Wutou decoction attenuates rheumatoid arthritis by modulating the Ahr/LOC101928120/SHC1 pathway.

CONTEXT: Wutou decoction (WTD) is a Chinese herbal formula alleviating rheumatoid arthritis (RA). SHC adaptor protein 1 (SHC1) regulates apoptosis, inflammation, and the production of reactive oxygen species (ROS). The LOC101928120 gene is located near the SHC1 gene. Bioinformatics analysis showed that the long non-coding RNA LOC101928120 binds to histone deacetylase HDAC1 that might regulate SHC1 expression. The LOC101928120 gene might be targeted by the transcriptional factor Aryl hydrocarbon receptor (Ahr). OBJECTIVE: This study determines the involvement of the Ahr/LOC101928120/SHC1 pathway in WTD alleviation of RA. MATERIALS AND METHODS: Wistar rats were injected with complete Freund's adjuvant in the hind footpad to construe the RA model. WTD (9.8 g/kg/day) was administered intragastrically for 15 days. The CHON-001 chondrocyte cells were treated with IL-1ß (10 ng/mL) alone or in combination with WTD (1 µg/mL). A RNA pull-down assay was performed to determine the interaction between LOC101928120 and HDAC1. Ahr targeting the LOC101928120 gene was detected using luciferase reporter and chromatin immunoprecipitation assays. RESULTS: WTD alleviated the swelling of the hind paw in rats with RA and suppressed the chondrocyte apoptosis and ROS production caused by IL-1ß. WTD decreased SHC1 but increased LOC101928120 in IL-1ß-treated chondrocytes. SHC1 knockdown and LOC101928120 overexpression also showed the protection. However, LOC101928120 knockdown attenuated the protective effects of WTD. WTD stimulated Ahr, which promoted LOC101928120 transcription. LOC101928120 recruited HDAC1 to the promoter region of the SHC1 gene, thereby decreasing SHC1. DISCUSSION AND CONCLUSION: This study revealed a new mechanism by which WTD alleviates RA by modulating the Ahr/LOC101928120/SHC1 pathway.

Interplay among H3K9-editing enzymes SUV39H1, JMJD2C and SRC-1 drives p66Shc transcription and vascular oxidative stress in obesity.

Aims: Accumulation of reactive oxygen species (ROS) promotes vascular disease in obesity, but the underlying molecular mechanisms remain poorly understood. The adaptor p66Shc is emerging as a key molecule responsible for ROS generation and vascular damage. This study investigates whether epigenetic regulation of p66Shc contributes to obesity-related vascular disease. Methods and results: ROS-driven endothelial dysfunction was observed in visceral fat arteries (VFAs) isolated from obese subjects when compared with normal weight controls. Gene profiling of chromatin-modifying enzymes in VFA revealed a significant dysregulation of methyltransferase SUV39H1 (fold change, -6.9, P < 0.01), demethylase JMJD2C (fold change, 3.2, P < 0.01), and acetyltransferase SRC-1 (fold change, 5.8, P < 0.01) in obese vs. control VFA. These changes were associated with reduced di-(H3K9me2) and trimethylation (H3K9me3) as well as acetylation (H3K9ac) of histone 3 lysine 9 (H3K9) on p66Shc promoter. Reprogramming SUV39H1, JMJD2C, and SRC-1 in isolated endothelial cells as well as in aortas from obese mice (LepOb/Ob) suppressed p66Shc-derived ROS, restored nitric oxide levels, and rescued endothelial dysfunction. Consistently, in vivo editing of chromatin remodellers blunted obesity-related vascular p66Shc expression. We show that SUV39H1 is the upstream effector orchestrating JMJD2C/SRC-1 recruitment to p66Shc promoter. Indeed, SUV39H1 overexpression in obese mice erased H3K9-related changes on p66Shc promoter, while SUV39H1 genetic deletion in lean mice recapitulated obesity-induced H3K9 remodelling and p66Shc transcription. Conclusion: These results uncover a novel epigenetic mechanism underlying endothelial dysfunction in obesity. Targeting SUV39H1 may attenuate oxidative transcriptional programmes and thus prevent vascular disease in obese individuals.

Hyperglycaemia-induced epigenetic changes drive persistent cardiac dysfunction via the adaptor p66Shc.

AIMS: Hyperglycaemia-induced reactive oxygen species (ROS) are key mediators of cardiac dysfunction. Intensive glycaemic control (IGC) has failed to reduce risk of heart failure in patients with diabetes but the underlying mechanisms remain to be elucidated. The present study investigates whether epigenetic regulation of the pro-oxidant adaptor p66Shc contributes to persistent myocardial dysfunction despite IGC. METHODS AND RESULTS: p66Shc expression was increased in the heart of diabetic mice, and 3-week IGC by slow-release insulin implants did not revert this phenomenon. Sustained p66Shc upregulation was associated with oxidative stress, myocardial inflammation and left ventricular dysfunction, as assessed by conventional and 2D speckle-tracking echocardiography. In vivo gene silencing of p66Shc, performed during IGC, inhibited ROS production and restored cardiac function. Furthermore, we show that dysregulation of methyltransferase DNMT3b and deacetylase SIRT1 causes CpG demethylation and histone 3 acetylation on p66Shc promoter, leading to persistent transcription of the adaptor. Altered DNMT3b/SIRT1 axis in the diabetic heart was explained by upregulation of miR-218 and miR-34a. Indeed, in human cardiomyocytes exposed to high glucose, inhibition of these miRNAs restored the expression of DNMT3b and SIRT1 and erased the adverse epigenetic signatures on p66Shc promoter. Consistently, reprogramming miR-218 and miR-34a attenuated persistent p66Shc expression and ROS generation. CONCLUSIONS: In diabetic left ventricular dysfunction, a complex epigenetic mechanism linking miRNAs and chromatin modifying enzymes drives persistent p66Shc transcription and ROS generation. Our results set the stage for pharmacological targeting of epigenetic networks to alleviate the clinical burden of diabetic cardiomyopathy.

Oxidative stress induces B lymphocyte DNA damage and apoptosis by upregulating p66shc.

OBJECTIVE: B lymphoma is a type of malignant tumor originating from the lymphatic hematopoietic system. The pathogenesis and treatment methods are not clear. The change of oxidative stress is closely related to the cell DNA damage and cell apoptosis, which may be served as a target for cancer treatment. This study aims to illustrate the role of oxidative stress in the regulation of B lymphocytoma. PATIENTS AND METHODS: The tumor tissue was collected from patients with B lymphocytoma. The p66shc level was detected by Western blot. Hydrogen peroxide (H2O2) was assessed by the kit. The oxidative stress model of B lymphoma cell was established by H2O2 treatment. ROS inhibitor or RNAi was used to regulate ROS level. ROS level was determined by flow cytometry. 8-OHdG level (DNA damage product) was tested by the kit. Cell apoptosis was evaluated by annexin V-PI. RESULTS: P66shc expression was significantly reduced, while H2O2 production was significantly decreased in the tumor tissue of B lymphoma compared with adjacent normal control. H2O2 stimulation markedly elevated ROS level and p66shc expression (p < 0.05), accompanied by the aggravation of DNA damage and increase of apoptosis. ROS inhibitor or p66shc RNAi treatment significantly attenuated DNA damage and declined cell apoptosis (p < 0.05). CONCLUSIONS: ROS production promoted p66shc expression, induced DNA damage, and facilitated cell apoptosis. Upregulation of p66shc by oxidative stress could be treated as a new therapeutic target for B lymphoma.

Activation of miR-34a-5p/Sirt1/p66shc pathway contributes to doxorubicin-induced cardiotoxicity.

The molecular mechanisms underlying anthracyclines-induced cardiotoxicity have not been well elucidated. MiRNAs were revealed dysregulated in the myocardium and plasma of rats received Dox treatment. MicroRNA-34a-5p (miR-34a-5p) was verified increased in the myocardium and plasma of Dox-treated rats, but was reversed in rats received Dox plus DEX treatments. Human miR-34a-5p was also observed increased in the plasma of patients with diffuse large B-cell lymphoma after 9- and 16-week epirubicin therapy. Up-regulation of miR-34a-5p was observed in Dox-induced rat cardiomyocyte H9c2 cells. MiR-34a-5p could augment Bax expression, but inhibited Bcl-2 expression, along with the increases of the activated caspase-3 and mitochondrial potentials in H9C2 cells. MiR-34a-5p was verified to modulate Sirt1 expression post-transcriptionally. In parallel to Sirt1 siRNA, miR-34a-5p could enhance p66shc expression, accompanied by increases of Bax and the activated caspase-3 and a decrease of Bcl-2 in H9c2 cells. Moreover, enforced expression of Sirt1 alleviated Dox-induced apoptosis of H9c2 cells, with suppressing levels of p66shc, Bax, the activated caspase-3 and miR-34a-5p, and enhancing Bcl-2 expression. Therefore, miR-34a-5p enhances cardiomyocyte apoptosis by targeting Sirt1, activation of miR-34a-5p/Sirt1/p66shc pathway contributes to Dox-induced cardiotoxicity, and blockage of this pathway represents a potential cardioprotective effect against anthracyclines.

Nicotine Exposure Augments Renal Toxicity of 5-aza-cytidine Through p66shc: Prevention by Resveratrol.

BACKGROUND/AIM: We have shown that either chronic nicotine (NIC) exposure or 5-aza-cytidine (AZA) augments oxidative stress-dependent injury through stimulating p66shc in renal cells. Hence, NIC could exacerbate adverse effects of AZA while antioxidants such as resveratrol (RES) could prevent it. MATERIALS AND METHODS: Renal proximal tubule cells (NRK52E) were treated with 20 µM RES prior to 200 µM NIC plus 100 nM AZA and cell injury (LDH release) was determined. Reporter luciferase assays determined p66shc activation and RES-induced antioxidant responses. Genetic manipulations identified the mechanism of RES action. RESULTS: NIC exacerbated AZA-dependent injury via augmenting p66shc transcription. While RES suppressed NIC+AZA-mediated injury, -surprisingly-it further enhanced activity of the p66shc promoter. RES protected cells via the cytoplasmic p66shc/Nrf2/heme oxygenase-1 (HO-1) axis. CONCLUSION: RES can protect the kidney from adverse effects of NIC in patients undergoing anticancer therapy.

Downregulation of a novel long noncoding RNA TRPM2-AS promotes apoptosis in non-small cell lung cancer.

Non-small cell lung cancer is one of the most common types of cancer, and the prognosis of non-small cell lung cancer is still poor. Recent evidence has proved that long noncoding RNA is involved in tumorigenesis. For non-small cell lung cancer, the expression profile of long noncoding RNA has been studied. Here, we identified a novel long noncoding RNA TRPM2-AS from published dataset and found TRPM2-AS is widely upregulated in non-small cell lung cancer tissues compared with adjacent non-tumor tissues. Higher expression level of TRPM2-AS was correlated with higher TNM stages and larger tumor size. Patients with high TRPM2-AS expression level had poor survival than those with low TRPM2-AS level. We silenced TRPM2-AS by small interfering RNA and found that cell proliferation was significantly inhibited after knockdown of TRPM2-AS. Annexin V/propidium iodide staining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay confirmed that cell apoptosis increased after TRPM2-AS knockdown. Further experiments showed that silence of TRPM2-AS upregulated SHC1 and silence of SHC1 partially reversed cell apoptosis after TRPM2-AS knockdown. In summary, the novel long noncoding RNA TRPM2-AS upregulated in non-small cell lung cancer, and downregulation of TRPM2-AS promotes apoptosis in vitro.

Novel miR-5582-5p functions as a tumor suppressor by inducing apoptosis and cell cycle arrest in cancer cells through direct targeting of GAB1, SHC1, and CDK2.

MicroRNAs (miRNAs) play pivotal roles in tumorigenesis as either tumor suppressors or oncogenes. In the present study, we discovered and demonstrated the tumor suppressive function of a novel miRNA miR-5582-5p. miR-5582-5p induced apoptosis and cell cycle arrest in cancer cells, but not in normal cells. GAB1, SHC1, and CDK2 were identified as direct targets of miR-5582-5p. Knockdown of GAB1/SHC1 or CDK2 phenocopied the apoptotic or cell cycle arrest-inducing function of miR-5582-5p, respectively. The expression of miR-5582-5p was lower in tumor tissues than in adjacent normal tissues of colorectal cancer patients, while the expression of the target proteins exhibited patterns opposite to that of miR-5582-5p. Intratumoral injection of a miR-5582-5p mimic or induced expression of miR-5582-5p in tumor cells suppressed tumor growth in HCT116 xenografts. Collectively, our results suggest a novel tumor suppressive function for miR-5582-5p and its potential applicability for tumor control.