Mutant p53R211* ameliorates inflammatory arthritis in AIA rats via inhibition of TBK1-IRF3 innate immune response.

BACKGROUND: Rheumatoid arthritis (RA) is an autoimmune inflammation disease characterized by imbalance of immune homeostasis. p53 mutants are commonly described as the guardian of cancer cells by conferring them drug-resistance and immune evasion. Importantly, p53 mutations have also been identified in RA patients, and this prompts the investigation of its role in RA pathogenesis. METHODS: The cytotoxicity of disease-modifying anti-rheumatic drugs (DMARDs) against p53 wild-type (WT)/mutant-transfected RA fibroblast-like synoviocytes (RAFLSs) was evaluated by MTT assay. Adeno-associated virus (AAV) was employed to establish p53 WT/R211* adjuvant-induced arthritis (AIA) rat model. The arthritic condition of rats was assessed by various parameters such as micro-CT analysis. Knee joint samples were isolated for total RNA sequencing analysis. The expressions of cytokines and immune-related genes were examined by qPCR, ELISA assay and immunofluorescence. The mechanistic pathway was determined by immunoprecipitation and Western blotting in vitro and in vivo. RESULTS: Among p53 mutants, p53R213* exhibited remarkable DMARD-resistance in RAFLSs. However, AAV-induced p53R211* overexpression ameliorated inflammatory arthritis in AIA rats without Methotrexate (MTX)-resistance, and our results discovered the immunomodulatory effect of p53R211* via suppression of T-cell activation and T helper 17 cell (Th17) infiltration in rat joint, and finally downregulated expressions of pro-inflammatory cytokines. Total RNA sequencing analysis identified the correlation of p53R211* with immune-related pathways. Further mechanistic studies revealed that p53R213*/R211* instead of wild-type p53 interacted with TANK-binding kinase 1 (TBK1) and suppressed the innate immune TBK1-Interferon regulatory factor 3 (IRF3)-Stimulator of interferon genes (STING) cascade. CONCLUSIONS: This study unravels the role of p53R213* mutant in RA pathogenesis, and identifies TBK1 as a potential anti-inflammatory target.

The role of non-coding RNAs (miRNA and lncRNA) in the clinical management of rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic systemic inflammatory disorder which is associated with the dysregulation of autoimmune response. In recent years, early diagnosis, aggressive treatment and alternative therapeutic options of disease-modifying anti-rheumatic drugs (DMARDs) markedly improve both the management and long-term prognosis of RA. Since the discovery of non-coding RNA (ncRNA) including microRNA (miRNA), long non-coding RNA (lncRNA) and others, their altered expressions have been unraveled to be deregulated in various diseases including RA. Several lines of evidence are emerging that ncRNA may contribute to the pathogenesis, disease progression and treatment of RA. For example, SNP rs2850711 within lnc00305 was indicated to associate with RA development susceptibility, whereas a higher level of miR-10a represented a good response to methotrexate (MTX) treatment in RA patients. In the aspect of refractory RA, ncRNA also plays an important role by affecting or regulating drug sensitivity in RA patients. Of note, lower expression of miR-20a in rheumatoid arthritis synovial fibroblast (RASFs) was demonstrated to activate the Janus Kinase (JAK)- signal transducer and activator of transcription 3(STAT3)-mediated inflammation, thereby promoting cell proliferation and apoptosis-resistant. In this review, we have illustrated the changes of ncRNAs and their underlying mechanisms in the whole developing period of RA pathogenesis and disease progression, as well as highlighted the novel therapeutic targets/strategies and bio-markers for RA therapy.

[Retracted] miR­141­3p suppresses proliferation and promotes apoptosis by targeting GLI2 in osteosarcoma cells.

Following the publication of the above article, a concerned reader drew to the Editor's attention that, in the above paper, they had identified multiple instances of overlapping data panels comparing the TUNEL assay data shown in Fig. 2C and D on p. 750 with that shown in Fig. 4C on p. 752, suggesting that data purportedly showing results obtained under different experimental conditions had been derived from a smaller number of original sources. Upon informing the authors about this matter, they consulted their original data and requested a corrigendum to take account of the overlapping data in Figs. 2 and 4; however, given the number of instances of overlapping data panels that were identified comparing these two figures, the Editor of Oncology Reports has decided that this article should be retracted from the publication owing to a lack of overall confidence in the presented data. Upon informing the authors of this decision, they did not accepted the decision to retract this article. The Editor apologizes to the readership for any inconvenience resulting from the retraction of this article. [Oncology Reports 39: 747­754, 2018; DOI: 10.3892/or.2017.6150].

N-Acetylcysteine overcomes epalrestat-mediated increase of toxic 4-hydroxy-2-nonenal and potentiates the anti-arthritic effect of epalrestat in AIA model.

Epalrestat, an aldose reductase inhibitor (ARI), has been clinically adopted in treating diabetic neuropathy in China and Japan. Apart from the involvement in diabetic complications, AR has been implicated in inflammation. Here, we seek to investigate the feasibility of clinically approved ARI, epalrestat, for the treatment of rheumatoid arthritis (RA). The mRNA level of AR was markedly upregulated in the peripheral blood mononuclear cells (PBMCs) of RA patients when compared to those of healthy donors. Besides, the disease activity of RA patients is positively correlated with AR expression. Epalrestat significantly suppressed lipopolysaccharide (LPS) induced TNF-α, IL-1ß, and IL-6 in the human RA fibroblast-like synoviocytes (RAFLSs). Unexpectedly, epalrestat treatment alone markedly exaggerated the disease severity in adjuvant induced arthritic (AIA) rats with elevated Th17 cell proportion and increased inflammatory markers, probably resulting from the increased levels of 4-hydroxy-2-nonenal (4-HNE) and malondialdehyde (MDA). Interestingly, the combined treatment of epalrestat with N-Acetylcysteine (NAC), an anti-oxidant, to AIA rats dramatically suppressed the production of 4-HNE, MDA and inflammatory cytokines, and significantly improved the arthritic condition. Taken together, the anti-arthritic effect of epalrestat was diminished or even overridden by the excessive accumulation of toxic 4-HNE or other reactive aldehydes in AIA rats due to AR inhibition. Co-treatment with NAC significantly reversed epalrestat-induced upregulation of 4-HNE level and potentiated the anti-arthritic effect of epalrestat, suggesting that the combined therapy of epalrestat with NAC may sever as a potential approach in treating RA. Importantly, it could be regarded as a safe intervention for RA patients who need epalrestat for the treatment of diabetic complications.

Age-related self-DNA accumulation may accelerate arthritis in rats and in human rheumatoid arthritis.

The incidence of rheumatoid arthritis (RA) is increasing with age. DNA fragments is known to accumulate in certain autoimmune diseases, but the mechanistic relationship among ageing, DNA fragments and RA pathogenesis remain unexplored. Here we show that the accumulation of DNA fragments, increasing with age and regulated by the exonuclease TREX1, promotes abnormal activation of the immune system in an adjuvant-induced arthritis (AIA) rat model. Local overexpression of TREX1 suppresses synovial inflammation in rats, while conditional genomic deletion of TREX1 in AIA rats result in higher levels of circulating free (cf) DNA and hence abnormal immune activation, leading to more severe symptoms. The dysregulation of the heterodimeric transcription factor AP-1, formed by c-Jun and c-Fos, appear to regulate both TREX1 expression and SASP induction. Thus, our results confirm that DNA fragments are inflammatory mediators, and TREX1, downstream of AP-1, may serve as regulator of cellular immunity in health and in RA.

Knockdown of miR-222 inhibits inflammation and the apoptosis of LPS-stimulated human intervertebral disc nucleus pulposus cells.

It has been demonstrated that miR­222 is upregulated in human intervertebral disc (IVD) degeneration tissues; however, the underlying mechanisms remain unclear. In this study, we aimed to elucidate the mechanisms of action of miR­222 in IVD tissues. Nucleus pulposus (NP) cells were treated with lipopolysaccharide (LPS) to simulate IVD degeneration. The expression level of miR­222 was detected by reverse transcription­quantitative polymerase chain reaction (RT­qPCR) in cells and tissues. Cell apoptosis was analyzed by flow cytometry. Additionally, western blot analysis was used to determine the levels of Toll­like receptor 4 (TLR4), Iκß­alpha (IκBα) and p65. Interleukin (IL)­1ß, tumor necrosis factor­α (TNF­α) and IL­6 protein expression levels were determined by enzyme­linked immunosorbent assay (ELISA). The target gene of miR­222 was determined by TargetScan7.2 and dual luciferase reporter gene analysis. Western blot analysis and RT­qPCR were used to determine the mRNA and protein levels of tissue inhibitor of metalloproteinase 3 (TIMP3). The mRNA expression level of miR­222 was found to be increased in IVD tissues and in LPS­stimulated cells, and its expression was positively associated with the clinical MRI grade. In vitro, apoptosis was promoted/inhibited by miR­222 mimics/inhibitors. Transfection with miR­222 mimics/inhibitors significantly increased/decreased the production of TNF­α, IL­1ß and IL­6 and suppressed/enhanced collagen II and aggrecan expression. The protein levels of TLR4, p­IκΒα and p­p65 were upregulated/downregulated by transfection with the mimics/inhibitors. In addition, it was demonstrated that TIMP3 was a direct target gene of miR­222, and was negatively regulated by miR­222 in NP cells. The silencing of TIMP3 reversed the inhibitory effects of miR­222 inhibitor on cell apoptosis, which was induced by LPS. Thus, on the whole, the findings of this study demonstrate that miR­222 functions as a promoter of IVD development, partly via the regulation of TIMP3.

miR-141-3p suppresses proliferation and promotes apoptosis by targeting GLI2 in osteosarcoma cells.

MicroRNAs (miRNAs) have been reported as key regulators in numerous diseases including osteosarcoma. The function of microRNA-141-3p (miR-141-3p) and whether this function is achieved by regulation of GLI family zinc finger 2 (GLI2) in osteosarcoma remain unclear. In the present study, we found decreased expression of miR-141-3p, but increased expression of GLI2 in osteosarcoma tissues and cell lines. In addition, we demonstrated a negative correlation between miR-141-3p and GLI2. Furthermore, we revealed that elevation of miR-141-3p resulted in a marked inhibition of proliferation and promotion of apoptosis as well as an obviously decrease in GLI2 in osteosarcoma cell lines. Furthermore, we determined that GLI2 is a target of miR-141-3p by a constructed luciferase assay. In addition, we showed that miR-141-3p could negatively regulate GLI2 and its downstream parathyroid hormone-related protein 1 (PTHRP1). Finally, through a series of antisense experiments we confirmed that the effect of miR-141-3p on proliferation and apoptosis was achieved through the GLI2 pathway in osteosarcoma cells. The findings of the present study may provide a new target for treating osteosarcoma.