Contributed equally
Intervertebral disc degeneration (IDD) is a major cause of lower back pain. The high morbidity associated with this disease diminishes the quality of life of those who are affected. MicroRNAs (miRs) play crucial roles in various diseases, including IDD. However, the mechanism via which miR-200c-3p plays a role in the development of IDD remains unknown. The present study aimed to investigate the effect of miR-200c-3p on the progression of IDD and the underlying mechanism. The expression level of miR-200c-3p was evaluated in intervertebral disc tissues from patients with IDD. To construct the IDD cell model, the nucleus pulposus (NP) cells were treated with lipopolysaccharide (LPS) 24 h following transfection with miR-200c-3p mimic or inhibitor. A luciferase activity assay was performed, while reverse transcription-quantitative PCR and western blotting were conducted to determine the RNA and protein expression levels, respectively. The expression level of miR-200c-3p in the intervertebral disc tissues of patients with IDD was lower than that of normal subjects. LPS treatment reduced the expression level of miR-200c-3p in NP cells. Moreover, miR-200c-3p mimic inhibited LPS-induced NP cell apoptosis. It was found that miR-200c-3p attenuated inflammatory cytokine levels and extracellular matrix (ECM) degradation in NP cells. Furthermore, miR-200c-3p targeted Ras-related protein 2C (RAP2C) in NP cells. RAP2C promoted apoptosis, inflammatory cytokine levels and ECM degradation by activating ERK signaling. Knockdown of RAP2C and inhibition of ERK signaling by SCH772984 partially reversed the proinflammatory effect of the miR-200c-3p inhibitor on LPS-treated NP cells. Thus, miR-200c-3p inhibits NP cell apoptosis, inflammatory cytokine levels and ECM degradation in IDD by targeting RAP2C/ERK signaling.
Intervertebral disc degeneration (IDD) is a prevalent complicated disease of the spine that reduces the quality of life of patients (
MicroRNAs (miRNAs/miRs) are small non-coding RNAs that modulate their target genes by regulating mRNA stability and translation, as well as controlling gene expression at the post-transcriptional level by pairing with target mRNAs at the 3′ untranslated region (3′UTR) (
Ras-related proteins (RAPs) organize various biological processes, including metabolic turnover, cytoskeletal organization, cell cycle, differentiation and cell adhesion (
Thus, the present study aimed to investigate the role and underlying mechanism of miR-200c-3p in the modulation of IDD development.
A total of 22 intervertebral disc tissues from patients with IDD (age, 41–64 years; 10 male and 12 female) and 9 normal samples (age, 40–49 years; 4 male and 5 female) used in this study were obtained from Qingdao No. 6 People's Hospital between May 2018 and December 2019. All patients with chronic lower back pain (lasting for >3 months) underwent MRI examinations (
Human NP cells were obtained from ScienCell Research Laboratories, Inc. The cells were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) containing 15% FBS (Gibco; Thermo Fisher Scientific, Inc.), 0.1 mg/ml streptomycin (Beijing Solarbio Science & Technology Co., Ltd.) and 100 U/ml penicillin (Beijing Solarbio Science & Technology Co., Ltd.) at 37°C with 5% CO2.
Lentiviral plasmids (GV248) carrying RAP2C short hairpin RNA (shRNA) and the corresponding negative control (NC) shRNA were synthesized and obtained from Shanghai GenePharma Co., Ltd. The sequence of RAP2C shRNA was 5′-GAAGCAAGAUCAGUGUUGU-3′ and the sequence of the NC shRNA was 5′-TTCTCCGAACGTGTCACGT-3′. The ratio of the lentiviral plasmid, packaging vector (Shanghai GenePharma Co., Ltd.) and envelope vector (Shanghai GenePharma Co., Ltd.) was 4:3:3. A second-generation system was used. Briefly, 293T cells (American Type Culture Collection) were plated into 6-well plates (1×106/well), which were used as the interim cell line, and they were transfected with 20 µg lentiviral plasmid using Lipofectamine® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C for 12 h. Lentiviral particles were collected using 72,000 × g/min centrifugation for 120 min. NP cells were plated into 24-well plates (2×105/well) and subsequently infected with the lentiviral supernatant (1×108 TU/ml) at a multiplicity of infection of 10 at 37°C for 12 h using TR-1003-G (Sigma-Aldrich; Merck KGaA). After 48 h of cell transduction, these cells were selected for in the presence of puromycin (1 µg/ml; Beyotime Institute of Biotechnology) for 3 days between transfection and experimentation at 37°C to generate stable NC and RAP2C-knockdown cells. Puromycin (0.5 µg/ml) was used for maintenance.
pcDNA3.1-RAP2C overexpression vector (OE-RAP2C) and empty pcDNA3.1 plasmid were synthesized and obtained from GenScript. miR-200c-3p mimic, miR-200c-3p inhibitor and corresponding scrambled controls were synthesized and obtained from Shanghai GenePharma Co., Ltd. NP cells (2×104/well) were transfected with 100 pmol pcDNA-3.1 vectors or 50 nM miRNA constructs using Lipofectamine® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions, at 37°C for 6 h. The sequences of each construct were as follows: miR-200c-3p mimic sense, 5′-UAAUACUGCCGGGUAAUGAUGGA-3′ and antisense, 5′-CAUCAUUACCCGGCAGUAUUAUU-3′; mimic NC sense, 5′-UUCUCCGAACGUGUCACGUTT-3′ and antisense, 5′-ACGUGACACGUUCGGAGAATT-3′; miR-200c-3p inhibitor, 5′-UCCAUCAUUACCCGGCAGUAUUA-3′; and inhibitor NC, 5′-CAGUACUUUUGUGUAGUACAA-3′. NP cells were harvested 24 h after transfection for use in subsequent experiments.
To construct the IDD cell model, NP cells were plated into 6-well plates (3×105/well) and treated with LPS (1 µg/ml; Sigma-Aldrich; Merck KGaA) for 24 h at 37°C after 24 h of transfection. The ERK inhibitor, SCH772984 (Selleck Chemicals), was used at a dosage of 10 µmol/l for 24 h at 37°C.
Total RNAs were extracted using TRIzol® (Invitrogen; Thermo Fisher Scientific, Inc.) and were treated with RQ1 RNase-Free DNase (Promega Corporation) for 30 min at 37°C. The quality of total RNA was detected at an absorbance 260/280 nm ratio using NanoDrop life spectrophotometer (Thermo Fisher Scientific, Inc.). cDNA was synthesized with the Maxima First-strand cDNA Synthesis kit according to the manufacturer's instructions (Thermo Fisher Scientific, Inc.). qPCR was performed using the SYBR Real-time PCR I kit (Takara Bio, Inc.). The thermocycling conditions used for the qPCR were as follows: Initial denaturation for 3 min at 95°C; followed by 37 cycles of denaturation at 94°C for 1 min, annealing at 60°C for 1 min and extension at 72°C for 1 min, followed by a final extension step at 72°C for 7 min. Relative quantification was performed using the 2−ΔΔCq method (
The levels of TNF-α (cat. no. KHC3013), IL-6 (cat. no. KAC1261) and IL-1β (cat. no. KAC1211) were analyzed using ELISA kits (eBioscience; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. All standards and samples were tested on a SpectraMax M5 microplate reader device (Molecular Devices, LLC) at an absorbance of 450 nm. A standard curve was prepared, and the concentration of the samples was calculated according to the absorbance value. Data are presented as the mean ± SD of three experiments.
In total, ~2×105 NP cells were plated into 6-well plates. Apoptosis was analyzed using the Annexin V-FITC Apoptosis Detection kit (Cell Signaling Technology, Inc.) according to the manufacturer's instructions. Briefly, ~2×105 NP cells were collected and washed with binding buffer, followed by flow cytometry analysis on a flow cytometer (BD Accuri™ C6 Plus; BD Biosciences). The apoptotic rate was calculated as the percentage of early + late apoptotic cells using FlowJo software (v10.7; FlowJo, LLC). Data are presented as the mean ± SD of three experiments.
TargetScan (
Total proteins were extracted from cells using RIPA buffer (Cell Signaling Technology, Inc.). Protein concentrations were measured using the BCA Protein Quantification kit (Abbkine Scientific Co., Ltd.). An equivalent concentration of protein (50 µg/lane) was separated via SDS-PAGE (12% polyacrylamide gels) and transferred onto PVDF membranes (MilliporeSigma). The membranes were blocked with 5% milk at 25°C for 60 min and incubated overnight at 4°C with the primary antibodies for RAP2C (1:3,000; cat. no. ab97805; Abcam), ERK (1:1,000; cat. no. ab32537; Abcam), phosphorylated (p)-ERK (1:1,000; cat. no. ab194776; Abcam) and β-actin (1:5,000; cat. no. ab8227; Abcam), in which β-actin served as the control. Then, the goat anti-rabbit IgG H&L (HRP) secondary antibodies (1:5,000; cat. no. ab7090; Abcam) were added to membranes for 1 h at 25°C, followed by visualization using an Odyssey CLx Infrared Imaging system (LI-COR Biosciences). Image Studio™ Lite (v4.0; LI-COR Biosciences) was used for analysis. Data are presented as the mean ± SD of three experiments.
Data are presented as the mean ± SD of three experiments and statistical analysis was performed using GraphPad Prism 7 (GraphPad Software, Inc.). An unpaired Student's t-test was used to compare two groups. One-way ANOVA was used for multiple group comparisons and Tukey's test was used for post hoc analysis. P<0.05 was considered to indicate a statistically significant difference.
To assess the potential association between miR-200c-3p and IDD, the expression level of miR-200c-3p was analyzed in patients with IDD and normal controls. The expression level of miR-200c-3p was decreased in the intervertebral disc tissues of patients with IDD (n=22) compared with that in normal subjects (n=9) (
To evaluate the role of miR-200c-3p in the development of IDD
Since inflammatory factors have been shown to be involved in the pathological processes of IDD (
miR-200c-3p has a recognized target site in RAP2C 3′UTR, as determined using TargetScan (
LPS-treated NP cells were infected with lentiviral plasmids carrying RAP2C shRNA or corresponding control shRNA or transfected with the vector carrying the complete RAP2C coding sequence for overexpression or the control vector. The efficiency of RAP2C shRNA and RAP2C overexpression was validated in the cells (
It has been reported that the activation of ERK signaling contributes to the development of IDD (
Knockdown of miR-200c-3p with a miR-200c-3p inhibitor promoted the expression of RAP2C. However, SCH772984 treatment blocked miR-200c-3p inhibitor-induced RAP2C expression in NP cells. The miR-200c-3p mimic inhibited the expression of RAP2C (
IDD is a complex disorder characterized by genotypic and phenotypic alterations, such as ECM degradation, apoptosis and inflammation (
RAP2C is an evolutionarily conserved Ras-like GTPase that serves as a molecular switch that pairs receptor signaling to remodel the actin cytoskeleton, cell adhesion and cell polarity (
ECM degradation plays a critical role in the development of IDD, in which collagen II and aggrecan serve as markers of ECM degradation (
The current study has some limitations. The expression levels of aggrecan and collagen II were determined only via RT-qPCR, while levels of inflammatory factors were assessed using ELISA alone. In future studies, the results of the present study should be verified using
In conclusion, the present study demonstrated that miR-200c-3p inhibited NP cell apoptosis, inflammatory cytokine levels and ECM degradation in IDD by targeting RAP2C/ERK signaling. Moreover, miR-200c-3p and RAP2C may serve as potential targets for IDD therapy.
Not applicable.
This study was supported in part by the Qingdao Medical Research Guidance Program in 2018: Three-dimensional Decompression Comprehensive Therapy for the Treatment of Lumbar Disc Herniation and Degeneration (grant no. 2018-WJZD113).
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
JC and KX designed the study. MJ performed the experiments. HR analyzed data. JC and KX confirm the authenticity of all the raw data. KX wrote the paper. All authors read and approved the final manuscript.
The protocol of this research has been approved by the Ethics Committee of Qingdao No.6 People's Hospital. All patients have signed written informed consent.
Not applicable.
The authors declare that they have competing interests.
intervertebral disc degeneration
nucleus pulposus
extracellular matrix
lipopolysaccharide
microRNAs
Ras-related proteins
miR-200c-3p inhibits LPS-induced NP cell apoptosis. (A) Expression levels of miR-200c-3p were measured via RT-qPCR in the intervertebral disc tissues of patients with IDD (n=22) and normal cases (n=9). (B) Expression levels of miR-200c-3p were assessed via RT-qPCR in NP cells treated with LPS (1 µg/ml). *P<0.05, **P<0.01 vs. NC. NP cells were treated with LPS (1 µg/ml) or co-treated with LPS (1 µg/ml) and miR-200c-3p mimic, inhibitor or corresponding control. (C) Expression levels of miR-200c-3p were analyzed via RT-qPCR. (D) Cell apoptosis was tested using flow cytometry analysis. Data are presented as the mean ± SD. *P<0.05, **P<0.01 vs. control; #P<0.05, ##P<0.01 vs. LPS. LPS, lipopolysaccharide; RT-qPCR, reverse transcription-quantitative PCR; miR, microRNA; NC, negative control; IDD, intervertebral disc degeneration; NP, nucleus pulposus.
miR-200c-3p attenuates inflammatory cytokine levels and extracellular matrix degradation in LPS-treated NP cells. NP cells were treated with LPS (1 µg/ml) or co-treated with LPS (1 µg/ml) and miR-200c-3p mimic, inhibitor. Levels of (A) TNF-α, (B) IL-6 and (C) IL-1β in the culture medium of the cells were analyzed using ELISAs. Expression levels of (D) collagen II and (E) aggrecan were examined via reverse transcription-quantitative PCR. Data are presented as the mean ± SD. *P<0.05, **P<0.01 vs. control; #P<0.05, ##P<0.01 vs. LPS. LPS. LPS, lipopolysaccharide; miR, microRNA; NP, nucleus pulposus.
miR-200c-3p targets RAP2C in nucleus pulposus cells. (A) Interaction of miR-200c-3p and RAP2C 3′UTR was identified via bioinformatics analysis using TargetScan. (B) NP cells were transfected with control mimic or miR-200c-3p mimic. (C) Luciferase activities of RAP2C WT and RAP2C with the miR-200c-3p-binding site mutant (RAP2C MUT) were determined using luciferase reporter gene assays. (D) mRNA expression level of RAP2C was assessed using reverse transcription-quantitative PCR in the cells. (E) Protein expression level of RAP2C was measured via western blotting. Data are presented as the mean ± SD. *P<0.05, **P<0.01 vs. control. ns, no significance; miR, microRNA; WT, wild-type; MUT, mutant; RAP2C, Ras-related protein 2C; UTR, untranslated region.
RAP2C promotes apoptosis, inflammatory cytokine levels and ECM degradation in LPS-treated NP cells. LPS (1 µg/ml)-treated NP cells were infected with lentiviral plasmids carrying RAP2C shRNA or the corresponding control shRNA, or transfected with the control vector or the vector carrying the complete RAP2C coding sequence for overexpression. (A) Expression levels of RAP2C were measured via western blotting. **P<0.01 vs. vector; ##P<0.01 vs. NC. (B) Cell apoptosis was measured using flow cytometry analysis. Levels of (C) TNF-α, (D) IL-6 and (E) IL-1β in the culture medium of the cells were analyzed using ELISAs. Expression levels of (F) collagen II and (G) aggrecan were examined via reverse transcription-quantitative PCR. Data are presented as the mean ± SD. **P<0.01 vs. control; #P<0.05, ##P<0.01 vs. LPS. LPS, lipopolysaccharide; shRNA/sh, short hairpin RNA; NC, negative control; RAP2C, Ras-related protein 2C; NP, nucleus pulposus; OE, overexpression vector.
RAP2C enhances apoptosis, inflammatory cytokine levels and extracellular matrix degradation by activating ERK signaling. (A) NP cells were infected with the lentiviral plasmids carrying RAP2C shRNA or the corresponding control shRNA, transfected with the control vector or the vector carrying the complete RAP2C coding sequence for overexpression or co-treated with the RAP2C overexpression vector and the ERK inhibitor SCH772984 (10 µmol/l). The expression levels of RAP2C, ERK and β-actin, and the phosphorylation of ERK (p-ERK) were examined via western blotting. *P<0.05, **P<0.01 vs. vector; #P<0.05 vs. NC. LPS (1 µg/ml)-treated NP cells were transfected with the control vector, or the vector carrying the complete RAP2C coding sequence for overexpression or co-treated with RAP2C overexpression vector and the ERK inhibitor SCH772984 (10 µmol/l). (B) Cell apoptosis was examined via flow cytometry analysis. Levels of (C) TNF-α, (D) IL-6 and (E) IL-1β in the culture medium of the cells were tested using ELISAs. Expression levels of (F) collagen II and (G) aggrecan were determined using reverse transcription-quantitative PCR. Data are presented as the mean ± SD. *P<0.05, **P<0.01 vs. control; #P<0.05, ##P<0.01 vs. LPS. LPS, lipopolysaccharide; shRNA/sh, short hairpin RNA; NC, negative control; RAP2C, Ras-related protein 2C; NP, nucleus pulposus; OE, overexpression vector; p-, phosphorylated.
miR-200c-3p suppresses IDD by targeting RAP2C/ERK signaling. (A) NP cells were transfected with miR-200c-3p mimic, inhibitor or corresponding control, or co-treated with miR-200c-3p inhibitor and the ERK inhibitor SCH772984 (10 µmol/l). The expression levels of RAP2C, ERK and β-actin, and the phosphorylation of ERK (p-ERK) were examined via western blotting. LPS (1 µg/ml)-treated NP cells were transfected with miRNA control inhibitor or miR-200c-3p inhibitor, or co-transfected with miR-200c-3p inhibitor and the lentiviral plasmids carrying RAP2C shRNA, or co-transfected with miR-200c-3p inhibitor and the ERK inhibitor SCH772984 (10 µmol/l). *P<0.05, **P<0.01 vs. vector; #P<0.05 vs. NC. (B) Cell apoptosis was analyzed using flow cytometry. The levels of (C) TNF-α, (D) IL-6 and (E) IL-1β in the culture medium of the cells were assessed using ELISA. The expression levels of (F) collagen II and (G) aggrecan were examined using reverse transcription-quantitative PCR. (H) The regulatory pathway of miR-200c-3p in LPS-treated NP cells. Data are presented as the mean ± SD. **P<0.01 vs. control; #P<0.05, ##P<0.01 vs. LPS. IDD, intervertebral disc degeneration; LPS, lipopolysaccharide; shRNA/sh, short hairpin RNA; NC, negative control; RAP2C, Ras-related protein 2C; NP, nucleus pulposus; p-, phosphorylated; miRNA/miR, microRNA.