Overexpression of FTO alleviates osteoarthritis by regulating the processing of miR-515-5p and the TLR4/MyD88/NF-κB axis.

Osteoarthritis (OA) is regarded as the most prevalent chronic joint disease. Fat-mass and obesity-associated gene (FTO) is involved in OA alleviation. This study elucidated the role of FTO in OA and the associated mechanism. We established a cell injury model by stimulating human normal chondrocytes (C28/I2) with lipopolysaccharide (LPS), and measured cell viability, apoptosis, and inflammatory cytokines using CCK-8, flow cytometry, Western blot, and ELISA. TLR4, MyD88, p/t-p65, and p/t-IκBα levels, FTO, COX-2, and iNOS mRNA levels, and m6A methylation levels were measured by Western blot, RT-qPCR, and colorimetry. RNA immunoprecipitation and co-immunoprecipitation were conducted to confirm the interaction between FTO and DGCR8. pri-miR-515-5p process was regulated in an m6A-dependent manner. After predicting the presence of several binding sites between miR-515-5p and TLR4 on Targetscan, we further confirmed their relationship by dual-luciferase assay. OA rat models were established by monosodium iodoacetate injection. The pathological changes in knee joint were observed by HE staining. FTO was diminished in LPS-induced C28/I2 cells. With the increase of LPS concentration, cell viability was repressed, apoptosis rate was increased, and inflammatory markers were promoted, which were annulled by FTO overexpression. FTO interacted with DGCR8 and modulated the pri-miR-515-5p processing in an m6A-dependent manner. miR-515-5p silencing partially averted the inhibitory effect of FTO on LPS-induced cell injury. Given that TLR4 was a direct target of miR-515-5p, miR-515-5p inactivated the MyD88/NF-κB pathway by targeting TLR4. FTO overexpression improved cartilage structure in OA rats, reduced apoptosis, inhibited inflammation in synovial fluid, and repressed the TLR4/MyD88/NF-κB axis. FTO alleviated OA in an m6A-dependent manner via the miR-515-5p/TLR4/MyD88/NF-κB axis.

Cai D ，Zhang J ，Yang J ，Lv Q ，Zhong C ... -  《-》

[miR-515-5p targeting Toll-like receptor 4 regulates myeloid differentiation primary response gene 88/nuclear factor-kappa B pathway to inhibit apoptosis and inflammatory response of osteoarthritis chondrocytes].

Cai D ，Yang Z ，Zhong C ，Zhang J ，Hong S ... -  《-》

FTO-mediated m6A demethylation of pri-miR-3591 alleviates osteoarthritis progression.

Increasing evidence have demonstrated the N6-methyladenosine (m6A) plays critical roles in osteoarthritis (OA) progression, but the role of m6A in OA has not been completely illuminated. Herein, we investigated the function and underlying mechanism of m6A demethylase fat mass and obesity-associated protein (FTO) in OA progression. The FTO expression was detected in mice OA cartilage tissues and lipopolysaccharide (LPS)-stimulated chondrocytes. Gain-of-function assays was used to evaluate the role of FTO in OA cartilage injury in vitro and in vivo. The miRNA-sequencing, RNA-binding protein immunoprecipitation (RIP), luciferase reporter assay, and in vitro pri-miRNA processing assays were conducted to confirm that FTO modulated the pri-miR-3591 process in an m6A-dependent manner and then the binding sites of miR-3591-5p with PRKAA2. FTO was outstandingly downregulated in LPS-stimulated chondrocytes and OA cartilage tissues. FTO overexpression enhanced the proliferation, suppressed apoptosis, and decreased degradation of extracellular matrix in LPS-induced chondrocytes, whereas FTO knockdown contributed to the opposite effects. In vivo animal experiments showed that FTO overexpression markedly alleviated OA mice cartilage injury. Mechanically, FTO-mediated m6A demethylation of pri-miR-3591 leaded to a maturation block of miR-3591-5p, which relieved the inhibitory effect of miR-3591-5p on PRKAA2 and then promoted the increase of PRKAA2, thereby alleviating OA cartilage damage. Our results attested that FTO alleviated the OA cartilage damage by mediating FTO/miR-3591-5p/PRKAA2 axis, which provided fresh insights into the therapeutic strategies for OA.

Liu W ，Jiang T ，Zheng W ，Zhang J ，Li A ，Lu C ，Lin Z ... -  《-》

LncRNA ARFRP1 knockdown inhibits LPS-induced the injury of chondrocytes by regulation of NF-κB pathway through modulating miR-15a-5p/TLR4 axis.

Long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) have been reported as the important regulators in osteoarthritis (OA). However, the detailed mechanism is implicated. The aim of this study is to reveal the functional mechanism of lncRNA ARFRP1 and miR-15a-5p in osteoarthritis. The expression level of genes was detected by quantitative real time polymerase chain reaction (qRT-PCR) or western blot assay. Cell Counting Kit-8 (CCK-8) was used to assess cell viability. Cell apoptosis rate was analyzed by flow cytometry analysis. Furthermore, Enzyme-linked immunosorbent assay (ELISA) was performed to measure tumor necrosis factor-α (TNF-α), interleukin (IL)-6 and IL-1β contents. The interaction between miR-15a-5p and ARFRP1 or Toll-like receptor 4 (TLR4) was predicted by miRcode or PITA, and then confirmed by the dual luciferase reporter assay or pull down assay. Besides, NF-κB-driven luciferase activity was determined using NF-κB luciferase reporter assay. ARFRP1 and TLR4 levels were increased and miR-15a-5p level was decreased in OA cartilage tissues and lipopolysaccharides (LPS)-induced chondrocytes. ARFRP1 knockdown inhibited LPS-induced the injury of chondrocytes. Interestingly, miR-15a-5p downregulated by ARFRP1 negatively modulated TLR4 expression through interaction. ARFRP1 mediated LPS-induced the injury of chondrocytes via regulating miR-15a-5p/TLR4 axis. Furthermore, ARFRP1 exerted function by modulation of NF-κB pathway. Our findings confirmed that ARFRP1 mediated LPS-induced the injury of chondrocytes through regulating NF-κB pathway by modulation of miR-15a-5p/TLR4 axis, providing theoretical basis for the treatment of OA patients.

Zhang G ，Zhang Q ，Zhu J ，Tang J ，Nie M ... -  《-》

The protective role of microRNA-140-5p in synovial injury of rats with knee osteoarthritis via inactivating the TLR4/Myd88/NF-κB signaling pathway.

Huang X ，Qiao F ，Xue P 《-》