MiRNA-Embedded ShRNAs for Radiation-Inducible LGMN Knockdown and the Antitumor Effects on Breast Cancer.

Legumain (LGMN) is highly expressed in breast cancer (BC) and other solid tumors and is a potential anticancer target. Here we investigate the anti-tumor effects of short hairpin RNAs (shRNAs) targeting LGMN embedded in a microRNA-155 (miR-155) architecture, which is driven by a radiation-inducible chimeric RNA polymerase II (Pol II) promoter. Lentiviral vectors were generated with the chimeric promoter which controlled the expression of downstream shRNA-miR-155 cassette. Fluorescence was observed by using confocal microscopy. Real-time quantitative PCR and Western blotting were used to determine the expression level of LGMN, MMP2, and MMP9. Furthermore, the proliferation and invasive ability of BC cells was analyzed via plate colony formation and invasion assays. Here we demonstrated that the chimeric promoter could be effectively induced by radiation treatment. Furthermore, the shRNA-miR-155 cassette targeting LGMN could be effectively activated by the chimeric promoter. Radiation plus knockdown of LGMN impairs colony formation and dampens cell migration and invasion in BC cells. Inhibition of LGMN downregulates MMP2 and MMP9 expression in BC cells. Pol II-driven shRNA-miR-155 could effectively suppress the growth and invasiveness of BC cells, and that the interference effects could be regulated by radiation doses. Moreover, knockdown of LGMN alleviates the aggressive phenotype of BC cells through modulating MMPs expression.

Tumor Invasiveness, Not Lymphangiogenesis, Is Correlated with Lymph Node Metastasis and Unfavorable Prognosis in Young Breast Cancer Patients (≤35 Years).

The morbidity rate of breast cancer is on the rise, and the age of onset appears to be trending toward a young age. Breast cancer in young women (BCYW) has a number of distinctive features that differ from breast cancer in middle-aged or elderly women (BCMEW). Lymphatic metastasis plays an important role in the spread of BCYW; however, the mechanisms of lymph node metastasis (LNM) in BCYW are not clear. This study aimed to investigate the mechanism of lymphatic metastasis in BCYW and to evaluate the relationships between lymphangiogenesis, the expression of matrix metalloproteinase 9 (MMP-9) and vascular endothelial growth factor C (VEGF-C) expression, clinicopathological characteristics, and prognosis. Using immunohistochemistry, MMP-9, VEGF-C and the level of lymphatic microvessel density (LMVD) were analyzed in 106 cases of breast invasive ductal carcinoma and 20 cases of breast proliferative lesions. Compared with BCMEW, BCYW had higher MMP-9 expression, higher LNM, and more adverse prognoses. In BCYW, high MMP-9 expression was positively correlated with LNM and impaired survival time. However, in BCMEW, MMP-9 expression was not correlated with LNM or survival time. In addition, high VEGF-C expression was positively correlated with a high level of LMVD in both BCYW and BCMEW. Nevertheless, a high level of LMVD was not correlated with LNM or survival time in the two groups. More importantly, univariate and multivariate survival analysis showed that MMP-9 expression and LNM were independent prognostic factors in BCYW. Our present study indicates that lymphangiogenesis induced by VEGF-C is augmented in breast cancer; however, a higher level of lymphangiogenesis has no significant impact on LNM or survival time. We suggest that tumor invasiveness, rather than lymphangiogenesis, plays an important role in LNM among BCYW. Moreover, MMP-9 and LNM were independent prognostic factors for BCYW.

Highly enantioselective reaction of 2-oxindoles with (3-indolyl)methanols by cooperative catalysis of a Lewis acid and organocatalyst.

An efficient cooperative biscinchona alkaloid and Lewis acid catalytic system was developed in the enantioselective α-alkylation of 2-oxindoles with (3-indolyl)(phenyl)methanols to provide (2-oxindole)-linker-indole derivatives in good yields (70-83%) with high enantioselectivities (81%-92%).

Stabilizing radical cation and dication of a tetrathiafulvalene derivative by a weakly coordinating anion.

After the chemical oxidation of the neutral tetrakis(methylthio)tetrathiafulvalene (TMT-TTF, 1) by specific oxidation agents with weakly coordinating anion, [Al(ORF)4](-) [ORF = OC(CF3)3], the radical cation TMT-TTF(•+) (1(•+)) and dication TMT-TTF(2+) (1(2+)) were successfully stabilized and isolated. All the compounds are well-soluble in some solvents and have been systematically investigated by absorption spectra, (1)H NMR, electron paramagnetic resonance (EPR) measurements. Their crystal structures and electronic properties have been studied in conjunction with theoretical calculation. The synthetic approach for chemical oxidation by specific salts of weakly coordinating anions is useful for stable radical cations of tetrathiafulvalene (TTF) and its derivatives in both solution and solid state, which will extend the further research, including structure-property relations on stable radicals for TTF derivatives and new functional materials based on them.

[Oxidative stress induced the expression of Bax and p53 during the apoptosis of alveolar type II epithelial cell].

OBJECTIVE: To investigate the effects of oxidative stress on the survival and apoptosis of alveolar epithelial type II (ATII) cells, as well as the mechanisms of apoptosis. METHODS: 500 mumol/L H(2)O(2) was added into primary ATII cells at different times and cell viability, apoptotic ratio and the expression of Bax and p53 were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometry (FCM) and Western blotting analysis, respectively. The change in mitochondrial membrane potential (MMP) was detected by fluorescence microscopy and FCM. RESULTS: The cell viability and MMP were decreased by H(2)O(2) compared with the controls (F(1)=85.211, F(2)=72.453, respectively, both P<0.05). The cell apoptotic ratios were increased with the time of the stimulation prolonged compared with the controls (F=54.002, P<0.05). H(2)O(2) increased Bax and p53 protein levels (F(1)=28.118, F(2)=43.456, both P<0.05). CONCLUSION: High level of oxidative stress can inhibit ATII cells proliferation, and induce cells apoptosis and decrease the MMP. Up-regulation of the expression of Bax and p53 may contribute to its apoptosis effects.

[Apoptosis of alveolar type II epithelial cells regulated by extracellular signal-regulated kinase under oxidative stress].

OBJECTIVE: To investigate survival and apoptotic responses of alveolar type II epithelial cells (AT II cells) under oxidative stress and the regulation mechanism mediated by extracellular signal-regulated kinase (ERK). METHODS: Primary passage of cultured rat AT II cells were challenged with hydrogen peroxide (H(2)O(2)), and the cells were pretreated with specific inhibitor of ERK (PD 98059) in another group. Cell viability, apoptotic rate and the expression of phosphorylated ERK1/2 (p-ERK1/2)were measured by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, flow cytometry and Western blotting analysis, respectively. RESULTS: Compared with control group, decreased cell viability and increased apoptotic rate in AT II cells occurred in dose-dependent manner when treated with H(2)O(2) 500 and 1,000 micromol/L (all P<0.05), but no differences were found when H(2)O(2) were 10 and 100 micromol/L in concentrations. When the cells were treated with 500 micromol/L H(2)O(2) for 30 minutes, no differences in cell viability and apoptotic rate were found compared with control group,but reduced cell viability and increased apoptotic rate were found when the duration was 180 minutes, and it was in time-dependent manner (both P<0.05). The expression of p-ERK peaked at 30 minutes after stimulation by 500 micromol/L H(2)O(2). When PD 98059 was added, it enhanced apoptotic rate after H(2)O(2)-exposure. CONCLUSION: Apoptosis can be induced by H(2)O(2) in AT II cells in dose-and time-dependent manners. ERK signaling pathway plays a role in the regulation of apoptosis and may be protective for AT II cells under oxidative stress.

[Study on the relationship between cytosolic phospholipase A2-gamma activation and myocardial cell injury during cardiopulmonary bypass].

OBJECTIVE: To investigate the relationship between cytosolic phospholipase A(2)-gamma(cPLA(2)-gamma) activation and alteration of myocardial ultrastructure during cardiopulmonary bypass (CPB) in the operation of ventricular septal defect. METHODS: Myocardial tissues from the right atria of 12 patients undergone ventricular septal defect were collected before and after CPB, cPLA(2)-gamma gene expression was detected by reverse transcription-polymerase chain reaction (RT-PCR) and analysis was carried out using gel image analysis software. Meanwhile, alteration of myocardial ultrastructure was observed under electron microscopy. RESULTS: cPLA(2)-gamma gene expressions were statistically significant between two groups (P<0.05), and they were higher in patients after CPB than those prior to CPB. The alteration of myocardial ultrastructure after CPB included hypertrophy of nucleus, swelling under plasma membrane, chromatin margination and dilatation of smooth endoplasmic reticulum (SER) in myocardial cell, structures degeneration, hyperplasia of mitochondria as well as swelling, and myocardial fibre dissolve, etc. CONCLUSION: Increased cPLA(2)-gamma gene expression in myocardial tissue after CPB might play an important role in damage of membrane integrity, energy decompensation and myocardial contraction dysfunction.

[Study on effect of dexamethasone on the expression of matrix metalloproteinase and its tissue inhibitors in hyperoxia-induced lung injury].

OBJECTIVE: To observe the effect of dexamethasone on the mRNA expression of matrix metalloproteinase (MMPs) and tissue inhibitor of metalloproteinase (TIMPs) in the lung tissue and to explore the protective mechanism of dexamethasone in hyperoxia-induced lung injury. METHODS: Thirty-two two-week old Wistar rats were randomly divided into atmospheric-air group (n=16) and hyperoxia group (n=16). After 7 days of continuous exposure to high concentration O (2)(>95%), the lung wet/dry(W/D) ratio, the protein content in bronchoalveolar lavage fluid(BALF) and histopathological changes of the lung were measured in 16 rats(8 in each group). The lung tissue specimens of the other 16 rats were cultured, 8 among which served as atmospheric-air control group, the remainder in the hyperoxia group were divided into hyperoxia control group,hyperoxia+dexamethasone (1 x 10(-8) mol/L) group, hyperoxia+dexamethasone (1 x 10(-6) mol/L) group, and hyperoxia+dexamethasone (1 x 10(-4) mol/L) group. Eight samples were examined in each group. After cultured for 24 hours, the lung tissue were collected and its mRNA expression of MMP-2, MMP-9, TIMP-1 and TIMP-2 were determined by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: 1. Pulmonary edema, hemorrhage and extensive inflammatory cells infiltration were observed in hyperoxia group, but no such changes were found in the atmospheric-air group. The lung W/D and the protein content in BALF in hyperoxia group were significantly higher than those in atmospheric air groups. 2. The mRNA expressions of MMP-2, MMP-9, TIMP-1, TIMP-2 and the ratio of MMP-2/TIMP-2, MMP-9/TIMP-1 were significantly higher in the hyperoxic group than those in the atmospheric-air group. 3. Dexamethasone could down-regulate the mRNA expressions of MMP-2 and MMP-9 in a concentration dependent manner. The mRNA expressions of TIMP-1, TIMP-2 also could be reduced by dexamethasone. Decreasing ratios of MMP-2/TIMP-2 and MMP-9/TIMP-1 were found in correlation with increasing concentration of dexamethasone. CONCLUSION: Dexamethasone can reduce the mRNA expressions of MMPs as well as regulate the balance of MMPs/TIMPs, which may be one of the mechanism of its protective effect on hyperoxia-induced lung injury.

Effect of polydatin on phospholipase A2 in lung tissues in rats with endotoxic shock.

OBJECTIVE: To study the effect of polydatin on phospholipase A(2) in lung tissues in rats with endotoxic shock. METHODS: Thirty-two healthy male Wistar rats were employed in this study. A total of 8 rats received normal saline intravenously (control group), 8 rats received 10 mg/kg of endotoxin (endotoxic shock group), 8 rats received 1 mg/kg of polydatin after endotoxin injection (polydatin treatment group), and 8 rats received 1 mg/kg of polydatin (polydatin prevention group) 30 minutes before endotoxin injection. Mean arterial pressure was measured once half an hour. Lung tissues were collected 6 hours later. Phospholipase A(2) activity was measured with acid titration. The gene expression of secretory phospholipase A(2) type IIA was detected with reverse transcription polymerase chain reaction. Meanwhile, the histological changes of the lungs among four groups were compared through microscopic examination. RESULTS: Phospholipase A(2) activity and the gene expression of secretory phospholipase A(2) type IIA increased after endotoxin injection, but polydatin could inhibit these effects of endotoxin. Obvious morphological evidence could be found in the lung pathological sections and the protective effect of polydatin was most significant in the polydatin prevention group. CONCLUSIONS: Polydatin has prophylactic and therapeutic effects (the former is more distinct than the latter) on acutely injured lungs in rats with endotoxic shock and which suggests that polydatin may be a phospholipase A(2) inhibitor.

[Change in the level of inflammatory cytokines and their relationship to the indicator in evaluating renal tubules injury of urinary in neonates with asphyxia].

OBJECTIVE: To explore the relationship between amount of inflammatory cytokines in urine and neonatal postasphyxia renal tubules injury. METHODS: The level of inflammatory cytokines such as interleukin (IL-8, IL-6), tumor necrosis factor-alpha (TNF-alpha) and the indicators of evaluating renal tubules injury [N-acetyl-glucosaminidase(NAG), gamma-glutamyltranspeptidase (gamma-GT), beta(2)-microglobulin (beta(2)-MG)] in urine were detected in neonates with asphyxia. RESULTS: Compared with control, the levels of IL-8, IL-6, TNF-alpha and NAG, gamma-GT, beta2-MG were obviously increased in mild asphyxia group. In severe asphyxia group, the parameters above were all significantly increased compared with mild asphyxia group and the control group. Within the asphyxia group, there were positive relationship between inflammatory cytokines and the indicator of evaluating renal tubules injury. CONCLUSION: The asphyxia may induce systemic inflammatory response syndrome (SIRS), which result in postasphyxia renal injury in neonates. The level of inflammatory cytokines in urine may be used as the indicators of evaluating the severity of asphyxia and postasphyxia renal injury in neonates.