transfected cells. Furthermore, compared with the control negative, miR-7b transfection significantly reduced luciferase reporter activity when miR-7b mimics were cotransfected into 293T cells with luciferase reporters. We observed no difference in luciferase reporter activity between miR-7b mimics and the negative control after mutation of the predicted binding sites for miR-7b (Fig. 4H). Taken together, these results confirmed that circAkap17b could serve as the miR-7 sponge and indirectly regulate target genes. Construction of pmirGLO-circAkap17-WT and pmirGLO-circAkap17-MUT reporter plasmid and detailed information for the Ago2 RIP are provided in Supplementary material 2 and 3.

Figure 4
Figure 4

circAkap17b may function as a sponge for miR-7. (A) Schematic model showing the putative binding sites of 8 miRNA candidates correlated with circAkap17b (total score ≥150). (B) Binding sites of miR-7a-5p and miR-7b in circAkap17b and mutated binding sites. (C and D) miR-7 expression increased following circAkap17b knockdown by siRNA and decreased after circAkap17b overexpression. (E and F) circAkap17b expression decreased in the presence of miR-7 mimic and increased after miR-7 inhibition. (G) The Ago2 RIP assay showed that Ago2 significantly enriched miR-7 and circAkap17b. (H) Luciferase assays were performed to detect the luciferase activities of 293T cells to confirm the interaction between miR-7 and circAkap17b. At least three replicates of each experiment were performed. Mean values and s.d. of the data are shown. One-way ANOVA and the Chi-square test were applied to analyze statistical significance. P < 0.05 was considered significant. *P < 0.05; **P <0.01, ***P < 0.001. Different letters (a, b and c) indicate significant differences between groups (P < 0.05). A full color version of this figure is available at https://doi.org/10.1530/JME-20-0036.

Citation: Journal of Molecular Endocrinology 65, 4; 10.1530/JME-20-0036

miR-7b inhibits FSH secretion by targeting Fshb

To verify whether miR-7b could target Fshb, we first predicted the interaction site between miR-7b and Fshb using the Targetscan (http://www.targetscan.org/vert_71/) and RNAhybrid programs. Interestingly, miR-7b and miR-7a-5p were consistent with the interaction site of Fshb (Fig. 5A). Next, we cloned the 200 bp upstream and downstream of the Fshb 3′ UTR and miR-7 binding sites between the XhoI (CTCGAG) and SalI (GTCGAC) sites of the pmirGLO plasmid to obtain the pmirGLO-Fshb 3′UTR-WT plasmid. We further mutated the predicted target sequence, forming the pmirGLO-Fshb 3′UTR-MUT reporter plasmid. A sequencing map confirmed that the target sequences were mutated successfully (Fig. 5B). Thereafter, a double luciferase reporter assay was performed to further confirm the interaction between miR-7b and Fshb. We observed significantly reduced luciferase activity after cotransfection of pmirGLO-Fshb-3′UTR WT plasmid and miR-7b mimics into 293T cells compared with the miRNA control negative. No significant difference was detected after cotransfection of the pmirGLO-Fshb-3′UTR mutated (MUT) plasmid and miR-7b into 293T cells (Fig. 5C). Furthermore, the expression levels of Fshb were significantly decreased after 24 h of transfecting miR-7b mimic into primary pituitary cells, while the relative expression of FSHb increased by 1.49-fold after transfection with a miR-7b inhibitor (Fig. 5D). Consistent with the RT-qPCR results, the ELISA showed that miR-7b mimic suppressed FSH secretion, while miR-7b inhibitor facilitated FSH secretion (Fig. 5E). To detect the effect of cells caused by miRNA transfection, we performed flow cytometry to examine cell apoptosis. The results revealed no significant difference among the four groups (NC, miRNA mimic, I-NC, and inhibitor), which indicated that the transfection experiment had no effect on cell viability (Supplementary material 4). Collectively, these results confirmed that miR-7b bound to the FSHb 3′ UTR in the cytoplasm and inhibited FSH secretion.

Figure 5
Figure 5

miR-7b inhibits FSH secretion by targeting Fshb. (A) A schematic drawing showed the predicted binding sites of miR-7 with respect to the Fshb 3’ UTR. (B) A sequencing map showing the target sequence of pmirGLO- Fshb-WT and mutated target sequence from AGAAAGGCAAGTCTTGCCA to GAGGGAATGGACTCCATTG. (C) Luciferase activity assays were performed after transfection of 293T cells with different vectors. (D) The relative Fshb expression levels transfected with miR-7b were analyzed by qRT-PCR. (E) The FSH concentration of supernatant transfected with miR-7b was measured by ELISA. At least three replicates of each experiment were performed. Mean values and standard deviations (SDs) of the data are shown. One-way ANOVA and the Chi-square test were applied to analyze statistical significance. Different letters (a and b) indicate significant differences between groups (P < 0.05). P < 0.05 was considered significant. A full color version of this figure is available at https://doi.org/10.1530/JME-20-0036.

Citation: Journal of Molecular Endocrinology 65, 4; 10.1530/JME-20-0036

circAkap17b facilitates FSH secretion through the miR-7-Fshb pathway

We conducted rescue experiments to confirm that circAkap17b could regulate biological progression through the circRNA-miRNA-mRNA axis. We found that knockdown of circAkap17b inhibited Fshb expression, while it alleviated inhibition efficiency caused by circAkap17b after cotransfection of circAkap17b siRNA and miR-7 inhibitor (Fig. 6A). In contrast, the relative expression level of Fshb and FSH secretion significantly increased after overexpression of circAkap17b, while cotransfection of pCD2.1-circAkap17b plasmid and miR-7 mimic counteracted its facilitating effect (Fig. 6B). In addition, the ELISA results showed that the miR-7 inhibitor alleviated the inhibitory effect caused by si-circAkap17b and facilitating effect caused by overexpression of circAkap17b (Fig. 6C and D). These results indicated that circAkap17b could promote Fshb expression and FSH secretion via the circAkap17b-miR-7-Fshb axis.

Figure 6
Figure 6

circAkap17b facilitates FSH secretion through the miR-7-Fshb pathway. (A) Relative Fshb expression levels transfected with si-circAkap17b or cotransfected with si-circAkap17b and miR-7 inhibitor were analyzed by qRT-PCR. (B) Relative Fshb expression levels transfected with circAkap17b OE plasmid or cotransfected with circAkap17b OE plasmid and miR-7. (C) FSH secretion levels transfected with si-circAkap17b or cotransfected with si-circAkap17b and miR-7 inhibitor were analyzed by ELISA. (D) FSH secretion levels transfected with circAkap17b OE or cotransfected with circAkap17b OE and miR-7. At least three replicates of each experiment were performed. Mean values and s.d. of the data are shown. One-way ANOVA and the Chi-square test were applied to analyze statistical significance. Different letters (a and b) indicate significant differences between groups (P < 0.05). P < 0.05 was considered significant. *P < 0.05.

Citation: Journal of Molecular Endocrinology 65, 4; 10.1530/JME-20-0036

Discussion

circRNAs, generated from precursor mRNA (pre-mRNA) back-splicing of thousands of genes (Li et al. 2018), have received extensive attention worldwide due to their cyclization structural specificity and enrichment in the eukaryotic transcriptome (Qu et al. 2015). circRNAs can be classified into three subclasses based on pathways by which they are generated: exonic circular RNAs (ecRNA), intronic circular RNAs (ciRNA), and exon–intron circular RNAs (EIciRNA) (Quan & Li 2018). The present study demonstrated that a large number of circRNAs were derived from coding exons, whereas they are less commonly derived from intronic or intergenic regions (Memczak et al. 2013). We identified a new circRNA in the rat pituitary gland, circAkap17b, which might be involved in the regulation of animal reproduction. We affirmed location of circAkap17b in the rat chromosome using the UCSC (http://genome.ucsc.edu/) and Ensemble (http://asia.ensembl.org/index.html) genome browser according to the circAkap17b sequence. circAkap17b is an exonic circular RNA consisting of four exons from Akap17b exon 3 to exon 6. A subsequent series of experiments confirmed that circAkap17b was indeed a stable cytoplasmic circular RNA molecule. In the nucleus, exons 3–6 of the Akap17b gene are circularized to form circAkap17b and miR-7 genes produce pri-miR-7 through the action of RNA polymerase II transcribing miRNAs. Pri‐miR-7 are processed into 70–120nt stem‐loop pre-miR-7 via Drosha endonuclease and the DGCR8 complex, and are exported to the cytoplasm through Exportin 5. In the cytoplasm, mature miR-7 are produced from pre‐miRNAs through the RNase endonuclease Dicer. Mature miR-7 are incorporated into the RISC, binding to Fshb and inhibiting Fshb translation. However, cytoplasmic circAkap17b can competitively bind to miR-7 and block the inhibitory effect of miR-7 on FSH, promoting Fshb expression and FSH secretion (Fig. 7).

Figure 7
Figure 7

Schematic illustration of the circAkap17b/miR-7/Fshb axis. Schematic diagram showing the underlying circAkap17b mechanism as a ceRNA for miR-7. A full color version of this figure is available at https://doi.org/10.1530/JME-20-0036.

Citation: Journal of Molecular Endocrinology 65, 4; 10.1530/JME-20-0036

circRNAs are generally expressed at low levels and often exhibit complex stage- and tissue-specific expression patterns (Salzman et al. 2013, Guo et al. 2014, Szabo et al. 2015). circRNAs are more ubiquitously expressed, and they are especially abundant in the mammalian brain (Rybak-Wolf et al. 2015, Szabo et al. 2015, You et al. 2015). In this study, we found that circAkap17b expression was downregulated in the nonsexual stage. Furthermore, circAkap17b showed widespread expression in different rat tissues and was relatively enriched in spleen. These results indicated that circAkap17b might play an important regulatory role in the spleen. We focused on the expression of circAkap17b in the pituitary. Compared with primary pituitary cells, the expression level of circAkap17b was significantly reduced in pituitary tumor cells (GH3, MMQ). In summary, these results show that circAkap17b exhibits stage-specific, tissue-specific, and cell-type-specific expression patterns, suggesting a potentially regulatory role for circAkap17b in the rat pituitary.

Gonadotropin follicle-stimulating hormone (FSH), composed of a common α subunit and a unique β subunit, plays a key role in regulating mammalian reproductive development (Ulloa-Aguirre et al. 1995, Lamminen et al. 2005). Many factors can regulate Fshb expression and influence FSH secretion, such as gonadotropin releasing hormone (GnRH) (Miller et al. 2002), inhibin (De Jong 1988), testosterone (Noguchi et al. 1996), and miRNAs (Han et al. 2017b, 2018b, Wang et al. 2019a). However, the underlying functions and regulatory mechanisms of ncRNAs, particularly circRNAs, in FSH secretion have rarely been reported. Here we found that knockdown/overexpression of circAkap17b could affect Fshb expression and FSH secretion. Our results demonstrated that circAkap17b might regulate the expression levels of Fshb through certain underlying mechanisms.

Endogenous circRNAs exert their functions through distinct mechanisms, such as promoting transcription of their parental gene, competitive binding to miRNA response elements (MREs), interactions with RNA-binding proteins, competition for linear splicing, and protein encoding genes (Ebbesen et al. 2017, Li et al. 2018). There is a popular trend in which the circRNA functions as a miRNA or RNA-binding protein sponge to regulate various physiological activities and diseases. In cancer, an increasing number of studies have described circRNAs functioning as ceRNAs to participate in cancer progression and malignancy (Zhao & Shen 2017, Cui et al. 2018, Qu et al. 2018, Shang et al. 2019). circRNAs act as miRNA sponges to mediate the progression of diverse tumor types, such as non-small cell lung cancer (Chen et al. 2019b), bladder cancer (Li et al. 2017), colorectal cancer (Weng et al. 2017), human oral squamous cell carcinoma (OSCC) (Chen et al. 2017), hepatocellular carcinoma (Yu et al. 2018), gastric cancer (Zhang et al. 2019c), and osteosarcoma (Wu et al. 2019b). The occurrence of tumor cell proliferation, migration, invasion, apoptosis, and other processes are frequently associated with circRNA aberrant expression (Han et al. 2017a, Meng et al. 2017, Yang et al. 2017a, 2019, Zhong et al. 2017, Zeng et al. 2018). In addition to functioning as a ceRNA in cancer, circRNAs play an important role in the regulation of physiological processes. For example, circRNA_0046366 inhibits hepatocellular steatosis through miR-34a-PPAR signaling (Guo et al. 2018), circHIPK3 regulates cell growth by sponging multiple miRNAs (Yuan et al. 2016), circRNA (HRCR) directly targets miR-223 to protect the heart from pathological hypertrophy and heart failure (Wang et al. 2016), and circVMA21 protects against intervertebral disc degeneration through the miR-200c-XIAP pathway (Cheng et al. 2018), among others (Wei et al. 2017, Han et al. 2018a, Liu et al. 2019c). However, the function and regulatory mechanism of circRNA in the pituitary is unclear. In the present study, we found that circAkap17b could act as a miR-7 sponge, as detected via dual luciferase reporter analysis, RIP, and RT-qPCR. These results suggested that circAkap17b could competitively bind to miR-7 and might alleviate the inhibitory effect of miR-7 on target genes.

In comparison to circRNAs, miRNA (miRNAs) are a large family of short (~22 nt) noncoding single-stranded ribonucleic acids (Yates et al. 2013). miRNAs guide the Argonaute protein Ago2 to mRNAs of encoding genes and form a ribonucleoprotein complex called the RNA-induced silencing complex (RISC) (Mohr & Mott 2015, Lu & Rothenberg 2018). In the cytoplasm, miRNAs can suppress the expression level of most mRNAs by recognizing the 3’ UTR sequence of target messenger RNAs to regulate a variety of biological functions (Ambros 2004, Bartel 2009). In addition, certain miRNAs appear to have distinct tissue specificities (e.g. miR-124a, nervous systems; miR-122, liver; miR-206, muscles; miR-126, blood vessels and heart; miR-200a, lateral line system and sensory organs; miR-30c, pronephros (Wienholds et al. 2005, Landgraf et al. 2007)). miR-7 is abundant in the pituitary and brain, and it has been reported to be involved in various physiological processes (Li et al. 2016, Zhang et al. 2017b, Luo et al. 2018) as well as tumor diseases (Comi 1993, Wan et al. 2017, Wu et al. 2017, Xia et al. 2018). In our previous study, we demonstrated that miR-7a-5p can suppress the expression level of FSH by targeting Fshb (Wang et al. 2019a). Additionally, considering that miR-7b is a member of the miR-7 family, we demonstrated that miR-7b also can suppress Fshb expression and FSH secretion by targeting Fshb. These findings have enriched research on the functional mechanism underlying the regulatory role of miRNA in hormone secretion in the pituitary.

Originally identified as star circRNA, Cdr1as/ciRS-7 is highly abundant in the mammalian brain (Hansen et al. 2013). Surprisingly, it strongly alters the free concentration of miR-7, resulting in increased levels of miR-7 targets (Memczak et al. 2013, Piwecka et al. 2017). There is emerging evidence supporting that ciRS-7 serves as a miR-7 sponge to regulate biological processes (Xu et al. 2015, Geng et al. 2016, Li et al. 2019b, Wang et al. 2019b) and cancer progression (Liu et al. 2018, Li et al. 2019a, Liu et al. 2019b, Wang et al. 2019b). circAkap17b, the function of which is consistent with Cdr1as, can act as a ceRNA to regulate gene transcription. In this study, we identified, for the first time, that circAkap17b acts as a miR-7 sponge in rat pituitary.

Taken together, our results illustrate the molecular mechanism through which circAkap17b regulates Fshb expression in rat pituitary at the posttranscriptional level and contributes to pituitary development and reproduction. These results confirm that circAkap17b can base pair with and competitively bind to miR-7 and prevent its enrichment in target mRNA, thus facilitating Fshb transcription and FSH secretion in the rat pituitary. These findings contribute to reproductive development and enrich circRNA potential function.

Supplementary materials

This is linked to the online version of the paper at https://doi.org/10.1530/JME-20-0036.

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) and the Science and Technology Project of Jilin Province (20190201166JC).

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    Figure 1

    Identification and characterization of circAkap17b in rat pituitary cells. (A) Schematic illustration demonstrating the formation of circAkap17b. circAkap17b is produced by the Akap17b gene of exons 3–6. (B) The presence of circAkap17b was validated by RT-PCR and agarose gel electrophoresis, followed by Sanger sequencing. The back-splice junction of circAkap17b is indicated by the black arrow. (C) RT-PCR analysis of the presence of circAkap17b in the pituitary. circAkap17b was amplified by divergent primers from cDNA but not gDNA. (D) The relative circAkap17b levels were analyzed by RT-qPCR after reverse transcription with random hexamer or oligo (dT)18 primers. (E) RT-qPCR analysis of circAkap17b and Akap17b mRNA relative expression after treatment with RNase R. (F) RNA FISH for circAkap17b. circAkap17b probes were labeled with Cy3. Nuclei were stained with DAPI. At least three replicates of each experiment were performed. Mean values and standard deviations (SDs) of the data are shown. One-way ANOVA and the Chi-square test were applied to analyze statistical significance. P < 0.05 was considered significant. *P < 0.05. A full color version of this figure is available at https://doi.org/10.1530/JME-20-0036.

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    Figure 2

    circAkap17b expression pattern in rat anterior pituitary. (A and D) RT-qPCR analysis of circAkap17b and Akap17b mRNA relative expression in different stages. (B and E) The relative expression level of circAkap17b and mAkap17b in mature rat different tissues. (C and F) circAkap17b and mAkap17b expression in pituitary primary cells and GH3, MMQ cell lines. At least three replicates of each experiment were performed. Mean values and s.d. of the data are shown. One-way ANOVA and the Chi-square test were applied to analyze statistical significance. P < 0.05 was considered significant. Different letters (a, b and c) indicate significant differences between groups (P < 0.05).

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    Figure 3

    Effects of circAkap17b overexpression/blockade on Fshb transcription. (A) Schematic illustration showing three siRNAs at the back-splicing junction site of circAkap17b. Schematic illustration showing siRNAs and circPRKCI expression vectors. (B) The expressions of circRNA-MYLK were determined by RT-qPCR using pituitary cells were transfected with siRNAs. RT-qPCR (C) and ELISA experiment (D) revealed Fshb expression and FSH secretion after transfection with circAkap17b siRNA-1. (E) Schematic illustration showing the circAkap17b vector structure. (F and H) The Fshb expression and FSH concentration analysis after transfection of the circAkap17b overexpression vector. At least three replicates of each experiment were performed. Mean values and s.d. of the data are shown. One-way ANOVA and the Chi-square test were applied to analyze statistical significance. P < 0.05 was considered significant. *P < 0.05. A full color version of this figure is available at https://doi.org/10.1530/JME-20-0036.

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    Figure 4

    circAkap17b may function as a sponge for miR-7. (A) Schematic model showing the putative binding sites of 8 miRNA candidates correlated with circAkap17b (total score ≥150). (B) Binding sites of miR-7a-5p and miR-7b in circAkap17b and mutated binding sites. (C and D) miR-7 expression increased following circAkap17b knockdown by siRNA and decreased after circAkap17b overexpression. (E and F) circAkap17b expression decreased in the presence of miR-7 mimic and increased after miR-7 inhibition. (G) The Ago2 RIP assay showed that Ago2 significantly enriched miR-7 and circAkap17b. (H) Luciferase assays were performed to detect the luciferase activities of 293T cells to confirm the interaction between miR-7 and circAkap17b. At least three replicates of each experiment were performed. Mean values and s.d. of the data are shown. One-way ANOVA and the Chi-square test were applied to analyze statistical significance. P < 0.05 was considered significant. *P < 0.05; **P <0.01, ***P < 0.001. Different letters (a, b and c) indicate significant differences between groups (P < 0.05). A full color version of this figure is available at https://doi.org/10.1530/JME-20-0036.

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    Figure 5

    miR-7b inhibits FSH secretion by targeting Fshb. (A) A schematic drawing showed the predicted binding sites of miR-7 with respect to the Fshb 3’ UTR. (B) A sequencing map showing the target sequence of pmirGLO- Fshb-WT and mutated target sequence from AGAAAGGCAAGTCTTGCCA to GAGGGAATGGACTCCATTG. (C) Luciferase activity assays were performed after transfection of 293T cells with different vectors. (D) The relative Fshb expression levels transfected with miR-7b were analyzed by qRT-PCR. (E) The FSH concentration of supernatant transfected with miR-7b was measured by ELISA. At least three replicates of each experiment were performed. Mean values and standard deviations (SDs) of the data are shown. One-way ANOVA and the Chi-square test were applied to analyze statistical significance. Different letters (a and b) indicate significant differences between groups (P < 0.05). P < 0.05 was considered significant. A full color version of this figure is available at https://doi.org/10.1530/JME-20-0036.

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    Figure 6

    circAkap17b facilitates FSH secretion through the miR-7-Fshb pathway. (A) Relative Fshb expression levels transfected with si-circAkap17b or cotransfected with si-circAkap17b and miR-7 inhibitor were analyzed by qRT-PCR. (B) Relative Fshb expression levels transfected with circAkap17b OE plasmid or cotransfected with circAkap17b OE plasmid and miR-7. (C) FSH secretion levels transfected with si-circAkap17b or cotransfected with si-circAkap17b and miR-7 inhibitor were analyzed by ELISA. (D) FSH secretion levels transfected with circAkap17b OE or cotransfected with circAkap17b OE and miR-7. At least three replicates of each experiment were performed. Mean values and s.d. of the data are shown. One-way ANOVA and the Chi-square test were applied to analyze statistical significance. Different letters (a and b) indicate significant differences between groups (P < 0.05). P < 0.05 was considered significant. *P < 0.05.

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    Figure 7

    Schematic illustration of the circAkap17b/miR-7/Fshb axis. Schematic diagram showing the underlying circAkap17b mechanism as a ceRNA for miR-7. A full color version of this figure is available at https://doi.org/10.1530/JME-20-0036.

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