[Effect of A20 gene induced silencing on the biological behaviors of human nasopharyngeal carcinoma cell].

Objective: To observe the effect of knockdown A20 gene expression on the proliferation, invasion and metastasis of human nasopharyngeal carcinoma cell in vivo and in vivo. Methods: Human nasopharyngeal carcinoma cell 5-8F-H3 was transfected with A20-specific shRNA Tet-on inducible plasmid vectors, and A20 silenced cells were screened by Puromycin. Quantitative RT-PCR and Western blot analysis were used to detect the mRNA level and protein of A20. The cell proliferation was detected by cell counting kit-8 (CCK8) and plate colony formation assays. The cell cycle and apoptosis were measured by flow cytometry. And the ability of cell invasion was measured using Boyden chamber assay in vivo. Subcutaneous tumor formation and liver metastasis in vivo were examined with whole-body fluorescence imaging system to observe the influence of silencing A20 gene expression in nude mice. Results: The stable A20 inducible silencing cells line 5-8F-H3/A20-shRNA was established successfully. Down-regulation of A20 mRNA and protein expression were observed in 5-8F-H3/A20-shRNA cells treated with DOX(both P<0.01). The results of CCK-8 assay (F=18.542, P=0.003), clone formation experiment (F=40.080, P<0.001) and flow cytometry analysis (F=7.398, P=0.024) in vivo showed that the cell proliferation of 5-8F-H3 was remarkably inhibited by down-regulation of A20 gene expression. The results of Boyden chamber assay showed that A20 gene silencing could inhibit the cell invasion ability (F=26.157, P<0.001). Silencing of A20 inhibited tumorigenesis and metastasis via subcutaneous tumor formation and liver metastasis experiments in nude mice. Conclusion: A20 gene is closely related to the malignant biological behaviors of nasopharyngeal carcinoma, and it may serve as a potential molecular target for the treatment of nasopharyngeal carcinoma.

[Effect of paraquat on the expression of a disintegrin and metalloproteinase-17 in A549 cells].

Objective: Construct a paraquat (PQ) cell fibrosis model in vitro, observe the effect of PQ on the expression of a disintegrin and metalloproteinase-17 (ADAM17) in A549 cells, and explore the role of ADAM17 in the pulmonary fibrosis induced by PQ poisoning. Methods: A549 cells are divided into normal control group, different concentration of PQ groups, CCK-8 is used to detect cell viability, screening concentration and time of PQ, cell morphology is observed under microscope; Enzyme-linked immunosorbent assay (ELISA) detectes fibrosis markers of collagen type I (Col I) and fibronectin (FN) expression. Establishment of cell model of fibrosis; distribution by immunocytochemical detection of ADAM17 in A549 cells, Reverse transcription-polymerase chain reaction and Western blot are used to detect the expression of ADAM17 mRNA and protein. Results: 1. With the increase of PQ concentration and the prolongation of the action time, the activity of A549 cells decreased (P< 0.05) , which is dose-dependent and time dependent. 2.The normal A549 cells fusion is paving stone growth and arranged more closely. After PQ induction, the cell arrangement was loose, the intercellular connection became loose, and some cells dissolved and died. 3.ELISA showed that with the increase of PQ concentration, the expression of Col I and FN increased (P<0.05) , and Col I and FN expression gradually increased with the prolongation of PQ time (P<0.05) , and the fibroblast model is successfully established. 4. Immunocytochemistry showes that ADAM17 is expressed in the cytoplasm of A549 cells. 5. RT-PCR and Western blot showed that the expression of ADAM17 mRNA and protein increased significantly with the increase of PQ concentration (P<0.05) , which is most obvious at PQ 200 µmol/L. With the prolonged action of PQ, the expression level of ADAM17 mRNA and protein also increased significantly (P<0.05) , and reached the peak in 24 h. Conclusion: PQ can induce morphological changes of alveolar epithelial cells, cause cell damage, and successfully establish a cell fibrosis model, which has a dose and time dependence on the toxicity of A549 cells. ADAM17 is overexpressed in the A549 cells induced by PQ and may be involved in the process of pulmonary fibrosis induced by paraquat.

A study on the expression of FGF-21 and NF-κB pathway in the tissues of atherosclerotic mice.

OBJECTIVE: To study the relationship between the expressions of fibroblast growth factor (FGF)-21 and NF-κB signal transduction pathway in the tissues of atherosclerotic mice. MATERIALS AND METHODS: A total of 40 apoE-/- male mice at 8 weeks were selected and randomly divided into 4 groups. 10 mice in group A were normally fed with diet. 10 mice in group B were fed with high-fat diet. 10 mice in group C were fed with high-fat diet + pravastatin. 10 mice in group D were fed with high-fat diet + subcutaneous injection of exogenous recombinant FGF-21 protein. Another 10 C57BL/6J mice at 8 weeks were normally fed with diet (group E). They were killed after 12 weeks to collect retinal venous blood. ELISA method was applied to detect the levels of serum FGF-21, NF-κB, monocyte chemo attractant protein (MCP-1), matrix metalloproteinase (MMP)-9 and TNF-α. Immunohistochemical staining and RT-PCR method were applied to detect the expression of FGF-21 in aortic arch and liver tissues. RT-PCR method and Western blot method were applied to detect the expression of NF-κB, MCP-1, MMP-9 and TNF-α in aortic arch and liver tissues. RESULTS: The levels of serum FGF-21, NF-κB, MCP-1, MMP-9 and TNF-α in group B were higher than those of group A and group E, and those of group C and group D were lower than those of group B (except FGF-21 in group D). The differences had statistical significance (p<0.05). The positive staining rates of FGF-21 in endothelial cells of aortic arch and liver tissues in group B were higher than those group A and group E, and those of group C and group D were lower than those of group B. The differences had statistical significance (p<0.05). The expression levels of FGF-21mRNA, NF-κB, MCP-1, MMP-9, TNF-αmRNA and protein in endothelial cells of aortic arch and liver tissues in group B were higher than those group A and group E, and those of group C and group D were lower than those of group B. The differences had statistical significance (p<0.05). CONCLUSIONS: FGF-21 may participate in the occurrence of atherosclerosis (AS), which is related to the activation of the NF-κB pathway. Lipid-lowering therapy can inhibit the activation of FGF-21 and NF-κB. Exogenous FGF-21 can also lower the activation of NF-κB and interpose in atherosclerosis process.

Targeting HDAC3, a new partner protein of AKT in the reversal of chemoresistance in acute myeloid leukemia via DNA damage response.

Resistance to cytotoxic chemotherapy drugs remains as the major cause of treatment failure in acute myeloid leukemia. Histone deacetylases (HDAC) are important regulators to maintain chromatin structure and control DNA damage; nevertheless, how each HDAC regulates genome stability remains unclear, especially under genome stress conditions. Here, we identified a mechanism by which HDAC3 regulates DNA damage repair and mediates resistance to chemotherapy drugs. In addition to inducing DNA damage, chemotherapy drugs trigger upregulation of HDAC3 expression in leukemia cells. Using genetic and pharmacological approaches, we show that HDAC3 contributes to chemotherapy resistance by regulating the activation of AKT, a well-documented factor in drug resistance development. HDAC3 binds to AKT and deacetylates it at the site Lys20, thereby promoting the phosphorylation of AKT. Chemotherapy drug exposure enhances the interaction between HDAC3 and AKT, resulting in decrease in AKT acetylation and increase in AKT phosphorylation. Whereas HDAC3 depletion or inhibition abrogates these responses and meanwhile sensitizes leukemia cells to chemotoxicity-induced apoptosis. Importantly, in vivo HDAC3 suppression reduces leukemia progression and sensitizes MLL-AF9+ leukemia to chemotherapy. Our findings suggest that combination therapy with HDAC3 inhibitor and genotoxic agents may constitute a successful strategy for overcoming chemotherapy resistance.

Novel Rechargeable M3V2(PO4)3//Zinc (M = Li, Na) Hybrid Aqueous Batteries with Excellent Cycling Performance.

A rechargeable hybrid aqueous battery (ReHAB) containing NASICON-type M3V2(PO4)3 (M = Li, Na) as the cathodes and Zinc metal as the anode, working in Li2SO4-ZnSO4 aqueous electrolyte, has been studied. Both of Li3V2(PO4)3 and Na3V2(PO4)3 cathodes can be reversibly charge/discharge with the initial discharge capacity of 128 mAh g(-1) and 96 mAh g(-1) at 0.2C, respectively, with high up to 84% of capacity retention ratio after 200 cycles. The electrochemical assisted ex-XRD confirm that Li3V2(PO4)3 and Na3V2(PO4)3 are relative stable in aqueous electrolyte, and Na3V2(PO4)3 showed more complicated electrochemical mechanism due to the co-insertion of Li(+) and Na(+). The effect of pH of aqueous electrolyte and the dendrite of Zn on the cycling performance of as designed MVP/Zn ReHABs were investigated, and weak acidic aqueous electrolyte with pH around 4.0-4.5 was optimized. The float current test confirmed that the designed batteries are stable in aqueous electrolytes. The MVP//Zn ReHABs could be a potential candidate for future rechargeable aqueous battery due to their high safety, fast dynamic speed and adaptable electrochemical window. Moreover, this hybrid battery broadens the scope of battery material research from single-ion-involving to double-ions -involving rechargeable batteries.

Comparison of platinum chromium everolimus-eluting stent with cobalt chromium everolimus-eluting stent in unselected patients undergoing percutaneous coronary intervention.

OBJECTIVE: The recent PLATINUM trial has demonstrated that the use of the new generation platinum chromium everolimus-eluting stents (PtCr-EES) yield clinical outcomes similar to those obtained by the use of cobalt chromium everolimus-eluting stents (CoCr-EES) in selected patients with 1 or 2 de novo coronary artery lesions. This study aimed to compare the safety and efficacy of the PtCr-EES and CoCr-EES in unselected patients from a real-life single-center registry. PATIENTS AND METHODS:   From July 2009 through November 2010, 788 consecutive patients in our institution with symptomatic coronary artery disease who were treated with the CoCr-EES (n = 410) or PtCr-EES (n = 378) were enrolled into this study. The primary endpoint of the study was target-lesion failure (TLF) at 12-month follow-up and the secondary endpoints were major adverse cardiovascular events and stent thrombosis. RESULTS: The prevalence of TLF in the PtCr-EES group (4.5%) was similar to that in the CoCr-EES group (3.9%). In addition, there were no significant differences in the 12-month rates of cardiac death (2.1% vs. 1.5%), myocardial infarction (2.4% vs. 3.9%), ischemia-driven target lesion revascularization (2.4% vs. 2.2%), and definite or probable stent thrombosis (0.5% vs. 1.5%, all p > 0.05). CONCLUSIONS: At 12-month follow-up, the PtCr-EES is comparable in safety and efficacy to the CoCr-EES in unselected patients with coronary artery diseases.

Efficacy of drug-eluting stent for chronic total coronary occlusions at different follow-up duration: a systematic review and meta-analysis.

OBJECTIVE: DESs have been proved to be beneficial for patients with chronic total coronary occlusions (CTO) in terms of cardiac function and other prognosis. We aim to compare the efficacy and safety of drug-eluting stent (DES) and bare-metal stent (BMS) in CTO recanalization at different follow-up duration. METHODS: Articles comparing outcomes between DES and BMS implantation in patients with CTO was searched. A fixed-effect (inverse-variance weighted) and random-effect (DerSimonian and Laird) model were used to analyze the pooling results. RESULTS: A total of 29 comparative studies including 24 cohort studies and 5 randomized controlled studies were identified with a total of 9140 patients (5008 received BMS and 4132 received DES). The risk of all cause death for DES was higher at 6 months and lower at 12 months than BMS, and no significant difference was shown at 24, 36 and 60 months. DES group had lower risk of MI after 12 months implantation, and no difference was shown at 6, 24, 36 and 60 months. Major adverse cardiovascular event (MACE)-free survival was clinically and significantly improved by 73%, 68%, 49%, 40% and 37% respectively in DES group at 6,12, 24, 36, and 60 months. CONCLUSIONS: DES is superior to BMS in binary restenosis, reocclusion and MACE-free survival during long-term follow up. The occurrences of all-cause death and MI show that the risk rate of BMS is higher than that of DES at 12 months. The frequency of all-cause death of DES is higher than BMS at 6 months. DES has higher risk of in-stent thrombosis than BMS at 36 months of implantation.

EBV-miR-BART7-3p promotes the EMT and metastasis of nasopharyngeal carcinoma cells by suppressing the tumor suppressor PTEN.

The epithelial-mesenchymal transition (EMT) is crucial to cancer progression and metastasis. Although multiple cellular miRNAs have been identified to regulate the EMT and metastasis in cancers, the role of viral miRNAs in cancer progression remains largely unknown. Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV)-associated malignancy typically characterized by its early metastasis. In the present study, we have discovered the involvement of a viral miRNA, EBV-miR-BART7-3p, in the EMT and metastasis of NPC cells. Initially, we observed that EBV-miR-BART7-3p was highly expressed in NPC and positively correlated with lymph node metastasis and clinical stage of NPC. Subsequently, we demonstrated that EBV-miR-BART7-3p enhanced cell migration/invasion in vitro, cancer metastasis in vivo, and particularly the EMT characterized by loss of epithelial markers and gain of mesenchymal features in NPC cells. Furthermore, mechanistic studies disclosed that EBV-miR-BART7-3p targeted a major human tumor suppressor PTEN, modulating PI3K/Akt/GSK-3ß signaling and eventually leading to the high expression and nuclear accumulation of Snail and ß-catenin, which favor EMT. Knockdown of PTEN could phenocopy the effect of EBV-miR-BART7-3p, whereas re-expression of PTEN resulted in a phenotypic reversion. Moreover, these findings were supported by an observation of an EBV-positive cell model in which silencing of endogenous EBV-miR-BART7-3p partially attenuated cell migration/invasion and altered EMT protein expression pattern via reverting PI3K/Akt, Snail and ß-catenin expression. Thus, this study suggests a novel mechanism by which EBV-miR-BART7-3p modulates the EMT and metastasis of NPC cells, and a clinical implication of EBV-miR-BART7-3p as a potential biomarker or therapeutic target.

BRAF mutation correlates with recurrent papillary thyroid carcinoma in Chinese patients.

PURPOSE: We investigated correlations of somatic BRAF V600E mutation and RET/PTC1 rearrangement with recurrent disease in Chinese patients with papillary thyroid carcinoma (ptc). METHODS: This prospective study included 214 patients with ptc histologically confirmed between November 2009 and May 2011 at a single institute. RESULTS: We found somatic BRAF V600E mutation in 68.7% and RET/PTC1 rearrangement in 25.7% of the patients. Although BRAF mutation was not significantly associated with clinicopathologic features such as patient sex or age, multicentric disease, thyroid capsule invasion, tumour stage, or nodal metastasis, it was significantly associated with recurrent disease. Multivariate analysis revealed that BRAF mutation and tumour size were independent risk factors associated with recurrent disease, with odds ratios of 9.072 and 2.387 respectively. The area under the receiver operating characteristic curve increased 8.3% when BRAF mutation was added to the traditional prognostic factors, but that effect was statistically nonsignificant (0.663 vs. 0.746, p = 0.124). RET/PTC1 rearrangement and nodal metastasis were significantly associated in all patients (p = 0.042), marginally associated in ptc patients (p = 0.051), but not associated in microptc patients (p = 0.700). RET/PTC1 rearrangement was not significantly associated with recurrent disease. CONCLUSIONS: BRAF positivity is an independent predictor of recurrent disease in ptc.

MiR-99a exerts anti-metastasis through inhibiting myotubularin-related protein 3 expression in oral cancer.

OBJECTIVE: We aimed at studying the role of the most deregulated miR-99a, identifying its downstream targets, and exploring the clinical potential of miR-99a and its target(s) in oral cancer. SUBJECTS AND METHODS: Following confirmation of miR-99a deregulation in nine oral lines and 26 pairwise clinical specimens, miR-99a-manipulated oral cancer cells were subjected to cell proliferation, migration, invasion, and in vivo murine metastasis assays. We characterized putative miR-99a target(s) using luciferase reporter assays and genetic manipulation. The inverse relation of miR-99a and its target(s) was examined in clinical specimens using real-time PCR and Western blot analysis. RESULTS: MiR-99a down-regulation was confirmed both in tested oral cancer cell lines and clinical specimens. Ectopic miR-99a expression inhibited oral cancer cell migration and invasion. Anti-miR-99a, silencing miR-99a functions, had the opposite effect. Myotubularin-related protein 3 (MTMR3) with one evolutionarily conserved seed region in the 3'-untranslated region was a novel miR-99a target. Depleting MTMR3 expression significantly reduced cell proliferation, migration, or invasion. There was an inverse expression of miR-99a and MTMR3 protein in oral cancer lines and clinical specimens. CONCLUSION: miR-99a repressed oral cancer cell migration and invasion partly through decreasing MTMR3 expression. MTMR3 may serve as a therapeutic target for oral cancer treatment.

Percutaneous balloon mitral valvuloplasty. An observation of 50 patients.

Percutaneous balloon mitral valvuloplasty (PBMV) was successfully performed in 50 selected patients with mitral stenosis by using Inoue pillow-shaped balloon and Inoue technique. The average diameter of balloon used was 26.9 +/- 0.9 mm. 90% (45/50) of cases had either double or single mitral commissura split. Of the rest 5 cases, 1 had a mitral score 13 and 4 had a history of mitral valve commissurotomy. Totally they had a mean mitral valve area increase from 1.13 +/- 0.32 to 2.21 +/- 0.43 cm2, left atrial pressure decrease from 31.8 +/- 9.3 to 14.7 +/- 5.6 mmHg, left atrial diameter reduction from 44.9 +/- 7.7 to 37.4 +/- 4.9 mm, and transmitral gradient decrease from 21.7 +/- 9.8 to 4.0 +/- 5.2 mmHg. Most patients had a obvious cardiac function improvement, especially in patients with mitral score of 8 or less. 30% patients (15/50) had a mild mitral regurgitation, but relieved 3-6 months after procedure. During one year of follow up, the majority of patients (16/20) were found in a good cardiac function, mitral area and the left atrial diameter, except in 4 patients with a high mitral score of more than 10. It is suggested that for patient with lower mitral morphological score and good general health, a larger diameter balloon might be suitable for effectively improving patient's symptom, but for patients with a previous surgical mitral commissurotomy, PBMV should not be selected.

Antithrombotic effects of Andrographis paniculata nees in preventing myocardial infarction.

In 16 dogs the endothelium of the left anterior descending coronary artery was injured mechanically. Then a copper wire was inserted into the lumen as a choke of flow and the vessel was slightly compressed from outside by a constrictor. Eight dogs had been treated beforehand with a preparation of flavone extracted from the root of the Chinese medicinal herb Andrographis paniculata (TFAP). In the control group, saline solution was given, the epicardially recorded ST segment started to elevate within 15 minutes, the platelet aggregation rate and the plasma levels of TXB2 increased rapidly, whereas the level of 6-k-PGF1 alpha remained stable. Platelet cGMP rose continuously; however, platelet cAMP rose only at 60 minutes. Histological findings confirmed the occurrence of arterial thrombus and myocardial necrosis. Contrariwise, in the pretreated group there was no elevation of the ST segment, plasma 6-k-PGF1 alpha and platelet cAMP were increased, the production of TXB2 and aggregation of platelets were inhibited, and no thrombus or myocardial infarction was induced. All data suggest that TFAP might promote the synthesis of PGI2, inhibit the production of TXA2, stimulate the synthesis of cAMP in platelets, impede aggregation of platelets, and thereby prevent the formation of thrombi as well as the development of myocardial infarction.

Protective effects of andrographis paniculata nees on post-infarction myocardium in experimental dogs.

One hour after the development of myocardial infarction by formation of thrombus, watery extract of Andrographis paniculata Nees (APN) was injected intravenously to 7 dogs for observing the protective effect of this drug on ischemic myocardium, 6 dogs served as control group. In the treated group PGI2 increased remarkably, synthesis of TXA2 was inhibited, cAMP in platelets was elevated, CK-MB peak was lowered and appeared earlier, ELT was shortened, release of platelet beta-GT was decreased, platelet maximum aggregation rate was inhibited, the size of ischemic area recorded by epicardial ECG was reduced, the amplitude of ST segment elevation was lower than that in the control group, Q wave appeared in only one dog. Pathologically, the myocardial structure surrounding the initially appearing ischemic area, i.e, the reversibly damaged area, became relatively normal, while the degree of myocardial degeneration and necrosis in the central part of the ischemic area was mild. These data suggest that APN may limit the expansion of ischemic focus, exert marked protective effect on reversibly ischemic myocardium and demonstrate a weak fibrinolytic action.

Study on experimental model of coronary artery thrombogenesis and acute myocardial infarction: changes in epicardial ECG, platelet function and biochemistry, and pathology.

A model was produced for formation of coronary artery thrombosis and development of acute myocardial infarction (AMI) in experimental dogs (n = 8) by injuring endothelium of coronary artery with a steel wire, inserting a copper wire into the lumen, and producing local stenosis with a constrictor. The results showed that ST segment of epicardial ECG began to elevate 15-30 min after handling the coronary artery, platelets aggregation rate and plasma TXB2 increased rapidly, plasma 6-k-PGF1 alpha did not change, platelets cGMP rose continuously, while platelets cAMP rose only at 60 min after handling the coronary artery. Histological findings confirmed the occurrence of artery thrombus and myocardial necrosis. The model may be helpful in investigating the whole process of activation of platelet, thrombogenesis and development of AMI, with the changes in biochemistry, structure, pathophsiology and ECG monitored during the period.

The effects of dauricine and verapamil as calcium channel blockers on the plasma concentration of 6-keto-prostaglandin F1 alpha and thromboxane B2 during cardiopulmonary bypass.

Three groups of ten dogs underwent 80 minutes cardiopulmonary bypass. Two of the three groups were administered dauricine, as a new calcium channel blocker, and verapamil, as a generally recognized calcium channel blocker, respectively, from 15 minutes pre-bypass to the end of the bypass procedure (a period of 95 minutes). By means of radioimmunoassay plasma 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) and thromboxane B2 (TxB2) as the stable metabolites of prostacyclin and thromboxane A2 were surveyed. During the aortic clamping the hemodynamic changes were recorded. The dauricine and verapamil groups showed markedly inhibited generation of excessive 6-keto-PGF1 and TxB2, a narrowed ratio of TxB2 to 6-keto-PGF1 alpha, as well as a reduced total systemic peripheral resistance within the perfusion period. These results suggest that dauricine and verapamil have a salutary effect in extracorporeal circulation. The possible mechanisms are discussed.