miR-146a靶向TRAF6继而抑制AR42J细胞的增殖及凋亡

目的 探究miR-146a对胰腺腺泡细胞AR42J增殖及凋亡的影响。方法 通过雨蛙素诱导AR42J细胞构建急性胰腺炎模型,酶联免疫吸附实验（ELISA）检测细胞上清液中淀粉酶、TNF-α、IL-6含量验证模型构建,qRT-PCR与Western blot分别检测各组中miR-146a和TRAF6表达量的改变。转染敲低AR42J细胞中miR-146a表达量,采用Edu检测细胞敲低后增殖改变,流式细胞术检测凋亡能力改变。结果 与未处理组相比,雨蛙素组细胞miR-146a表达量显著降低（P <0.05）,TRAF6表达量明显升高（P <0.05）,并且敲低miR-146a后AR42J细胞增殖能力明显降低,凋亡能力明显升高。结论 miR-146a可能通过抑制TRAF6表达从而降低AR42J细胞凋亡并促进细胞增殖能力。     

Inhibition of Cdc42–intersectin interaction by small molecule ZCL367 impedes cancer cell cycle progression,proliferation, migration,and tumor growth

Cdc42 is a member of the Rho family of small GTPases that are at the crossroads of major oncogenic signaling pathways involved in both lung and prostate cancers. However, the therapeutic potential of Cdc42 regulation is still unclear due to the lack of pharmacological tools. Herein, we report that ZCL367 is a bona fide Cdc42 inhibitor that suppressed cancer development and ZCL278 can act as a partial Cdc42 agonist. In lung cancer cell lines with varying EGFR and Ras mutations as well as both androgen-independent and androgen-dependent prostate cancer cell lines, ZCL367 impeded cell cycle progression, reduced proliferation, and suppressed migration. ZCL367 decreased Cdc42–intersectin interactions and reduced Cdc42-mediated filopodia formation. ZCL367 showed increased potency and selectivity for Cdc42 when compared to Rac1 and RhoA. ZCL367 reduced A549 tumorigenesis in a xenograft mouse model. Altogether, ZCL367 is a selective Cdc42 inhibitor and an excellent candidate for lead compound optimization for further anticancer studies.     

In vivo and in vitro effects of 42-hydroxy-palytoxin on mouse skeletal muscle: Structural and functional impairment

Palytoxins (PLTXs) are known seafood contaminants and their entrance into the food chain raises concern about possible effects on human health. The increasing number of analogs being identified in edible marine organisms complicates the estimation of the real hazard associated with the presence of PLTX-like compounds. So far, 42-OH-PLTX is one of the few congeners available, and the study of its toxicity represents an important step toward a better comprehension of the mechanism of action of this family of compounds. From this perspective, the aim of this work was to investigate the in vivo and in vitro effect of 42-OH-PLTX on skeletal muscle, one of the most sensitive targets for PLTXs. Our results demonstrate that 42-OH-PLTX causes damage at the skeletal muscle level with a cytotoxic potency similar to that of PLTX. 42-OH-PLTX induces cytotoxicity and cell swelling in a Na⁺-dependent manner similar to the parent compound. However, the limited Ca²⁺-dependence of the toxic insult induced by 42-OH-PLTX suggests a specific mechanism of action for this analog. Our results also suggest an impaired response to the physiological agonist acetylcholine and altered cell elasticity.     

Interacting with α7 nAChR is a new mechanism for AChE to enhance the inflammatory response in macrophages

Acetylcholine (ACh) regulates inflammation via α7 nicotinic acetylcholine receptor (α7 nAChR). Acetylcholinesterase (AChE), an enzyme hydrolyzing ACh, is expressed in immune cells suggesting non-classical function in inflammatory responses. Here, the expression of PRiMA-linked G4 AChE was identified on the surface of macrophages. In lipopolysaccharide-induced inflammatory processes, AChE was upregulated by the binding of NF-κB onto the ACHE promotor. Conversely, the overexpression of G4 AChE inhibited ACh-suppressed cytokine release and cell migration, which was in contrast to that of applied AChE inhibitors. AChEmt, a DNA construct without enzymatic activity, was adopted to identify the protein role of AChE in immune system. Overexpression of G4 AChEmt induced cell migration and inhibited ACh-suppressed cell migration. The co-localization of α7 nAChR and AChE was found in macrophages, suggesting the potential interaction of α7 nAChR and AChE. Besides, immunoprecipitation showed a close association of α7 nAChR and AChE protein in cell membrane. Hence, the novel function of AChE in macrophage by interacting with α7 nAChR was determined. Together with hydrolysis of ACh, AChE plays a direct role in the regulation of inflammatory response. As such, AChE could serve as a novel target to treat age-related diseases by anti-inflammatory responses.     

IL-8对肺癌NCI-H157细胞增殖和迁移的影响

目的 研究IL-8对肺癌细胞增殖和迁移的影响,初步探讨IL-8调控肺癌细胞的分子机制.方法 体外培养肺癌NCI-H157细胞,用不同浓度的IL-8刺激肺癌细胞,分别用MTT法检测IL-8对肺癌细胞的增殖作用;划痕损伤实验和Transwdl小室两种方法检测肺癌细胞的迁移能力;Western blot检测Rac1和Cdc 42蛋白的表达变化.结果 IL-8促进NCI-H157细胞增殖,但随浓度增加,细胞增殖活性差异无统计学意义;细胞划痕损伤和Transwell实验均表明IL-8可诱导NCI-H157细胞迁移,且具有浓度依赖性;Western blot结果显示,随着IL-8浓度增加,Rac1和Cdc 42的表达水平逐渐升高,以Cdc42变化最为显著.结论 IL-8可促进肺癌细胞增殖和迁移,可能与Rac1和Cdc42表达有关.     

Desensitization pattern of cardiac β-adrenoceptor subtypes by prolonged in vivo infusion of pindolol and celiprolol in rats

Summary The regulatory effects of pindolol and celiprolol on cardiac -adrenoceptor density were studied in vivo in order to assess the subtype selectivity of their partial agonistic activity (PAA). The substances were continuously administered to rats for 1 week by means of implanted osmotic minipumps. The density of -adrenoceptor subtypes were estimated from ICYP saturation binding experiments performed on cardiac ventricular plasma membranes in the presence of a highly selective antagonist (CGP 20172 A or ICI 118,551). Both antagonists were employed at concentrations as high as to block one subtype only without affecting the complementary subtype. For control purposes, rats were also treated with isoprenaline (0.4 mg/kg/h) and propranolol (0.15 mg/kg/h), or vehicle. Pindolol (0.036 mg/kg/h) and celiprolol (0.36 mg/kg/h) reduced the density of ventricular ₂-adrenoceptors by 46% and 23%, respectively, which — in the case of pindolol — was significant when compared to the non-treated controls. Both compounds, however, produced a small, but distinct increase in the number of ₁-adrenoceptors by approximately 26%. This finding is in contrast to the propranolol-induced upregulation of both ₁- and ₂-adrenoceptors by approximately 80%. Since supramaximal doses of each drug were administered, a significant smaller increase of ₁-adrenoceptors by pindolol and celiprolol —as compared to the increase produced by propranolol — can be interpreted as evidence for a PAA of pindolol and celiprolol on ₁-adrenoceptors as well. Isoprenaline as a full agonist caused a marked loss of of both -adrenoceptor subtypes. Although it exhibits equal affinity at both subtypes the decrease amounted to 80% of the ₂- but only to 54% of the ₁-adrenoceptors density. This indicates that the down-regulation of cardiac -adrenoceptors in general seems to be more pronounced at the ₂-than at the ₁-adrenoceptors population. We conclude that the subtype desensitization pattern of agents with intrinsic activity precludes the determination of subtype-selectivity, since ₁- and ₂-adrenoceptors appear to differ in their sensitivity presumably as a result of subtype specific baseline desensitization produced by endogenous catecholamines.     

Differential gene expression analysis of tubule forming and non-tubule forming endothelial cells: CDC42GAP as a counter-regulator in tubule formation

The formation of new tubular structures from a quiescent endothelial lining is one of the hallmarks of sprouting angiogenesis. This process can be mimicked in vitro by inducing capillary-like tubular structures in a three-dimensional (3D) fibrin matrix. We aimed to analyze the differential mRNA expression in two phenotypically distinct cell populations from the same culture, namely in tubule-forming endothelial cells and monolayer endothelial cells not participating in tubule formation. A fibrin-rich 3D matrix derived from human plasma was used to facilitate tubule formation by human foreskin microvascular endothelial cells (hMVEC). After 7 days of stimulation with VEGF, bFGF, and TNF-alpha, the culture consisted of a monolayer and capillary-like sprouts that had grown into the fibrinous matrix. A method was developed to separate the monolayer and tubule-forming populations of hMVEC, keeping their cellular integrity intact to ensure mRNA extraction and cDNA production. Subsequent array analysis resulted in an inventory of differentially expressed genes that were associated with either tube-forming (angiogenic) or non-angiogenic capacity. Differential gene expression was verified by real-time PCR on the original RNA samples as well as on RNA obtained from laser-capture microdissected cross sections of monolayers and capillary structures in the 3D fibrinous matrix. The expression of CDC42GAP, an inhibitor of active-state small Rho GTPases, was reduced in tubular hMVEC. Overexpression of CDC42GAP in hMVEC attenuated endothelial tubule formation, while its suppression by siRNA slightly enhanced this process. Thus, CDC42GAP was identified as a counter-regulatory mediator for tubule formation.     

TNK2 promoted esophageal cancer progression via activating egfr‐akt signaling

BackgroundThis study investigated the clinical implication of TNK2 expression in esophageal cancer patients’ cancer tissue samples.MethodsThe expression of TNK2 in esophageal cancer tissues and para‐carcinoma tissue was assessed with immunohistochemistry and Western blot analysis; besides, the proteins of CDC42, EGFR, and Akt were also analyzed. Then, Kaplan‐Meier survival curves of TNK2 protein expression level were assayed with 184 esophageal cancer patients from TCGA database. Moreover, with multiple linear regression analysis, we detected the correlations of TNK2 expression associated with tumor differentiation degree and metastasis status.ResultsIt revealed that TNK2 was highly expressed in the cytoplasm of esophageal cancer tissues compared with para‐carcinoma tissue; besides, the proteins of CDC42, EGFR, and Akt were also up‐regulated in different levels of esophageal cancer tissues. However, there was no significant difference of the overall survival time of TNK2 protein expression in 184 esophageal cancer patients from TCGA database (p = 0.37). But, in the included study samples of our study, there was positive coefficience between TNK2 protein expression and differentiation degree in esophageal cancer with multiple linear regression analysis [R = 0.928, 95% confidence interval (0.085‐0.12)].ConclusionOur results indicated that TNK2 was a potential diagnostic marker and promoted esophageal cancer progression through activating EGFR‐AKT signaling.     

一例早发癫痫性脑病42型患儿的临床表型及基因变异分析

目的探讨一例早发癫痫性脑病42型患儿的基因型与表型特征。方法详细询问患儿的病史,结合其临床表型、影像学及遗传学特征进行临床诊断,并对其父母进行Sanger测序验证,明确致病变异的来源。结果患儿无意识头向一侧轻度歪斜,眼球向同侧斜视,脑电图异常放电。磁共振成像显示左额后皮层可疑异常信号,伴右侧上颌窦及筛窦炎症。全外显子组测序提示患儿携带CACNA1A基因c.5789G>A杂合变异,Sanger测序提示父母双方并未携带相同的变异,提示其为新发变异。结论先证者CACNA1A基因c.5789G>A杂合变异可能是导致其早发癫痫性脑病42型的原因。