CircRNA-9119 suppresses poly I:C induced inflammation in Leydig and Sertoli cells via TLR3 and RIG-I signal pathways
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Circular RNAs (circRNAs) contribute to the epigenetic modulation of pathological and physiological conditions. The understanding of the impact of circRNAs on generation of testicular inflammatory reactions is insufficient.
Our research adopted a poly I:C-triggered testicular inflammation murine model and cell assays.
Microarray data and quantitative evaluation revealed the elevation in the concentrations of Toll-like receptor 3 (TLR3), circRNA-9119, and retinoic acid inducible gene-I (RIG-I) and repression in the levels of miR-136 and miR-26a. Inhibition of circRNA-9119 expression impaired the inflammatory reactions in the separated Leydig and Sertoli cells subjected to poly I:C treatment. CircRNA-9119 suppressed the expression of miR-136 and miR-26a by acting as a microRNA sponge. miR-136 and miR-26a repressed the expression of RIG-I and TLR3 through the expected target region in Leydig and Sertoli cells in vitro. Inhibition of miR-136 and miR-26a expression, at least in part, restored the expression of inflammatory cytokines, which were inhibited upon circRNA-9119 expression silencing. Furthermore, the expression of circRNA-9119 was positively associated with RIG-I and TLR3 mRNA and protein levels. The expression of inflammatory genes triggered by poly I:C treatment was noticeably suppressed after RIG-I and TLR3 knockout.
Our results suggest that circRNA-9119 may serve as a competing endogenous RNA that insulated miR-136 and miR-26a and consequently defended RIG-I and TLR3 mRNAs against miR-26a/miR-136-mediated inhibition of testicular cells. Moreover, RIG-I and TLR3 contributed to the modulation of poly I:C-triggered inflammatory cytokine generation during orchitis in testicular cells.
KeywordsOrchitis Inflammation circRNA-9119 miR-26a miR-136 TLR3 RIG-I
Analysis of variance
Dual-luciferase reporter assay
Extracellular signal-regulated kinase
Intercellular adhesion molecule 1
Interferon regulator factor 3
Monocyte chemoattractant protein-1
Nuclear factor kappa B
Pathogen-associated molecule patterns
Protein phosphatase 2 regulatory subunit B alpha
PATTERN recognition receptors
Prostaglandin-endoperoxide synthase 2
Retinoic acid inducible gene-I
Reverse-transcription polymerase chain reaction
Toll-like receptor 3
Toll-like receptor 4
Tumor necrosis factor
In the process of generation of mammalian sperms, germ cells give rise to numerous innovative antigens after the development of immunocompetence (Yule et al. 1988). In the testis, no damaging immune reactions are stimulated via antigens under physiological circumstances. However, in several pathological circumstances such as trauma, infection, and inflammatory reactions, immunosuppression could be counteracted and may lead to chronic autoimmune orchitis, which may contribute to male infertility (Schuppe et al. 2008). Therefore, the understanding of the etiology of orchitis may assist in the development of innovative strategies to avoid and treat this illness.
Circular RNAs (circRNAs) are regarded as RNAs with loop structures produced via abnormal transcript splicing (Jeck et al. 2013; Rybak-Wolf et al. 2015). CircRNAs are thought to perform crucial functions in various biological reactions, including cell migration, proliferation, and differentiation (Chen et al. 2015; Ebbesen et al. 2016). Few studies have proved the impact as well as mechanisms of circRNAs in orchitis. CircRNAs have been demonstrated to contribute to inflammatory reactions in other cells or tissues. For instance, circRNA-ITCH is displayed in sense orientation to the known protein-coding gene ITCH. ITCH was recognized when ITCH disturbance triggered a lethal autoimmune inflammation (Perry et al. 1998). Ng et al. revealed that the circRNA RasGEF1B acts as a positive modulator of intercellular adhesion molecule 1 (ICAM-1) of the Toll-like receptor 4 (TLR4)/lipopolysaccharide (LPS) pathway, an essential signaling pathway in inflammatory reactions (Ng et al. 2016). The majority of assays have been conducted using cells in steady conditions, which may not highlight the expression of circRNAs in various types of inflammatory reactions (Perry et al. 1998) (Ng et al. 2016). One of these include the transcriptomic reaction of testicular cells after exposure to inflammation triggers. MicroRNAs (miRNAs) modulate gene expression via binding to the 3′-untranslated region (UTR) of the complementary nucleotides of their target mRNAs (Fujii et al. 2018; Hayes et al. 2014). CircRNAs may serve as miRNA sponges and regulate their transcription during various disease development (Rong et al. 2017). Furthermore, miRNAs serve as crucial contributors of orchitis. For instance, it was previously revealed that multiple inflammation-associated miRNAs were inhibited in response to LPS exposure in murine testis models (Parker and Palladino 2017). Moreover, let-7, miR-17, and miR-449a exerted overlapping regulatory effects on male fertility. These miRNAs modulate male germ cell generation and self-renewal (miR-34a/c, let-7, and miR-200c), avoid germ cell death (miR-34c), and target multiple steps of spermatogenesis (miR-34a/c) (Bouhallier et al. 2010; Yao et al. 2015). As a consequence, it is suggested that infections and immune stimulation in testes may induce inflammatory reactions through the modulation of miRNA levels that may be crucial for the normal functioning of testes.
To investigate whether circRNA and miRNA exert crucial role in inflammation and functional development of testes, here we evaluate circRNA expression profiles between non-treated testicular cells (Leydig and Sertoli cells) and testicular cells after treatment with poly I:C, which simulates products of RNA virus replication (Li et al. 2005). Moreover, the association between circRNA and some essential inflammatory sensors, including TLR3 and retinoic acid inducible gene-I (RIG-I), was explored.
C57BL/6 mice were acquired from Laboratory Animal Center of Peking Union Medical College (Beijing, China). RIG-I and TLR3 (RIG-I−/− and TLR3−/−) mice were obtained from Jackson Laboratories (Bar Harbor, ME, USA). Mice were kept in pathogen-free conditions of 12 h light/dark cycle with humidity and temperature in control and food and water ad libitum. All procedures associated with mice were in conformity to guidelines of Care and Use of Laboratory Animals approved via Chinese Council on Animal Care.
Injection of poly I:C into the testis
Poly I:C was locally injected into the testis of mice following the previous report (Zhu et al. 2013). Briefly, pentobarbital sodium (50 mg/kg) was used to anesthetize mice. Testes were removed via operations. One testis was injected with a mixture of 1 μL of lipofectamine RNAiMAX, 0.3 μg of poly I:C, and 10 μL of phosphate-buffered saline (PBS). Testes were rebuilt 6 h subsequent to the injection of poly I:C to evaluate the mRNA and protein expression of inflammatory markers. Every procedure was approved by the Animal Care and Use Committee of the The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University and was in conformity with the guidelines of National Institute of Health.
Male germ cells were separated from mice aged 5 weeks as mentioned before (Shang et al. 2011; Zhu et al. 2013). Briefly, testes of three mice were separated from capsules and incubated for 15 min with 0.5 mg/mL of collagenase type 1 (Sigma) in DMEM at room temperature under gentle shaking condition. An 80-μm copper mesh was used to filter the suspension and to isolate interstitial cells from seminiferous tubules. Leydig cells were treated with 0.125% trypsin for 5 min to allow detachment after 24 h of incubation. Detachment protocol was not applied to testicular macrophages. Macrophages in Leydig cell preparation were less than 5%, as per F4/80 immunostaining specific for macrophages. Nonadherent cells were obtained to culture germ cells. Purity of germ cells was over 95%, as evident from the morphology of nucleus in 4′,6-diamidino-2-phenylindole (DAPI) staining (Scarpino et al. 1998). Leydig cell purity was > 92% based on staining for 3β-hydroxysteroid dehydrogenase, a marker of Leydig cells (Klinefelter et al. 1987). The germ cell purity was > 95% based on cell nuclear morphology after staining with DDX4. The purity of Sertoli cells was > 95% based on the immunostaining for Wilms tumor nuclear protein 1, a marker of Sertoli cells (Sharpe et al. 2003).
For poly I:C treatment, Leydig and Sertoli cells (5 × 105 cells/well) were seeded in 60-mm or 6-well plates and cultured for 24 h. Cells were subjected to serum starvation for 2 h prior to transfection with 2 μg/mL of poly I:C as instructed. Freshly separated germ cells were transfected with 2 μg/mL poly I:C, using Lipofectamine 2000 reagent (Invitrogen), under optimized conditions (Manna et al. 2009).
For gene silencing using small-interfering RNA (siRNA), six-well plates were seeded with Leydig and Sertoli cells cells (2 × 105 cells/well) and the cells were transfected with 50 nM of siRNA using Lipofectamine 2000 reagent (Invitrogen), under optimized conditions (Manna et al. 2009) and incubated for 24 h.
Microarray and quantitative assessment
Separated testicular tissues were shock-frozen at once with the help of liquid nitrogen. The specimens (three poly I:C-supplemented and three non-supplemented) were homogenized with TRIzol reagent (Invitrogen). NanoDrop ND-1000 was used to quantify total RNA in every specimen.
Total RNA in every specimen was amplified and transcribed to fluorescent cRNA using random primers, as per Arraystar Super RNA Labeling protocol (Arraystar Inc.). Arraystar Human circRNA Array was used to hybridized the labeled cRNAs. Agilent G2505C Scanner was used to scan arrays after washing the slides.
Real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR)
Trizol reagent was used for total RNA isolation. DNase 1 without RNase (Invitrogen) was added to the RNA solution to remove any DNA contaminant, and the product was verified with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) PCR amplification and two micrograms of RNA was reverse transcribed. Power SYBR® Green PCR master mix kit (Applied Biosystems, CA, USA) was used to perform PCR on ABI PRISM 7300 real-time cycler (Applied Biosystems). Relative expression levels of transcripts were quantified according to comparative 2−ΔΔCT method mentioned in Applied Biosystems User Bulletin No. 2 (P/N 4303859) (Livak and Schmittgen 2001).
Western blot analysis
The proteins (15 μg/well) were separated on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis gel and the separated bands were electronically transferred onto PVDF membranes. Membranes were blocked for 1 h with Tris-buffered saline containing 5% skim milk at room temperature, then was incubated with primary antibodies at 4 °C. Primary antibodies includes: anti-IRF3 antibody (1:1000, SAB4501564, Sigma), anti-phospho-IRF3 antibody (1:1000, SAB4504031, Sigma), anti-p65 antibody (1:2000, ABE347, Sigma), anti-phospho-p65 antibody (1:1000, SAB4300009, Sigma), anti-Actin antibody (1:5000, A2066, Sigma), anti-TLR3 antibody (1:2500, sc-32,232, Santa Cruz), and anti-RIG-I antibody (1:2500, SAB1305861, Sigma). TBS including 0.1% Tween-20 was used to wash the membranes twice before incubation with secondary antibodies. Enhanced chemiluminescence detection kit (Zhongshan Biotechnology Co.) was used to observe the Ag/Ab complex. β-Actin served as the loading control.
Indirect immunofluorescence assay (IFA)
For IFA of cultivated testicular cells, cells were permeabilized with 0.2% Triton X-100 in PBS for 15 min and then fixed with paraformaldehyde at 25 °C for 30 min. Cells were blocked with 5% normal goat serum and incubated with primary antibodies. Cells were washed twice with PBS and incubated with appropriate secondary antibodies conjugated to fluorescein isothiocyanate (FITC)/tetramethylrhodamine (TRITC) (Zhongshan Biotechnology Co.) for 60 min. Neutral balsam was used to mount cells and cells were visualized under a fluorescence microscope BX-51 (Olympus, Tokyo, Japan).
Dual-luciferase reporter assay (DLRA)
Luciferase reporter assay was performed to verify miR-136 and miR-26a target genes. The wild-type (WT) and mutant 3′-UTR of TLR3, RIG-I, and circRNA-9119 were adopted. Calibration of luminescence (Luc) was carried out based on firefly luciferase sequence. Renilla luciferase was used as a reporter. Cells were transfected with miR mimic or NC and luminescence vectors and incubated for 36 h.
The severity of inflammation was analyzed in PBS-injected and Poly I:C-injected testis. Testis were processed routinely, embedded in paraffin wax and 3–5 μm thick serial sections were prepared. The first slide was stained with hematoxylin and eosin and the subsequent section was used for immunohistochemistry. Two veterinary anatomic pathologists independently and blindly scored the histologic lesion severity as previously described (Clancy et al. 2018).
Data are expressed as mean ± standard deviation (SD). Differences among groups were evaluated with one-way analysis of variance (ANOVA) and two-tailed Student’s t-test. A value of P < 0.05 was considered significant.
Poly I:C triggered the expression of inflammatory cytokines in mouse testes and separated testicular cells
CircRNA expression profile in the testes stimulated with poly I:C
CircRNA-9119 was crucial for inflammatory reactions in testicular cells
CircRNA-9119 modulates miR-26a and miR-136 expression
Inflammatory activity of circRNA-9119 is associated with miR-136 and miR-26a
miR-26a and miR-136 modulates the activities of LPS-triggered chondrocytes by targeting TLR3 and RIG-I, respectively
Both TLR3 and RIG-I modulate circRNA-9119-mediated inflammatory reactions in testicular cells
CircRNAs are gene regulators that participate in multiple physiological functions and pathological reactions, and may serve as miRNA sponges (Hansen et al. 2013). Several circRNAs have been recently identified and shown to exert important regulatory effects as miRNA sponges (Rong et al. 2017). However, most studies on circRNAs have focused on tumorigenesis, and no studies have reported the role of circRNAs in testis-related pathologies. In the present study, we found that circRNA-9119 is a regulatory circRNA involved in the inflammation of testes and serves as a sponge for miR-26a and miR-136 in Sertoli and Leydig cells in response to poly I:C stimulation.
Both miR-26a and miR-136 are conserved miRNAs that play an important role in cell differentiation, development, and growth (Zhang et al. 2015; Yang et al. 2012). It has been reported that miR-26a modulates cell proliferation by mediating TLR9 expression (Jiang et al. 2014b) and regulates the survival of various cells by targeting SODD gene (Reuland et al. 2013). miR-26a also reversely modulates TLR3 expression in murine macrophages and attenuates pristine-triggered arthritis (Jiang et al. 2014a). miR-136 exerts a regulatory role in non-small cell lung cancer cells by stimulating the extracellular signal-regulated kinase (ERK)1/2 pathway through protein phosphatase 2 regulatory subunit B alpha (PPP2R2A) (Shen et al. 2014). Overexpression of miR-136 results in the inhibition of metastasis-associated traits in lung adenocarcinoma cells via Smad2 and Smad3 (Yang et al. 2014). In addition, miR-136 serves as a modulator of RIG-I-mediated innate immunity, which counteracts influenza A virus H5N1 replication in A549 cells (Zhao et al. 2015). Together, these researches indicate that miR-26a and miR-136 possess important functions in different biological and pathological processes. We confirmed the low abundance of miR-26a and miR-136 and high levels of circRNA-9119 in inflammatory testicular cells. qRT-PCR results further indicated that both miRNAs were downregulated by circRNA-9119. Furthermore, miR-26a and miR-136 expression inhibition reversed the pro-inflammatory effects of circRNA-9119. Thus, we hypothesize that circRNA-9119 may contribute to the modulation of orchitis by regulating the expression levels of miR-26a and miR-136 in testicle cell lines in vitro.
Testes are vulnerable to multiple pathogens arising from blood or those from the genitourinary tract (Zhao et al. 2014). Testicular cells must get over the immune privilege aiming at exerting a proper and efficient local reaction counteracting invading pathogens. This reaction occurs through appropriate antimicrobial innate immune reactions (Zhao et al. 2014). Innate immunity of testicles is extremely crucial during impaired systemic immunity (Zhao et al. 2014). Macrophages are a major population of cells that represent approximately 20% of the total testicular interstitial cells in mice under physiological conditions (Hedger 2002). The macrophages have an important function in regulating the development and steroidogenesis of Leydig cells in rats (Hutson 2006). Macrophages belong to the family of antigen-presenting cells. However, testicular macrophages exhibit relatively low inflammatory responses and high immunosuppressive properties compared with the macrophages located in other tissues (Kern et al. 1995). Therefore, we did not choose macrophages in the present study. But its molecular mechanism on inflammation will be researched in future. Both Sertoli cells and Leydig cells are different functional testicular cell types. But their manifestation during testicular innate immune was reported to be regulated by TLR and RLR similarly (Shang et al. 2011; Riccioli et al. 2006; Yoneyama et al. 2005; Wu et al. 2016). Pattern recognition receptors (PRRs) are a group of receptors that are stimulated via conserved structures of microorganisms, also called as pathogen-associated molecule patterns (PAMPs). TLRs are well investigated and 13 TLRs have been identified in mammals. As a double-stranded RNA sensor in the cytoplasm, RIG-I-like receptors comprise two functional parts, MDA5 and RIG-I. Murine Leydig cells exhibit expression and activities of TLR3 and TLR4, while RIG-I and MDA5 expression is frequent in mouse Leydig cells. MDA5 was also identified in spermatids (Zhu et al. 2013). Poly I:C stimulates innate immune reactions in Sertoli and Leydig cells via TLR3 and RIG-I-mediated pathways (Zhu et al. 2013; Zhang et al. 2013; Palladino et al. 2007). Our research demonstrates that poly I:C injection and transfection drastically upregulated the expression of pro-inflammatory cytokines, chemokines, and IFNs not only in Leydig but also in Sertoli cells. Moreover, stimulation of testicular cells derived from TLR3−/− and RIG−/− mice with poly I:C resulted in the reduction in the expression of inflammatory cytokines as compared with the cells from the WT mice, suggestive of the important contribution of TLR3 and RIG-I in the initiation of testicular immune reaction. We confirmed that the expression miR-26a and miR-136 negatively correlated with the concentrations of TLR3 and RIG-I, which positively correlated with circRNA-9119 level in the inflammatory testicular cells. DLRA results indicated that the 3′-UTR of TLR3 and RIG-I was targeted by miR-26a and miR-136, respectively. Therefore, TLR3 and RIG-I upregulation may contribute to the pro-inflammatory activities of circRNA-9119 in orchitis.
In conclusion, we demonstrate the pro-inflammatory function of circRNA-9119 in the testes and isolated Sertoli and Leydig cells in response to poly I:C stimulation. circRNA-9119 participates in testicular immune reaction that counteracts mimetic viral infections. circRNA-9119 acted as a sponge for both miR-26a and miR-136, which in turn target TLR3 and RIG-I, respectively, the two essential molecules in orchitis. The present study results provide an evidence of the significance of the inflammation-related circRNA-9119-miR-26a/miR-136-TLR3/RIG-I axis for innate immune reactions of testes.
Thanks For The Sustentation Fund from Pediatric Surgery Department, the Second Affiliated Hospital of Wenzhou Medical University (Provincial Key Discipline of Zhejiang Province).
Study design/planning: LQ, JL, XX. Data collection/entry: JL, XX. Data analysis/statistics: XX. Data interpretation: LQ. Preparation of manuscript: JL. Literature analysis/search: LQ, JL, XX. All authors read and approved the final manuscript.
Ethics approval and consent to participate
Every procedure was approved by the Animal Care and Use Committee of the The Second Affiliated Hospital and Yuying Children’s Hospital of Wenzhou Medical University and was in conformity with the guidelines of National Institute of Health.
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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