miR-195-5p exerts tumor-suppressive functions in human lung cancer cells through targeting TrxR2.

miR-195-5p has been widely explored in various cancers and is considered as a tumor-suppressive microRNA. However, its roles in human lung cancer pathogenesis are not fully elucidated. In this study, we aimed to explore how miR-195-5p is involved in malignant behaviors of lung adenocarcinoma (LUAD) cells. miR-195-5p expression was examined in the tumor tissues of patients with LUAD and human LUAD cell lines including A549 and PC-9. Thioredoxin reductase 2 (TrxR2) was predicted to be an mRNA target of miR-195-5p using online tools and validated by the Dual-Luciferase Reporter Assay. Lentivirus infection was used for gene overexpression, while gene knockdown was achieved by RNA interference. Cell proliferation was determined by Cell Counting Kit-8 and 5-ethynyl-2'-deoxyuridine methods, and cell migration and invasion were assayed with transwell experiments. Cell apoptosis was determined by annexin V staining-based flow cytometry. The antitumor effects of miR-195-5p were also evaluated in nude mice xenografted with A549 cells. We found that miR-195-5p was lowly expressed in human LUAD cells, and its overexpression markedly suppressed cell proliferation, migration, and invasion and increased the apoptosis of LUAD cells in vitro. TrxR2 knockdown phenocopied the tumor-suppressive effects of miR-195-5p overexpression, while simultaneous TrxR2 overexpression remarkably reversed the effects of miR-195-5p overexpression on malignant behaviors of A549 and PC-9 cells. Additionally, miR-195-5p overexpression inhibited the growth of xenografted A549 tumor in nude mice. Our work verified that miR-195-5p exerts tumor-suppressive functions in LUAD cells through targeting TrxR2 and suggested that the miR-195-5p/TrxR2 axis is a potential biomarker for LUAD therapy.

p53R2 regulates thioredoxin reductase activity through interaction with TrxR2.

Ribonucleotide reductase small subunit p53R2 is a member of the ribonucleotide reductase family that supplies dNTPs for nuclear and mitochondrial DNA replication and repair. Here, we have identified a mitochondrial thioredoxin reductase 2 (TrxR2) as a novel p53R2-binding protein. We demonstrated a direct interaction between the two, and observed that p53R2 stimulated the enzymatic activity of TrxR in vitro. Moreover, TrxR2 activity was significantly lower in p53R2 knockdown cells, and increased when p53R2 was overexpressed, effects that were independent of p53. Furthermore, p53R2 knockdown suppressed UV-induced TrxR activity. These findings suggest that p53R2 acts as a positive regulator of TrxR2 activity in mitochondria both under normal physiological conditions and during the cellular response to DNA damage.

Comparison of age-dependent alterations in thioredoxin 2 and thioredoxin reductase 2 expressions in hippocampi between mice and rats.

BACKGROUND: Aging is one of major causes triggering neurophysiological changes in many brain substructures, including the hippocampus, which has a major role in learning and memory. Thioredoxin (Trx) is a class of small redox proteins. Among the Trx family, Trx2 plays an important role in the regulation of mitochondrial membrane potential and is controlled by TrxR2. Hitherto, age-dependent alterations in Trx2 and TrxR2 in aged hippocampi have been poorly investigated. Therefore, the aim of this study was to examine changes in Trx2 and TrxR2 in mouse and rat hippocampi by age and to compare their differences between mice and rats. RESULTS: Trx2 and TrxR2 levels using Western blots in mice were the highest at young age and gradually reduced with time, showing that no significant differences in the levels were found between the two subfields. In rats, however, their expression levels were the lowest at young age and gradually increased with time. Nevertheless, there were no differences in cellular distribution and morphology in their hippocampi when it was observed by cresyl violet staining. In addition, both Trx2 and TrxR2 immunoreactivities in the CA1-3 fields were mainly shown in pyramidal cells (principal cells), showing that their immunoreactivities were altered like changes in their protein levels. CONCLUSIONS: Our current findings suggest that Trx2 and TrxR2 expressions in the brain may be different according to brain regions, age and species. Therefore, further studies are needed to examine the reasons of the differences of Trx2 and TrxR2 expressions in the hippocampus between mice and rats.

Smart mitochondrial-targeted cancer therapy: Subcellular distribution, selective TrxR2 inhibition accompany with declined antioxidant capacity.

Targeting mitochondrial redox homeostasis is an appealing methodology for cancer therapeutics because of the upregulated antioxidant capacity in drug resistance cases. By coupling triphenylamine (TPA) with an excellent fluorescent group BODIPY, a novel mitochondrial-targeted fluorescent probe, BODIPY-TPA (BTPA), was synthesized and characterized. Confocal microscopic colocalization imaging indicated that BTPA exhibited a subcellular mitochondrial distribution. Cytotoxicity experiments suggested that BTPA exhibited selective anticancer activity via the induction of mitochondrial dysfunction in BGC-823 cancer cells. BTPA induced alterations in mitochondrial redox homeostasis because of the electron-donating property of TPA and mitochondrial selectivity. In further studies, TrxR2 in the mitochondria was alternatively inhibited, which contributed to MtROS accumulation further attenuated PI3K/Akt signaling pathway. The resultant decline in mitochondrial antioxidant capacity aggravated mitochondrial oxidative stress, which is responsible for cytochrome C release and caspase-9 activation. NAC completely reversed BTPA-induced ROS-dependent mitochondrial-mediated intrinsic apoptosis. Therefore, BTPA was designed as a superior fluorescent cancer-imaging probe and a mitochondrial redox-targeting anticancer agent.

The study on polymorphisms of Sep15 and TrxR2 and the expression of AP-1 signaling pathway in Kashin-Beck disease.

The aim of the study was to investigate the association between rs5859 in Sep15, rs1139793 in TrxR2 polymorphisms with the risks of KBD and to detect the expression of AP-1 pathway in KBD subjects and in vitro. 208 KBD and 206 control subjects were included. PCR-Restriction Fragment Length Polymorphism (RFLP), Amplification Refractory Mutation Specific-PCR (ARMS-PCR) and Western Blotting were conducted. The results showed the minor A-allele frequency of rs5859 in KBD was statistically significantly higher than that in the control group (P < 0.05). The cases carrying A-allele had a 2-fold (95%CI: 1.064-3.956) increased risk of developing KBD compared with the G-allele carriers. There was no significant difference in genotype and allele distribution of rs1139793 between KBD patients and controls (P > 0.05). The frequency of the minor A allele of rs5859 was significantly different in Chinese healthy population compared with European, African and American. The frequency of the minor A allele of rs1139793 showed significant difference when compared with African and American. The levels of JunB, JunD, P65 proteins in KBD group were higher than those in control group (P < 0.0001). The expression of JunB, JunD, P65 proteins all increased in tBHP-induced C28/I2 oxidative damage model compared with control group (P < 0.05) and decreased after Se supplementation. Our finding indicated Sep15 is a possible candidate susceptibility gene for KBD. Combined with the in vitro study, our studies reveal novel insights into the mechanism of Se supplementation as an antioxidant via inhibiting the AP-1 signaling pathway in patients with KBD.

Inhibition of TrxR2 suppressed NSCLC cell proliferation, metabolism and induced cell apoptosis through decreasing antioxidant activity.

AIMS: This study aims to analyze the effect of thioredoxin reductase 2 (TrxR2) on lung cancer cell proliferation, apoptosis, invasion and migration in vitro. MAIN METHODS: Real-time PCR was used to measure the expression of TrxR2 in NSCLC tumor tissues. After pAd-TrxR2 or shRNA-TrxR2 was transfected into A549 or NCI-H1299 cells, the cell proliferation was measured by CCK-8 method; cell apoptosis was measured by flow cytometry; cell invasion and migration was measured by Transwell method. The production of ROS was measured by DCFH-DA method; the activity of SOD, CAT and GSH-Px was measured by relative ELISA kit. KEY FINDINGS: The results showed that TrxR2 was up-regulated in NSCLC tumor tissues. Inhibition of TrxR2 suppressed NSCLC cell proliferation and induced apoptosis, and inhibited cell invasion and migration. However, overexpression of TrxR2 showed the opposite effect. Furthermore, when cells were transfected with shRNA-TrxR2, the production of ROS was significantly increased, and SOD, CAT and GSH-Px activity was decreased. Conversely, pAd-TrxR2 transfection showed the opposite effect. SIGNIFICANCE: Taken together, our results suggest that TrxR2 acts as an oncogenic gene in the context of lung cancer progression. The inhibition of TrxR2 suppressed lung cancer cell proliferation, invasion and migration and induced cell apoptosis by inducing ROS production and decreasing antioxidant activity. TrxR2 may be a potential target for NSCLC treatment.

Mitochondrial thioredoxin reductase 2 is elevated in long-lived primate as well as rodent species and extends fly mean lifespan.

In a survey of enzymes related to protein oxidation and cellular redox state, we found activity of the redox enzyme thioredoxin reductase (TXNRD) to be elevated in cells from long-lived species of rodents, primates, and birds. Elevated TXNRD activity in long-lived species reflected increases in the mitochondrial form, TXNRD2, rather than the cytosolic forms TXNRD1 and TXNRD3. Analysis of published RNA-Seq data showed elevated TXNRD2 mRNA in multiple organs of longer-lived primates, suggesting that the phenomenon is not limited to skin-derived fibroblasts. Elevation of TXNRD2 activity and protein levels was also noted in liver of three different long-lived mutant mice, and in normal male mice treated with a drug that extends lifespan in males. Overexpression of mitochondrial TXNRD2 in Drosophila melanogaster extended median (but not maximum) lifespan in female flies with a small lifespan extension in males; in contrast, overexpression of the cytosolic form, TXNRD1, did not produce a lifespan extension.

Brain mitochondria from DJ-1 knockout mice show increased respiration-dependent hydrogen peroxide consumption.

Mutations in the DJ-1 gene have been shown to cause a rare autosomal-recessive genetic form of Parkinson's disease (PD). The function of DJ-1 and its role in PD development has been linked to multiple pathways, however its exact role in the development of PD has remained elusive. It is thought that DJ-1 may play a role in regulating reactive oxygen species (ROS) formation and overall oxidative stress in cells through directly scavenging ROS itself, or through the regulation of ROS scavenging systems such as glutathione (GSH) or thioredoxin (Trx) or ROS producing complexes such as complex I of the electron transport chain. Previous work in this laboratory has demonstrated that isolated brain mitochondria consume H2O2 predominantly by the Trx/Thioredoxin Reductase (TrxR)/Peroxiredoxin (Prx) system in a respiration dependent manner (Drechsel et al., Journal of Biological Chemistry, 2010). Therefore we wanted to determine if mitochondrial H2O2 consumption was altered in brains from DJ-1 deficient mice (DJ-1(-/-)). Surprisingly, DJ-1(-/-) mice showed an increase in mitochondrial respiration-dependent H2O2 consumption compared to controls. To determine the basis of the increased H2O2 consumption in DJ1(-/-) mice, the activities of Trx, Thioredoxin Reductase (TrxR), GSH, glutathione disulfide (GSSG) and glutathione reductase (GR) were measured. Compared to control mice, brains from DJ-1(-/-) mice showed an increase in (1) mitochondrial Trx activity, (2) GSH and GSSG levels and (3) mitochondrial glutaredoxin (GRX) activity. Brains from DJ-1(-/-) mice showed a decrease in mitochondrial GR activity compared to controls. The increase in the enzymatic activities of mitochondrial Trx and total GSH levels may account for the increased H2O2 consumption observed in the brain mitochondria in DJ-1(-/-) mice perhaps as an adaptive response to chronic DJ-1 deficiency.

Alcohol induces mitochondrial redox imbalance in alveolar macrophages.

Alcohol abuse suppresses the immune responses of alveolar macrophages (AMs) and increases the risk of a respiratory infection via chronic oxidative stress and depletion of critical antioxidants within alveolar cells and the alveolar lining fluid. Although alcohol-induced mitochondrial oxidative stress has been demonstrated, the oxidation of the mitochondrial thioredoxin redox circuit in response to alcohol has not been examined. In vitro ethanol exposure of a mouse AM cell line and AMs from ethanol-fed mice demonstrated NADPH depletion concomitant with oxidation of mitochondrial glutathione and oxidation of the thioredoxin redox circuit system including thioredoxin 2 (Trx2) and thioredoxin 2 reductase (Trx2R). Mitochondrial peroxiredoxins (Prdx's), which are critical for the reduction of the thioredoxin circuit, were irreversibly hyperoxidized to an inactive form. Ethanol also decreased the mRNAs for Trx2, Trx2R, Prdx3, and Prdx5 plus the mitochondrial thiol-disulfide proteins glutaredoxin 2, glutathione reductase, and glutathione peroxidase 2. Thus, the mitochondrial thioredoxin circuit was highly oxidized by ethanol, thereby compromising the mitochondrial antioxidant capacity and ability to detoxify mitochondrial reactive oxygen species. Oxidation of the mitochondrial thioredoxin redox circuit would further compromise the transient oxidation of thiol groups within specific proteins, the basis of redox signaling, and the processes by which cells respond to oxidants. Impaired mitochondria can then jeopardize cellular function of AMs, such as phagocytosis, which may explain the increased risk of respiratory infection in subjects with an alcohol use disorder.

Mechanosensitive microRNAs-role in endothelial responses to shear stress and redox state.

Endothelial functions are highly regulated by imposed shear stress in vivo. The characteristics of shear stress determine mechanotransduction events that regulate phenotypic outcomes including redox and inflammatory states. Recent data indicate that microRNAs (miRs) in vascular endothelial cells play an essential role in shear stress-regulated endothelial responses. More specifically, atheroprotective pulsatile flow (PS) induces miRs that inhibit mediators of oxidative stress and inflammation while promoting those involved in maintaining vascular homeostasis. Conversely, oscillatory flow (OS) elicits the opposing networks. This is exemplified by the PS-responsive transcription factor Krüppel-like factor 2 (KLF2), which regulates miR expression but is also regulated by OS-sensitive miRs to ultimately regulate the oxidative and inflammatory state of the endothelium. In this review, we outline important findings demonstrating the multifaceted roles of shear stress-regulated miRs in endothelial redox and inflammatory balance. Furthermore, we discuss the use of algorithms in deciphering signaling networks differentially regulated by PS and OS.