下调microRNA-448通过上调Matrilin-3抑制IL-1β诱导的人软骨细胞软骨降解资源-CSDN文库

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RES E A R C H Open Access

Downregulation of microRNA-448 inhibits

IL-1β-induced cartilage degradation in

human chondrocytes via upregulation of

matrilin-3

Hao Yang

,DiWu

, Hua Li

, Nan Chen

and Yongjun Shang

* Correspondence:

shangyongjundalian@yahoo.com

Department of Orthopedics, Dalian

University Affiliated Xinhua Hospital,

No. 156 Xinhua Street, Shahekou

District, Dalian 116021, Liaoning

Province, People’s Republic of China

Full list of author information is

available at the end of the article

Abstract

Background: Osteoarthritis is characterized by the continuous degradation of the

articular cartilage. The microRNA miR-448 has been found to be broadly involved in

cellular processes, including proliferation, apoptosis, invasion and EMT. While aberrant

expression of miR-448 has been found in multiple cancers, its level in osteoarthritis

cartilage and its role in the progression of this disease are still unknown. Here, we

examined the functional roles of miR-448 and its expression in osteoarthritis tissues,

including IL-1β-stimulated osteoarthritis chondrocytes.

Methods: Chondrocytes were isolated from human articular cartilage and stimulated

with IL-1β. The expression levels of miR-448 in the cartilage and chondrocytes were

both determined. After transfection with an miR-448 mimic or inhibitor, the mRNA

levels of aggrecan, type II collagen and MMP-13 were determined. Luciferase reporter

assay, qRT-PCR and western blot were performed to explore whether matrilin-3 was a

target of miR-448. Furthermore, we co-transfected chondrocytes with miR-448 inhibitor

and siRNA for matrilin-3 and then stimulated them with IL-1β to determine whether

miR-448-mediated IL-1β-induced cartilage matrix degradation resulted from directly

targeting matrilin-3.

Results: The level of miR-448 was significantly higher and matrilin-3 expression was

significantly lower in osteoarthritis cartilage and IL-1β-induced chondrocytes than in

normal tissues and cells. Furthermore, matrilin-3 expression was reduced by miR-448

overexpression. MiR-448 downregulation s ign ifican tly alleviated the IL-1β-induced

downregulation of aggrecan and type II collagen expression, and upregulation of

MMP-13 expression. MiR-448 overexpression had the opposite effects. Knockdown of

matrilin-3 reversed the effects of the miR-448 inhibitor on the expressions of aggrecan,

type II collagen and MMP-13.

Conclusion: The findings showed that miR-448 contributed to the progression of

osteoarthritis by directly targeting matrilin-3. This indicates that it has potential as a

therapeutic target for the treatment of osteoarthritis.

Keywords: Osteoarthritis, MicroRNA-448, Interleukin-1 beta, Chondrocyte, Extracellular

matrix, Matrilin-3

Cellular & Molecular

Biolo

Letters

License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium,

provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and

indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/

publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Yang et al. Cellular & Molecular Biology Letters (2018) 23:7

https://doi.org/10.1186/s11658-018-0072-6

Background

Osteoarthritis (OA), which is characterized by articular cartilage degradation, is the most

common type of degenerative joint disease [1]. Chondrocytes are the only cells found in

the articular cartilage and they play a critical role in maintaining matrix integrity and tissue

homeostasis [2]. During the progress of osteoarthritis, they are metabolically activated,

whereupon they subsequently disrupt the metabolic homeostasis of the extracellular matrix

(ECM) by increasing the production of matrix-degrading enzymes such as matrix metallo-

proteinase 13 (MMP-13) and decreasing the synthesis of aggrecan and type II collagen [3].

This results in the degradation of the ECM. Developing effective therapeutic strategies to

treat osteoarthritis will certainly require an understanding of chondrocyte injuries and

related mechanisms.

It has been reported that interleukin-1 beta (IL-1β), one of the main cytokines involved in

the pathogenesis of osteoarthritis, can inhibit cell proliferation, reduce the synthesis of aggre-

can and type II collagen, and increase the expression of MMP-13 in chondrocytes [2, 3].

MicroRNAs (miRNAs) are a class of non-coding small RNA identified as critical

post-transcriptional regul ators. They play critical roles in several biological processes ,

including cell proliferation, migration, invasion and differentiation [4, 5]. An increasing

number of studies have indicated that miRNAs participate in the pathogenesis of osteo-

arthritis, with confirmation for miR-139, miR-140, miR-23a-3p and miR-365 [6–9].

It has been reported that miR-448 is broadly involved in cellular processes, including prolif-

eration, apoptosis, invasion and epithelial–mesenchymal transition (EMT) [10, 11]. Aberrant

miR-448 expression has been confirmed in many cancers [12–14]. However, the levels of

miR-448 in osteoarthritis cartilage and its role in progression of this disease are still unclear.

Earlier studies have also demonstrated that the expression of matrilin-3 is dramatic-

ally lower in osteoarthritis cartilage than in normal cartilage [15]. Thus, miRNAs and

matrilin-3 clearly both have the important roles in the pathogenesis of osteoarthritis.

The online database microRNA.org was used to predict that matrilin-3 is a binding

target of miR-448. This study focuses on whethe r miR-448 plays a significant role in

progression of osteoarthritis by targeting matrilin-3.

Our data show that the miR-448 level significantly increased and the expression of

matrilin-3 significantly decreased in osteoarthritis cartilage compared to normal cartilage.

Furthermore, we confirmed that miR-448 could directly target the matrilin-3 gene. The

functional analysis showed that knockdown of miR-448 upregulated IL-1β-inhibited the

expressions of aggrecan and type II collagen, and downregulated IL-1β-induced MMP-13

expression by increasing matrilin-3 expression, contributing to the protection of chondro-

cytes from IL-1β-induced degradation of ECM. To confirm these results, the functional

roles of miR-448 were studied in osteoarthritis chondrocytes. As expected, miR-448 also

had similar effects on IL-1β-induced chondrocytes. According to the findings, miR-448 is

a promising target for prevention and treatment of osteoarthritis.

Methods

Specimen selection

Human cartilage samples were collected from 10 osteoarthritis patients undergoing total

knee replacement surgery and from 10 traumatic amputees without rheumatoid arthritis

or osteoarthritis. Patients with osteoarthritis were diagnosed according to the American

Yang et al. Cellular & Molecular Biology Letters (2018) 23:7 Page 2 of 14

College of Rheumatology criteria. All samples were collected from the Department of

Orthopedics, Dalian University Affiliated Xinhua Hospital. Informed consent was obtained

from all patients and the study was approved by the Ethics Committee of Dalian University

Affiliated Xinhua Hospital.

Cell culture and treatment

To isolate the primary osteoarthritis and normal chondrocytes, osteoarthritis and normal

articular cartilage slices were chopped finely with a scalpel blade. Cartilage tissue was

predigested with trypsin (0.1%, w/v; Sigma) for 10 min at 37 °C. After removing the tryp-

sin solution, the tissue slices were treated overnight with type IV Clostridium collagenase

in Dulbecco’s modified Eagle medium (DMEM) with 5% fetal bovine serum (FBS). Then

the sample was filtered to remove the undigested cartilage and the chondrocyte cells were

pelleted at 2000 x g for 5 min before being resuspended. After the initial isolation, the

cells were maintained in high-density cultures in DMEM supplemented with 10% FBS, L-

glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin (GIBCO). First passage

chondrocytes were obtained after 2 weeks. All the experiments were done within 3 days

of the passage 1 culture.

Primary osteoarthritis and normal chondrocytes were transfected with miR-448

mimic or inhibitor (RiboBio) at a 100 nM concentration by using Lipofectamine 3000

reagent (Invitrogen) following the manufacturer’s instructions. After transfection for

48 h, the medium was removed, and cells were stimulated with 5 ng/ml IL-1β for 24 h

or were not stimulated, and then total RNA prepared from chondrocytes was used to

check the expressions of miR-448, matrilin-3, aggrecan, type II collagen and MMP-13.

Cell supernatants were harvested and MMP-13 production was quantified via ELISA.

RNA extraction and quantitative real-time PCR

Total RNA of chondrocytes was extracted for the analysis of miRNA and mRNA fol-

lowing the manufacturer’s instructions. For quantification of miR-448, the TaqMan

MicroRNA Reverse Transcription Kit and TaqMan miRNA assay (Qiagen) were re-

spectively used to perfo rm reverse transcription and PCR following the manufacturer’s

instructions. U6 was used as the internal control. The gene expressions of matrilin-3,

aggrecan, type II collagen and MMP-13 were detected using the SYBR Green PCR kits

(TAKARA). GAPDH served as an internal control. All PCR assays were performed in

CFX96 Touch (Bio-Rad). The following primers were used:

 Matrilin-3 forward, 5’-TCTCCCGGATAATCGACACTC-3′, reverse, 5’-CA

AGGGTGTGATTCGACCCA-3’

 Aggrecan forward, 5’-ACCAGACGGGCCTCCCAGAC-3′, reverse, 5’-TGG

CTCTGCCCCAGAGGGAC-3′

 Type II collagen forward, 5’-TGAGGGCGCGGTAGAGACCC-3′, reverse, 5’-TG

CACACAGCTGCCAGCCTC-3’

 MMP-13 forward, 5’-ATGCGGGGTTCCTGATGTGG-3′, reverse, 5’-GG

CCCAGGAGGAAAAGCATG-3’

 GAPDH forward, 5’-ACAACTTTGGTATCGTGGAAGG-3′, reverse, 5’-GC

CATCACGCCACAGTTTC-3’

Yang et al. Cellular & Molecular Biology Letters (2018) 23:7 Page 3 of 14

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