Identification and Verification of Endoplasmic Reticulum Stress-Related Genes as Novel Signatures for Osteoarthritis Diagnosis and Therapy: A Bioinformatics Analysis-Oriented Pilot Study.

BACKGROUND AND PURPOSE: Endoplasmic reticulum stress (ERS) has been reported to be closely associated with the development of osteoarthritis (OA), but the underlying mechanisms are not fully delineated. The present study was designed to investigate the involvement of ERS-related genes in regulating OA progression. METHODS: The expression profiles of OA patients and normal people were downloaded from the gene expression omnibus (GEO) database. The differentially expressed genes (DEGs) in datasets GSE55457 and GSE55235 were screened and identified by R software with the construction of the protein-protein interaction (PPI) networks. Through the STRING and Venn diagram analysis, hub ERS-related genes were obtained. Gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) enrichment analyses were performed. Biomarkers with high diagnostic values of osteoarthritis (OA) were studied. The hematoxylin and eosin (H&E) staining and micro-CT were applied to evaluate the establishment of the OA model. The expression levels of biomarkers were validated with the use of reverse transcription­quantitative polymerase chain reaction (RT-qPCR) and western blot. Finally, we evaluated the correlations of hub ERS-related genes with the immune infiltration cells via the CIBERSORT algorithm. RESULTS: A total of 60 downregulated and 52 upregulated DEGs were identified, and the following GO and KEGG pathway analyses verified that those DEGs were mainly enriched in biological process (BP), cellular component (CC), molecular function (MF), and inflammation-associated signal pathways. Interestingly, among all the DEGs, six ER stress-associated genes, including activating transcription factor 3 (ATF3), DEAD-Box Helicase 3 X-Linked (DDX3X), AP-1 transcription factor subunit (JUN), eukaryotic initiation factor 4 (EIF4A1), KDEL endoplasmic reticulum protein retention receptor 3 (KDELR3), and vascular endothelial growth factor A (VEGFA), were found to be closely associated with OA progression, and the following RT-qPCR and Western Blot analysis confirmed that DDX3X, JUN, and VEGFA were upregulated, whereas KDELR3, EIF4A1, and ATF3 were downregulated in OA rats tissues compared to the normal tissues, which were in accordance with our bioinformatics findings. Furthermore, our receiver operating characteristic (ROC) curve analysis verified that the above six ER stress-associated genes could be used as ideal biomarkers for OA diagnosis and those genes also potentially regulated immune responses by influencing the biological functions of mast cells and macrophages. CONCLUSION: Collectively, the present study firstly identified six ER stress-associated genes (ATF3, DDX3X, JUN, EIF4A1, KDELR3, and VEGFA) that may play critical role in regulating the progression of OA.

FGF1 reduces cartilage injury in osteoarthritis via regulating AMPK/Nrf2 pathway.

Osteoarthritis (OA) is a systemic joint degenerative disease involving a variety of cytokines and growth factors. In this study, we investigated the protective effect of fibroblast growth factor 1 (FGF1) knockdown on OA and its underlying mechanisms in vitro. In addition, we evaluated the effect of FGF1 knockout on the destabilization of the medial meniscus (DMM) and examined the anterior and posterior cruciate ligament model in vivo. FGF1 affects OA cartilage destruction by increasing the protein expression of Nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1), which is associated with the phosphorylation of AMPK and its substrates. Our study showed that FGF1 knockdown could reverse the oxidative damage associated with osteoarthritis. Nrf2 knockdown eliminated the antioxidant effect of FGF1 knockdown on chondrocytes. Furthermore, AMPK knockdown could stop the impact of FGF1 knockdown on osteoarthritis. These findings suggested that FGF1 knockdown could effectively prevent and reverse osteoarthritis by activating AMPK and Nrf2 in articular chondrocytes.

LncRNA LINC00963 promotes osteogenic differentiation of hBMSCs and alleviates osteoporosis progression by targeting miRNA-760/ETS1 axis.

Although long non-coding RNA LINC00963 has been reported to play a crucial regulatory role in osteoporosis (OP), its specific mechanism has not been well studied. Cell viability of human bone marrow mesenchymal stem cells (hBMSCs) transfected with short hairpin RNA targeting LINC00963 (sh-LINC00963) and negative control (sh-NC) was analysed by cell counting kit-8 (CCK-8) assay. Alkaline phosphatase (ALP) activity in hBMSCs transfected with sh-LINC00963 and sh-NC after induction by osteogenic medium (OM) on day 7 was detected. The protein expression levels of osteocalcin (OCN) and osteopontin (OPN) in hBMSCs transfected with sh-LINC00963 and sh-NC during OM induction on day 3 were detected by western blot. The relationship among LINC00963, miR-760, and E26 transformation specific-1 (ETS1) was determined by bioinformatics analysis, luciferase reporter assay, and RNA-binding protein immunoprecipitation (RIP) assay. A rat model with OP was established to confirm the role of LINC00963 in vivo. The expression level of LINC00963 was much lower in hBMSCs isolated from the discarded femoral head tissues of OP patients compared with that in health patients. Meanwhile, the expression level of LINC00963 was significantly increased and the expression level of miR-760 was decreased in hBMSCs during osteogenic induction. LINC00963 could bind to the 3'-untranslated region (3'-UTR) of miR-760 and negatively regulate the expression of miR-760, then promote the osteogenic differentiation in hBMSCs. ETS1 was identified as a target of miR-760. Moreover, overexpression of LINC00963 obviously reduced bone mineral density (BMD) of the left femur in OP rats and alleviated OP progression in vivo. Our results demonstrated that LINC00963 positively regulated the expression of ETS1 by directly targeting miR-760, and then promoted osteogenic differentiation of hBMSCs in vitro, and also attenuated OP progression in vivo, suggesting that LINC00963 might be a potential therapeutic target for OP.

LINC01534 Promotes the Aberrant Metabolic Dysfunction and Inflammation in IL-1ß-Simulated Osteoarthritic Chondrocytes by Targeting miR-140-5p.

OBJECTIVE: Long non-coding RNA 01534 (LINC01534) is highly expressed in the tissues of patients with osteoarthritis (OA). This study investigated the mechanism of LINC01534 on abnormal metabolic dysfunction in OA chondrocytes induced by interleukin-1ß (IL-1ß). METHODS: The quantitative real-time polymerase chain reaction (qRT-PCR) was used to determine the expressions of LINC01534, aggrecan, collagen II, and matrix metalloproteinase (MMPs) in OA cartilage tissue or OA chondrocyte model induced by IL-1ß. The expressions of aggrecan and collagen II in the chondrocyte were detected by Western blot. The levels of tumor necrosis factor-α (TNF-α), IL-8, IL-6, MMP-13, MMP-9, MMP-3, and prostaglandin E2 (PGE2) in chondrocyte were determined by enzyme-linked immunosorbernt assay. Bioinformatics, dual luciferin gene reporting, RNA pulldown, and Northern blot were used to determine the interaction between LINC01534 and miR-140-5p. RESULTS: The results showed that LINC01534 was upregulated in both OA cartilage tissue and OA chondrocyte model. In addition, silencing LINC01534 significantly alleviated the inhibitory effect of IL-1ß on expressions of aggrecan and collagen II in chondrocytes, and significantly downregulated the expression of matrix metalloproteinases in IL-1ß-induced chondrocytes. Meanwhile, silencing LINC01534 also significantly inhibited the productions of proinflammatory factors NO, PGE2, TNF-α, IL-6, and IL-8 in the IL-1ß-induced chondrocytes. Furthermore, miR-140-5p was confirmed to be a direct target of LINC01534. More importantly, inhibition of miR-140-5p significantly reversed the inhibitory effect of silencing LINC01534 on abnormal matrix degradation in the IL-1ß-induced chondrocyte model of OA. CONCLUSION: Therefore, LINC01534 could promote the abnormal matrix degradation and inflammatory response of OA chondrocytes through the targeted binding of miR-140-5p.