CircEYA3 aggravates intervertebral disc degeneration through miR-196a-5p/EBF1 axis and NF-κB signaling pathway

doi:10.21203/rs.3.rs-3106032/v1

Download PDF

Research Article

CircEYA3 aggravates intervertebral disc degeneration through miR-196a-5p/EBF1 axis and NF-κB signaling pathway

https://doi.org/10.21203/rs.3.rs-3106032/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Background

Intervertebral disc degeneration (IDD) is one of the most frequent causes of disability. Currently, there is an incomplete understanding of the molecular mechanism that underlies the pathogenesis and progression of IDD..Regulatory non-coding RNAs, including circular RNAs (circRNAs) and microRNAs (miRNAs) play an important role in IDD progression. This study aimed to examine the role and molecular mechanism of circEYA3 in IDD.

Methods

In order to gain a deeper understanding of the potential regulatory effects of circRNAs, miRNAs, and mRNAs in IDD, all expression matrices underwent standardized analyses. The involvement of the circEYA3/miR-196a-5p/EBF1 axis in IDD was confirmed through both in vivo and in vitro experiments. The molecular mechanism of EBF1 in IDD was further elucidated through various methods, including Chip-seq analysis and Immunofluorescence staining.

Results

Firstly, a circRNA/miRNA/mRNA network in IDD was constructed according to the standardized analyses of all expression matrixes. We identified differential expression of transcription factor EBF1, circEYA3 and microRNA-196a-5p in normal and IDD NP tissues. Alteration of circEYA3 mediated the degradation of extracellular mechanisms (ECM), apoptosis and proliferation of NP cells (NPC). MiR-196a-5p was identified as a direct regulatory target of circEYA3 and EBF1. Functional analysis showed that circEYA3 and EBF1 modulated ECM degradation, apoptosis and proliferation of NPC, which could be reversed by miR-196a-5p. EBF1 regulated the activity of the NF-кB signaling pathway by activating the promoter region of IKKβ. Collectively, The circEYA3 modulated the progression of IDD and mediated the activity of the NF-kB signaling pathway by regulating the miR196a-5p/EBF1 axis.

Conclusions

Our research proposed a new molecular mechanism for the development of IDD and provided a prospective therapeutic target for IDD.

Intervertebral disc degeneration

circEYA3

miR-196a-5p

EBF1

NF-kB signaling pathway

Intervertebral disc degeneration (IDD), common in the aging population, is responsible for increasing obesity rate worldwide and a leading cause of disability.[1–3] As IDD aggravates over a period of time patients suffer from mechanical pain, loss of myodynamia and reduced quality of life.[4, 5] Surgical therapy places a great burden on wider social economy. However, non-surgical therapeutic options are limited and unstable due to the lack of understanding of the molecular mechanisms of pathogenesis.[6–8] Exacerbation of IDD leads to the reduction in the number of nucleus pulposus cells (NPC) and degradation of the extracellular matrix (ECM).[9–11] Thus, there is an urgent need to elucidate the molecular mechanisms of IDD and understand the role of NPC.

Recent studies have revealed that endogenous non-coding RNAs (ncRNAs), including circular RNAs (circRNAs) and microRNAs (miRNAs), participate in numerous biological and pathological processes.[12–16] Significantly, circRNAs have a complete circular structure with the characteristics of stabilization and biological functional diversity. Several studies have indicated that circRNAs act as sponges for miRNAs and specifically bind to them.[17–19] The RNA-induced silencingcomplex is involved in the development of numerous diseases by regulating the expression of downstream target genes of miRNAs. However, the roles of circRNAs and circRNA/miRNA axis in IDD progression remain unclear.

Early B-cell factor-1 (EBF1) is a key B cell-specific transcription factor involved in numerous diseases and is functionally required for various phenotypic mutations in vitro and in vivo, highlighting its importance in IDD development.[20–22] The nuclear factor kappa beta (NF-κB) signaling pathway is a classical pro-survival signaling pathway in cells. The abnormal phosphorylation of IκBα disassociates NF-κB from IκB and allows NF-κB to translocate to the nucleus, leading to the activation of NF-κB-related genes and pathological changes in cells.[23–25] Unnatural modulation of the NF-κB signaling pathway can result in cell death and abnormal cell function in NPC.[26–28] A previous study revealed that EBF1 is involved in the activation of the NF-κB signaling pathway.[29] Nevertheless, the function of EBF1 in NPC remains relatively unknown.

In the present study, we constructed the regulation network of circRNAs/miRNAs/mRNAs axis by analyzing NPC in IDD using bioinformatics. Further experiments were carried out to elucidate the mechanism of circEYA3 and its downstream miR-196a-5p/EBF1/IKKβ axis.

Bioinformatics Analysis

The Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/) was searched for OA datasets. Microarray datawas collected from GEO datasets (GSE70362, GSE67566 and GSE116726). Raw files of microarray data were downloaded and normalised using a robust multiarray averaging method with ‘affy’ and ‘simpleaffy’ packages of ‘R’ software (www.R-project.org/). The processed gene expression matrix is provided in Supplementary Table S1, Supplementary Table S2, and Supplementary Table S3. The ‘pheatmap’ and ‘ggplot2’ packages of ‘R’ were applied to visualize the data. The enrichment analyses was calculated by datasets of GO (geneontology.org). Cytoscape (cytoscape.org/) was used to map the regulatory network based on the base pairing predicted by Starbase (starbase.sysu.edu.cn/) (Supplementary Table S4). The data of ChIP-seq was collected from Cistrome Data Browser (cistrome.org/db/#/). After searching for the keyword ‘EBF1’, data was analyzed in the current study (Fig. S1).

Clinical samples

Between October 2019 and March 2022, we procured NP tissues from a cohort of patients who had undergone Transforaminal Lumbar Interbody Fusion (n = 33, mean age 66.8 ± 2.1 years). Additionally, we obtained NP tissues from a group of patients who had undergone percutaneous endoscopic lumbar discectomy without IDD (n = 6, mean age 32.7 ± 2.5 years). Following surgical procedures, tissue samples were cryopreserved in liquid nitrogen prior to experimentation. A subset of tissues from healthy patients were subjected to direct digestion for subsequent cell culture.

Real-time PCR Analysis

The total RNA was isolated by RNeasy Mini Kit (74106, Qiagen, Valencia, CA) from NP tissues and cultured NPC. The cDNA was synthesized by QuantiTect Reverse Transcription Kit (205314, Qiagen, Valencia, CA). Quantitative real-time PCR (q-PCR) was done by using QuantiFast SYBR Green PCR Kit (204056, Qiagen, Valencia, CA). Relative RNA expression was calculated using the 2^−ΔΔCTmethod with normalization to U6 small nuclear RNA. Each experiment was performed in triplicate.

NPC culture

We extracted NPC from normal patients’ NP tissues as previously described. Briefly, NP tissue was cut into as small pieces as possible and then digested with 0.1% trypsin (15400054, Gibco Company, USA) for 20 minutes. Thereafter, it was digested with a Type II collagenase (17101015, Gibco Company, USA) in Dulbecco’s modified Eagle’s medium (DMEM, 11965092, Gibco Company, USA) in the water bath at 37°C for 30 minutes. Undigested tissue was filtered using a 40 nm strainer. The NPC were then cultured in DMEM containing 10% fetal bovine serum (30044333, Gibco Company, USA). The second and third passage cultured NPC were used for the experiment.

Cell transfection

The cDNA of EBF1, circEYA3 and IKKβ were cloned into the multiple cloning site of the pcDNA3.1 vector (Invitrogen, USA). MiR-196a-5p mimic, negative control oligonucleotides (miR-NC), miR-196a-5p inhibitor, negative control oligonucleotide (NC inhibitor), small interfering RNA of EBF1, circEYA3 or IKKβ (siEBF1, sicirc-1, sicirc-2, sicirc-3, siIKKβ), scramble siRNA of EBF1 circEYA3 or IKKβ (siSCR) were purchased from RiboBio (Guangzhou, China). Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) was used for the transfection assay in 6-well plate with seeded chondrocytes. Q-PCR was used to detect the expression level of mRNA. Transfection efficiency was detected by fluorescence microscope (OLYPAS, Japan). After 48h, chondrocytes were stimulated with IL-1β (10ng/ml) for the 24h and used for further analysis.

Western blot assay

Cytoplasmic protein and nuclear protein were exacted using a Nuclear and Cytoplasmic Extraction Reagents kit (78835, Thermo Fisher Scientific, USA). The protein concentration was measured by the BCA Assay Kit (23223, Thermo Scientific, USA). The protein was electrophorsed with 10% SDS-PAGE gels, and subsequently transferred to the polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). Specific primary antibodies (ab181602, ab32535, ab39012, ab188570, ab214429, ab32561, ab76429, ab124957, ab32536, ab32511, Abcam, Cambridge, UK) (PA5-61136, Invitrogen, USA) were co-incubated with the membrane overnight at 4°C. Rewarming the membranes for 2h, and then incubated with anti-rabbit IgG (ab97051, Abcam, Cambridge, UK) at 37°C for 2h. ECL Western blot kit (32209, Thermo Fisher Scientific, USA) were used to detect the bands. GAPDH and Lamin B were used as control.

Immunofluorescence (IF) staining

The NPC were cultured in a 12-well plate and stimulated with IL-1β for 24h. Primitively, cells were fixed with 4% paraformaldehyde for 20 min. Subsequently, the cells were treated with 0.2% Triton X-100 for 3 min, and blocking with 5% BSA for 1h. The cells were incubated with primary antibody (Abcam, Cambridge, UK) overnight at 4℃. The chondrocytes were washed with PBS and incubated with goat anti-rabbit IgG (SA00013-4, SA00013-2, Proteintech, China) for 1h at room temperature. Lastly, DAPI(4',6- diamidino-2-phenylindole) (c0065, Solarbio, China) was used for nuclear staining. The fluorescence was observed by with fluorescence microscope (OLYPAS, Japan).

Flow cytometry

PE Annexin V Apoptosis Detection Kit I (559763, BD Pharmingen, USA) was used to determine the apoptotic rate of treated chondrocytes. Subsequently, the results were analyzed with the fluorescence-activated cell sorting (FACS) flow cytometer (BD Biosciences, USA). Each experiment was performed in triplicate.

Dual luciferase reporter gene assay

The pmirGLO Dual-Luciferase miRNA Target Expression Vector was obtained from GenePharma (China). The amplified DNA sequences were cloned to the pmirGLO reporter plasmid to form wild type EBF1 3′-UTR (WT) and mutated EBF1 3′-UTR (MUT) luciferase vectors. The pmiRGLO luciferase reporter vectors of the circEYA3 were constructed as above. Human chondrocytes were plated (5× 10⁴ cells per well) in 24-well plates overnight. Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) was used to transfect the NPC with plasmid and miR-196a-5p mimic or the control. The luciferase activity was measured by the dual-luciferase reporter gene assay system (E1910, Promega, USA) after 48h. Data were shown as the mean value ± SD and each experiment was performed in triplicate.

RNA immunoprecipitation (RIP) assay

Magna RIPTM RNA Binding Protein Immunoprecipitation Kit (17–700, Millipore, USA) was used to conduct the RIP assay. The endogenous miR-196a-5p, combined with circEYA3, was pulled down. Briefly, cultured chondrocytes were collected and resuspended in RIPA buffer (R0020, Solarbio, China). Then incubated the cell extracts with RIP buffer containing magnetic beads conjugated with human anti-Ago2 antibody (07-590, Millipore, USA) or mouse IgG (CBL610, Millipore, USA) negative control overnight at 4°C. The next morning, proteinase K was used to co-incubate with the magnetic beads after washing three times. Subsequently, total RNAs was isolated from the extracts using the TRIzol reagent. The relative enrichment of circEYA3 and miR-196a-5p were determined by RT-qPCR analysis.

Fluorescence in situ hybridization (FISH) assays

The FISH assays were done by FISH kit (F03402, GenePharma, China). In Brief, the probes specific to circEYA3 and miR-196a-5p were added to NPC and then pre-denatured at 78℃ for 5 minutes. Then, hybridization was carried out at 42℃ overnight. DAPI (c0065, Solarbio, China) was used to counterstaining the nuclei with 20 min in darkness. The sample was scanned and photographed under Olympus microscope.

Chromatin Immunoprecipitation (ChIP)

Chip assay was performed using the EZ-Magna ChIP kit (17-10086, EMD Millipore, GER). Human chondrocytes were fixed with 4% paraformaldehyde and incubated with glycine for 10 min to generate DNA–protein cross-links. Then, the cells were lysed with Cell Lysis Buffer and Nuclear Lysis Buffer and sonicated to generate chromatin fragments of 400–800 bp. The lysates were immunoprecipitated with Magnetic Protein A Beads conjugated with EBF1 antibody (ab108369, Abcam, Cambridge, UK) or IgG. Finally, the precipitated DNA was analyzed by PCR.

Rat model of IDD

All animal experimental procedures were ratified by the Animal Ethics Committee of Dalian Medical University. The study was experimented in strict compliance with the Guidelines for the Care and Use of Laboratory Animals published in 2011. The rats were purchased from the Experimental Animal Center of Dalian Medical University. The IDD model was made as described previously. Briefly, the rats were placed in the supine position to locate the lumbar disc behind the bifurcation of the inferior vena cava after anesthesia. The annulus fibrosus is then punctured with a syringe and left for 1 minute. Finally, the rat abdomen was sutured layer by layer. MiR-NC, miR-196a-5p agomir (50µM) and lentivirus (1×10⁹ PFU, 20µl) expressing EBF1 or circEYA3 were injected into the lumbar disc of recipient rats (20µL per joint per rat twice a week for 4 weeks) (n = 12 per group). Eight weeks after surgery, rats lumbar were scanned and used for subsequent experiments.

Histology staining

The rats’ lumbar were fixed in 4% paraformaldehyde for paraffin. Decalcification of tissue using EDTA decalcification solution for two months. The section was pre-treated with drying, deparaffining and rehydrating. Then cut the tissues into 4µm slices. Safranin-O & fast green-stained (G1371, Solarbio, China) sections were used to evaluate the degeneration of the NP and the Morphology of the lumbar.

Statistical analysis

SPSS 17.0 software was used to analyze the data. Data were presented as means ± standard deviation (SD). Student ’s t-test was used to compare the significant difference of two groups. Standard deviation (SD) represented the variation of data values. The one-way analysis of variance (ANOVA) was used to determine the significant difference of multiple groups. Pearson Correlation Coefficient was used to identify the correlation. Each experiment was carried out thrice at least. Statistical significance was defined as P value < 0.05.

CircEYA3 modulated the progression of IDD

Initially, standardized analyses of all expression matrices were performed. The heatmap depicts differential expression of circRNA in IDD (Fig. 1A). CircRNAs with logFC > 1 were selected for further investigation. As shown in Fig. 1B, C, circEYA3 (has_circ_0007895) is formed by reverse splicing from Exon2 to Exon6 of EYA3. CircEYA3 was stably expressed in nucleus pulposus by PCR validation of reverse splicing sequences (Fig. 1D). Results of q-PCR analysis showed that the expression level of circEYA3 in the IDD group was significantly higher than that of normal (Fig. 1E). Three interfering RNAs which were designed to down-regulate circEYA3 in NPC, significantly down-regulated the expression level of circEYA3 (Fig. 1F). Furthermore, the use of the first interfering RNA, sicirc-1 in the following experiment did not affect the expression level of EYA3 in NPC. (Fig. 1G). Western blotting and immunofluorescence showed that circEYA3 promoted interleukin (IL)-1β-induced ECM degradation, inflammatory response, and apoptosis in NPC. In contrast, downregulation of circEYA3 expression reversed the regulatory effects (Fig. 1H, I). Additionally, we determined the proliferative ability of NPC by ki-67 immunofluorescence staining (Fig. 1J). Flow cytometry analysis showed that circEYA3 promoted IL-1β-induced NPC apoptosis (Fig. 1K). These findings indicate that abnormal circEYA3 mediates IDD progression by regulating ECM degradation, proliferation, and apoptosis of NPC.

Construction of circRNA/miRNA/mRNA network in IDD

MiRNAs with logFC > 2 (Fig. 2A) and mRNAs with logFC > 0.6 (Fig. 2C) were selected as genes of the network. Furthermore, gene ontology (GO) analysis was performed to study the differences in cellular components (CC), molecular functions (MF), and biological processes (BP) between the IDD and normal groups based on the variant expression matrices of miRNAs (Fig. 2B), and mRNAs (Fig. 2D). To better understand the relationship between circRNA, miRNA, and mRNAs, a circRNA/miRNA/mRNA network was constructed by combining circRNA/miRNA/mRNA interactions (Fig. 2E).

CircEYA3 regulated the expression of EBF1 by acting as a sponge for miR-196a-5p

Based on the aforementioned results, the circRNA EYA3/miR-196a-5p/EBF1 network was selected for further analysis (Fig. 3A). Results of q-PCR analysis showed significant reduction in the expression level of miR-196a-5p in IDD (Fig. 3B). In contrast, expression level of EBF1 in IDD was high (Fig. 3C). Correlation analysis showed that the expression levels of circEYA3 and EBF1 negatively correlated with that of miR-196a-5p (Fig. 3D). The locations of circEYA3 and miR-196a-5p in the cytoplasm were determined by fluorescence in situ hybridization (FISH) (Fig. 3E). The binding sites between circEYA3 and miR-196a-5p were predicted using the online website (starbase.sysu.edu.cn/) (Fig. 3F). Dual-luciferase reporter gene determination confirmed that circEYA3 was a direct target of miR-196a-5p in NPC (Fig. 3G). Moreover, anti-Argonaute 2 (Ago2) RIP analysis verified the endogenous interaction between circEYA3 and miR-195a-5p (Fig. 3H). Additionally, EBF1 was deemed to be a direct target of miR-196a-5p in NPC through bioinformatics and dual-luciferase reporter gene determination (Fig. 3I, J). The expression of EBF1 in NPC is jointly regulated by circEYA3 and miR-196a-5p (Fig. 3K, L). Moreover, the result of the immunofluorescence analysis was similar to that of western blotting. (Fig. 3M). As described above, circEYA3 sponges miR-196a-5p and indirectly regulates the expression of EBF1 in NPC.

CircEYA3/miR-196a-5p/EBF1 network mediates IDD progression in ECM degradation, proliferation, and apoptosis

Combined analyses of the aforementioned data was used to perform rescue experiments to analyze the function of the circEYA3/miR-196a-5p/EBF1 axis in IDD progression. As shown in Fig. 4A and B, overexpression of circEYA3 and EBF1 promoted ECM degradation, inflammatory response, and apoptosis in NPC, whereas overexpression of miR-196a-5p reversed these effects. Nevertheless, the expression levels of MMP-13, IL-6, and PARPin NPC were reduced due to the depletion of circEYA3 or EBF1 levels, whereas the inhibition of miR-196a-5p effectively blocked this effect. Additionally, miR-196a-5p effectively reversed the circEYA3/EBF1-mediated antiproliferative and apoptotic effects, whereas the sicirc-1/siEBF1-mediated proliferative and antiapoptotic effects were nullified by the miR-196a-5p inhibitor (Fig. 4C, D). Further, the regulatory role of the circEYA3/miR-196a-5p/EBF1 axis in IDD progression was investigated in Sprague-Dawley (SD) rats. Various reagents were injected as treatment into the knee joints of SD rats. The vertebral bodies of the SD rats were harvested by surgery and analysed eight weeks after treatment. As shown in Fig. 4E, complete intervertebral discs were determined by MRI and safranin-O and fast green staining. The group with overexpression of circEYA3/EBF1 indicated severe degeneration of NP tissues, which promoted IDD severity compared with the vector group. These data illustrate that the circEYA3/miR-196a-5p/EBF1 axis mediates ECM degradation, inflammatory response, proliferation, and apoptosis in IDD progression and provides promising therapeutic targets for the rehabilitation of individuals with IDD.

EBF1 promoted the activation of NF-κB signaling pathway

As shown in Fig. 5A and B, EBF1 promoted the phosphorylation of IκBα, which promoted the entrance of p65 into the nucleus. Immunofluorescence analysis provided strong evidence for this (Fig. 5C). To understand the exact mechanism of EBF1 and NF-κB signaling pathway, a motif model illustration was constructed (Fig. 5D). According to the Chip-seq database of the online website, there may be binding sites between EBF1 and the promoter region of the inhibitor of nuclear factor kappa B kinase beta (IKBKB) (Fig. S1). As shown in Fig. 5E and F, based on the transcription direction of IKBKB, we predicted four possible binding sites. The results of the ChIP experiment verified that EBF1 could bind to the promoter region of IKBKB in IL-1β-treated NPC (Fig. 5G, H). Western blotting indicated that EBF1 promoted the expression of IKKβ (Fig. 5I). Furthermore, the upregulated IKKβ promoted the phosphorylation of IκBα and played a role in the entry of p65 into the nucleus. As shown in Fig. 5J, K, p65 was activated and entered the nucleus in transfected-circEYA3 NPC. However, these effects were effectively reversed by co-transfection of NPC with miR-196a-5p. The miR-196a-5p inhibitor abolished the inhibitory effect mediated by sicirc-1 on NF-кB (Fig. 5L). These data show that the circEYA3/miR-196a-5p/EBF1 axis modulates the activation of the NF-кB pathway.

Molecular mechanism of circEYA3 and miR-196a-5p/EBF1/IKKβ axis in NPC

The exact molecular mechanism uncovered by this study is shown in Fig. 7. As a sponge of miR-196a-5p, circEYA3 inhibits its inhibitory effect on EBF1 in NPC. The circEYA3 modulates the progression of IDD and mediates the activity of the NF-κB signaling pathway by regulating the miR196a-5p/EBF1/IKBKB axis.

Intervertebral disc degeneration is an age-related degenerative disease. The degeneration of the nucleus pulposus of the intervertebral disc begins earlier than the degeneration of muscles and bone tissues.[30] Intervertebral disc degeneration can cause a decrease in the height of the intervertebral disc and lead to various spinal diseases as a result of changes in mechanical properties. Currently, low back pain is the fifth most common reason for medical treatment worldwide, and many patients experience great pain. These diseases not only significantly reduce the quality of life of patients but are also a huge burden on medical resources. Therefore, there is an urgent need for research to understand the underlying mechanisms of IDD to provide new therapeutic solutions.

The ncRNAs, including miRNAs, long ncRNAs (lncRNAs), and the recently discovered circRNAs, play an important role in a variety of cellular processes as regulatory elements encoded by the genome[31–33]. Previous studies have shown that the upregulation of circ-4099 in NPC may act as a self-protective mechanism against the development of IDD by regulating the miR-616-5p/Sox9 pathway [34]. Liang et al CircEYA3 promotes the progression of PDAC[35].In the current study, we constructed a circRNA/miRNA/mRNA interaction network in IDD. The circEYA3 was confirmed to be present at high levels in IDD. Through functional experiments, we found that circEYA3 promoted IL-1β-induced ECM degradation and apoptosis while inhibiting NPC proliferation. This indicates that circEYA3 plays a critical role in IDD progression.

CircRNAs are single-stranded RNAs that form covalently closed rings and regulate the activation of downstream pathways through competing endogenous RNAs (CeRNAs)[36–39]. It was previously discovered that circRNA_104670 regulates NPC survival and promotes IDD development by sponging Mir-17-3p[40]. This study found that the expression levels of circEYA3 and EBF1 in NPC are negatively correlated with Mir-196a-5p. Moreover, the results of dual-luciferase analysis proved that circEYA3 could sponge mir-196a-5p, which regulates the expression of downstream genes. Similarly, the binding between mir-196a-5p and EBF1 mRNA specifically inhibited the expression level of EBF1. Furthermore, circEYA3 and mir-196a-5p could co-regulate the expression of EBF1 in NPC. The rescue experiments determined that circEYA3/mir-196a-5p/EBF1 mediated IDD in numerous aspects including ECM degradation, inflammatory response, cell proliferation, and apoptosis.

EBF1 is a transcription factor with a unique DNA-binding structure that binds to the IPT/ TiG-like domain using atypical zinc fingers [41–43]. In this study, EBF1 levels were found to be abnormally high in IDD tissues. Using ChIP assay, we found that EBF1 directly binds to the promoter region of IKBKB to promote its transcription, thus increasing its expression level. IKKβ, the translated protein of IKBKB, promotes the phosphorylation of IκBα, thereby promoting the activation of the NF-κB signaling pathway. Additionally, we verified the regulatory role of circEYA3/mir-196a-5p/EBF1 axis in NF-κB signaling, and the rescue experiments showed that this axis mediates the activity of p65.

In summary, we believe that circEYA3 competitively binds miR-196a-5p to regulate the expression of EBF1, thereby affecting the activity of the NF-κB signaling pathway and IDD progression. The circEYA3/miR-196a-5p/EBF1 axis may provide new therapeutic targets for the non-surgical treatment of IDD.

Availability of data and materials

Datasets used and/or analyzed during the present study can be obtained from the corresponding author upon reasonable request.

Funding Statement

This work was supported by the Natural Science Foundation of Liaoning Province [grant numbers 2020-MS-25]

Acknowledgments

We would like to acknowledge the professors and colleagues who provide suggestions for improvements in present research.

Author information

Authors and Affiliations

Department of Spine Surgery, The Second Hospital of Dalian Medical University, No. 467, Zhongshan Road, Shahekou District, Dalian, 116023 Liaoning, China.

Tianfu Wang, Dehui Song, Yingxia Li, Zhengwei Li & Dapeng Feng.

Contributions

T.Wang. and D.Feng. conceived research ideas and designed experiments. T.Wang. wrote the main manuscript text. D.Song. and Y.Li. prepared figures 1-3. T.Wang. and Z.Li. prepared figures 4-6. D.Feng. edited the manuscript. All authors reviewed the manuscript.

Corresponding author

Correspondence to Dapeng Feng.

Ethics declarations

Ethics approval and consent to participate

The present trials were approved by respective ethics committees and conducted according to the Helsinki Declaration. The protocol was approved by the Ethics Committee of The Second Hospital of Dalian Medical University. The clinical and medical monitoring of the study was continuous throughout the study, with all patients providing written informed consent.

Consent for publication

Not applicable.

Conflicts of Interest

The authors declare that they have no conflict of interest.

Supplementary Materials

Supplementary Table S1. The gene expression matrix of circRNAs.

Supplementary Table S2. The gene expression matrix of miRNAs.

Supplementary Table S3. The gene expression matrix of mRNAs.

Supplementary Table S4. The regulatory network of circRNA/miRNA/mRNA.

Figure S1. The ChIP-seq data of EBF1 from Cistrome Data Browser.

Rashid M, Kristofferzon ML, Nilsson A, Heiden M: Factors associated with return to work among people on work absence due to long-term neck or back pain: a narrative systematic review. BMJ Open 2017, 7(6):e014939.
Gupta N, Rasmussen CL, Hartvigsen J, Mortensen OS, Clays E, Bultmann U, Holtermann A: Physical Activity Advice for Prevention and Rehabilitation of Low Back Pain- Same or Different? A Study on Device-Measured Physical Activity and Register-Based Sickness Absence. J Occup Rehabil 2022, 32(2):284-294.
Watanabe S, Takahashi T, Takeba J, Miura H: Factors associated with the prevalence of back pain and work absence in shipyard workers. BMC Musculoskelet Disord 2018, 19(1):12.
Wynne-Jones G, Cowen J, Jordan JL, Uthman O, Main CJ, Glozier N, van der Windt D: Absence from work and return to work in people with back pain: a systematic review and meta-analysis. Occup Environ Med 2014, 71(6):448-456.
Croft PR, Papageorgiou AC, Ferry S, Thomas E, Jayson MI, Silman AJ: Psychologic distress and low back pain. Evidence from a prospective study in the general population. Spine (Phila Pa 1976) 1995, 20(24):2731-2737.
Yasuoka H, Asazuma T, Nakanishi K, Yoshihara Y, Sugihara A, Tomiya M, Okabayashi T, Nemoto K: Effects of reloading after simulated microgravity on proteoglycan metabolism in the nucleus pulposus and anulus fibrosus of the lumbar intervertebral disc: an experimental study using a rat tail suspension model. Spine (Phila Pa 1976) 2007, 32(25):E734-740.
Roughley PJ: Biology of intervertebral disc aging and degeneration: involvement of the extracellular matrix. Spine (Phila Pa 1976) 2004, 29(23):2691-2699.
Zhao CQ, Wang LM, Jiang LS, Dai LY: The cell biology of intervertebral disc aging and degeneration. Ageing Res Rev 2007, 6(3):247-261.
Setton LA, Chen J: Cell mechanics and mechanobiology in the intervertebral disc. Spine (Phila Pa 1976) 2004, 29(23):2710-2723.
Cui S, Zhang L: circ_001653 Silencing Promotes the Proliferation and ECM Synthesis of NPCs in IDD by Downregulating miR-486-3p-Mediated CEMIP. Mol Ther Nucleic Acids 2020, 20:385-399.
Song J, Chen ZH, Zheng CJ, Song KH, Xu GY, Xu S, Zou F, Ma XS, Wang HL, Jiang JY: Exosome-Transported circRNA_0000253 Competitively Adsorbs MicroRNA-141-5p and Increases IDD. Mol Ther Nucleic Acids 2020, 21:1087-1099.
Salzman J: Circular RNA Expression: Its Potential Regulation and Function. Trends Genet 2016, 32(5):309-316.
Yu B, Zhu Z, Hu T, Lu J, Shen B, Wu T, Guo K, Chaudhary SK, Feng H, Zhao W et al: Construction of a circular RNA-based competing endogenous RNA network to screen biomarkers related to intervertebral disc degeneration. BMC Musculoskelet Disord 2022, 23(1):675.
Meng GD, Xu BS: Circular RNA hsa_circ_0001658 Inhibits Intervertebral Disc Degeneration Development by Regulating hsa-miR-181c-5p/FAS. Comput Math Methods Med 2021, 2021:7853335.
Li Y, Zhou S, Peng P, Wang X, Du L, Huo Z, Xu B: Emerging role of circular RNA in intervertebral disc degeneration: Knowns and unknowns (Review). Mol Med Rep 2020, 22(4):3057-3065.
Mezher M, Abdallah S, Ashekyan O, Shoukari AA, Choubassy H, Kurdi A, Temraz S, Nasr R: Insights on the Biomarker Potential of Exosomal Non-Coding RNAs in Colorectal Cancer: An In Silico Characterization of Related Exosomal lncRNA/circRNA-miRNA-Target Axis. Cells 2023, 12(7).
Guo W, Mu K, Zhang B, Sun C, Zhao L, Li HR, Dong ZY, Cui Q: The circular RNA circ-GRB10 participates in the molecular circuitry inhibiting human intervertebral disc degeneration. Cell Death Dis 2020, 11(8):612.
Guo W, Mu K, Zhang B, Sun C, Zhao L, Dong ZY, Cui Q: The circular RNA FAM169A functions as a competitive endogenous RNA and regulates intervertebral disc degeneration by targeting miR-583 and BTRC. Cell Death Dis 2020, 11(5):315.
Li L, Wang N, Wang J, Li J: Hsa_circRNA_001859 regulates pancreatic cancer progression and epithelial-mesenchymal transition through the miR-21-5p/SLC38A2 pathway. Cancer Biomark 2023, 37(1):39-52.
Treiber N, Treiber T, Zocher G, Grosschedl R: Structure of an Ebf1:DNA complex reveals unusual DNA recognition and structural homology with Rel proteins. Genes Dev 2010, 24(20):2270-2275.
Bohle V, Doring C, Hansmann ML, Kuppers R: Role of early B-cell factor 1 (EBF1) in Hodgkin lymphoma. Leukemia 2013, 27(3):671-679.
Nelson T, Velazquez H, Troiano N, Fretz JA: Early B Cell Factor 1 (EBF1) Regulates Glomerular Development by Controlling Mesangial Maturation and Consequently COX-2 Expression. J Am Soc Nephrol 2019, 30(9):1559-1572.
Zhang GZ, Liu MQ, Chen HW, Wu ZL, Gao YC, Ma ZJ, He XG, Kang XW: NF-kappaB signalling pathways in nucleus pulposus cell function and intervertebral disc degeneration. Cell Prolif 2021, 54(7):e13057.
Shao Z, Wang B, Shi Y, Xie C, Huang C, Chen B, Zhang H, Zeng G, Liang H, Wu Y et al: Senolytic agent Quercetin ameliorates intervertebral disc degeneration via the Nrf2/NF-kappaB axis. Osteoarthritis Cartilage 2021, 29(3):413-422.
Li F, Sun X, Zheng B, Sun K, Zhu J, Ji C, Lin F, Huan L, Luo X, Yan C et al: Arginase II Promotes Intervertebral Disc Degeneration Through Exacerbating Senescence and Apoptosis Caused by Oxidative Stress and Inflammation via the NF-kappaB Pathway. Front Cell Dev Biol 2021, 9:737809.
Shen L, Xiao Y, Wu Q, Liu L, Zhang C, Pan X: TLR4/NF-kappaB axis signaling pathway-dependent up-regulation of miR-625-5p contributes to human intervertebral disc degeneration by targeting COL1A1. Am J Transl Res 2019, 11(3):1374-1388.
Chen T, Li P, Qiu J, Hu W, Li S, Shi H, Qiu X, Huang D, Gao W, Liang A: Aloin Regulates Matrix Metabolism and Apoptosis in Human Nucleus Pulposus Cells via the TAK1/NF-kappaB/NLRP3 Signaling Pathway. Stem Cells Int 2022, 2022:5865011.
Zhang Y, Zhang YS, Li XJ, Huang CR, Yu HJ, Yang XX, Wang BX: Overexpression of miR-150 Inhibits the NF-kappaB Signal Pathway in Intervertebral Disc Degeneration through Targeting P2X7. Cells Tissues Organs 2019, 207(3-4):165-176.
Okosun J, Bodor C, Wang J, Araf S, Yang CY, Pan C, Boller S, Cittaro D, Bozek M, Iqbal S et al: Integrated genomic analysis identifies recurrent mutations and evolution patterns driving the initiation and progression of follicular lymphoma. Nat Genet 2014, 46(2):176-181.
Boxberger JI, Orlansky AS, Sen S, Elliott DM: Reduced nucleus pulposus glycosaminoglycan content alters intervertebral disc dynamic viscoelastic mechanics. J Biomech 2009, 42(12):1941-1946.
Salvatori B, Biscarini S, Morlando M: Non-coding RNAs in Nervous System Development and Disease. Front Cell Dev Biol 2020, 8:273.
Garcia-Padilla C, Aranega A, Franco D: The role of long non-coding RNAs in cardiac development and disease. AIMS Genet 2018, 5(2):124-140.
Fan L, Yao L, Li Z, Wan Z, Sun W, Qiu S, Zhang W, Xiao D, Song L, Yang G et al: Exosome-Based Mitochondrial Delivery of circRNA mSCAR Alleviates Sepsis by Orchestrating Macrophage Activation. Adv Sci (Weinh) 2023, 10(14):e2205692.
Wang H, He P, Pan H, Long J, Wang J, Li Z, Liu H, Jiang W, Zheng Z: Circular RNA circ-4099 is induced by TNF-alpha and regulates ECM synthesis by blocking miR-616-5p inhibition of Sox9 in intervertebral disc degeneration. Exp Mol Med 2018, 50(4):1-14.
Rong Z, Shi S, Tan Z, Xu J, Meng Q, Hua J, Liu J, Zhang B, Wang W, Yu X et al: Circular RNA CircEYA3 induces energy production to promote pancreatic ductal adenocarcinoma progression through the miR-1294/c-Myc axis. Mol Cancer 2021, 20(1):106.
Barrett SP, Salzman J: Circular RNAs: analysis, expression and potential functions. Development 2016, 143(11):1838-1847.
Arnaiz E, Sole C, Manterola L, Iparraguirre L, Otaegui D, Lawrie CH: CircRNAs and cancer: Biomarkers and master regulators. Semin Cancer Biol 2019, 58:90-99.
Xu S, Zhou L, Ponnusamy M, Zhang L, Dong Y, Zhang Y, Wang Q, Liu J, Wang K: A comprehensive review of circRNA: from purification and identification to disease marker potential. PeerJ 2018, 6:e5503.
Xue Q, Huang Y, Chang J, Cheng C, Wang Y, Wang X, Miao C: CircRNA-mediated ceRNA mechanism in Osteoarthritis: Special emphasis on circRNAs in exosomes and the crosstalk of circRNAs and RNA methylation. Biochem Pharmacol 2023, 212:115580.
Song J, Wang HL, Song KH, Ding ZW, Wang HL, Ma XS, Lu FZ, Xia XL, Wang YW, Fei Z et al: CircularRNA_104670 plays a critical role in intervertebral disc degeneration by functioning as a ceRNA. Exp Mol Med 2018, 50(8):1-12.
El-Magd MA, Allen S, McGonnell I, Mansour AA, Otto A, Patel K: Shh regulates chick Ebf1 gene expression in somite development. Gene 2015, 554(1):87-95.
Georgopoulos K: Ebf1 in DNA repair and leukemogenesis. Blood 2015, 125(26):3969-3971.
Vilagos B, Hoffmann M, Souabni A, Sun Q, Werner B, Medvedovic J, Bilic I, Minnich M, Axelsson E, Jaritz M et al: Essential role of EBF1 in the generation and function of distinct mature B cell types. J Exp Med 2012, 209(4):775-792.

No competing interests reported.

FigureS1.jpg
Figure S1. The ChIP-seq data of EBF1 from Cistrome Data Browser.
SupplementaryTableS1.txt
Supplementary Table S1. The gene expression matrix of circRNAs.
SupplementaryTableS2.txt
Supplementary Table S2. The gene expression matrix of miRNAs.
SupplementaryTableS3.txt
Supplementary Table S3. The gene expression matrix of mRNAs.
SupplementaryTableS4.txt
Supplementary Table S4. The regulatory network of circRNA/miRNA/mRNA.

Download PDF

Version 1

posted

You are reading this latest preprint version

CircEYA3 aggravates intervertebral disc degeneration through miR-196a-5p/EBF1 axis and NF-κB signaling pathway

Status:

Version 1

Abstract

Figures

Introduction

Materials and Methods

Bioinformatics Analysis

Clinical samples

Real-time PCR Analysis

NPC culture

Cell transfection

Western blot assay

Immunofluorescence (IF) staining

Flow cytometry

Dual luciferase reporter gene assay

RNA immunoprecipitation (RIP) assay

Fluorescence in situ hybridization (FISH) assays

Chromatin Immunoprecipitation (ChIP)

Rat model of IDD

Histology staining

Statistical analysis

Results

CircEYA3 modulated the progression of IDD

Construction of circRNA/miRNA/mRNA network in IDD

CircEYA3 regulated the expression of EBF1 by acting as a sponge for miR-196a-5p

CircEYA3/miR-196a-5p/EBF1 network mediates IDD progression in ECM degradation, proliferation, and apoptosis

EBF1 promoted the activation of NF-κB signaling pathway

Molecular mechanism of circEYA3 and miR-196a-5p/EBF1/IKKβ axis in NPC

Discussion

Declarations

Funding Statement

Acknowledgments

Conflicts of Interest

Supplementary Materials

References

Additional Declarations

Supplementary Files

Status:

Version 1