Long Non‑Coding RNA HOTAIRM1‑1 Silencing in Cartilage Tissue Induces Osteoarthritis Through Microrna‑125B

EXPERIMENTAL AND THERAPEUTIC MEDICINE 22: 933, 2021 Long non‑coding RNA HOTAIRM1‑1 silencing in cartilage tissue induces osteoarthritis through microRNA‑125b WEN‑BIN LIU1*, QI‑JIN FENG2*, GUI‑SHI LI3, PENG SHEN4, YA‑NAN LI5 and FU‑JIANG ZHANG1 1Department of Joint Surgery, Tianjin Hospital, Tianjin 300211; 2Department of Orthopedics, Second Affiliated Hospital of Tianjin University of Traditional Chinese Medicine, Tianjin 300150; 3Department of Joint Surgery, Yuhuangding Hospital, Yantai, Shandong 264000; 4Department of Rheumatology and Immunology, Tianjin First Center Hospital, Tianjin 300192; 5Department of Orthopedics, Tianjin Dongli Hospital, Tianjin 300300, P.R. China Received September 4, 2020; Accepted March 11, 2021 DOI: 10.3892/etm.2021.10365 Abstract. Aberrations in long noncoding RNA (lncRNA) Introduction expression have been recognized in numerous human diseases. In the present study, the of role the long noncoding Osteoarthritis (OA) is the most common type of arthritis, and RNA HOX antisense intergenic RNA myeloid 1 variant it affects >10% of the adult population (1). Physicians consider (HOTAIRM1‑1) in regulating the pathological progression the disease to be a degenerative disease that involves all the of osteoarthritis (OA) was investigated. The aberrant expres‑ tissues of the joint. In OA, cartilage within a joint begins to sion of HOTAIRM1‑1 in OA was demonstrated, but the break down, and the underlying bone begins to change (2). OA molecular mechanisms require further analysis. The aim of can induce substantial limb pain, stiffness, disability, and even the present study was to explore the function of miR‑125b loss of whole‑body mobility. OA occurs most frequently in the in modulating chondrocyte viability and apoptosis, and to hands, hips and knees, leading to difficulties in patients' daily address the functional association between HOTAIRM1‑1 activities (3). Therefore, numerous management strategies to and miR‑125b as potential targets. A miR‑125b inhibitor prevent cartilage degeneration, such as microfracture surgery was used, which laid the foundation for the following to stimulate a healing response, stem cell therapy to repair investigation. The study confirmed that HOTAIRM1‑1 and damaged articular cartilage, cartilage transplantation, and miR‑125b are inversely expressed in chondrocytes. The complementary treatments, have been developed (4‑6). expression of HOTAIRM1‑1 was downregulated and the Long noncoding RNAs (lncRNAs) are transcripts expression of miR‑125b was upregulated in tissues from longer than 200 nucleotides and are noncoding RNAs. patients with OA. HOTAIRM1‑1 directly interacted with Aberrant expression of lncRNAs participate in numerous miR‑125b in chondrocytes. HOTAIRM1‑1 knockdown was biological processes and various diseases by posttranscrip‑ associated with chondrocyte proliferation and extracellular tional or posttranslational regulation (7,8). Jiang et al (9) and matrix degradation. Furthermore, miR‑125b reversed the Marques‑Rocha et al (10) reported that patients with OA have effect of HOTAIRM1‑1 on cell proliferation and apoptosis. aberrant expression of certain lncRNAs, which indicates that In conclusion, the present study indicates that the loss of lncRNAs may play a key role in articular cartilage degeneration. HOTAIRM1‑1 function leads to aberrant increases in the Recently, the novel lncRNA HOX antisense intergenic RNA proliferation and apoptosis of chondrocytes. miR‑125b myeloid 1 (HOTAIRM1) was found to be expressed in cells of may be a potential downstream mechanism that regulates the myeloid lineage (11,12). Additional evidence has confirmed the function of HOTAIRM1‑1, and this finding provides a that HOTAIRM1 plays a crucial role in the pathological progres‑ therapeutic strategy for OA. sion of colorectal cancer and several other diseases, such as acute myeloid leukemia (13‑15). Nonetheless, the potential function of HOTAIRM1 in the development of osteoarthritis remains unknown. HOTAIRM1 is located between the HOXA1 and HOXA2 loci and has been identified as a myeloid‑specific regulator in Correspondence to: Dr Fu‑Jiang Zhang, Department of Joint the HOXA gene family. HOTAIRM1 targets gene transcrip‑ Surgery, Tianjin Hospital, 406 Jiefangnan Road, Tianjin 300211, P. R. Ch i na tion during the chromosome remodeling that occurs when E‑mail: [email protected] myeloid cells are induced to undergo chondrogenic differentia‑ tion (16). Furthermore, previous studies in mesenchymal stem *Contributed equally cells showed that miR‑125b is a miRNA target of HOTAIRM1 during osteoblastic differentiation (16,17). In the present study, Key words: cartilage, osteoarthritis, HOXA, gene the hypothesis that HOTAIRM1‑1 and miR‑125b have a func‑ tional association and that miR‑125b is a potential therapeutic target of articular cartilage denegation was tested. 2 LIU et al: NEW TARGETING THERAPEUTIC STRATEGY FOR OA Materials and methods at every 1˚C/sec. Relative quantification was performed using the 2‑ΔΔCq method (19). U6 was used as the endogenous refer‑ Tissue collection. The present study was approved by the ence gene for all experiments with ncRNA. GAPDH was used human ethics committee of Tianjin Hospital. All the patients as normalization for matrix metalloprotease (MMP)13 and provided written informed consent and participated in the interleukin (IL)‑10. study according to their own will. A total of 15 articular carti‑ lage samples were obtained from patients with OA undergoing Cell proliferation. To analyze cell proliferation, Cell Counting total knee arthroplasty (n=15; mean age, 65.8 ± 6.2 years; Kit‑8 (Dojindo Molecular Technologies, Inc.) was used. The 5 males and 10 females). Healthy control articular cartilage cells were seeded into 96‑well plates at ~1x10 cells in each samples were collected from trauma patients who underwent well. Cell proliferation was determined at 0, 24, 48 72 and amputation and did not have a history of rheumatoid arthritis 96 h. The 96‑well plates were analyzed on a microplate reader or OA (n=8; mean age, 41.2 ± 9.1 years; 5 male and 3 female). at OD 450 nm. There was no significant difference between the OA group and the control group in terms of sex or age. Luciferase reporter gene assay. To construct 3'‑untrans‑ lated region (UTR) Green Renilla Luciferase reporters Cell culture. Chondrocytes from the cartilage of patients (Thermo Fisher Scientific, Inc.), mutant and wild‑type with OA were cultured following a previously published sequences were simultaneously constructed. Chondrocytes protocol (18). In brief, small sections of OA cartilage tissues were cultured in 24‑well plates, and then, the miR‑125b were digested with trypsin (0.25%). Then, type II colla‑ mimics, miR‑NC and mutant or wild‑type HOTAIRM1‑1 genase (0.2%; Gibco; Thermo Fisher Scientific, Inc.) was were transfected into the chondrocytes using a transfection incubated with the samples. The chondrocytes were cultured kit (Lipofectamine 2000®; Thermo Fisher Scientific, Inc.) in Dulbecco's modified Eagle's medium (DMEM; Gibco; at 37˚C for 4 h. After 48 h, luciferase reporter gene assays Thermo Fisher Scientific, Inc.) containing 10% FBS and main‑ were performed using a pierce renilla luciferase glow assay tained in a cell incubator containing 5% CO2 at 37˚C. kit (Thermo Fisher Scientific, Inc.) to measure the luciferase activity. The results are shown as the firefly/Renilla ratio Cell transfection. The plasmids containing small interfering normalized to the Renilla luciferase activity. (si)RNA targeting HOTAIRM1‑1 (si‑HOTAIRM1‑1, 5'‑CAC CGG AGA CTG GTA GCT TAT TAT TCA AGA GAT AAT AAG Western blotting. Proteins were obtained from cultured cells CTA CCA GTC TCC TTT TTTG‑3') or a si‑HOTAIRM1‑1 with radioimmunoprecipitation assay (RIPA) buffer (Pierce; negative control sequence (si‑NC, 5'‑CAC CGT TCT CCG AAC Thermo Fisher Scientific, Inc.) on ice for 30 min. The protein GTC ACG TCA AGA GAT TAC GTG ACA CGT TCG GAG AAT concentration of each cell lysate was determined by a BCA Assay TTT TT‑3') were purchased from Gene‑Pharma (Shanghai kit (Thermo Fisher Scientific, Inc.). The protein samples (50 µg) GenePharma Co., Ltd.). Briefly, siRNA was transfected were separated on 8% polyacrylamide gels by electrophoresis into cultured chondrocytes by using Lipofectamine® 2000 for 5 min at 50 V, and then the voltage was increased to 100 V (Thermo Fisher Scientific, Inc.) at a concentration of 50 nM, for 1 h. Then, the proteins were transferred from the gels onto following the manufacturer's instructions, which were plated polyvinylidene difluoride (PVDF) membranes (EMD Millipore) into 6‑well plates the day before transfection to achieve followed by blocking with 5% non‑fat milk in buffer [10 mM 60‑70% confluency. After 48 h, the transfected cells were used Tris‑HCl (pH 7.6), 100 mM NaCl and 0.1% Tween‑20] at 23˚C for further analysis. for 1 h. The membranes were incubated with anti‑collagen II Synthetic miRNA mimics miR‑125b, miR‑125b inhibitor (cat. no. ab185430; 1:500 dilution; Abcam) and anti‑aggrecan and their negative control (custom synthesized by Shanghai (cat. no. ab36861, 1:1,000 dilution; Abcam) primary antibodies GenePharma Co., Ltd.) were transfected into cells following for 4 h at room temperature. The membrane was washed twice the Lipofectamine® RNAiMAX Reagent (Thermo Fisher and then incubated with Goat Anti‑Rabbit IgG H&L secondary Scientific, Inc.) transfection protocol. After 48 h, the trans‑ antibodies (cat. no. ab205718; 1:1,000; Abcam) for 60 min at fected cells were used for further

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