HOPX is methylated and exerts tumour‐suppressive function through Ras‐induced senescence in human lung cancer

HOPX acts as a tumour suppressor in various cancers. However, the regulation of HOPX in human lung cancer as well as the mechanism underlying its tumour‐suppressive function has not yet been well elucidated. Here we investigated the epigenetic regulation and molecular mechanism by which HOPX exerts growth inhibitory effects. We found that HOPX was down‐regulated in 12 out of 13 lung cancer cell lines and in 69 out of 120 primary lung tumours at mRNA and protein levels. Patients with lung adenocarcinoma (ADC) exhibited significantly more positive staining of HOPX protein compared with lung squamous cell carcinoma (SCC) (p =0.036). Again in ADC, patients with higher HOPX expression had a significantly longer disease‐free survival (p =0.001). Methylation analysis showed that down‐regulation of HOPX was associated with DNA methylation (p =0.011). To analyse the function of HOPX in lung cancer cells, stable transfection with an expression vector of HOPX was performed. It turned out that HOPX inhibited tumour cell proliferation rate, migration, and invasion, and, more interestingly, forced expression of HOPX enhanced cellular senescence via activation of oncogenic Ras and the downstream MAPK pathway, which in turn led to decreased MDM2 and increased p21. On the contrary, knockdown of HOPX by siRNA resulted in reduced Ras activity, inactivation of the MAPK pathway, and decreased p21 levels, accompanied by reduced cellular senescence. Additionally, the HOPX‐induced senescence pathway was also active in human bronchial epithelial cells. Taken together, our data suggest that down‐regulation of HOPX was related to DNA methylation and that HOPX exerts tumour‐suppressive activity by oncogenic Ras‐induced cellular senescence in lung cancer cells. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

ERK1/2通路及其介导多发性硬化发病的研究进展

丝裂原活化蛋白激酶1/2（ERK1/2）通路是第一个被发现的细胞信号转导通路,由Ras、Raf、MEK1/2和ERK1/2组成。ERK1/2通路激活后可以将细胞外信号从细胞膜转到细胞核,参与了细胞的多种生理病理功能,如细胞的生长、增殖、分化和凋亡等,并且与多种疾病的发病有关,包括多发性硬化（MS）和实验性自身免疫性脑脊髓炎（EAE）。ERK1/2通路的激活可以引起星形胶质细胞、MG、T细胞、巨噬细胞等活化,释放多种炎性因子,引起髓鞘损伤,导致MS/EAE的发病。多项研究表明,通过抑制ERK1/2通路可以减少炎性因子的释放,减轻髓鞘损伤,改善MS/EAE的病情,为治疗MS药物的开发提供了重要的靶点。     

基于Ras/Raf-1/ERK信号转导通路调控VEGF的表达探讨活血解毒方对糖尿病大鼠视网膜的影响

目的：观察活血解毒方对糖尿病大鼠视网膜血管形态的影响并探讨其可能的作用机制。方法：一次性腹腔注射链脲佐菌素(Streptozotocin,STZ,65 mg/kg)诱导糖尿病大鼠模型,造模后随机分为模型组和活血解毒方组。另设正常对照组。药物干预12周后,应用视网膜消化铺片法观察视网膜血管形态;应用Western blot法检测视网膜VEGF蛋白的表达,应用Real-Time PCR法检测视网膜VEGF、Ras、Raf-1与ERK mRNA的表达。结果：模型组视网膜毛细血管面积密度及内皮细胞/周细胞比例与较正常组升高(P<0.001),VEGF蛋白及VEGF、Ras、ERKmRNA表达升高(P<0.05),Raf-1mRNA表达升高(P>0.05)。与模型组比较,活血解毒方组视网膜毛细血管面积密度和内皮细胞/周细胞比例降低(P<0.01和P<0.001),VEGF蛋白与VEGF、Ras、Raf-1、ERK mRNA表达下降(P<0.05)。结论：活血解毒方能明显抑制视网膜毛细血管和内皮细胞的增生,其机制可能是通过干预Ras/Raf-1/ERK信号转导通路,下调VEGF在视网膜中的表达,抑制血管新生,从而改善DR。     

肝细胞癌组织中Ras相关结构域家族1A基因甲基化与环境因素的关系

目的 探讨肝细胞癌(UCC)组织中Ras相关结构域家族1A( RASSF1A)基因甲基化与环境因素的关系.方法 采用甲基化特异性聚合酶链反应检测80例HCC组织中RASSF1A基因甲基化状态,免疫组织化学法检测HCC组织中黄曲霉素B1( AFB1 )-DNA加合物和多环芳烃(PAHs) -DNA加合物的水平,并结合临床资料进行统计分析.结果 RASSF1A基因甲基化率在肝癌组织中(60/80)显著高于癌旁组织(33/80,P＜0.01).HCC组织中RASSF1A基因甲基化率在AFB1-DNA加合物阳性组(43/51)高于AFB1-DNA加合物阴性组(17/29,P＜0.05);在PAHs-DNA加合物阳性组(39/45)高于PAH-DNA加合物阴性组(21/35,P＜ 0.05).结论 HCC组织中RASSF1A基因甲基化与环境致癌因素AFBI -DNA加合物、PAHs-DNA加合物水平密切相关.     

RhoA、RhoB和RhoC在肺癌细胞中的表达及意义

目的 通过研究Rho亚家族RhoA、RhoB和RhoC在肺癌组织及细胞系中表达水平的变化,探讨Rho亚家族与肺癌发生、发展及转移的关系。方法：应用荧光定量RT-PCR法、Western blotting法研究四种肺癌细胞系中RhoA、RhoB和RhoC mRNA和蛋白的表达水平变化,免疫组织化学法检测45例人肺癌组织中RhoA、RhoB和RhoC蛋白的表达。结果 四种肺癌细胞系中RhoA和RhoB mRNA和蛋白表达水平显著高于对照组(均P<0.05),肺癌细胞系组间比较它们的表达水平差异无统计学意义(均P>0.05),而RhoC mRNA和蛋白表达水平各组间差异无统计学意义(均P>0.05)。RhoA、RhoB蛋白在人肺癌组织中呈阳性表达,在中分化鳞癌组阳性率与非中分化鳞癌组阳性率差异无统计学意义(均P>0.05),而RhoC 全部呈阴性表达。结论 Rho亚家族RhoA、RhoB在肺癌中异常高表达,提示它们参与了肺癌的发生、分化和转移过程。     

Octopamine-mediated circuit mechanism underlying controlled appetite for palatable food in Drosophila

The easy accessibility of energy-rich palatable food makes it difficult to resist food temptation. Drosophila larvae are surrounded by sugar-rich food most of their lives, raising the question of how these animals modulate food-seeking behaviors in tune with physiological needs. Here we describe a circuit mechanism defined by neurons expressing tdc2-Gal4 (a tyrosine decarboxylase 2 promoter-directed driver) that selectively drives a distinct foraging strategy in food-deprived larvae. Stimulation of this otherwise functionally latent circuit in tdc2-Gal4 neurons was sufficient to induce exuberant feeding of liquid food in fed animals, whereas targeted lesions in a small subset of tdc2-Gal4 neurons in the subesophageal ganglion blocked hunger-driven increases in the feeding response. Furthermore, regulation of feeding rate enhancement by tdc2-Gal4 neurons requires a novel signaling mechanism involving the VEGF2-like receptor, octopamine, and its receptor. Our findings provide fresh insight for the neurobiology and evolution of appetitive motivation.The adaptive control of foraging decisions is crucial to survival and reproduction and is mediated by complex brain mechanisms. For example, in hungry animals, feeding behaviors can be modulated by diverse neural systems including those responsible for receiving and processing sensory properties and assigning reward and motivational significance of food stimuli (1–3). At present, elucidation of molecular and circuit mechanisms underlying the adaptive control of feeding behavior remains highly challenging.Our previous studies have shown that Drosophila larvae, like mammals, display diverse adaptive foraging strategies in response to appetizing odors or satiety state and food quality (4–6). For example, larvae fed for ad libitum intake tend to prefer soft, liquid sugar media that contain readily ingestible sugar solution but decline solid media in which sugar solution is embedded in gelled agar and is less accessible (5). However, as food deprivation is prolonged, larvae will become increasingly persistent in extracting the sugar solution from solid media (7). We have also shown that an evolutionarily conserved signaling cascade, involving neuropeptide F (NPF, the fly homolog of neuropeptide Y, or NPY) and insulin-like peptides (dILPs), selectively integrates motivational state (hunger) with persistence to pulverize solid food (5, 7).The observation that the conserved NPY-like system selectively promotes food acquisition behaviors that require high energetic cost has led us to postulate that fly larvae may use other conserved neural mechanisms to regulate acquisition of readily accessible palatable food. In this work, we provide evidence which supports this hypothesis. We show that an octopamine (OA)/β-adrenergic-like receptor (Octß3R)-dependent circuit mechanism selectively regulates appetite for soft sugar media. This circuit mechanism seems to involve two subsets of tdc2-Gal4 neurons in the subesophogeal ganglia (SOG). One of them mediates the hunger-driven increase of feeding and is modulated by a novel activity of the VEGF2-like receptor pathway (8). The other is required for preventing excessive appetite in fed larvae. This and our previous findings provide fresh mechanistic insights into how brain mechanisms differentially organize appetitive motivations in responses to high- and low-quality food sources under different energy states.     

ras癌基因第12密码子点突变和胃癌患者预后关系的研究

对福尔马林固定，石蜡包埋的胃癌组织使用移聚酶链式反应－限制性片段长度多态性（简称ＰＣＲ－ＲＦＬＰ）技术同时进行ｃ－Ｈａ－ｒａｓ基因第１２位和６１位，Ｎ－ｒａｓ基因第１２位，ｋ－ｒａｓ第１２位和１３位密码子点突变的研究，发现３３．３％（１４／４２），的胃癌有ｃ－Ｈａ－ｒａｓ基因第１２位密码子的点突变；４．８％（２／４２）的病例有ｋ－ｒａｓ基因第１２位密码子的点突变，点突变的发生与患者的预后，淋巴结转     

Recovery of a rare clone from a population of unstable retroviral vector-expressing mammalian cells using a new RNA extraction and slot-blot protocol

Although a useful and important method of gene transfer, retroviral vectors can be genetically unstable. In the course of experiments using DOEJS, a retroviral vector able to confer expression of a H-ras oncogene and a neomycin resistance gene (neo) on mammalian cells (Compere et al., 1989), it was found that the vast majority of infected rat embryo fibroblasts, recovered on the basis of neo activity (i.e., G418 resistance), did not express ras mRNA. It was subsequently observed that most cells in the ψ2 cell line used to propagate DOEJS failed to produce virus capable of expressing both ras and neo in primary rat embryo fibroblasts. A simplified RNA extraction and slot-blot technique was developed to screen mRNA from several hundred fibroblast clones and, in doing so, infected fibroblast clones producing both neo and ras mRNA were identified at low frequency. The DOEJS/ψ2 packaging line was subsequently subcloned and individual clones screened for their ability to confer appropriate gene expression on target cells. Subclone DOEJS/ψ2-B6 was eventually isolated after screening 24 DOEJS subclones and 240 infected rat embryo fibroblast colonies. DOEJS/ψ2-B6 was shown to induce reliably phenotypic transformation, G418 resistance, and ras and neo mRNA expression in primary rat embryo fibroblasts. The RNA extraction and screening procedure was thus useful for recovering an infrequent subclone producing a retrovirus with the original properties.     

肺鳞癌、肺腺癌Ki-ras和p53基因序列分析

目的 :研究肺鳞癌、腺癌与Ki-ras基因外显子 1,2及p5 3基因外显子 7,8突变谱并观察肺鳞癌、腺癌预后同Ki-ras和 p5 3基因突变的关系。方法 :用PCR和测序方法分析 33例肺鳞癌和 2 7例肺腺癌Ki-ras基因外显子 1,2和 p5 3基因外显子 7,8错义突变。并追踪观察突变者和非突变者的二年存活率。结果 :肺鳞癌中发现 5例 (15 .16 % )Ki-ras基因突变和 12例 (36 .37% )p5 3基因突变 ;肺腺癌中发现 2例 (7.4 1% )Ki-ras基因突变和 7例 (2 5 .93% ) p5 3基因突变 ,两型肺癌间Ki-ras和p5 3基因突变阳性率差异均无统计学意义 (P>0 .0 5 ) ;肺鳞癌、腺癌中 p5 3基因突变总阳性率为 31.6 7% ,Ki-ras基因突变总阳性率仅 11.6 7% ,p5 3基因突变率高于Ki-ras基因突变率 (P <0 .0 5 )。p5 3基因突变谱与一般报道相似。Ki-ras基因突变情况与一般报道有所不同 ,未发现一般文献报道较多的第 12位等密码子突变 ,发现有第 6位密码子纯合性突变和第 31位和第 79位密码子杂合性突变。预后追踪显示 :无突变的病例 2年生存率为 4 0 % ,存在突变的病例 2年生存率仅 12 %。结论 :肺鳞癌、腺癌的Ki-ras和 p5 3基因突变谱较广 ,肺鳞癌和腺癌的预后均与这两个基因突变有关。