Silencing of long‑chain non‑coding RNA GAS5 in osteoarthritic chondrocytes is mediated by targeting the miR‑34a/Bcl‑2 axis

  • Authors:
    • Qinghui Ji
    • Xiaofeng Qiao
    • Yongxiang Liu
    • Dawei Wang
    • Jinglong Yan
  • View Affiliations

  • Published online on: December 20, 2019     https://doi.org/10.3892/mmr.2019.10900
  • Pages: 1310-1319
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Abstract

The present study aimed to investigate the effects of the long non‑coding RNA (lncRNA) growth arrest‑specific transcript 5 (GAS5) on proliferation, apoptosis and the inflammatory response of osteoarthritic chondrocytes (OACs) and its associated mechanism of action. Primary chondrocytes were isolated from cartilage tissues of osteoarthritis (OA) patients for subculture. GAS5 was silenced in OACs by liposome transfection. The effects of GAS5 silencing on proliferation, apoptosis, stromal metabolism and inflammatory response of OACs were analyzed. The association of GAS5 with its target microRNA‑34a (miR‑34a) and the downstream target gene Bcl‑2 was verified by luciferase reporter assays. The results indicated that GAS5 silencing promoted the proliferation, inhibited cell apoptosis and caused G1 arrest of OACs compared with the control group (P<0.05). The expression levels of TNF‑α and IL‑6 in the supernatant of OACs in the si‑GAS5 group were significantly lower than those of the control group (P<0.05). The results of the double luciferase reporter assays indicated that overexpression of GAS5 downregulated miR‑34a and upregulated Bcl‑2 levels (P<0.05) compared with the expression levels of these markers in the control group. In contrast to GAS5 overexpression, knockdown of this RNA caused a significant upregulation of miR‑34a levels and a significant downregulation in the levels of Bcl‑2 (P<0.05). Moreover, GAS5 overexpression could counteract the inhibition of apoptosis by overexpression of miR‑34a (P<0.05). The data indicated that GAS5 participated in the development of OA by regulating the biological behavior of chondrocytes via the miR‑34a/Bcl‑2 axis.

Introduction

Osteoarthritis (OA) is a degenerative disease characterized by cartilage degeneration, osteophyte formation, and narrowing of the joint space. This disease has become the fourth most disabling cause in the world (1). A major focus in bone research has been the understanding of the etiology and pathogenesis of OA. Recent studies have revealed that long non-coding RNAs (lncRNAs) are associated with a variety of diseases, including OA (24). The lncRNA growth arrest-specific transcript 5 (GAS5) is one of the most important lncRNAs noted in human T lymphocytes and in non-transformed lymphocytes, and its current functional annotation is that of a tumor suppressor gene currently (5). LncRNAs can exert their biological functions by targeting miRNAs. Previous studies that investigated GAS5 and its target miRNAs have mainly focused on cancer (6,7). A limited number of studies have explored the role of GAS5 in the development of OA (8). In the present study, GAS5 expression was investigated with regard to chondrocyte proliferation, apoptosis, extracellular matrix (ECM) metabolism and inflammatory response by silencing its expression in osteoarthritic chondrocytes (OACs).

Materials and methods

Source of specimens

A total of 30 patients who underwent knee arthroplasty from January 2016 to June 2018 at the Second Affiliated Hospital of Harbin Medical University were enrolled (OA group). In addition, 30 patients with artificial hip arthroplasty due to femoral neck fracture were selected as the control group. Cartilage tissue samples of OA and control patients were obtained during surgery. The cartilage tissue was stored in a sample bottle of phosphate-buffered saline (PBS) solution containing 20% medium and 5% calf serum for chondrocyte culture. The present study was approved by the Second Affiliated Hospital of Harbin Medical University Ethics Committee and all subjects signed the relevant informed consent.

Isolation and culture of chondrocytes

The cartilage tissue was cut into small pieces of approximately 1 mm3 in diameter using a pair of ophthalmic scissors. The tissue fragments were digested and centrifuged (250 × g for 15 min at room temperature), the supernatant was discarded and the primary chondrocytes were isolated from the precipitate. The collected primary chondrocytes were resuspended in RPMI-1640 complete medium containing 10% fetal bovine serum, 100 U/ml penicillin and 100 U/ml streptomycin. The cells were cultured at 37°C, in the presence of 5% CO2. The culture medium was replaced every two days. The chondrocytes were identified by toluidine blue staining and immunocytochemical analysis. The subculture was carried out when the cells were grown to 80% confluence. The samples from the second-generation chondrocytes were obtained for subsequent experiments.

Toluidine blue staining

Following routine digestion, the chondrocytes were inoculated in 24-well culture plates and pre-plated with glass slides. The chondrocytes were collected and the culture solution was discarded. The cells were placed in 4% paraformaldehyde and fixed at 4°C for 1 h. The slides were washed with tap water for 15 min and placed in toluidine blue dye solution for 2 h. Excess dye solution was removed and the slides were placed under an inverted microscope for visualization.

Transfection

The second generation OACs were obtained for further experiments. The following groups were included: Control, si-NC and si-GAS5. The cells were transfected with GAS5 siRNA and the corresponding GAS5 siRNA negative sequences using Lipofectamine 2000 transfection reagent. The molecular mechanism of GAS5 that affected chondrocyte apoptosis in OA was examined. miR-34a overexpression or inhibitory cell lines (miR-34a mimic and its negative control mimic-NC, as well as miR-34a inhibitor and its negative control inhibitor-NC) were used for further experiments. In addition, a GAS5 overexpression or knockout cell line (transfection of OA articular chondrocytes with GAS5 and sh-GAS5 plasmids to overexpress or knockdown intracellular GAS5) and a co-transfected cell model that overexpressed GAS5 and miR-34a (miR-34a mimic and its negative control mimic-NC, as well as GAS5 and its negative control sh-GAS5) were established. All protocols were carried out in accordance with the manufacturer's instructions. Following transfection and incubation for 24 h, the transfection efficiency was verified by reverse transcription-quantitative PCR (RT-qPCR) detection of GAS5.

RT-qPCR

Total RNA was extracted following cell transfection. RNA extraction was conducted using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.) in an RNAase-free environment. The total RNA concentration and purity were detected by the ultra-micro nuclear protein assay. A quantitative PCR amplification instrument was used to detect the expression levels of the objective gene. The primers sequences of GAS5,miR-34a and Bcl-2 were designed and synthesized by Shanghai Sangon Biotech (Table I). The reaction conditions were as follows: preheating at 50°C for 2 min and initial denaturation at 95°C for 10 min. A total of 35 cycles were performed that included the following steps: Denaturation at 95°C for 10 sec, annealing at 60°C for 1 min and extension at 72°C for 55 sec. The analysis was carried out by the 2−ΔΔCq (9) method.

Table I.

Primer sequences.

Table I.

Primer sequences.

Primer nameSequences
GAS5(F) 5′-TCGGCTTGACTACACTGTGT-3′
(R) 5′-GGAGGCTGAGGATCACTTGA-3′
Bcl-2(F) 5′-GAGCATCTCACACTCGTTG-3′
(R) 5′-GAAAGAGGGATGCTGTCTCG-3′
β-actin(F) 5′-CATGGAATCTGTGGCATGG-3′
(R) 5′-TGATCTTCATGGTGCTGGGA-3′
miR-34aStem loop: 5′-AGCTCAGAAGCTGCCACAAT-3′
(F) 5′-TTCAAGAACACCTGCACAGC
(R) 5′-GGAAAGTACGCAGCCAAGTC

[i] F, forward; R, reverse.

MTT assay for the detection of chondrocyte growth

The cells were cultured for 48 h following transfection. The cell density was adjusted at 2×104 cells/ml and the cells were transferred into 96-well plates. A total of 5 replicate wells were set in each group and cultured at 37°C in the presence of 5% CO2. A total of 20 µl MTT solution (5 mg/ml) was added to each well. Following continuous culture for 4 h, 150 µl of dimethyl sulfoxide (DMSO) was added to each well, shaken and mixed for 10 min at room temperature. The absorbance value (OD) of each group of cells was measured at 490 nm. The measurements were repeated three times per well.

Flow cytometry (FCM) for apoptosis detection and cell cycle analysis

The cells were cultured for 48 h following transfection. The samples were centrifuged at 1,000 × g for 5 min at room temperature and 500 µl of buffer was added to adjust the cell suspension concentration to 1×106 cells/ml. A total of 5 µl of Annexin V-FITC was added and mixed with the samples. The samples were incubated at 4°C for 15 min in the dark. A total of 5 µl of PI staining solution was added and the samples were incubated at 4°C for 5 min in the dark. Apoptosis was detected by FCM. The cell suspension was transferred to a 1.5-ml centrifuge tube, and 95 µl pre-cooled 75% ethanol was added. The final samples were incubated at 4°C for 24 h. Following centrifugation at 1,000 × g for 5 min at room temperature, the supernatant was discarded and the cells were resuspended in PBS. A total of 500 µl PI staining solution (0.5%) was added and the cells were resuspended and incubated in the dark. The cell cycle was detected by FCM.

Western blot analysis

Following transfection, each group of cells was collected. The cells were lysed with RIPA cell lysate (Beyotime Institute of Biotechnology) and centrifuged at 12,000 × g for 10 min at 4°C. The protein concentration was determined by BCA. Protein (40 µg) was separated by 10% SDS-PAGE and transferred into polyvinylidene difluoride (PVDF) membranes. The membranes were blocked by TBST (0.25%Tween) with 5% skimmed milk for 2 h at room temperature, and incubated with the corresponding primary antibodies (Invitrogen; Thermo Fisher Scientific, Inc.): Anti-Bcl2 (1:1,000, cat. no. 138800), Aanti-Bax (1:1,000, cat. no. BMS163), anti-MMP3 (1:5,000, cat. no. MA514247), anti-Collagen II (1:5,000, cat. no. MA512789), anti-Aggrecan (1:1,000, cat. no. MA316888), anti-Ki-67 (1:200, MA514520), anti-PCNA (1:100, MA511358), anti-CDK2 (1:2,000, cat. no. MA532017), Anti-p53 (1:1,000, cat. no. MA512557), at 4°C overnight. The secondary antibodies Invitrogen; Thermo Fisher Scientific, Inc.): Goat anti-mouse HRP-IgG (1:10,000, cat. no. G21040) or Goat anti-rabbit HRP-IgG (1:10,000, cat. no. G21234) were incubated for 2 h at room temperature. The ECL luminescent agent (Tiangen Biotech Co., Ltd.) was added for film development in a dark room. β-actin was used as an internal reference. The analysis was performed by Quantity-One software (v4.6.6; Bio-Rad Laboratories, Inc.).

Enzyme-linked immunosorbent assay (ELISA)

The expression levels of the inflammatory factors IL-6 and TNF-α were assessed by ELISA in chondrocytes derived from osteoarthritis patients. The experiments were performed step by step in strict accordance with the kit instructions.

Luciferase reporter assay

The binding site of GAS5 on miR-34a was predicted by the Starbase v2 software (http://starbase.sysu.edu.cn/index.php), and the binding site of miR-34a and Bcl-2 was predicted by TargetScan (http://www.targetscan.org/vert_72/). PCR amplified a fragment of the miR-34a binding site in GAS5 and Bcl-2, respectively. The corresponding fragments were inserted into the pcDNA-Report vector, respectively. The cells were co-transfected with the miR-34a mimic or the mimic-NC using GAS5-MUT (or GAS5-WT) and Bcl-2-MUT (or Bcl-2-WT) plasmids, respectively. Fluorescence intensity was determined according to the Dual Luciferase reporter kit (Invitrogen; Thermo Fisher Scientific, Inc.). All experiments were carried out in strict accordance with the manufacturer's instructions.

Statistical analysis

All data in this study were processed using SPSS 20.0 statistical software (IBM Corp.). All data are expressed as the mean ± standard deviation (SD). Comparison among multi-groups were conducted by ANOVA and pairwise comparisons were performed using the LSD t-test. P<0.05 was considered to indicate a statistically significant difference.

Results

Primary culture and identification of chondrocytes

The morphology of articular chondrocytes in the two groups and in the second-generation chondrocytes were stained with toluidine blue and observed using microscopy. The chondrocytes in the two groups were normal in morphology and exhibited a long fusiform structure (Fig. 1A). Following staining with toluidine blue, the nuclei of the cells were stained dark blue and the nucleolus was visible (Fig. 1B). The data indicated that the chondrocytes in the normal group exhibited higher density than those of the OA group.

Comparison of lncRNA GAS5 expression in normal chondrocytes and OACs

The expression of GAS5 in primary cultured chondrocytes was detected by RT-qPCR. GAS5 was expressed in both articular and normal chondrocytes and its expression levels in the osteoarthritic chondrocytes was significantly higher than those noted in normal chondrocytes (2.37±1.12 vs. 1.07±0.22) (P<0.05, data not shown).

Transfection efficacy

The transfection efficacy was verified by RT-qPCR. The expression levels of GAS5 in the si-GAS5 group were significantly lower compared with those of the control group (P<0.05). The expression levels in the si-NC and control groups exhibited no significant difference (P>0.05, Fig. 2A).

The expression levels of miR-34a were significantly higher in the miR-34a mimic group, while the miR-34a inhibitor group exhibited significantly lower miR-34a levels than those of the control group (Fig. 2B). No significant differences were noted in the expression levels of miR-34a between the mimic-NC, the inhibitor-NC and the control groups (P>0.05). GAS5 overexpression or knockout in transfected cell lines (GAS5 or sh-GAS5) was successfully established in chondrocytes (Fig. 2C).

Effects of GAS5 silencing on chondrocyte proliferation, cell cycle and apoptosis induction

The damage caused in articular cartilage is considered the pathological basis of OA. During that process, chondrocyte proliferation and apoptosis play an important role (10). MTT assay demonstrated that the proliferation of cells in the si-GAS5 group was significantly increased compared with that of the control group (P<0.05). Western blot analysis indicated that the expression levels of proliferating cell nuclear antigen (PCNA) and Ki-67 in the si-GAS5 group were significantly higher than those of the control group (P<0.05, Fig. 3A). The results suggested that GAS5 silencing could promote the proliferation of OACs.

FCM analysis indicated that the apoptosis rate of OACs in the si-GAS5 group was significantly lower than that of the control group following transfection and cell culture for 48 h (Fig. 3B, P<0.05). The expression levels of Bax in the si-GAS5 group were significantly increased, while the expression levels of Bcl-2 were significantly decreased (P<0.05, Fig. 3B). The results of the FCM analysis further demonstrated that the percentage of G0/G1 phase cells in the si-GAS5 group was increased, while the number of cells in the G2/M phase decreased significantly compared with that of the control group (Fig. 3C, P<0.05). Western blot analysis indicated that the expression levels of CDK2 in the si-GAS5 group was significantly lower than that in the control group, while the expression of p53 was higher than that in the control group (Fig. 3C, P<0.05). These results indicated that silencing of GAS5 could cause G1 arrest, promote proliferation of OACs and inhibit the induction of apoptosis.

Effects of GAS5 silencing on ECM metabolism and inflammatory response in OACs

Articular cartilage ECM protects chondrocytes from mechanical stress. The most important component is collagen II, followed by proteoglycans. Excessive degradation and loss of ECM is one of the important signs of the onset of OA (11). The results indicated that the expression levels of type II collagen and aggrecan were significantly increased in the si-GAS5 group compared with those of the control group, while the expression levels of MMP-13 were significantly decreased (P<0.05, Fig. 4A), indicating that GAS5 silencing inhibited the degradation of the cartilage matrix.

The inflammatory response plays an important role in the development of OA (12). ELISA indicated that the levels of TNF-α and IL-6 in the si-GAS5 group were significantly lower than those of the control group (P<0.05, Fig. 4B), indicating that GAS5 silencing could reduce the severity of the inflammatory response of OACs.

GAS5 targets miR-34a and regulates the expression of Bcl-2

It was predicted by TargetScan that miR-34a may be a target of GAS5 and Bcl-2 a candidate target gene of miR-34a (Fig. 5A). The results of the dual luciferase reporter assay demonstrated that luciferase activity was significantly decreased in cells co-transfected with GAS5-WT and miR-34a mimic (P<0.05, Fig. 5B). Bcl-2-WT and miR-34a mimic co-transfection resulted in a significant decrease in luciferase activity (P<0.05). GAS5 overexpression or knockout cell lines were established. Moreover, miR-34a overexpression was achieved by transfection of miR-34a to the cells, and miR-34a inhibition by addition of a miR-34a inhibitor. The results indicated that miR-34a was downregulated in the GAS5 group (Fig. 5C), while the expression levels of the Bcl-2 protein were increased compared with those of the control group (P<0.05, Fig. 5C). The effect noted in the sh-GAS5 group was contradictory to these findings. The expression levels of Bcl-2 in the miR-34a mimic and miR-34a inhibitor groups were lower and higher than those of the control group, respectively (P<0.05, Fig. 5D). These results indicated that GAS5 could indirectly regulate the expression levels of Bcl-2 by targeting miR-34a.

GAS5 targets miR-34a/Bcl-2 to regulate apoptosis

Cell lines that were co-transfected with GAS5 and miR-34a were established. The results of the FCM analysis indicated that the apoptotic rate of the GAS5 group was significantly increased compared with that of the control group (P<0.05, Fig. 6A), suggesting that GAS5 overexpression promoted the induction of apoptosis. The apoptotic rate in the miR-34a mimic with GAS5 overexpression group was lower than that of the GAS5 group alone and higher than that of the miR-34a mimic group alone (P<0.05). Consequently, the expression levels of Bcl-2 were significantly increased and significantly decreased in the GAS5 and in the miR-34a mimic groups, respectively (P<0.05, Fig. 6B). These findings indicated that the induction of OAC apoptosis by GAS5 was reduced by miR-34a and that the Bcl-2-mediated inhibition of miR-34a expression was reduced by GAS5. Collectively, the data indicated that GAS5 regulated the induction of OAC apoptosis by targeting the miR-34a/Bcl-2 axis.

Discussion

The main process involved in the pathogenesis of OA is the degeneration of articular cartilage, in which chondrocyte proliferation and apoptosis play an important role (13,14). Under normal circumstances, the processes of proliferation and apoptosis of chondrocytes are coordinated in an orderly manner in cartilage tissues. During excessive induction of chondrocyte apoptosis, internal cartilage disorders may occur, leading to abnormal cartilage function (15). Blanco et al (16) demonstrated that the proportion of apoptotic chondrocytes in the osteoarthritic cartilage was significantly higher than that noted in normal tissues (11% vs. 5.1%, P<0.01), which confirmed that chondrocyte apoptosis was associated with OA. Several lncRNAs have been revealed to be closely associated with the proliferation and apoptosis of chondrocytes. Li et al (17) revealed that PVT1 expression in OACs was significantly increased, while inhibition of PVIT1 could inhibit cell apoptosis. Zhang et al (18) indicated that the expression of UFC in OACs was downregulated and that this process could inhibit chondrocyte proliferation and promote apoptosis by targeting miR-34a. A previous study revealed that GAS5 overexpression played an important role in cell proliferation, apoptosis and growth (19). In radiation-induced thymic lymphoma, upregulation of GAS5 expression was involved in the regulation of cell proliferation and colony formation by participating in the chromosomal rearrangement of Notch1 (20). In non-small cell lung cancer, GAS5 could inhibit cell development by regulation of cell cycle progression via the p53/E2F1 signaling pathway, which acted as a tumor suppressor (21). Moreover, previous studies have revealed that GAS5 blockers accelerate cell proliferation and reduce apoptosis by promoting cell cycle progression (22). However, the role of GAS5 on OACs is not very clear. In the present study, differential expression of GAS5 in OACs and normal chondrocytes was observed. Silencing of GAS5 promoted cell proliferation and inhibited apoptosis, which revealed that GAS5 may be involved in the progression of OA by regulating OAC cell cycle progression.

The metabolic balance of the cartilage matrix guarantees the normal function of the cartilage tissue. Degradation of type II collagen in the ECM of chondrocytes can lead to abnormal cartilage morphology, as well as subchondral bone and trabecular bone structure (23). The direct cause of cartilage matrix degradation is mainly caused by increased protease activity of matrix metalloproteinases (MMPs) (24). Recent studies have revealed that lncRNAs play an important role in the ECM balance of chondrocytes, including lncRNA CIR (4,25) and MSR (26). In the present study, GAS5 silencing significantly inhibited the expression levels of MMP-13 in OACs. In addition, it increased the content of type II collagen and aggrecan in OACs, indicating that GAS5 may be involved in the development of OA by the regulation of chondrocyte ECM metabolism. OA is considered a degenerative and an inflammatory disease (12). Proinflammatory cytokines act on chondrocytes in order to cause chondrocyte metabolism and secretion disorders as well as chondrocyte proliferation and apoptosis abnormalities (27). Previous studies have suggested that lncRNAs may be considered a bridge between inflammatory factors and cartilage destruction (2,28,29). Therefore, lncRNAs can be used as anti-inflammatory drugs for OA and can replace the use of glucocorticoid drugs, which exhibit several adverse reactions. The present study demonstrated that silencing of GAS5 reduced the levels of IL-6 and TNF-α in OACs, indicating that GAS5 may be associated with the regulation of several inflammatory factors in the osteoarthritic cartilage.

miR-34a expression is downregulated in various tumors, such as prostate cancer (30) and breast cancer (31), indicating tumor suppressor functions of this RNA. Previous studies have revealed that miR-34a can inhibit cell proliferation and promote cell apoptosis (32). Bcl-2 is a target of miR-34a and are both involved in the regulation of apoptosis (33). In a previous study, bioinformatics analysis revealed binding of GAS5 and miR-34a and of miR-34a and Bcl-2, indicating that these molecules may be involved in the induction of OAC apoptosis. Moreover, luciferase reporter assays were used to test this hypothesis and it was revealed that GAS5 overexpression in OACs downregulated the expression of miR-34a, while it upregulated Bcl-2 levels in order to promote apoptosis induction. GAS5 and miR-34a were co-transfected in OACs to further validate the association between GAS5 and the miR-34a/Bcl-2 pathway. The results indicated that GAS5 overexpression could reverse the effects of miR-34a overexpression caused on the induction of apoptosis and on the expression of Bcl-2. Collectively, the results demonstrated that GAS5 could regulate the induction of OAC apoptosis by targeting the miR-34a/Bcl-2 axis.

In summary, the present study indicated that the expression levels of GAS5 were upregulated in OACs. GAS5 may participate in the development of OA by regulating apoptosis, cartilage ECM metabolism and chondrocyte inflammatory response. The mechanism of these processes may be associated with the regulation of the miR-34a/Bcl-2 pathway.

Acknowledgements

Not applicable.

Funding

No funding was received.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

JY contributed to the conception and design of the present study, QJ contributed to the acquisition, analysis and interpretation of data, and drafted the manuscript. XQ contributed to the case collection, statistical analysis of clinical data and revised the manuscript critically for important intellectual content. YL and DW contributed to data collection and the performance of basic experiments. QJ contributed to the final manuscript revision and all authors agreed to be accountable for all aspects of the present study in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Ethics approval and consent to participate

The present study was approved by The Second Affiliated Hospital of Harbin Medical University Ethics Committee and all subjects signed the relevant informed consent.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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March-2020
Volume 21 Issue 3

Print ISSN: 1791-2997
Online ISSN:1791-3004

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Spandidos Publications style
Ji Q, Qiao X, Liu Y, Wang D and Yan J: Silencing of long‑chain non‑coding RNA GAS5 in osteoarthritic chondrocytes is mediated by targeting the miR‑34a/Bcl‑2 axis. Mol Med Rep 21: 1310-1319, 2020
APA
Ji, Q., Qiao, X., Liu, Y., Wang, D., & Yan, J. (2020). Silencing of long‑chain non‑coding RNA GAS5 in osteoarthritic chondrocytes is mediated by targeting the miR‑34a/Bcl‑2 axis. Molecular Medicine Reports, 21, 1310-1319. https://doi.org/10.3892/mmr.2019.10900
MLA
Ji, Q., Qiao, X., Liu, Y., Wang, D., Yan, J."Silencing of long‑chain non‑coding RNA GAS5 in osteoarthritic chondrocytes is mediated by targeting the miR‑34a/Bcl‑2 axis". Molecular Medicine Reports 21.3 (2020): 1310-1319.
Chicago
Ji, Q., Qiao, X., Liu, Y., Wang, D., Yan, J."Silencing of long‑chain non‑coding RNA GAS5 in osteoarthritic chondrocytes is mediated by targeting the miR‑34a/Bcl‑2 axis". Molecular Medicine Reports 21, no. 3 (2020): 1310-1319. https://doi.org/10.3892/mmr.2019.10900