Hyun Ah Kim; Hyun A Jung; Tae Young Kim

doi:10.4078/jrd.2011.18.4.264

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Kim, Jung, and Kim: Identification of Genes Regulated by IL-1β Using Integrative microRNA and mRNA Genomic Analysis in Human Articular Chondrocytes

Original Article

J Rheum Dis 2011;18(4):264-275.

Published online: 31 December 2011

DOI: https://doi.org/10.4078/jrd.2011.18.4.264

Identification of Genes Regulated by IL-1β Using Integrative microRNA and mRNA Genomic Analysis in Human Articular Chondrocytes

Hyun Ah Kim¹, Hyun A Jung¹, Tae Young Kim²

¹Department of Internal Medicine, Hallym University Sacred Heart Hospital, Anyang, Korea

²Department of Orthopedic Surgery, Hallym University Sacred Heart Hospital, Anyang, Korea

Correspondence : Hyun Ah Kim Division of Rheumatology, Department of Internal Medicine, Hallym University Sacred Heart Hospital, 896, Pyongchon-dong, Dongan-gu, Anyang 431-070, Korea E-mail : kimha@hallym.ac.kr

Received 12 April 201120 July 2011 Accepted 22 July 2011

Abstract

Objective

The physiological and pathogenetic role of microRNAs (miRNAs) in the maintenance of joint homeostasis and in the development of arthritis is recently being elucidated. In this study, we attempted to identify differentially expressed miRNAs in human osteoarthritis (OA) chondrocytes in response to interleukin (IL)-1β. In addition, simultaneous profiling of miRNA and mRNA expression was performed to get an integrated analysis of miRNA and mRNA expression.

Methods

Monolayer cultured chondrocytes obtained from knee cartilages of OA patients were stimulated with IL-1β for 4 hours and RNA was isolated. One microgram of total RNA was polyadenylated and converted to cDNA and miRNA microarray was performed. Seven hundred thirty five oligos were used, corresponding to 470 well-annotated human miRNA sequences and 265 potential miRNAs that were identified recently. mRNA microarray was performed simultaneously using the RNA samples that were used for miRNA array. Both sequence and expression information was used to identify regulatory relationship between miRNA and mRNA pairs.

Results

Expression profiling of miRNA extracted from IL-1β treated chondrocytes identified 25 miRNA which showed differential expression. We also identified 7190 mRNAs differentially regulated by IL-1β treatment. Among the 25 miRNAs differentially regulated, 13 miRNAs had targets searched by MiRANDA scheme. By combining target search and miRNA-mRNA pairing, we could identify 1043 miRNA-mRNA target pairs. MiR-200a was found to be expressed in human OA and normal cartilages, with downregulation in OA lesion cartilages.

Conclusion

It is suggested that miRNA may play a role in the regulation of cartilage degradation in OA.

Keywords: Chondrocytes, Interleukin-1, microRNA, miR- 200a, mRNA, Osteoarthritis

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Figure 1.

Real-time RT-PCR results for miR-200a in chondrocytes in response to IL-1β. Total RNA was isolated from IL-1β treated monolayer chondrocytes obtained from donors different from those that were used in miRNA microarray. The RNA, U6 small nuclear 2 (RNU6-2) gene was used as a control to normalize differences in total RNA levels in each sample. The value of each control sample was set at 100 and was used to calculate the fold change in IL-1β treated chondrocytes. Data are from triplicate experiments using chondrocytes from 3 different donors. ∗denoted p<0.05 compared to control by using Mann-Whitney U test.

Figure 2.

RT-PCR results for tensin-1 and Tbx-15 in chondrocytes in response to IL-1β. Total RNA was isolated from IL-1β treated monolayer chondrocytes obtained from donors different from those that were used in mRNA microarray. GAPDH gene was used as a control to normalize differences in total RNA levels in each sample. Data are representative of duplicate experiments using chondrocytes from 3 different donors.

Figure 3.

In situ hybridization for miR-200a in normal and OA cartilages. Cartilage tissues obtained from OA patients at the time of joint replacement surgery were obtained from both lesion and non-lesion areas (defined as areas with preserved cartilage surface and thickness). For normal cartilage, macro and microscopically normal cartilage samples obtained from the femoral head of patients with femoral neck fracture were used. Data are representative of specimens from 4 different donors each for normal and OA (×100, original magnification).

Table 1.

MiRNAs with differential expression between IL-1β treated and control chondrocytes from OA cartilages (n=6)

miRNA	Chromosomal location	Fold change
tHS_15.1	Unknown	2.15
HS_176	11	2.01
HS_221	15	1.63
hsa-miR-202	10	1.52
hsa-miR-335	7	1.48
hsa-miR-563	3	1.46
hsa-miR-362	×	1.39
hsa-miR-452	×	1.38
HS_149	9	1.36
hsa-miR-603	10	1.35
HS_175	11	1.29
hsa-miR-425	3	1.24
HS_219	15	1.16
hsa-miR-507	×	1.16
hsa-miR-766	×	1.15
HS_145.1	9	1.08
hsa-let-7e	19	1.04
hsa-miR-31	9	-1.06
hsa-miR-454-3p	17	-1.11
hsa-miR-181b	9	-1.25
hsa-miR-565	3	-1.28
hsa-miR-200a	1	-1.42
hsa-miR-449	5	-1.42
HS_141	10	-1.46
hsa-miR-594	7	-1.53

Positive fold change values indicate elevated expression and negative fold change values indicate decreased expression in IL-1β treated chondrocytes compared to control.

Table 2.

MiRNAs upregulated by IL-1β and their target mRNAs identified by integrative analysis

MicroRNA (upregulated)	mRNA (downregulated)	Comments
Let-7e	GSPT1 (G1 to S phase transition 1)	Controls G1 to the S phase transition; downregulated in chondrocyte redifferentiation in 3D pellet culture (12)
	SOCS1 (suppressors of cytokine signaling 1)	Induced by FGF; upregulated in the synovial membranes from patients with RA (13)
	SNX19 (sorting nexin 19)	Expressed in the limb cartilage of mouse embryos and in the degraded cartilage of adult mouse knee joints during osteoarthritis progression; a potent chondrogenic stimulator (14)
	CLP1 (cleavage and polyadenylation factor I subunit homolog)	Stimulates cell proliferation in rabbit articular chondrocytes (15)
miR-335	CASP7 (caspase7)	Elicited in human articular chondrocytes by sodium nitroprusside (16)
	Hoxd13 (homeobox D13)	Development of the forelimb and hindlimb autopod in mice (17)
	TLR1 (Toll-like receptor 1)	Pattern recognition receptor involved in innate immunity
	TIAM1 (T-cell lymphoma invasion and metastasis 1)	Down-regulated in high pressure culture and upregulated in cyclic stretching culture in chondrosarcoma line (18); linked with hyaluronan metabolism (19)
miR-425	TNC (Tenascin)	Oligomeric glycoprotein of the extracellular matrix; upregulated in tissues at developmental stages, during wound healing, and during tumorigenesis; upregulated in neonatal cartilage (20)
	IL4R (IL4 receptor)	Involved in the anabolic response of chondrocytes from normal articular cartilage; chondroprotective in cartilage biology and play a central role in the maintenance of the extracellular matrix (21)
	VIM (Vimentin)	A member of the intermediate filament family, which make up the cytoskeleton; maintains cell shape, integrity of the cytoplasm, and stabilizes cytoskeletal interactions; decreased in chondrosarcoma cells in cyclic and continuous hydrostatic pressure (19); higher expression in nucleus pulposus than in articular cartilage of rat (22)
miR-563	FGF18 (fibroblast growth factor 18)	Possess broad mitogenic and cell survival activities, and are involved in embryonic development, cell growth, morphogenesis, tissue repair, tumor growth, and invasion; plays a role in the maintenance of chondrocyte properties (23); stimulates chondrogenesis and cartilage repair in a rat model of injury-induced osteoarthritis (24)
	HAS3 (hyaluronan synthase 3)	Involved in the synthesis of the unbranched glycosaminoglycan hyaluronan, or hyaluronic acid, which is a major constituent of the extracellular matrix;upregulated by IL-1 in bovine chondrocytes (25)
	IL7R (IL-7 receptor)	Blocking apoptosis during differentiation and activation of T lymphocytes; respond to IL-7 stimulation with increased production of matrix metalloproteinase-13 and with proteoglycan release from cartilage explants (26)
	GSPT1 (G1 to S phase transition 1)
	FOXC2 (forkhead box C2)	The forkhead family of transcription factors which may play a role in the development of mesenchymal tissues; associated with the early stage of chondrogenic differentiation; regulated by BMP in skeletal precursor cells (27)
miR-603	Lyn (v-yes-1 Yamaguchi sarcoma viral related oncogene homolog)	A Src kinase gene which upon T-cell antigen receptor (TCR) stimulation, functions in the initial step of TCR-mediated signal transduction; inhibition by Src kinase inhibitor promotes chondrogenic gene expression and morphology in monolayer culture (28)
	MAX (MYC associated factor X)	A member of the basic helix-loop-helix leucine zipper family of transcription factors, which forms homodimers and heterodimers with Mad, Mxi1 and Myc; expressed mainly in the nucleus of proliferative chondrocytes and decreased as the chondrocytes mature (29)
	POR (P450 (cytochrome) oxidoreductase)	An endoplasmic reticulum membrane oxidoreductase which donate electrons directly from NADPH to all microsomal P450 enzymes; mutations in this gene have been associated with amenorrhea and disordered steroidogenesis, congenital adrenal hyperplasia and Antley-Bixler syndrome; specific inhibition leads to decreased cell proliferation and differentiation and induction of apoptosis in rat chondrocytes with decreased intracellular cholesterol content and Indian hedgehog expression (30)
	NKX3-1 (NK3 homeobox 1)	A transcription factor which is a putative prostate tumor suppressor expressed in a largely prostate-specific and androgen-regulated manner; act together with Sox9 to maintain the chondrogenic cell fate and promote early differentiation (31)
miR-766	PTGER2 (prostaglandin E receptor 2)	A receptor for prostaglandin E2; decreased by IL-1 in human chondrocytes (32); stimulation of human articular chondrocytes with PGE2 through the EP2 receptor suppresses proteoglycan accumulation and synthesis, aggrecan gene expression and decreases the type II collagen:type I collagen ratio; expressed at higher levels in knee cartilage than in ankle cartilage and in a OA grade-dependent manner (33); a specific EP2 agonist promotes regeneration of cartilage tissues with a physiological osteochondral boundary in rabbit injury models (34)
	SDC4 (Syndecan 4)	A transmembrane (type I) heparan sulfate proteoglycan that functions as a receptor in intracellular signaling; the mRNA level increase in cartilage tissue from heavily damaged area of human OA (35); specifically induced in type X collagen-producing chondrocytes both in human and in murine OA; knock-out mice are protected from cartilage damage in a surgically induced model of OA by decrease in ADAMTS-5 activity (36); interact with MMP-13 C-terminal domain in cultured human chondrocytes (37)
	SAT1 (spermidine/spermine N1-acetyltransferase 1)	A rate-limiting enzyme in the catabolic pathway of polyamine metabolism catalyzing the acetylation of spermidine and spermine, and involved in the regulation of the intracellular concentration of polyamines and their transport out of cells; increase in activity by parathyroid hormone (PTH) in rabbit costal chondrocytes (38)

Table 3.

MiRNAs downregulated by IL-1β and their target mRNAs identified by integrative analysis

MicroRNA downregulated	mRNA upregulated	Comments
miR-181b	GPI (glucose phosphate isomerase)	Multifunctional phosphoglucose isomerase protein involved in energy pathways; implicated as an autoantigen in rheumatoid arthritis; conformational changes under acidic conditions, such as those encountered in the synovial fluid of arthritic joints, results in increased association and deposition of fibronectin fibrils on the joint surface (39)
	GSTA-4 (glutathione S-transferase alpha 4)	Involved in cellular defense against toxic, carcinogenic, and pharmacologically active electrophilic compounds; ablation augments 4-hydroxynonenal cytotoxicity in human OA chondrocytes (40)
	P4HA2 (prolyl 4-hydroxylase, alpha polypeptide II)	A key enzyme in collagen synthesis which catalyzes the formation of 4-hydroxyproline essential to the proper three-dimensional folding of newly synthesized procollagen chains;exposure of primary human articular chondrocytes to 1% oxygen increase transcript levels, while treatment with 2-methoxyestradiol reduce transcriptional activity (41)
	PTHR1 (parathyroid hormone receptor 1)	A receptor for parathyroid hormone(PTH) and for parathyroid hormone-like hormone; expressed principally by proliferative and prehypertrophic chondrocytes in endochondral growth (42); increase by co-culture of human articular chondrocytes with non-sclerotic subchondral osteoblast (43); activation of PTH/PTHrP receptor signaling by hPTH results in the inhibition of chondrogenic differentiation in full-thickness articular cartilage defects (44); delays chondrocyte hypertrophy mediated by both Runx2-dependent and -independent mechanisms (45)
	MAP3K6 (mitogen-activated protein kinase kinase kinase 6)	A member of the serine/threonine protein kinase family; weakly activate MAPK7, but not MAPK1/ERK or MAPK14
	SLC26A11 (solute carrier family 26, member 11)	A sodium independent sulfate transporter; a member of the solute linked carrier 26 family of anion exchangers;essential for homeostasis and intracellular electrolyte balance; expressed in bovine articular chondrocytes, and regulates sulfate uptake, an essential step in the pathway for sulphation of glycosaminoglycans (46)
miR-200a	RBL2 (retinoblastoma-like 2 (p130))	Contributes to cell cycle exit, and knock out leads to deregulated proliferation, reduced expression of Cbfa1, and reduced hypertrophic differentiation of chondrocytes (47); effectors of FGF-mediated growth inhibition in chondrocytes (48)
	STMN1 (stathmin 1)	A ubiquitous cytosolic phosphoprotein, involved in the regulation of the microtubule filament system by destabilizing microtubules; proliferating chondrocytes in vivo exhibit higher level of stathmin than either resting or growth zone in the growth plate (49)
	PBX1 (pre-B-cell leukemia homeobox 1)	A component of various protein complexes implicated in developmental gene expression; knock-out leads to embryonic death with widespread patterning defects of the axial and appendicular skeleton, decrease in chondrocyte proliferation, and an increase in hypertrophic chondrocytes (50)
	PTHR1
	TEN (Tensin 1)	Localizes to focal adhesions and crosslinks actin filaments; a substrate of calpain II; upregulated by BMP in bovine and murine chondrocytes (51)
	Tbx15 (T-box-15)	A phylogenetically conserved family of transcription factors that regulate a variety of developmental processes; expressed during limb development in the early limb bud, in prehypertrophic chondrocytes of cartilaginous templates, and in mesenchymal precursor cells and prehypertrophic chondrocytes of the vertebral column and the head of mouse embryo; mutants display a general reduction of bone size, changes of bone shape, reduction in the size of cartilaginous templates, and reduction in proliferation of prehypertrophic chondrocytes (52)
miR-31	HTRA1 (HtrA serine peptidase 1)	Member of the trypsin family of serine proteases; regulates the availability of insulin-like growth factors (IGFs) by cleaving IGF-binding proteins; increased in the knee and TM joints of mouse OA models in early stages of the disease (53); HtrA1-generated aggrecan fragments containing the VQTV (356) neoepitope were significantly more abundant in osteoarthritic cartilage compared with cartilage from healthy joints (54)
	FGFR3 (fibroblast growth factor receptor 3)	Binds acidic and basic fibroblast growth hormone and plays a role in bone development and maintenance; mutation leads to achondroplasia; induces premature senescence in chondrocytes, manifested as growth arrest, alteration of cellular shape, and loss of the extracellular matrix (55)
	CYTL1 (cytokine-like 1)	Expressed in bone marrow and cord blood mononuclear cells; regulates chondrogenesis as a novel autocrine factor, but not hypertrophic maturation of chondrocytes during cartilage development (56)
	HOXA2 (homeobox A2)	Encodes a DNA-binding transcription factor which may regulate morphogenesis, and differentiation; the encoded protein may be involved in the placement of hindbrain segments in the proper location along the anterior-posterior axis during development; persistent expression in chondrogenic cells results in overall chondrodysplasia with delayed cartilage hypertrophy, mineralization, and ossification in mouse embryo (57)
	GPX7 (glutathione peroxidase 7)	An antioxidant enzyme that disproportionate reactive oxygen species; increased by IL-1 and IL-6 dose and time-dependently in bovine chondrocytes (58)
	CDK4 (cyclin-dependent kinase 4)	A catalytic subunit of the protein kinase complex important for cell cycle G1 phase progression; overexpression leads to resistance to FGF-induced p107 dephosphorylation and growth arrest (59)
miR-565	ENG (endoglin)	A major glycoprotein of the vascular endothelium, and a component of the transforming growth factor (TGF) beta receptor complex; mutations in this gene cause hereditary hemorrhagic telangiectasia; expressed on human chondrocytes at levels comparable with endothelial cells and forms higher order complexes with the type I and II TGFβ receptors (60)
	BARX1 (BARX homeobox 1)	A member of the Bar subclass of homeobox transcription factors which play a role in developing teeth and craniofacial mesenchyme of neural crest origin; expressed in the developing joint and articular cartilage and has an inhibitory effect on chondrogenic initiation (61)
	CTSK (cathepsin K)	A lysosomal cysteine proteinase involved in bone remodeling and resorption; induced in phenotypically altered chondrocytes in human OA and animal OA model (62,63); chemical inhibitor results in mild to moderate beneficial effects on gross and histopathological parameters of OA and reduction of biomarkers of collagen type I and II degradation in the canine partial medial meniscectomy model of OA (64)

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