Effect of ubiquitin-specific proteinase 43 on ovarian serous adenocarcinoma and its clinical significance.

BACKGROUND: Ovarian cancer stands as a highly aggressive malignancy. The core aim of this investigation is to uncover genes pivotal to the progression and prognosis of ovarian cancer, while delving deep into the intricate mechanisms that govern their impact. METHODS: The study entailed the retrieval of RNA-seq data and survival data from the XENA database. Outliers were meticulously excluded in accordance with TCGA guidelines and through principal components analysis. The R package 'deseq2' was harnessed to extract differentially expressed genes. WGCNA was employed to prioritise these genes, and Cox regression analysis and survival analysis based on disease-specific time were conducted to identify significant genes. Immunohistochemistry validation was undertaken to confirm the distinct expression of USP43. Furthermore, the influence of USP43 on the biological functions of ovarian cancer cells was explored using techniques such as RNA interference, western blotting, scratch assays, and matrigel invasion assays. The examination of immune infiltration was facilitated via CIBERSORT. RESULTS: The study unearthed 5195 differentially expressed genes between ovarian cancer and normal tissue, comprising 3416 up-regulated and 1779 down-regulated genes. WGCNA pinpointed 204 genes most intimately tied to tumorigenesis. The previously undisclosed gene USP43 exhibited heightened expression in tumour tissues and exhibited associations with overall survival and disease-specific survival. USP43 emerged as a driver of cell migration (43.27 ± 3.91% vs 19.69 ± 1.94%) and invasion ability (314 ± 32 vs 131 ± 12) through the mechanism of epithelial mesenchymal transition, potentially mediated by the KRAS pathway. USP43 was also identified as a booster of CD4+ T memory resting cell infiltration, while concurrently reducing M1 macrophages within cancer, thereby fostering a milieu with relatively immune suppressive traits. Interestingly, USP43 demonstrated connections with epigenetically regulated-mRNAsi, although not with mRNAsi. CONCLUSION: This study underscores the role of USP43 in facilitating tumour migration and invasion. It postulates USP43 as a novel therapeutic target for ovarian cancer treatment.

Ovarian cancer is the most deadly tumour among all gynecological tumours. Thus we tried to explore the relevant mechanism of ovarian cancer because its occurrence and development mechanism has not been fully elucidated. We used bioinformatics methods to perform differential gene analysis on ovarian cancer tissues and normal tissues, and used methods such as WGCNA and COX regression analysis to find the gene USP43 related to tumour development and prognosis. USP43 is a gene that has not been studied in ovarian cancer before. Through RNA interference technology, we found that it can promote the migration and invasion ability of ovarian cancer and promote epithelial-mesenchymal transition of ovarian cancer cells. In addition, this gene has also been proven to be related to tumour immunity and tumour stemness. These results indicate that USP43 can promote the tumorigenesis of ovarian cancer and can be used as a drug target.

Resveratrol attenuates inflammation and fibrosis in rheumatoid arthritis-associated interstitial lung disease via the AKT/TMEM175 pathway.

BACKGROUND AND PURPOSE: Interstitial lung disease (ILD) represents a significant complication of rheumatoid arthritis (RA) that lacks effective treatment options. This study aimed to investigate the intrinsic mechanism by which resveratrol attenuates rheumatoid arthritis complicated with interstitial lung disease through the AKT/TMEM175 pathway. METHODS: We established an arthritis model by combining chicken type II collagen and complete Freund's adjuvant. Resveratrol treatment was administered via tube feeding for 10 days. Pathological changes in both the joints and lungs were evaluated using HE and Masson staining techniques. Protein expression of TGF-ß1, AKT, and TMEM175 was examined in lung tissue. MRC-5 cells were stimulated using IL-1ß in combination with TGF-ß1 as an in vitro model of RA-ILD, and agonists of AKT, metabolic inhibitors, and SiRNA of TMEM175 were used to explore the regulation and mechanism of action of resveratrol RA-ILD. RESULTS: Resveratrol mitigates fibrosis in rheumatoid arthritis-associated interstitial lung disease and reduces oxidative stress and inflammation in RA-ILD. Furthermore, resveratrol restored cellular autophagy. When combined with the in vitro model, it was further demonstrated that resveratrol could suppress TGF-ß1 expression, and reduce AKT metamorphic activation, consequently inhibiting the opening of AKT/MEM175 ion channels. This, in turn, lowers lysosomal pH and enhances the fusion of autophagosomes with lysosomes, ultimately ameliorating the progression of RA-ILD. CONCLUSION: In this study, we demonstrated that resveratrol restores autophagic flux through the AKT/MEM175 pathway to attenuate inflammation as well as fibrosis in RA-ILD by combining in vivo and in vitro experiments. It further provides a theoretical basis for the selection of therapeutic targets for RA-ILD.

The regulatory role and mechanism of lncTUG1 on cartilage apoptosis and inflammation in osteoarthritis.

BACKGROUND: Long-stranded non-coding RNA TUG1 is lowly expressed in osteoarthritic chondrocytes. This study aimed to elucidate the role of TUG1 in osteoarthritic cartilage damage and the underlying mechanisms. METHODS: Combined database analysis, using primary chondrocytes as well as the C28/I2 cell line, was performed by qRT-PCR, Western blotting, and immunofluorescence to determine the expression of TUG1, miR-144-3p, DUSP1, and other target proteins. Dual luciferase reporter gene and RIP to verify direct interaction of TUG1 with miR-144-3-p and miR-144-3-p with DUSP1, Annexin V-FITC/PI double staining to detect apoptosis. CCK-8 to detect cell proliferation. The biological significance of TUG1, miR-144-3p, and DUSP1 was assessed in vitro experiments using siRNA for TUG1, mimic and repressor for miR-144-3p, and overexpression plasmid for DUSP1. In this study, all data were subjected to a t-test or one-way analysis of variance with a p-value < 0.05 as the cutoff. RESULTS: TUG1 expression was closely associated with osteoarthritic chondrocyte damage, and knockdown of TUG1 significantly promoted chondrocyte apoptosis and inflammation. In the present study, we found that TUG1 inhibited chondrocyte apoptosis and inflammation by competitively binding miR-144-3p, deregulating the negative regulatory effect of miR-144-3p on DUSP1, promoting DUSP1 expression, and inhibiting the p38 MAPK signaling pathway. CONCLUSIONS: In conclusion, our study clarifies the role of the ceRNA regulatory network of TUG1/miR-144-3p/DUSP1/P38 MAPK in OA cartilage injury and provides an experimental and theoretical basis for genetic engineering tools to promote articular cartilage repair.

[Sinoline inhibits NLRP3 and downstream inflammatory factors to alleviate the arthritis symptoms in adjuvant arthritis rats].

Objective To investigate the anti-inflammatory effects of sinoline on adjuvant arthritis (AA) rats and the changes of NOD like receptor pyrin-domain containing 3 (NLRP3). Methods Wistar rats were randomly divided into control group, AA model group, (100, 200, 400) mg/kg sinomenine group and 100 mg/kg Tripterygium wilfordii group, with 10 rats in each group. Except for the control group, AA model was established by Freund complete adjuvant. 12 days after modeling, control group and model group were given the same amount of normal saline, and other groups were given drugs by intragastric administration, once a day, for consecutive 16 days. Joint conditions of AA rats were evaluated by multiple arthritis and joint swelling. The level of NLRP3 protein in synovial tissues was detected by Western blot, and the expression and distribution of NLRP3 in synovial tissues were detected by immunohistochemical staining. Serum levels of interleukin-1ß (IL-1ß), IL-6 and tumor necrosis factor α (TNF-α) were detected by ELISA. Results Compared with the control group, multiple arthritis and joint swelling significantly increased in model group, while those significantly decreased in sinomenine treatment groups and Tripterygium wilfordii group. Decreased expression of NLRP3 protein in synovial tissue was observed, along with the significantly reduced levels of IL-1ß, IL-6 and TNF-α in serum in sinoline treatment groups. Conclusion Sinolin can improve joint inflammation in AA rats by inhibiting NLRP3 and downstream inflammatory factors.

[N-acetylcysteine inhibits the proliferation of hydrogen peroxide treated fibroblast-like synoviocytes in rats with adjuvant arthritis (AA) via blocking Nrf2/Keap1 pathway].

Objective To investigate the effect of N-acetylcysteine (NAC) on the proliferation of fibroblast-like synoviocytes (FLS) treated with low concentration of hydrogen peroxide (H2O2) in rats with adjuvant arthritis (AA) and its mechanism. Methods Twenty SD rats were divided into a normal group and a model group (10 rats in each group). The model group was established by subcutaneous injection of Freund's complete adjuvant into the toe of rats, and the rats were sacrificed 28 days later. The contents of serum malondialdehyde (MDA) were detected by thiobarbituric acid method; the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were determined by hydroxylamine method and colorimetry respectively; and Nrf2 and Keap1 proteins in ankle synovial tissues of AA rats were detected by immunohistochemistry. AA-FLS were isolated, cultured, and identified by digestion of ankle joint slides of AA rats in vitro. The effects of NAC at different concentrations (final concentration 0, 0.3, 0.9, 3, 10, 30, 90, 180 µmol/L) on the activity of AA-FLS treated with H2O2 at low concentration (5 µmol/L) were detected by CCK-8 assay. The content of mitochondrial reactive oxygen species (ROS) in AA-FLS was detected by MitoSOX fluorescent probe. The effects of NAC (final concentration 0, 3, 10, 30 µmol/L) on Nrf2 and Keap1 protein expressions in AA-FLS treated with H2O2 at low concentration were detected by Western blotting. Results Compared with those in the control group, in AA model, the MDA level increased and SOD and GSH-Px levels decreased in serum, and the Nrf2 protein increased and the Keap1 protein decreased in synovial tissue. Immunocytochemical staining confirmed that the isolated and cultured cells were AA-FLS; NAC inhibited the proliferation of AA-FLS treated with H2O2 in a concentration-dependent manner, and the mitochondrial ROS content and the protein expressions of Nrf2 and Keap1 decreased. Conclusion NAC can inhibit the proliferation of AA-FLS treated with H2O2, which may be related to blocking Nrf2/Keap1 pathway.

Nrf2-Keap1 pathway-mediated effects of resveratrol on oxidative stress and apoptosis in hydrogen peroxide-treated rheumatoid arthritis fibroblast-like synoviocytes.

Resveratrol (Res) is a polyphenolic compound that has a variety of biological functions and activities. This study aimed to explore the mechanisms of the antioxidant and proapoptotic effects of Res in H2 O2 -treated rheumatoid arthritis fibroblast-like synoviocytes (RA-FLSs) by the Nrf2-Keap1 signaling pathway. We found that 5 µM H2 O2 promoted cell proliferation and increased intracellular reactive oxygen species (ROS) and malondialdehyde (MDA) content in RA-FLSs. However, Res could reverse these effects in 5 µMH2O2-treated RA-FLSs by (1) promoting expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1), (2) reducing expression of Kelch-like ECH-related protein 1 (Keap1), (3) inhibiting production of ROS and MDA, (4) blocking activation of nuclear factor-κB (NF-κB) p65, (5) inhibiting cell proliferation and migration, and (6) activating Bcl-2/Bax to induce apoptosis. After lentiviral silencing of Nrf2 (siNrf2) mRNA expression in RA-FLSs, Res addition did not increase the expression of Nrf2 or HO-1 to reduce the production of mitochondrial ROS caused by 5 µM H2 O2 . Res reduced the Bcl-2/Bax ratio, but siNrf2 reduced the ability of Res to promote apoptosis. We conclude that Res inhibits ROS production by activating the Nrf2 pathway, thereby inhibiting activation of NF-κB and proliferation and migration of RA-FLSs, to induce apoptosis. Targeting the Nrf2-Keap1 pathway may be a relevant aim of using Res in the treatment of RA.

Effect of lentivirus-mediated overexpression or silencing of MnSOD on apoptosis of resveratrol-treated fibroblast-like synoviocytes in rheumatoid arthritis.

Fibroblast-like synoviocytes in rheumatoid arthritis (RA-FLSs) play a key role in cartilage destruction. We previously found that resveratrol (Res) could promote FLSs apoptosis in adjuvant arthritis rats, but the underlying mechanism was unclear. According to our latest study, Res can suppress the expression of mitochondrial superoxide dismutase (MnSOD) and RA-FLSs proliferation. It was associated with elevated mitochondrial reactive oxygen species levels. Therefore, we hypothesized that Res-mediated RA-FLSs apoptosis might occur via the MnSOD- mitochondrial reactive oxygen species pathway. RA-FLSs were infected with lentiviruses and screened with puromycin at a concentration of 8 µg/ml. We divided the RA-FLSs into four groups: a control group, a negative control (NC) group, a MnSOD overexpression group, and a MnSOD RNAi group. The four groups of RA-FLSs were tested using confocal laser scanning microscopy, CCK-8 assays, flow cytometry, and western blotting were conducted to determine the involvement of the MnSOD-mitochondrial reactive oxygen species pathway. Compared with the NC group, the MnSOD overexpression group treated with different concentrations of Res (0, 25, 50, 100, or 200 µM) and 5 µM H2O2 showed reduced levels of mitochondrial reactive oxygen species, increased B-cell-lymphoma-2 (Bcl-2), reduced Bcl-2 Associated X protein (Bax), and fewer apoptotic cells. The MnSOD RNAi group showed the opposite results. Thus, we concluded that Res could facilitate RA-FLSs apoptosis by regulating MnSOD expression and mitochondrial reactive oxygen species levels. Our findings show a novel mechanism for the beneficial effects of Res, especially in relation to the MnSOD-mitochondrial reactive oxygen species signaling pathway in RA.

Reducing-Autophagy Derived Mitochondrial Dysfunction during Resveratrol Promotes Fibroblast-Like Synovial Cell Apoptosis.

In rheumatoid arthritis patients, the fibroblast-like synovial cells (FLS) growth is not controlled normally, but is similar to the tumor cells proliferation in histology. Our previous studies have shown that resveratrol inhibits the proliferation of FLS and promotes FLS apoptosis. However, the molecular mechanisms involved in resveratrol-induced FLS apoptosis have not been determined yet. Here, we showed that the FLS cell viability (following pretreatment with 5 µM H2 O2 for 24 hr) exhibited better proliferation performance than at other concentrations via the CCK-8 assay. The cell apoptotic rate increased with the increasing concentration of resveratrol (0, 40, 80, 160, 320 µM), as detected by TdT-mediated dUTP nick-end labeling (TUNEL) staining and western blotting. Furthermore, the expression level of autophagy-related proteins (LC3A/B, ATG-5) decreased with the increased concentration of resveratrol, as determined by immunofluorescence and western blot analysis. We also showed that resveratrol induced FLS mitochondrial morphology change. Moreover, mitochondrial function detection showed that the mitochondrial membrane potential was lost with the increased concentration of resveratrol as examined by the JC-1 assay. The production of ATP in cells was positively and negatively correlated with the resveratrol concentration. Simultaneously, the intracellular calcium release and calcium influx decreased gradually with the increase in resveratrol concentration. Therefore, we proposed that resveratrol can reduce the level of autophagy in FLS. The decrease in the autophagy level can lead to the accumulation of reactive oxygen species, which may result in mitochondrial dysfunction and promotion of FLS apoptosis. Anat Rec, 301:1179-1188, 2018. © 2018 Wiley Periodicals, Inc.