LAMTOR5 expression level is a biomarker for colorectal cancer and lncRNA LAMTOR5-AS1 predicting miRNA sponging effect

Molecular Biology Reports - Strong evidence indicated that high expression of HBXIP (also known as LAMTOR5) promotes cancer cells proliferation and helps cancer progression. Long non-coding RNAs...     

SIRT6 safeguards human mesenchymal stem cells from oxidative stress by coactivating NRF2

SIRT6 belongs to the mammalian homologs of Sir2 histone NAD⁺-dependent deacylase family. In rodents, SIRT6 deficiency leads to aging-associated degeneration of mesodermal tissues. It remains unknown whether human SIRT6 has a direct role in maintaining the homeostasis of mesodermal tissues. To this end, we generated SIRT6 knockout human mesenchymal stem cells (hMSCs) by targeted gene editing. SIRT6-deficient hMSCs exhibited accelerated functional decay, a feature distinct from typical premature cellular senescence. Rather than compromised chromosomal stability, SIRT6-null hMSCs were predominately characterized by dysregulated redox metabolism and increased sensitivity to the oxidative stress. In addition, we found SIRT6 in a protein complex with both nuclear factor erythroid 2-related factor 2 (NRF2) and RNA polymerase II, which was required for the transactivation of NRF2-regulated antioxidant genes, including heme oxygenase 1 (HO-1). Overexpression of HO-1 in SIRT6-null hMSCs rescued premature cellular attrition. Our study uncovers a novel function of SIRT6 in maintaining hMSC homeostasis by serving as a NRF2 coactivator, which represents a new layer of regulation of oxidative stress-associated stem cell decay.     

白假丝酵母CFL1基因通过转录调控参与氧化压力应答

【背景】CFL1基因是白假丝酵母高铁还原酶基因,介导胞外铁离子的还原,在白假丝酵母胞内铁稳态的维持方面发挥着重要作用。【目的】研究CFL1基因调节氧化压力应答的分子机制。【方法】采用液体培养及巨噬细胞模型,测定CFL1缺失对氧化压力耐受性和杀伤巨噬细胞能力的影响;使用羟基自由基清除剂二甲基亚砜(DMSO)分析其对缓解氧化压力敏感性的影响;采用实时荧光定量PCR分析CFL1缺失对氧化压力应答基因表达的影响;采用过氧化氢酶(CAT)活性测定方法研究CFL1缺失对CAT1基因表达的影响;通过构建WT-CAT1-GFP和cfl1Δ/Δ-CAT1-GFP菌株分析过氧化氢酶基因过表达对cfl1Δ/Δ氧化压力敏感性的影响。【结果】白假丝酵母CFL1基因的缺失会造成杀伤巨噬细胞能力的减弱,氧化压力应答基因表达的下降。过氧化氢酶基因的过表达则能恢复与野生型几乎一致的氧化压力水平。【结论】CFL1基因通过转录调控参与白假丝酵母氧化压力应答过程。     

DAPK2 regulates oxidative stress in cancer cells by preserving mitochondrial function

Death-associated protein kinase (DAPK) 2 is a serine/threonine kinase that belongs to the DAPK family. Although it shows significant structural differences from DAPK1, the founding member of this protein family, DAPK2 is also thought to be a putative tumour suppressor. Like DAPK1, it has been implicated in programmed cell death, the regulation of autophagy and diverse developmental processes. In contrast to DAPK1, however, few mechanistic studies have been carried out on DAPK2 and the majority of these have made use of tagged DAPK2, which almost invariably leads to overexpression of the protein. As a consequence, physiological roles of this kinase are still poorly understood. Using two genetically distinct cancer cell lines as models, we have identified a new role for DAPK2 in the regulation of mitochondrial integrity. RNA interference-mediated depletion of DAPK2 leads to fundamental metabolic changes, including significantly decreased rate of oxidative phosphorylation in combination with overall destabilised mitochondrial membrane potential. This phenotype is further corroborated by an increase in the production of mitochondrial superoxide anions and increased oxidative stress. This then leads to the activation of classical stress-activated kinases such as ERK, JNK and p38, which is observed on DAPK2 genetic ablation. Interestingly, the generation of oxidative stress is further enhanced on overexpression of a kinase-dead DAPK2 mutant indicating that it is the kinase domain of DAPK2 that is important to maintain mitochondrial integrity and, by inference, for cellular metabolism.Death-associated protein kinase (DAPK) 2 shares a high level of homology within its kinase domain with the other two DAPK family members, DAPK1 (DAPk) and DAPK3 (ZIPK/DLK). Since the identification of DAPK1 by Kimchi and co-workers¹ numerous studies have shown that DAPK1 functions as a tumour suppressor, is linked to key events in autophagy and is involved in mitochondrial maintenance² and metabolism.³ DAPK2, which was characterised in 1999,⁴ is significantly smaller than DAPK1, and it lacks ankyrin repeats, the cytoskeletal binding domain and the death domain, all of which are part of DAPK1''s unique structure.¹ Several functions have been ascribed to DAPK2 and they often coincide with those of DAPK1. Like DAPK1, DAPK2 is also involved in the formation of autophagic vesicles,^{5, 6} modulation of receptor induced cell death^{7, 8, 9} and several modes of intrinsic apoptotic cell death.⁶ While epigenetic silencing of DAPK1 has been reported in many different human cancers,^{10, 11} DAPK2 appears to be silenced mainly in haematological disorders,¹² although it has been shown to modulate TRAIL-induced apoptosis in several cancer cell lines of non-haematological origin.⁹ Most approaches used for studying the role of DAPK2 used tagged DAPK2 and it is, therefore, still unclear whether these functions are also carried out by the native protein, expressed at much lower, endogenous, levels.DAPK1 has been shown to regulate mitochondrial integrity and to modulate the mitochondrial membrane potential² but, to the best of our knowledge, no work has been carried out in this respect with regard to DAPK2. Since DAPK1 and DAPK2 appear to share many functions and both are thought to reside, at least partially, in the mitochondria, we hypothesised that DAPK2 depletion regulated mitochondrial metabolism. Mitochondrial dysfunction is characterised by the induction of reactive oxygen species (ROS) in the cell.¹³ Ultimately, dysfunctional mitochondria can no longer be powerhouses of use to the cell and are, therefore, targeted for degradation. Alternatively, their membranes can depolarise leading to the release of cytochrome c, an early apoptotic process.¹⁴ Using two distinct cancer cell types, namely U2OS osteosarcoma and A549 non-small cell lung cancer cells,^{9, 15} we show that DAPK2 depletion increases the levels of intracellular ROS, leads to mitochondrial depolarisation and impairs mitochondrial metabolism. DAPK2 thus exerts metabolic and mitochondria-regulating functions, which have not been described to date and that can explain why it is downregulated in haematological malignancies,^{12, 16, 17} and involved in modulating death-inducing signalling in solid tumours.⁹     

Melanocortin 1 receptor regulates melanoma cell migration by controlling syndecan-2 expression

The melanocortin 1 receptor (MC1R), a key regulator of melanogenesis, is known to control inflammation, acting in concert with the MC1R ligand α-melanocyte-stimulating hormone. Although cell migration is a key event in inflammation, few studies have addressed the function of MC1R in this context. Using highly motile melanoma cells, we found that the expression level of MC1R was associated with the extent of migration of mouse melanoma cells, suggesting that MC1R plays a functional role in controlling this migration. Overexpression of MC1R enhanced melanoma cell migration, whereas the opposite was true when MC1R levels were knocked down using small inhibitory RNAs. Interestingly, MC1R expression enhanced the synthesis of syndecan-2, a cell surface heparan sulfate proteoglycan known to be involved in melanoma cell migration. Knockdown of syndecan-2 expression decreased MC1R-mediated cell migration. Further, MC1R inhibited the activation of p38 MAPK, subsequently enhancing expression of sydnecan-2, in parallel with an increase in the extent of cell migration. Consistently, activation of p38 by H(2)O(2) inhibited syndecan-2 expression and cell migration, whereas inhibition of p38 activation enhanced syndecan-2 expression and cell migration. Finally, we found that α-melanocyte-stimulating hormone inhibited MC1R-mediated cell migration via activation of p38 and inhibition of syndecan-2 expression. Together, the data strongly suggest that MC1R regulates melanoma cell migration via inhibition of syndecan-2 expression.     

HMSCs exosome-derived miR-199a-5p attenuates sulfur mustard-associated oxidative stress via the CAV1/NRF2 signalling pathway

Sulfur mustard (SM) is a blister-producing chemical warfare agent which could lead to a cascade of systemic damage, especially severe acute lung injury. Oxidative stress is considered to be vital processes for the SM toxicity mechanism. We previously proved the therapeutic effect of exosomes derived from bone marrow mesenchymal stromal cells in promoting the repair of alveolar epithelial barrier and inhibiting apoptosis. However, the key functional components in exosomes and the underlying mechanisms have not been fully elaborated. This research shed light on the function of the key components of human umbilical cord mesenchymal stem cell-derived exosomes (HMSCs-Ex). We noted that HMSCs-Ex-derived miR-199a-5p played a vital role in reducing pneumonocyte oxidative stress and apoptosis by reducing reactive oxygen species, lipid peroxidation products and increasing the activities of antioxidant enzymes in BEAS-2B cells and mouse models after exposure to SM for 24 h. Furthermore, we demonstrated that the overexpression of miR-199a-5p in HMSCs-Ex treatment induced a further decrease of Caveolin1 and the activation of the mRNA and protein level of NRF2, HO1 and NQO1, compared with HMSCs-Ex administration. In summary, miR-199a-5p was one of the key molecules in HMSCs-Ex that attenuated SM-associated oxidative stress via regulating CAV1/NRF2 signalling pathway.     

CYP2E1-mediated oxidative stress regulates HO-1 and GST expression in maneb- and paraquat-treated rat polymorphonuclear leukocytes

Cytochrome P4502E1 (CYP2E1), glutathione-S-transferase A4-4 (GSTA4-4), and inducible nitric oxide synthase (iNOS) are implicated in maneb- and paraquat-induced toxicity leading to various pathological conditions. The study aimed to investigate the role of CYP2E1 in maneb- and paraquat-induced oxidative stress in rat polymorphonuclear leukocytes (PMNs) and its crosstalk with iNOS-mediated nitrosative stress and GSTA4-4-linked protective effect, if any and their consequent links with the nuclear factor erythoid 2-related factor 2 (Nrf2) activation and heme oxygenase-1 (HO-1) expression. Rats were treated with/without maneb and/or paraquat for 1, 2, and 3 weeks along with vehicle controls. Subsets of rats were also treated with diallyl sulfide (DAS) or aminoguanidine (AG) along with the respective controls. Maneb and paraquat augmented the reactive oxygen species (ROS), lipid peroxidation (LPO) and 4-hydroxy nonenal (4-HNE) contents, and superoxide dismutase (SOD) activity in the PMNs. However, maneb and paraquat attenuated the reduced glutathione (GSH) level and the expression/activity of total GST and GST-pi. Maneb and paraquat increased the expression/activity of CYP2E1, GSTA4-4, iNOS, Nrf2 and HO-1, and nitrite content. CYP2E1 inhibitor, DAS noticeably alleviated maneb- and paraquat-induced ROS, LPO, 4-HNE, SOD, Nrf2 and HO-1, GST, GSH, and GST-pi while iNOS, nitrite content and GSTA4-4 levels were unchanged. Conversely, AG, an iNOS inhibitor, attenuated maneb- and paraquat-directed changes in nitrite, LPO, iNOS but it did not alter ROS, GSH, SOD, GST, GST-pi, Nrf2, HO-1, CYP2E1, and GSTA4-4. The results demonstrate that CYP2E1 induces iNOS-independent free radical generation and subsequently modulates the Nrf2-dependent HO-1 and 4-HNE-mediated GST expression in maneb- and paraquat-treated PMNs.     

Catalytic inactive heme oxygenase-1 protein regulates its own expression in oxidative stress

Heme oxygenase-1 (HO-1) catalyzes the degradation of heme and forms antioxidant bile pigments as well as the signaling molecule carbon monoxide. HO-1 is inducible in response to a variety of chemical and physical stress conditions to function as a cytoprotective molecule. Therefore, it is important to maintain the basal level of HO-1 expression even when substrate availability is limited. We hypothesized that the HO-1 protein itself could regulate its own expression in a positive feedback manner, and that this positive feedback was important in the HO-1 gene induction in response to oxidative stress. In cultured NIH 3T3 cells, transfection of HO-1 cDNA or intracellular delivery of pure HO-1 protein resulted in activation of a 15-kb HO-1 promoter upstream of luciferase as visualized by bioluminescent technology and increased HO-1 mRNA and protein levels. These effects were independent of HO activity because an enzymatically inactive mutant form of HO-1 similarly activated the HO-1 promoter and incubation with HO inhibitor metalloporphyrin SnPP did not affect the promoter activation. In addition, HO-1-specific siRNA significantly reduced hemin and cadmium chloride-mediated HO-1 induction. Furthermore, deletion analyses demonstrated that the E1 and E2 distal enhancers of the HO-1 promoter are required for this HO-1 autoregulation. These experiments document feed-forward autoregulation of HO-1 in oxidative stress and suggest that HO-1 protein has a role in the induction process. We speculate that this mechanism may be useful for maintaining HO-1 expression when substrate is limited and may also serve to up-regulate other genes to promote cytoprotection and to modulate cell proliferation.