Circular RNA GRB10 as a competitive endogenous RNA regulating nucleus pulposus cells death in degenerative intervertebral disk

Guo, Wei; Zhang, Bin; Mu, Kun; Feng, Shi-Qing; Dong, Zhan-Yin; Ning, Guang-Zhi; Li, Hao-Ran; Liu, Shen; Zhao, Ling; Li, Yan; Yu, Bing-Bing; Duan, Hui-Quan; Sun, Chao; Li, Yong-**

doi:10.1038/s41419-017-0232-z

Circular RNA GRB10 as a competitive endogenous RNA regulating nucleus pulposus cells death in degenerative intervertebral disk

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Published: 23 February 2018

Volume 9, article number 319, (2018)
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Cell Death & Disease

Circular RNA GRB10 as a competitive endogenous RNA regulating nucleus pulposus cells death in degenerative intervertebral disk

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Wei Guo¹^na1^na2,
Bin Zhang²^na1,
Kun Mu³,
Shi-Qing Feng²,
Zhan-Yin Dong¹,
Guang-Zhi Ning²,
Hao-Ran Li¹,
Shen Liu²,
Ling Zhao¹,
Yan Li²,
Bing-Bing Yu²,
Hui-Quan Duan²,
Chao Sun² &
…
Yong-** Li²

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Abstract

Intervertebral disc degeneration (IDD) is an important factor leading to low back pain, but the underlying mechanisms remain poorly understood. Compared with normal nucleus pulposus (NP) tissues, the expression of circ-GRB10 was downregulated in IDD. Furthermore, overexpression of circ-GRB10 inhibited NP cell apoptosis. circ-GRB10 could sequester miR-328-5p, which could potentially lead to the upregulation of target genes related to cell proliferation via the ErbB pathway. In conclusion, the present study revealed that circ-GRB10/miR-328-5p/ERBB2 signaling pathway is involved in IDD development, suggesting that circ-GRB10 might be a novel therapeutic target for IDD.

The circular RNA circ-GRB10 participates in the molecular circuitry inhibiting human intervertebral disc degeneration

Article Open access 13 August 2020

The circular RNA FAM169A functions as a competitive endogenous RNA and regulates intervertebral disc degeneration by targeting miR-583 and BTRC

Article Open access 04 May 2020

Hsa_circ_0134111 promotes intervertebral disc degeneration via sponging miR-578

Article Open access 08 February 2022

Introduction

Intervertebral disc degeneration (IDD) is characterized by extracellular matrix breakdown and abnormal matrix synthesis leading to reduced hydration, loss of disc height, and decreased potential to absorb load^1,2. It is considered as the predominant cause for chronic low back pain and spine-related disease, leading to economic and social burden worldwide³. It is estimated that as much as 84% of the population suffers from low back pain at some point in their lifetime, whereas 10% are chronically disabled⁴. Nevertheless, current strategies for IDD treatment is hampered by an incomplete understanding of its pathogenesis. Nowadays, IDD treatment is limited to symptomatic interventions, which do not adequately improve outcomes since no disease-modifying drugs are available⁵. Consequently, the clinical management of diseases related to IDD remains severely limited. Therefore, unveiling the pathophysiology and molecular mechanisms underlying IDD is imperative for develo** novel therapeutic approaches.

The intervertebral disc is the largest avascular structure in the body⁶. Studies have shown that blood vessels do not enter the nucleus pulposus (NP) tissue, and NP cells in the center of an adult disk can be up to 8 mm away from the nearest blood vessel⁷. Accumulating evidence indicated that a variety of cellular events are dysregulated in the progression of IDD, ranging from NP cell apoptosis to cytokine expression^{7,8,9,Full size image}

Circ-GRB10 regulates the expression of ERBB2

Bioinformatics prediction showed that ERBB2 was the target of miR-328-5p, as predicted by three microRNA target gene databases (DIANA, miRDB, and miRTarbase) (Fig. 6a). To determine whether circ-GRB10 could regulate ERBB2 expression through miR-328-5p, we analyzed the mRNA and protein levels of ERBB2 in NP cells isolated from normal tissues. The expression of circ-GRB10 was attenuated after siRNA silencing (P < 0.001) and increased by circ-GRB10 overexpression (P < 0.001) (Supplemental Fig. 3a). The expression of miR-328-5p was upregulated after miR-328-5p mimic addition (P < 0.001) and downregulated after miR-328-5p inhibitor treatment (P < 0.01) (Supplemental Fig. 3b). Moreover, the expression of miR-328-5p was altered after circ-GRB10 silencing or overexpression (Supplemental Fig. 3c). As shown in Fig. 6b, c, the expression of ERBB2 was inhibited by miR-328-5p mimics and could be reversed by circ-GRB10 overexpression at both the mRNA and protein levels. Circ-GRB10 silencing resulted in a reduction of ERBB2 at both the mRNA and protein levels, as well as in the downregulation of miR-328-5p. Meanwhile, the expression of ERBB2 was elevated in NP cells treated with a miR-328-5p inhibitor (Fig. 6d, e). These results indicated that circ-GRB10 regulates ERBB2 through miR-328-5p.

**Fig. 6: The circ-GRB10/miR-328-5p axis is critical for cell function.**

Circ-GRB10-miR-328-5p-ERBB2 regulatory loop is critical for NP cells survival

As shown in Fig. 7a, b, the cell death rate in IDD NP tissues was significantly higher than in normal NP cells, as indicated by the TUNEL assay(P < 0.01). We next checked whether circ-GRB10 participated in NP cell apoptosis and survival. After nutrition deprivation, the constitutive expression of circ-GRB10 and ERBB2 was downregulated compared to normal cells (all P < 0.01, Fig. 8a, b). The apoptosis in NPs cells of the circ-GRB10 overexpression group was significantly suppressed compared with the mock circRNA vector group (P < 0.001); nevertheless, circ-GRB10 knockdown markedly increased the NP cell apoptosis rate (P < 0.001); circ-GRB10 could significantly stimulate NP cells survival under nutrient deprivation conditions (P < 0.01) (Fig. 8h). Moreover, the expression of apoptosis-related proteins was measured. As shown in Fig. 8k, i, the expression of cleaved caspase-3 was upregulated after circ-GRB10 silencing and by nutrient deprivation (both P < 0.001), and attenuated by circ-GRB10 overexpression (P < 0.05). Figure 8m, n shows that the expression of cleaved PARP1 was upregulated after circ-GRB10 silencing and by nutrient deprivation (both P < 0.001), and attenuated by circ-GRB10 overexpression (P < 0.01). The LC3-II/LC3-I ratio was increased after circ-GRB10 silencing and by nutrient deprivation (both P < 0.001), and decreased by circ-GRB10 overexpression (P < 0.01) (Fig. 8o, p). Moreover, Fig. 8q, r shows that the expression of p62 was upregulated after circ-GRB10 silencing and by nutrient deprivation (both P < 0.001), and attenuated by circ-GRB10 overexpression (P < 0.001).

**Fig. 8: NP cells apoptosis rate in each group after transfection.**

Discussion

The pathogenesis of IDD remains poorly understood. In this study, bioinformatics identified 104 differentially expressed circRNAs and 27 differentially expressed miRNAs in degenerative NP cells. Among them, circ-GRB10 could be a circRNA playing important roles in IDD. Dysregulated circ-GRB10 was a ceRNA-regulating ERBB2 expression by sponging miR-328-5p, which mediates the death of NP cells in IDD. Moreover, circ-GRB10 favored the survival of NP cells under nutrient deprivation conditions by upregulating the expression of ERBB2 in NP cells. These findings strongly suggest that circ-GRB10 play an essential role in the pathogenesis of IDD.

Intervertebral disc degeneration changes are clinically important as they are associated with back pain and morbidity. Cell death is a fundamental biological process that can be separated into three classes: apoptosis, autophagy, and necrosis²⁶. The deregulation of cell death is associated with the etiology, pathogenesis, and treatment of many diseases^18,27,28 such as cancer, Alzheimer’s disease, heart diseases, and Parkinson’s disease^29,30. Over the past years, increasing evidence has indicated that NP cell death contributes to spinal degenerative diseases and IDD^7,10,31.

The intervertebral disc is avascular and the NP cells depend on diffusion from blood vessels at the disc’s margins to supply the nutrients essential for cellular activity and viability and to remove metabolic wastes³². Loss of nutrient supply can lead to NP cell death, loss of matrix production, and matrix degradation, and consequently to disc degeneration³². Nutrient deprivation can increase autophagy, which promotes apoptosis or lead to “autophagic cell death”^32,33,34. In addition to nutrient deprivation, the regulation of autophagy involves multiple pathways and cellular stresses³⁵. Autophagy itself is a protective process through which the cells recycle cellular components to ensure their survival, but excessive autophagy triggers the apoptosis of NP cells under conditions of nutrient deprivation³⁴. In addition, nutrient deprivation may induce autophagosome formation³⁶ and NP cell autophagy by increasing the ratios of LC3-II/LC3-I and beclin-1/β-actin³⁴. Two well-characterized signaling cascades, including the target of rapamycin (TOR) and Ras-cAMP-dependent protein kinase A (PKA) pathways, sense nutrient status³⁵. TOR regulates nutrient sensing, cell growth, and autophagy³⁷. TOR activates downstream proteins, including Akt kinase (also known as protein kinase B), phosphoinositide-3 kinase (PI3K), and growth factor receptor³⁸. The inhibition of autophagy significantly decreases the rate of apoptosis in cells disrupted by H₂O₂³⁹. Hence, controlling the autophagy response in NP cells could enhance cell survival and probably delays disc degeneration.

The present study showed that circ-GRB10 overexpression significantly decreased apoptosis of NP cells under nutrient deprivation environment, suggesting that circ-GRB10 is beneficial for NP cells survival under nutrient deprivation conditions. In addition, the results suggested that apoptosis and autophagy are also involved in the pathogenesis of IDD, as indicated by alterations of cleaved caspase3, cleaved PARP1, LC3II/LC3I, and p62. Accordingly, circ-GRB10 knockdown increased the apoptosis rate of NP cells, and circ-GRB10 overexpression inhibited the death of NP cells in vitro.

A previous study demonstrated that ERBB2 inhibits autophagy via the formation of a complex with Beclin 1, which is a key regulator of autophagy⁴⁰. In addition, lapatinib, a dual tyrosine kinase inhibitor of EGFR and ERBB2, induced autophagic cell death in breast cancer cells overexpressing ERBB2⁴⁰, human hepatoma cells⁴¹, and acute myeloblastic leukemia⁴². In the present study, circ-GRB10 could regulate ERBB2 expression by sponging miR-328-5p. These findings indicated that dysregulation of circ-GRB10 may contribute to IDD progression by regulating NP cell apoptosis/survival. This could be a potentially novel therapeutic target for IDD.

Unfortunately, whether these results are due to circ-GRB10 acting as a sponge, to circ-GRB10 enhancing the degradation of miR-328-5p, or to decreased miR-328-5p expression remains to be elucidated. To do so, co-expression of both the circRNA and miRNA will be performed in the future⁴³. Nevertheless, the Pearson correlation analysis showed a fairly good anti-correlation between circ-GRB10 and miR-328-5p, highlighting their relationship but without revealing its nature. In addition, the FISH analysis revealed that both were localized in the same cellular compartment (cytoplasm), further supporting this correlation.

In conclusion, the present study demonstrated that circ-GRB10 facilitated the survival of NP cells during nutrient deprivation by upregulating ERBB2 expression through inactivating miR-328-5p, and possibly through a pathway involving mTOR. These findings may be valuable to better understand the molecular mechanisms involving the survival and death of the intervertebral disk cells, and suggested a promising therapeutic target for IDD treatment.

Materials and methods

Ethics statement

This study was approved and supervised by the Ethics Committee of Tian** Medical University General Hospital and Hebei Province Cangzhou Hospital of Integrated Traditional and Western Medicine. Human NP tissues were obtained from patients undergoing surgery at The Tian** Medical University General Hospital and Hebei Province Cangzhou Hospital of Integrated Traditional and Western Medicine. Written informed consents were obtained from all patients for the use of their tissues for research purposes.

Clinical specimens

Human lumbar degenerative NP specimens were obtained from 20 patients with IDD undergoing discectomy. The control samples were taken from 20 age- and sex-matched patients with fresh traumatic lumbar fracture who underwent anterior decompressive surgery because of neurological deficits. Table 2 presents the characteristics of the patients.

Table 2 Clinical features of study population

Full size table

Microarray data

CircRNA expression data set (GSE67566) and miRNA expression data set (GSE63492) were downloaded from the Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo)¹⁶. There were five human NP samples derived from patients with IDD and five samples derived from cadaveric disc as normal control. The platform was the GPL19978 Agilent-069978 Arraystar Human CircRNA microarray V1 for circRNA and GPL19449 Exiqon miRCURY LNA microRNA Array for miRNA. Probe annotation files were also acquired.

Preprocessing and differential analysis

Raw data were converted into recognizable format with the package affy of R. Missing values were imputed by a method based on the K nearest neighbors (KNNs). The KNN-based method selects genes with expression profiles similar to the gene of interest to impute missing values⁴⁴. After background correction and data normalization with the median method⁴⁵, differential analysis was performed using the limma package between degeneration samples and controls. The first is the design matrix, which indicates which RNA samples have been applied to each array. The second is the contrast matrix, which specifies which comparisons you would like to make between the RNA samples. For statistical analysis and assessing differential expression, limma uses an empirical Bayesian method to moderate the standard errors of the estimated log-fold changes. The basic statistic used for significance analysis is the moderated t-statistic, which is computed for each probe and for each contrast. Moderated t-statistics lead to P values in the same way that ordinary t-statistics do, except that the degrees of freedom are increased to reflect the greater reliability associated with the smoothed standard errors. Limma provides the topTable and decideTests functions, which summarize the results of the linear model, perform hypothesis tests, and adjust the P values for multiple testing. Results include (log) fold changes, standard errors, t-statistics, and P values⁴⁶. |Log(fold change)| >2 and adj. P value <0.05 were set as the cutoffs to identify differentially expressed RNAs.

Annotation for circRNA–miRNA interaction

The circRNA–miRNA interactions were predicted using miRNA target prediction based on the TargetScan⁴⁷ and miRanda⁴⁸ databases. To establish the circRNA–miRNA network, we searched MREs on circRNAs, then selected the miRNAs according to the seed match sequences. The interaction networks between circRNAs and predicted miRNAs were then visualized with Cytoscape.

Quantitative real-time RT-PCR

After RNA extraction, the M-MLV reverse transcriptase (Invitrogen, Carlsbad, CA) was used for complementary DNA (cDNA) synthesis, according to the manufacturer’s instructions. The expression of circ-GRB10 was evaluated by quantitative PCR using the SYBR Green assay. Specific divergent primers were designed to amplify the circular transcripts (forward: 5′-GCCGCCGCAAAGCAGATATTC-3′; reverse: 5′-ACAGACTCCAGCAGGGTCAG-3′). All primers used in this study are listed in Table 3. PCR was performed in a 10-μL reaction volume, including 2 μL of cDNA, 5 μL of 2× Master Mix, 0.5 μL of forward primer (10 μM), 0.5 μL of reverse primer (10 μM), and 2 μL of double-distilled water. The reaction was set at 95 °C for 10 min for pre-denaturation, then at 95 °C for 10 s and at 60 °C for 60 s, repeated for 40 cycles. GAPDH was used as a reference. The target and reference were amplified in triplicate. The relative expression of each circRNA was calculated using the 2^−△△Ct method⁴⁹.

Table 3 Sequences of primers for qRT-PCR and siRNA-related sequence

Full size table

Isolation and culture of human NP cells

Tissue specimens were washed twice with phosphate-buffered saline (PBS). Nucleus pulposus was separated from the annulus fibrosus using a stereotactic microscope and cut into pieces (2–3 mm³). Nucleus pulposus cells were released from tissues by incubation with 0.25 mg/mL of type II collagenase (Invitrogen, Carlsbad, CA) for 12 h at 37 °C in Dulbecco’s modified Eagle medium (DMEM/F12; GIBCO, Grand Island, NY). After isolation, NP cells were resuspended in DMEM/F12 containing 10% FBS (GIBCO), 100 mg/mL streptomycin, 100 U/mL penicillin, and 1% l-glutamine, and then incubated at 37 °C in a humidified atmosphere with 5% CO₂. The confluent cells were detached by trypsinization, seeded into 35-mm tissue culture dishes in complete culture medium (DMEM/F12 supplemented with 10% FBS, 100 mg/mL streptomycin, and 100 U/mL penicillin) in a 37 °C, 5% CO₂ environment. The medium was changed every 2 days. The cells at the second passage were used for subsequent experiments.

siRNA and circ-GRB10 overexpression plasmid construction

According to the circRNA sequences of circ-GRB10 (hsa_circ_0080210) in circBase, the siRNA of circ-GRB10 (each siRNA had three pairs of sequences) and negative controls (NCs) were designed and synthesized by Guangzhou Geenseed Biotech Co., Guangzhou, China. To induce GRB10 transcript formation in vitro by nonlinear splicing, the circ-GRB10 overexpression vector was constructed. The front and back circular frames were synthesized and added to pLCDH-ciR for the circularization of the transcripts. As previously reported, the front circular frame contains the endogenous flanking genomic sequence with the EcoR I restriction site, and the back circular frame contains part of the inverted upstream sequence with the BamH I restriction site⁵⁰. The cDNA encoding circ-GRB10 in Hela cells was amplified using the primers forward: cgGAATTCTGAAATATGCTATCTTACAGATATTCTGGAGGAAGGTGTGA and reverse: cgGGATCCTCAAGAAAAAATATATTCACCTGCTTTGCGGCGGCCTGGCTCGGAGGTAA. The 637-bp target fragment contains, in order, the EcoR I site, splice acceptor AG, circ-GRB10 sequence, splice donor GT, and BamH I site. Then, the amplified fragment was cloned into the vector between the two frames. We also established a mock vector only containing a non-sense sequence between the two circular frames without the circ-GRB10 cDNA. Vector construction was verified by direct sequencing. The vectors were constructed with the help of Guangzhou Geenseed Biotech Co., Guangzhou, China.

Fluorescence in situ hybridization

Fluorescence in situ hybridization was performed to detect subcellular location of circ-GRB10 and miR-328-5p according to the method described by Vautrot⁵¹. A FISH probe labeled with Alexa Fluor(^®) 488 for circ-GRB10 was designed to detect the splicing junction of two exons. The probe sequence was 5′-AGTCAAAGCGAATGTCAAGTGTCTGGCACCTCCC-3′. The probe for miR-328-5p was labeled with cy3; the probe sequence was 5′-CCCTGAGCCCCTCCTGCCCC C-3.

Cell transfection

The third-generation NP cells were used for transfection. Culture plates were incubated at 37 °C in a humidified atmosphere with 5% CO₂. Cells were transfected with corresponding plasmids or siRNAs using Lipofectamine 3000 (Invitrogen, Carlsbad, CA), according to the manufacturer’s recommendations. The cells were collected 48 h after transfection.

Dual-luciferase reporter assay

The binding site of circ-GRB10 (either wild type or mutated) were inserted into the KpnI and SacI sites of the pGL3 promoter vector (Realgene, Nan**g, China) in the dual-luciferase reporter assay. Firstly, cells were plated on 24-well plates. Then, 80 ng of plasmid, 5 ng of renilla luciferase vector pRL-SV40, 50 nM miR-328-5p mimics, and NCs were transfected into the cells using lipofectamine 3000 (Invitrogen, Shanghai, China). Cells were collected and measured according to the manufacturer’s instructions using the Dual-Luciferase Assay (Promega, Madison, WI, USA) after 48 h of transfection. All experiments were repeated three times independently.

Western blotting

Cultured cells were homogenized in lysis buffer (0.25 M Tris-HCl, pH 6.8, 20% glycerol, 4% SDS, 10% mercaptoethanol) supplemented with protease and phosphatase inhibitors. Samples containing equal amounts of protein (10 µg) were separated in SDS-polyacrylamide gels (SDS-PAGE, 10–12%). Proteins were transferred to polyvinylidene fluoride membranes. The membranes were blocked with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) at room temperature for 1 h. The membranes were incubated overnight at 4 °C with primary antibodies (1:3000) in TBST containing 5% non-fat milk. The secondary antibodies (1:6000) were incubated at room temperature for 1 h. The immunoblots were developed using an ECL system.

Flow cytometry

Nucleus pulposus cells were assigned to the NC group (cells transfected with empty vector and cultured with 10% FBS), circ-GRB10 si group (cells transfected with siRNA-circ-GRB10 and cultured with 10% FBS), circ-GRB10 overexpression (circ-GRB10 OE) group (cells transfected with pLCDH-cir-GRB10 and cultured with 10% FBS), nutrient deprivation group (cells transfected with empty vector and cultured with DMEM/F12), and function recovery group (cells transfected with pLCDH-cir-GRB10 and cultured with DMEM/F12).

After transfection for 48 h, the medium was discarded and the cells were washed with PBS and digested with 0.25% trypsin. PBS was used to resuspend the cells, followed by centrifugation at 2000 r.p.m. per min for 5 min. The precipitate was washed twice with PBS and cell concentration was adjusted to 1 × 10⁶ cells per mL. Cells were fixed with precooled 70% alcohol for 30 min, centrifuged, and stained with propidium iodide (PI) containing RNA enzyme (GR1-25, SBS Genetech, Bei**g, China) for 30 min. The samples were detected using a flow cytometer (FACS Calibur, Becton, Dickinson and Company, New Jersey, USA) and recorded at excitation wavelength of 488 nm.

Cells in the logarithmic growth phase were digested with 0.25% trypsin. Cells were washed three times with cool PBS, centrifuged, and resuspended with buffer. According to the instruction of the Annexin V-FITC/PI apoptosis kit (Abnova, Walnut, CA, USA), 5 μL of Annexin-V-FITC, and 50 μL of PI were added for reaction at 4 °C for 15 min. Then, 300 μL of binding buffer were added. Cell apoptosis was detected at excitation wavelength of 488 nm and emission wavelengths of 515 and 560 nm. The experiments were repeated three times and the average value was taken.

TUNNEL assay

Deparaffinization was achieved by serial gradient ethanol. Antigen retrieval was carried out using Proteinase K (Beyotime, China). Then, 0.3% Triton X-100 was used to permeated cell membrane. TdT and dUTP (2:29) were incubated at room temperature for 2 h. The cells were counter-stained with DAPI to visualize the nuclei under a fluorescence microscope (Olympus, Tokyo, Japan).

Statistical analysis

Each experiment was repeated at least three independent times, and the cells in each experiment were collected from a single isolation. The results of real-time quantitative RT-PCR and the gray values from western blotting are shown as mean ± SEM. Statistical significance was determined using a two-tailed Student’s t test (SPSS 22.0). Significance was set at *P < 0.05, **P < 0.01, ***P < 0.001.

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Acknowledgements

This study was supported by the State Key Program of National Natural Science Foundation of China (81330042), Special Program for Sino-Russian Joint Research Sponsored by the Ministry of Science and Technology, China (2014DFR31210), and International Cooperation Program of National Natural Science Foundation of China (81620108018).

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Wei Guo and Bin Zhang contributed equally to this work.
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Authors and Affiliations

Department of Orthopaedics, Hebei Province Cangzhou Hospital of Integrated Traditional and Western Medicine (Cangzhou No.2 Hospital), 31 Huanghe Road, Hebei, 061001, Cangzhou, China
Wei Guo, Zhan-Yin Dong, Hao-Ran Li & Ling Zhao
Department of Orthopaedics, Tian** Medical University General Hospital, 154 Anshan Road, He** District, 300052, Tian**, China
Bin Zhang, Shi-Qing Feng, Guang-Zhi Ning, Shen Liu, Yan Li, Bing-Bing Yu, Hui-Quan Duan, Chao Sun & Yong-** Li
Department of Breast Surgery, Hebei Province Cangzhou Hospital of Integrated Traditional and Western Medicine (Cangzhou No.2 Hospital), 31 Huanghe Road, Hebei, 061001, Cangzhou, China
Kun Mu

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Wei Guo
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Bin Zhang
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Kun Mu
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Shi-Qing Feng
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Hao-Ran Li
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Shen Liu
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Ling Zhao
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Yan Li
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Chao Sun
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Yong-** Li
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Guo, W., Zhang, B., Mu, K. et al. Circular RNA GRB10 as a competitive endogenous RNA regulating nucleus pulposus cells death in degenerative intervertebral disk. Cell Death Dis 9, 319 (2018). https://doi.org/10.1038/s41419-017-0232-z

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Received: 12 September 2017
Revised: 11 December 2017
Accepted: 14 December 2017
Published: 23 February 2018
DOI: https://doi.org/10.1038/s41419-017-0232-z
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Circular RNA GRB10 as a competitive endogenous RNA regulating nucleus pulposus cells death in degenerative intervertebral disk

Abstract

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Introduction

Circ-GRB10 regulates the expression of ERBB2

Circ-GRB10-miR-328-5p-ERBB2 regulatory loop is critical for NP cells survival

Discussion

Materials and methods

Ethics statement

Clinical specimens

Microarray data

Preprocessing and differential analysis

Annotation for circRNA–miRNA interaction

Quantitative real-time RT-PCR

Isolation and culture of human NP cells

siRNA and circ-GRB10 overexpression plasmid construction

Fluorescence in situ hybridization

Cell transfection

Dual-luciferase reporter assay

Western blotting

Flow cytometry

TUNNEL assay

Statistical analysis

References

Acknowledgements

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