MEG3 promotes proliferation and inhibits apoptosis in osteoarthritis chondrocytes by miR-361-5p/FOXO1 axis

doi:10.21203/rs.2.11513/v4

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MEG3 promotes proliferation and inhibits apoptosis in osteoarthritis chondrocytes by miR-361-5p/FOXO1 axis

https://doi.org/10.21203/rs.2.11513/v4

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published 30 Dec, 2019

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Background: This study aimed to investigate the role of long non-coding RNA (lncRNA) maternally expressed 3 (MEG3) and related molecular mechanisms in osteoarthritis (OA). Methods: Patients with OA and patients undergoing thigh amputation were involved in OA group and normal group, respectively. Cartilage tissues of all patients were isolated and cultured. Based on different transfection, MEG3 cells were grouped into Blank, pcDNA3.1-NC, pcDNA3.1-MEG3, si-NC, si-MEG3, pcDNA3.1-NC + mimics NC, pcDNA3.1-MEG3 + mimics NC, pcDNA3.1-NC + miR-361-5p mimics and pcDNA3.1-MEG3 + miR-361-5p mimics group. The RT-qPCR was used to detect the expression of MEG3, miR-361-5p and FOXO1. Moreover, western blot, luciferase reporter assay, RIP, CCK-8 and flow cytometry analysis were performed to reveal the morphology, proliferation and apoptosis in cartilage cells. Finally, the histological analysis and immunostaining were performed on OA rat model. Results: The expression of MEG3 and FOXO1 in OA was significantly decreased while miR-361-5p was increased compared with the normal group. MEG3 might serve as a ceRNA of miR-361-5p in OA chondrocytes. Moreover, the western blot and CCK-8 assay showed that MEG3, targeted miR-361-5p/FOXO1, might elevate cell proliferation and impair cell apoptosis. Finally, rat model analysis showed that MEG3 suppressed the cartilage matrix degradation. Conclusion: Taken together, MEG3 can contribute to cell proliferation, inhibit cell apoptosis and extracellular matrix (ECM) degradation via miR-361-5p/FOXO1 axis in OA chondrocytes.

Orthopedics

Molecular Biology

osteoarthritis

maternally expressed 3

miR-361-59

FOXO1

ECM degradation

Osteoarthritis (OA) is a disease increasing with age and even induces serious pain and disability ¹. It leads to various pathological changes including articular cartilage degradation, synovial inflammation and subchondral osteoblast activation ². Although various genetic, biological, and biomechanical components have been proved to be associated with OA ³, the molecular mechanisms underlying the progression of OA is not completely understood.

LncRNAs play important regulatory roles in oncogenic pathways ^4-8. The biological function of lncRNAs has been reported to be associated with knee OA progression ⁹. As a member of lncRNA, maternally expressed 3 (MEG3) has been proved to participate in the development of tumors ¹⁰. Recently, MEG3 is found to be a potential therapeutic target for OA, which has been confirmed to participate in various cancer including lung cancer ¹¹, breast cancer¹² and esophageal cancer ¹². Su et al. showed that MEG3 was suppressed and inversely related to vascular endothelial growth factor A levels in OA. ¹³. Prior publication performed by You et al. suggested that MEG3 suppressed chondrogenic differentiation of synovium-derived mesenchymal stem cells via epigenetically hindering TRIB2. Methylene blue can relieve the progression of OA through regulating the expression level of MEG3¹⁴. Down-regulation of MEG3 can result in the development of OA via miR-16/SMAD7 axis ¹⁵.Interestingly, MEG3 is proved to be down-regulated and inversely associated with VEGF levels in OA ¹⁶. Jin et al. indicated that the down-regulation of MEG3 leaded to OA progression via certain miRNA-target gene axis ¹⁷. Based on the above-mentioned researches, we realized that MEG3 might be involved in the OA development via multiple signal axises. To identify the novel molecular mechanism of MEG3 and complement the MEG3-centric regulatory network in OA, we conducted this work. In the current analysis, the mechanism of MEG3 and related molecules in OA was explored based on cartilage cells obtained from patients with OA (OA group) and artificial joint replacement (normal group). The RT-qPCR, western blot, luciferase reporter assay, RNA immunoprecipitation (RIP), cell counting kit 8 (CCK-8) and flow cytometry analysis were performed to reveal the effects of MEG3 in cartilage cells. Histological analysis and immunostaining were implemented on OA rat model. The study might provide a new target and theoretical basis for OA treatment.

Patients and grouping

The cartilage tissue of OA patients was derived from the knee joints of 30 patients who had undergone total knee arthroplasty. Meanwhile, the healthy cartilage tissues were obtained from 20 patients who had not undergone OA or RA (rheumatoid arthritis). There was no statistical difference in age and gender between OA patients and healthy volunteers. The age and sex of all the samples were matched. All patients voluntarily signed the informed notice. The current study obtained the approval of Ethics Committee of the hospital (ethic vote 198/203).

Cell culture

The femoral articular cartilage of the femur was obtained and cut. Then, it was digested by 0.2% type II collagenase, oscillation resolving for 40min at 37°C, washed by D-Hanks, and diluted in DMEM/F12 (Gibco, USA) combined with 10% FBS, 100 u/ml penicillin (Gibco, USA) as well as 100 mg/ml streptomycin (Gibco, USA). These cells were culture in a saturated humidity incubator (37°C, 5% CO₂). The solution was changed every two days until the chondrocytes grew into sheets and covered over 85% confluence ¹⁸. The cells of 2 or 3 generations were enrolled for the further investigation.

Cell transfection

The MEG3 overexpression vector pcDNA3.1-MEG3, si-MEG3 for MEG3 knockdown, si-EOXO1 used to decrease the expression of FOXO1 (Sangon Biotech, Shanghai, China), miR-361-5p mimics and miR-361-5p mimics NC (Guangzhou Reeber) were prepared, followed by cells transfection according to the instructions of the transfection reagent. All transfections were carried out by Lipfectamine 2000 transfection kit (Invirtrogn, USA) according to the instructions. After 48 hours, the samples were treated with IL-1β (10 ng / ml) for 24 hours. Finally, cells of each group were collected for following experiments.

Real-time fluorescent quantitative PCR

Total RNA (500 ng) from sample of each group was obtained as well as quantified using TRIzol reagent, and cDNA template was synthesized by PrimeScript RT kit (Takara biomedical Technology Co., Ltd., Beijing, China). GAPDH was used as reference (Table 1). The associated conditions were as follows: 95°C for 3 min, 39 cycles at 95 °C for 10 s, 55 °C. Fluorescence signals were collected at the end points of each cycle extension on the ABI7500 platform, followed by the amplification curve investigation. Relative expression of candidate genes were calculated by 2^-∆∆CT method ¹⁹.

Luciferase reporter assay

The regulatory relation between MEG3 and miR-361-5p was revealed by using StarBase as well as miR-361-5p and FOXO1. A wild type (MEG3-WT) containing the indicated fragment of the putative binding site between MEG3 3’UTR and miR-361-5p was constructed as well as mutant (MEG3-MUT) without the above mentioned fragment. Then, the wild type or mutant were cloned into the pmirGLO reporter vector (Promega, USA). After inoculation of human chondrocytes in 24-well plates (5 × 10⁵/well) for 24 hours, miR-361-5p mimic or mimics NC, and MEG3-WT and MEG3-MUT were co-transfected into human chondrocytes by Lipofectamine 3000 (Thermo Fisher Scientific), followed by dual-luciferase reporter gene assay after 48 hours transfection. Similar to the above steps, the FOXO1-WT and FOXO1-MUT were synthesized, and then, the correlation of miR-361-5p and FOXO1 was determined using dual-luciferase reporter gene assay.

RIP assay

RIP was determined by using a Magna RIPTM RNA kit (Millipore, USA). Briefly, the cultured chondrocytes were further suspended in RIP lysis buffer (Solarbio). Then, cell extract was incubated overnight with RIP buffer containing human anti-Ago2 antibody beads (Millipore) (Input and normal IgG served as controls). Finally, RNA samples were extract using TRIzol reagent, followed by relative enrichment of MEG3/FOXO1 and miR-361-5p detection.

Western blotting

Cultured cells and chondrocytes isolated from treated tissues were treated with protein lysate RIPA buffer to extract the whole proteins. Briefly, the extracted protein was quantified by bicinchoninic acid (BCA) method. A total of 50 g samples was investigated by 10% polyacrylamide gels, and transferred to polyvinylidenefluoride membranes (5% Skim milk/BSA). Samples was treated with primary antibodies including β-Catenin (#19807),MMP-13 (#94808), Collagen II (#34712), PCNA (#13110), Bax (#5023) and Bcl-2 (#4223) antibody (1:1000, Cell Signaling, Boston, USA), as well as Ki67 (ab92742), ADAMTS-5 (ab41037) and Aggrecan (ab36861) (Abcam, Cambridge, MA, USA) ¹⁷. Then, samples were treated with the HRP-conjugated secondary antibody (1:5000, #7074; Cell Signaling Technology, Danvers, MA,USA). Finally, the protein was developed with diaminobenzidine (DAB). All experiments were repeated for 3 times.

CCK-8 assay

All cells were placed in 24-well plates (2 × 10³ cells/well, 37 °C and 5% CO₂). After being incubated for 0, 24, 48, 72 h, a total of 10 μL CCK-8 (Sigma-Aldrich) reagent was added into each well for further incubation. OD₄₉₀ value was measured by enzyme-linked immunosorbent assay ²⁰.

Flow cytometry assay

Simply, the cells were treated by trypsin supplemented with 200 μL Annexin V-FITC, incubated for 10 min in dark, and then washed with 200 μL PBS and 10 μL PI. Cell apoptosis was then detected by using flow cytometry (Beckman Coulter), followed by the data analysis.

OA rat model construction

Male SD rats (200-250 g) were obtained from the Experimental Animal Center of Taishan Medical College. All rats were anesthetized with intramuscular injection of sodium pentobarbital (0.05 mg/g, Chuangdong Co., Chongqing, China). Then, SD rats were traversed by the medial collateral ligament and destabilized by the medial meniscus (DMM). One week after the operation, si-NC and si-MEG3 (1×10⁹ PFU, 20 μl) were injected into the knee joint of the recipient rat (n = 6 for each group, 20 μL per joint) twice a week for 4 weeks. Eight weeks after the operation, the rats were sacrificed with cervical dislocation method (external force dislocated the cervical vertebra of rat and disconnects the spinal cord from the cerebrospinal cord), and then the knee joints were harvested. All experiment was performed in Experimental Center of Taishan Medical College. This study was approved by the Laboratory Animal Ethics Committee of Taishan Medical College (No. 2019146), and all experiments abided by the guide for the care and use of laboratory animal.

Histological and immunostaining investigation

Cartilage samples was placed in paraformaldehyde (4%), embedded in paraffin and cut into sample slice (5μm/slice). The cartilage destruction was evaluated by using the Safranin ‘O’ staining. Histological scores were measured according to the International Osteoarthritis Research Association (OARSI) grading system, which ranging from 0 (normal) to 6 (>80% represented the cartilage loss). Scores were determined from multiple serial sections of the knee joint of each mouse.

Statistical Analysis

The SPSS 18.0 (Chicago, IL, USA) was selected as the software for data analysis. Meanwhile, all the data in current investigation were represented as the mean ± standard deviation (SD). Significant differences between two groups were assessed using Student’s t-test, while least significant difference between means (LSD)-t multiple comparison test was processed for more than two groups. Spearman analyses were performed to identify the correlations of miR-361-5p and MEG3 or FOXO1. P < 0.05 was considered as statistically significant ²¹.

MEG3 regulated OA cell proliferation, apoptosis, and cartilage matrix degradation

Compared with normal group, MEG3 in chondrocytes of OA group was significantly decreased (P < 0.001) (Figure 1A). After transfection with pcDNA3.1-MEG3 or MEG3 siRNA into OA chondrocytes, the expression of MEG3 in pcDNA3.1-MEG3 group was dramatically promoted compared with that in pcDNA3.1-NC group (Figure 1B) (P < 0.05). Moreover, compared with the si-NC group, MEG3 expression in the si-MEG3 group was significantly suppressed (Figure 1B) (P < 0.05). Moreover, silencing MEG3 significantly inhibited cell proliferation compared with si-NC (Figure 1C) (P < 0.05), while the cell proliferation ability of the pcDNA3.1-MEG3 was greatly promoted compared to the pcDNA3.1-NC group (Figure 1D) (P < 0.05). Moreover, the proliferative markers ki67 and PCNA showed the consistent results with CCK-8 analysis(Figure 1E) (P < 0.01). In addition, the apoptosis rate of si-MEG3 group was dramatically elevated than that of si-NC group, , and the apoptosis rate of pcDNA3.1-MEG3 group was significantly inhibited compared with pcDNA3.1-NC group (Figure 1F) (P < 0.01). Consistently, the apoptotic protein Bax was significantly increased by MEG3 knockdown and attenuated by high-regulated MEG3 expression. The anti-apoptosis protein Bcl-2 revealed the converse tendency (Figure 1G) (P < 0.01).

The rat OA model analysis showed that compared with si-NC group, MMP13 and ADAMTS-5 were up-regulated, while Collagen II and Aggrecan expression were down-regulated in si-MEG3 group (Figure 1H) (P < 0.01). Conversely, pcDNA3.1-MEG3 group revealed that MMP13 and ADAMTS-5 were repressed, while Collagen II and Aggrecan were enhanced(Figure 1H) (P < 0.01). These results demonstrated that MEG3 can promote proliferation, inhibit apoptosis and ECM degradation in OA.

MEG3 might act as a ceRNA of miR-361-5p in OA chondrocytes

The binding sites of MEG3 and miR-361-5p were predicted using StarBase (Figure 2A). The luciferase reporter assay demonstrated that MEG3 regulated miR-361-5p, MEG3-WT and miR-361-5p mimics co-transfected cells with significantly reduced luciferase activity (Figure 2B) (P < 0.01). Meanwhile, the RIP assay showed that compared with lgG control group, MEG3 and miR-361-5p were dramatically overexpressed in the Ago2 pellet (Figure 2C). After transfection of pcDNA3.1-MEG3 or si-MEG3 into OA chondrocytes, miR-361-5p was down-regulated when compared with pcDNA3.1-NC group, while miR-361-5p was significantly up-regulated when compared with si-NC group (Figure 2D) (P < 0.01). Moreover, miR-361-5p was dramatically promoted in chondrocytes of OA compared with normal group (Figure 2E) (P < 0.001). Furthermore, the Spearman correlation analysis showed a negative correlation between expression of MEG3 and miR-361-5p (r = -0.529, P = 0.0026) (Figure 2F). Thus, we hypothesized that MEG3 maybe a ceRNA of miR-361-5p in OA and negatively regulate the expression of miR-361-5p.

The effect of MEG3 on proliferation, apoptosis and cartilage matrix degradation of OA chondrocytes was reversed by miR-361-5p

Compared with pcDNA3.1-NC + mimics NC group, miR-361-5p was decreased in pcDNA3.1-MEG3 + mimics NC group, while miR-361-5p was promoted in pcDNA3.1-NC + miR-361-5p mimics group (Figure 3A) (P < 0.01). Moreover, the cell proliferation ability of pcDNA3.1-NC + miR-361-5p mimics group was inhibited than that of pcDNA3.1-NC + mimics NC group. The promoting effect of MEG3 on cell proliferation was reversed by miR-361-5p mimics (Figure 3B) (P < 0.05). Detection of the expression of PCNA and ki67 using western blot verified the data of CCK-8 assay (Figure 3C) (P < 0.01). Flow cytometry analysis revealed that miR-361-5p inhibited apoptosis and its addition to the cells transfected with MEG3 (pcDNA3.1-MEG3) promoted cell apoptosis. (Figure 3D) (P < 0.01). As expected, the expression level of Bax and Bcl-2 exhibited the consistent results as the flow cytometry assay (Figure 3E) (P < 0.05). In addition, the ECM degradation-related proteins including MMP13, ADAMTS-5, Collagen II and Aggrecan determined that the co-transfection of MEG3 and miR-361-5p mimics relieved the ECM degradation that aggravated by MEG3 (Figure 3F) (P < 0.05). In summary, these findings suggested that miR-361-5p can reverse the impacts of MEG3 on OA.

FOXO1 was a directly target of miR-361-5p and positively regulated by MEG3

Bioinformatics analysis employed starBase showed that the putative target of miR-361-5p was FOXO1, and the sequence of the binding site between miR-361-5p and FOXO1 was exhibited in Figure 4A. Results of dual-luciferase reporter assay illustrated that miR-361-5p significantly eliminated the luciferase activity of FOXO1-WT group, and the high-regulated MEG3 relieved this inhibitory effect induced by miR-361-5p. However, there was none differences in FOXO1-MUT group (Figure 4B) (P < 0.05). We next verified the association of miR-361-5p and FOXO1 via conducting RIP assay. As shown in Figure 4C, miR-361-5p and FOXO1 could be markedly pulled down by the anti-IgG in contrast to anti-Ago2 (P < 0.01). In addition, FOXO1 expression was significantly decreased in OA cartilage tissues compared with normal control (Figure 4D) (P < 0.001), which was similar to the expressional pattern of MEG3. Spearman analyses elucidated that FOXO1 was negatively regulated by miR-361-5p (r = -0.4015, P = 0.0279) (Figure 4E) while positively modulated by MEG3 (r = 0.7119, P < 0.001) (Figure 4F). Subsequently, FOXO1 mRNA expression was investigated in OA chondrocytes after MEG3 and si-MEG3 transfections using qRT-PCR method. The data showed that overexpression of MEG3 remarkably promoted the FOXO1 expression while the interference of si-MEG3 induced the down-regulation of FOXO1 (Figure 4G) (P < 0.01). Collectively, these above results disclosed that FOXO1 has a closely correlation with miR-361-5p and MEG3.

FOXO1 contributed to the impacts of MEG3 in OA

To assess whether FOXO1 affects the function of MEG3 on cell viability, apoptosis and ECM degradation in OA chondrocytes, OA chondrocytes firstly were transfected with MEG3, MEG3+si-FOXO1, si-FOXO1 and corresponding control. QRT-PCR analysis determined the FOXO1 expression levels in each group, which showed that MEG3 can elevate the FOXO1 expression (Figure 5A) (P < 0.05). CCK-8 assay revealed that down-regulation of FOXO1 significantly suppressed cell viability and its addition in OA chondrocytes with high-regulated MEG3 attenuated cell viability compared with MEG3+si-NC group (Figure 5B) (P < 0.01). Moreover, PCNA and ki67 expressions were also inhibited by si-FOXO1, and decreased the enhanced effects of MEG3 (Figure 5C, P < 0.05). Instead, flow cytometry results uncovered that si-FOXO1 facilitated apoptosis compared with MEG3+si-NC and MEG3+si-FOXO1 groups (Figure 5D) (P < 0.05). In NC+si-FOXO1 group, MMP13 and ADAMTS-5 were attenuated while Collagen II and Aggrecan were increased. Co-transfection of MEG3 and si-FOXO1 eliminated the influence of MEG3 and si-FOXO1 (Figure 5E) (P < 0.05). Taken together, these data demonstrated that FOXO1 can strengthen the effects of MEG3 in OA.

MEG3 relieved the cartilage matrix degradation in OA rats

To ensure the successful delivery of pcDNA3.1-NC or pcDNA3.1-MEG3 into the chondrocytes, we measured the MEG3 levels and the results showed that pcDNA3.1-MEG3 had been successfully delivered into OA rats (Figure 6A) (P < 0.01). Injection of pcDNA3.1-MEG3 effectively reduced the cartilage damage of the operation, protected the cartilage from degradation, and reduced the loss of proteoglycan and joint soft cell (Figure 6B) (P < 0.01). Moreover, based on the results of ECM degradation-related proteins expression levels, the interference of pcDNA3.1-MEG3 inhibited cartilage bone marrow matrix degradation (Figure 6C) (P < 0.05).

Although OA affecting about 237 million people worldwide ²², the detail molecule mechanism of OA is still not fully investigated until now. In the current study, the qRT-PCR analysis revealed that the MEG3 was significantly inhibited while miR-361-5p was increased. Meanwhile, StarBase prediction showed that the MEG3 was a sponge of miR-361-5p in OA chondrocytes. Moreover, functional experiments showed that MEG3 can promote cell proliferation while inhibit cell apoptosis by sponging miR361-5p.

As a maternally expressed imprinted gene, MEG3 has been found in various normal tissues except for tumor cells ¹¹^,²³. A previous study showed that the downregulation of MEG3 was associated with the expression of tumor genes in epithelial cells ²⁴. Ying et al. showed that the suppression of MEG3 was related with the progression of bladder cancer ²⁵. Prior publication showed that the low expression induced by gene knockdown can inhibit apoptosis of chondrocyte in OA rat model ²⁶. Actually, the low expression of MEG3 has also been reveled in OA. MEG3 has been proved to participate in the development of OA via miR-16/AMAD7 interaction ¹⁷. A previous study indicated that MEG3 was down-regulated and inversely associated with vascular endothelial growth factor A levels in OA. ¹³. In the current study, the qRT-PCR analysis showed that the expression of MEG3 in OA group was significantly decreased than that in normal group. Thus, we speculated that MEG3 might be involved in the progression of OA. Thus, we performed functional experiments including CCK-8 and flow cytometry assays and found that MEG3 promoted cell viability while inhibited apoptosis in OA. Additionally, OA is severe malignancy characterized by ECM degradation. Enhancement of ECM degradation can accelerate the progression of OA²⁷. The ECM is mainly composed of Collagen II and Aggrecan, which was degradated by proteases, such as matrix metalloproteinases (MMPs) for collagens degradation²⁸. MMP-13, a key collagenase in OA, has high-regulated expression in OA²⁹^,³⁰. The inhibition of ADAMTS-5 can relieve the symptoms of OA³¹. Herein, to further assess the effects of MEG3 in OA development, the ECM degradation-related factors expression levels were measured by western blot. The observations revealed that MEG3 suppressed the degradation of ECM, suggesting that MEG3 can protect chondrocytes from damaging caused by OA.

Extensive evidence suggested that miRNAs play a crucial role in OA progression. Wang et al. demonstrated that miR-140-5p was related with chondrocyte proliferation, apoptosis and inflammation in OA ³². MiR-203 inhibition could ameliorate OA cartilage degradation in postmenopausal rat model ³³. We used starBase to forecast the target miRNA of MEG3 and found that miR-361-5p was a target of MEG3. A lot of literatures have indicated that miR-361-5p can participate in multiple tumors, such as gastric cancer³⁴, liver cancer³⁵, cholangiocarcinoma³⁶ and so on. The present study described that miR-361-5p was highly regulated in OA. Furthermore, miR-361-5p can reverse the regulation of cell viability, apoptosis and ECM degradation caused by MEG3 in OA.

Previous studies have indicated that the FOXO molecules are an evolutionarily conserved family and exert important effects in development, aging as well as longevity. This family consist four members, for example, FOXO1³⁷^,³⁸. More importantly, silencing SGK1 can alleviate the chondrocyte anabolic and catabolic imbalance through stimulating FOXO1-mediated autophagy in human chondrocytes³⁹. Levinger et al. also detected the levels of FOXO1 in skeletal muscle, serum and synovial fluid in patients with knee OA⁴⁰. Importantly, bioinformatics analysis showed that FOXO1 might serve as a target gene of miR-361-5p, negatively modulated by miR-361-5p and positively regulated by MEG3. In agree with MEG3, FOXO1 elevated cell proliferation, while hindered apoptosis and the degradation of ECM.

In conclusion, MEG3 can positively modulate FOXO1 via sponging miR-361-5p in OA. Furthermore, MEG3 might promote cell proliferation, and impair cell apoptosis and ECM degradation via miR-361-5p/FOXO1 axis in OA chondrocytes. These data maybe shed new insights for improvement of OA treatment.

osteoarthritis OA

long noncoding RNA lncRNA

Long noncoding RNAs lncRNAs

maternally expressed 3 MEG3

Ethics approval and consent to participate: The ethics committee of The Third Hospital of Hebei Medical University approved the study. This study was written informed consent from the patients.

Consent for publication: Not applicable.

Availability of data and material: All data generated or analyzed during this study are included in this published article and its supplementary information files.

Competing interests: The authors declare that they have no competing interests.

Funding: Not applicable.

Authors' contributions: AYW, NXH and YFZ designed and analyzed the experiment, and was a major contributor in writing the manuscript. YZC, CHS, YL and YS performed the experiment. All authors read and approved the final manuscript.

Acknowledgements: Not applicable.

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Table 1 The amplification primer used for current RT-qPCR analysis

Name of primer	Sequences
LncRNA MEG3	5′-CTGCCCATCTACACCTCACG-3′
	5′-CTCTCCGCCGTCTGCGCTAGGGGCT-3′
GAPDH	5′-TGCACCACCAACTGCTTAGC-3′
	5′-GGCATGCACTGTGGTCATGAG-3′
miR-361-5p	5′-ATAAAGTGCTGACAGTGCAGATAGTG-3′
	5′-TCAAGTACCCACAGTGCGGT-3′
U6	5′-CTCGCTTCGGCAGCACA-3′
	5′-AACGCTTCACGAATTTGCGT-3′
FOXO1	5′-GAGGAGCCTCGATGTGGATG-3
	5′-CCGAGATTTGGGGGAACGAA-3

Notes: MEG3, maternally expressed 3; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RT-qPCR, real-time fluorescence quantitative polymerase chain reaction.

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MEG3 promotes proliferation and inhibits apoptosis in osteoarthritis chondrocytes by miR-361-5p/FOXO1 axis

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