The role of ferroptosis-related genes for overall survival prediction in breast cancer.

BACKGROUND: Ferroptosis is a novel iron-dependent form of cell death, which is implicated in various diseases including cancers. However, the influence of ferroptosis-related genes on the prognosis of breast cancer remains unclear. METHODS: RNA sequencing data of 1053 breast cancer tissue samples and 111 normal tissue samples from The Cancer Genome Atlas (TCGA) were analyzed. Expression levels of 259 ferroptosis-related genes were compared. Gene Ontology (GO) and the Kyoto Gene and Genomic Encyclopedia (KEGG) analyses were conducted on differentially expressed genes. Cox univariate analysis was conducted to explore the potential prognostic biomarkers of breast cancer. Infiltrating immune cell status was assessed. RESULTS: A total of 66 ferroptosis-related genes were differentially expressed in breast cancer tissues. The enriched GO terms included Biological Process (mainly included response to oxidative stress, cellular response to chemical stress, multicellular organismal homeostasis, cofactor metabolic process, response to metal ion, response to steroid hormone, cellular response to oxidative stress, transition metal ion homeostasis, iron ion homeostasis, and cellular iron ion homeostasis), Cellular Component (mainly included apical plasma membrane, early endosome, apical part of cell, lipid droplet, basolateral plasma membrane, blood microparticle, clathrin-coated pit, caveola, astrocyte projection, and pronucleus) and Molecular Function (mainly included iron ion binding, ubiquitin protein ligase binding, oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen, oxidoreductase activity, acting on the CH-OH group of donors, NAD or NADP as acceptor, ferric iron binding, aldo-keto reductase (NADP) activity, oxidoreductase activity, acting on single donors with incorporation of molecular oxygen, steroid dehydrogenase activity, alditol:NADP+1-oxidoreductase activity, and alcohol dehydrogenase (NADP+) activity). The enriched KEGG pathway mainly included the HIF-1 signaling pathway, NOD-like receptor signaling pathway, ferroptosis, IL-17 signaling pathway, central carbon metabolism in cancer, PPAR signaling pathway, PD-L1 expression, and PD-1 checkpoint pathway in cancer. Among them, 38 ferroptosis-related genes were significantly associated with the prognosis of breast cancer. The prognostic model was constructed, and breast cancer patients in low-risk group had a better prognosis. In addition, risk score of ferroptosis prognostic model was negatively correlated with B cells (r = -0.063, p = 0.049), CD8+ T cells (r = -0.083, p = 0.010), CD4+ T cells (r = -0.097, p = 0.002), neutrophils (r = -0.068, p = 0.033), and dendritic cells (r = 0.088, p = 0.006). CONCLUSIONS: The ferroptosis pathway plays a key role in breast cancer. Some differentially expressed ferroptosis-related genes can be used as prognostic biomarkers for breast cancer.

[Effect of retroviral vector mediating RNA interference on the release of tumor necrosis factor-alpha in lipopolysaccharide-stimulating macrophage].

OBJECTIVE: To explore the effect of small interfering RNA (siRNA) targeting NF-kappaB signal pathway on the expression level of tumor necrosis factor alpha (TNF-alpha) released by lipopolysaccharides (LPS)-stimulating-macrophages. METHODS: Human monocytic THP-1 cell was induced by phorbol myristate acetate (PMA) and transformed into macrophage. Two groups of macrophage were infected by siRNA retroviral expression vector specific to NF-kappaB functional subunit P65 (siRNA group) and Scramble control vector (Scramble control group) constructed by molecular cloning technology. Lipopolysaccharide (50 microg/ml) was used to treat the macrophages continuously. RT-PCR was performed to detect the expression level of NF-kappaB P65 mRNA and TNF-alpha mRNA at different time-points of LPS stimulation. Western blotting was used to analyze the protein level of NF-kappaB P65. Enzyme-linked immunosorbent assay was applied to analyze the expression level of TNF-alpha released by LPS-stimulated macrophages. RESULTS: At Hours 12 and 24 after LPS stimulation, the expression level of NF-kappaB P65 mRNA in siRNA group (0.97 +/- 0.02, 0.89 +/- 0.01) was significantly less than that in Scramble control group (1.01 +/- 0.03, 0.97 +/- 0.01, both P < 0.05). At Hours 24 and 72 after LPS stimulation, the expression level of NF-kappaB P65 protein in siRNA group (0.95 +/- 0.04, 0.94 +/- 0.01) was obviously less than that in Scramble control group (1.07 +/- 0.06, 1.03 +/- 0.05, both P < 0.05). At Hours 4, 8, 12 and 24 after LPS stimulation, TNF-alpha mRNA released by siRNA group macrophages was far less than that by Scramble control group macrophages (0.92 +/- 0.02 vs 0.98 +/- 0.01, 0.86 +/- 0.02 vs 1.00 +/- 0.01, 0.79 +/- 0.03 vs 1.01 +/- 0.01, 0.78 +/- 0.03 vs 1.02 +/- 0.01, all P < 0.05). At Hours 2, 4, 8, 24, 36, 48, 54 and 72 after LPS stimulation, the TNF-alpha content in culture medium supernatant in siRNA group macrophage was less than that in scramble control group (P < 0.05). CONCLUSION: NF-kappaB P65 siRNA inhibits the functional activity of NF-kappaB signal pathway in PMA-induced macrophage. Then it blocks the activation of macrophage and the excessive release of TNF-alpha due to endotoxin stimulation. The RNA interference technology may be applied to prevent and treat excessive inflammatory reaction in acute lung injury.

[Effect of depsides salts from salvia miltiorrhiza on functions of platelet and vascular endothelial cell of patients with chronic obstructive pulmonary disease in acute exacerbation stage].

OBJECTIVE: To investigate the influence of depsides salts from salvia miltiorrhiza (DSM) on the functions of platelet and vascular endothelial cell in severe chronic obstructive pulmonary diseases (COPD) patients with acute exacerbation. METHODS: Forty patients with severe COPD in acute exacerbation stage were randomly divided into two groups: conventional treatment (CT) group and DSM treatment (DSM) group, each consisting of 20 cases, and 20 healthy adults served as control group. All COPD patients were given conventional treatment, while for the patients in DSM group 0.2 g depsides DSM was given through intravenous drip everyday in addition for 2 weeks. The levels of the plasma platelet membrane glycoprotein 140 (GMP140) and von Willebrand factor (vMF) in the blood samples were determined with enzyme linked immunosorbent assay (ELISA) and the level of CD62P/CD61 with flow cytometry (FCM). RESULTS: The level of GMP140 [CT group: (17.51+/-2.75) microg/L, DSM group: (16.94+/-2.57) microg/L], vMF [CT group: (13.64+/-2.37) microg/L, DSM group: (14.14+/-2.17) microg/L] and CD62P/CD61 [CT group: (20.24+/-2.64)% , DSM group: (19.54+/-2.69)%] were elevated significantly in severe COPD patients with acute exacerbation compared to the control group before treatment [(11.21+/-1.11)%, (9.25+/-1.80) microg/L, (6.13+/-1.17) microg/L, all P<0.01]. After intervention, the levels of the above three indexes in both treatment groups were significantly decreased, and the decrease in DSM group [GMP140: (3.91+/- 0.57) microg/L , vWF: (3.86+/-0.71) microg/L, CD62P/CD61: (3.69+/-0.62)%] was more prominent than the CT group [GMP140: (2.30+/-0.33) microg/L, vWF: (2.72+/- 0.45) microg/L , CD62P/CD61: (2.24+/-0.45)%, all P<0.01], but they were higher than normal levels. CONCLUSION: DSM has the effect of inhibiting the activation of vascular endothelial cells and platelet. The medicine may be used to prevent thrombotic diseases.

LKB1 inactivation leads to centromere defects and genome instability via p53-dependent upregulation of survivin.

Inactivating mutations in the liver kinase B1 (LKB1) tumor suppressor gene underlie Peutz-Jeghers syndrome (PJS) and occur frequently in various human cancers. We previously showed that LKB1 regulates centrosome duplication via PLK1. Here, we report that LKB1 further helps to maintain genomic stability through negative regulation of survivin, a member of the chromosomal passenger complex (CPC) that mediates CPC targeting to the centromere. We found that loss of LKB1 led to accumulation of misaligned and lagging chromosomes at metaphase and anaphase and increased the appearance of multi- and micro-nucleated cells. Ectopic LKB1 expression reduced these features and improved mitotic fidelity in LKB1-deficient cells. Through pharmacological and genetic manipulations, we showed that LKB1-mediated repression of survivin is independent of AMPK, but requires p53. Consistent with the key influence of LKB1 on survivin expression, immunohistochemical analysis indicated that survivin is highly expressed in intestinal polyps from a PJS patient. Lastly, we reaffirm a potential therapeutic avenue to treat LKB1-mutated tumors by demonstrating the increased sensitivity to survivin inhibitors of LKB1-deficient cells.

[Effect of isoflurane delayed preconditioning on myocardial ischemia reperfusion injury in rabbits].

OBJECTIVE: To investigate the protective effect of isoflurane delayed preconditioning on myocardial ischemia reperfusion injury and the potential mechanism in rabbits. METHODS: Thirty New Zealand male white rabbits were randomly assigned to 3 groups: Control group; I/R group; and 2.0% isoflurane group. Isoflurane group was exposed to 2.0% isoflurane-100% oxygen for 2 hours. Control group and I/R group were exposed to 100% oxygen for 2 hours and served as untreated controls. Twenty-four hours later I/R group and isoflurane group underwent 40 minutes of coronary occlusion followed by 2 hours of reperfusion. Blood samples were taken from the arterial line at 20 minutes before the occlusion(T1), 20 minutes after the occlusion(T2), 40 minutes after the occlusion(T3), 1 hours after the reperfusion(T4), and 2 hours after the reperfusion(T5) to determine the plasma level of TNF-alpha. At the end of the reperfusion, infarct size and area at risk were defined by Evans and TTC staining. The heart was harvested and levels of the p38MAPK activity were determined by Western blot, and ultrastructures were observed under the electron microscope. RESULTS: The p38MAPK activity of isoflurane group was significantly lower than that of I/R group (P<0.05). Isoflurane significantly (P<0.05) reduced the infarct size(19.7%+/-2.8% in isoflurane group) of the left ventricular area at risk as compared with the controls (37.8%+/-1.7% in I/R group).The injury of I/R group was worse than that of isoflurane group under the light microscope. Isoflurane group had a lower level of TNF-alpha than I/R group. CONCLUSION: Isoflurane can inhibit p38MAPK activity during myocardial ischemia reperfusion and modulate the cytokine expression, which may be one of the molecular mechanisms of isoflurane delayed preconditioning on cardioprotection.

[Effect of ulinastain on the expression of hemeoxygenase-1 in oleic acid-induced acute lung injury in rats].

OBJECTIVE: To explore the effect of ulinastain on the expression of hemeoxy genase-1 (HO-1) in oil acid-induced acute lung injury in rats. METHODS: The animal model of acute lung injury was established by oil acid. Thirty SD rats were randomly divided into 3 groups: the blank control group (A), the acute lung injury group (B) and the acute lung injury group (C) followed by injecting 100 mL/kg ulinastatin. Each group consisted of 10 rats. Group A were given 0.2 mL/kg natural solution through the trial vein; Group B and C were given 0.2 mL/kg oil-acid through trial vein, while group C were injected 100mL/kg ulinastatin by the peritoneal cavity after injecting oil acid. After 4 hours, the rates of respiration were counted and blood samples were cramped out through the heart puncture for blood gas analysis. The expressions of hemeoxygenase-1 and the pathologic construction changes were determined by HE staining in the lower right lung of rats in the 3 groups. RESULTS: The respiration dysfunction caused by oil acid could be prominently improved by ulinastain. There was only a little expression of hemeoxygenase-1 in the lung of Group A, but the expression increased in Group B and significatively increased in Group C. CONCLUSION: Ulinastatin may protect the rats from acute lung injury through increasing the expression of HO-1.

Effect of siRNA against NF-κB on sepsis­induced acute lung injury in a mouse model.

The aim of the present study was to explore the protective effect of small interfering RNA (siRNA) against nuclear factor κB (NF-κB) p65 on sepsis-induced acute lung injury (ALI) in mice. In total, 70 male Kunming mice were randomly divided into a healthy control group, a sepsis group, a specific interfering group and a scrambled control group (Sc), and the latter three groups were divided into post-operational 6 and 12 h subgroups, each of which consisted of 10 mice. The mice were administered with NF-κB siRNA, scrambled siRNA and normal saline via tail vein injection. Following 1 h, a mouse model of septic ALI was produced by cecal ligation and puncture (CLP) in the two siRNA groups and the sepsis control group. At 6 and 12 h post­operation, the experimental mice were sacrificed and the lung tissue samples were collected. Histopathological changes, wet/dry ratio of lung weight, NF-κB protein and NF-κB p65 mRNA levels, matrix metalloproteinase-9 (MMP-9) mRNA and protein activity were detected. Compared with the sepsis group and the Sc at the corresponding time, the expression levels of NF-κB p65 mRNA, the lung injury of experimental mice, the wet/dry ratio and the levels of MMP-9 mRNA and protein activity decreased, and significant differences were observed at 6 h post-operation (P<0.05). RNA interference against NF-κB p65 was able to decrease the expression of NF-κB and further inhibit the early phasic excessive inflammatory reaction in sepsis, which may alleviate ALI.

[Analysis of microbial community of a Beijing simulator water distribution system].

Identification of compositions of the biofilm in a Beijing simulator water distribution system pipe networks by PCR and single strand conformation polymorphism (PCR-SSCP) technique and analysis of its heterotrophic bacterial by heterotrophic plate count (HPC) were performed. Results showed that when the water velocities were same, the count of heterotrophic bacterial on the zinficated steel wall was about five times of the PVC. On the other hand, when the pipe materials were zinficated steel, the count of heterotrophic bacterial in the deadwater pipe wall was about 1/5 of the 0.6 m/s region. The difference of the bacterial count maybe related to the smooth of the water pipe surface and the velocity of flow which affected the attachment of the microorganisms and the quantity of O2 and nourishment, respectively. Same SSCP electrophoresis profiles were observed between samples from different material pipes and water velocities. After sequencing and contrasting with the GenBank, the identity of three bands from the SSCP gel with Bacillus cereus (GenBank AB190077), Peudomonas sp. yged143 (GenBank EF419342) and a unclassified bacteria, Bacterium UASWS0134 (GenBank DQ190347) were 100%, 99% and 94%, respectively. The sameness of the microbial community structure may be induced by the samples which were from the same simulator system and the sampling regions were near and the microorganisms could transform in these regions easily. The observed potential pathogens of Bacillus cereus and Peudomonas sp. should lead to a consideration of the microbiology safety of drinking water.