1、FoxM1在幽门螺杆菌相关胃癌中的作用及其机制 2、miR-146a在吗啡诱导细胞凋亡中的作用及其机制
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摘要
研究背景
胃癌是世界范围内最常见的恶性肿瘤之一,在与癌症相关的死亡中,胃癌致死率居于第二位。胃癌的发生是一个多因素交叉、多步骤渐进的过程。其中,幽门螺杆菌(Helicobacter pylori, H.pylori)感染与胃癌的发生之间关系密切,1994年世界卫生组织已将其正式列为类致癌因子。研究显示在慢性胃炎、萎缩性胃炎、肠上皮化生、异型增生至胃癌的演变过程中,幽门螺杆菌可能作为一个先导性的始动因素参与到胃癌的早期事件。幽门螺杆菌及其毒力因子CagA参与到幽门螺杆菌感染所导致的病理过程中,但涉及的分子机制尚未明了,仍需大量的科学研究来进一步探索。
近年来的研究证明,感染、炎症与一些肿瘤的发生有着密切的关系。胃癌发生的一个重要原因是基于幽门螺杆菌感染所诱发的慢性胃炎的形成。炎症恶性转化的调控网络是由众多基因、非编码RNA、蛋白质和代谢分子所构成的。其中,叉头蛋白M1(Forkhead Box M1, FoxM1)是细胞周期相关正调控转录因子,其促进肿瘤细胞的增殖和侵袭转移,调控下游细胞周期相关蛋白的表达,广泛参与到多种癌症的发生和发展过程中。microRNA(miRNA)是近年来发现的一类长度约为18—24个核苷酸的非编码小分子RNA。它主要通过与靶基因3’UTR的完全或不完全配对,进而降解靶基因信使RNA (mRNA)或抑制其翻译,从而参与调控个体发育、细胞凋亡、增殖及分化等生命活动。miRNA在肿瘤细胞和肿瘤组织中异常表达,可以作为原癌基因或者抑癌基因,与肿瘤的发生发展关系密切。已经证实FoxM1和miRNAs在癌细胞及癌组织中异常表达,影响癌症的发生、发展及预后等,然而FoxM1及miRNAs在幽门螺杆菌相关胃癌发生的具体作用机制仍有待于进一步研究。
研究目的
本课题组以往的研究发现,增殖相关细胞周期转录因子FoxM1通过调控下游细胞周期相关蛋白P27kip1在胃癌发生中发挥作用。在此基础上,本研究的目的是探讨FoxM1幽门螺杆菌感染所致的慢性炎症到癌症的恶性转化中相关分子机制,为寻求新的胃癌防治生物靶标提供理论依据。
方法和结果
1. FoxM1在幽门螺杆菌感染胃部组织标本中的表达分析：通过实时定量PCR (Real-time PCR)及免疫组织化学(IHC)检测,在幽门螺杆菌感染阳性的癌旁正常组织、浅表性胃炎、萎缩性胃炎伴肠化生及胃癌组织病例标本中,FoxM1在肿瘤早期的炎性阶段已经被显著活化,并随着病程的深入表达逐渐升高。
2.体外分析幽门螺杆菌及其毒力因子CagA调控FoxMl对胃癌细胞增殖的影响：通过Real-time PCR及western blot检测,在多种胃癌细胞株中均存在FoxM1的表达,并且幽门螺杆菌感染可以时间依赖性和剂量依赖性地活化FoxMl。当用幽门螺杆菌的不同组分处理胃癌细胞株时,毒力因子CagA在调控FoxM1的表达中发挥重要作用,同时,利用CagA质粒转染细胞也得到相似的结果。利用幽门螺杆菌感染或者CagA质粒转染胃癌细胞株均能够降低P27kiP1mRNA以及蛋白水平的表达,同时抑制P27kiP1启动子的活性,并且可以显著增强细胞的克隆形成能力,提示二者能够促进肿瘤细胞增殖。
3.预测鉴定FoxMl是miR-370的直接靶基因并检测FoxM与miR-370在不同胃癌细胞株和胃癌组织样本中的表达水平及相关性：分析miR-370调控FoxM1在幽门螺杆菌对胃癌细胞增殖中的影响,microRNA芯片筛查及生物信息学软件预测均提示miR-370可靶向作用于FoxM1。组织标本中,Real-time PCR检测表明在炎症向癌症转化的过程中miR-370表达呈下降趋势,与FoxM1的表达呈现负相关关系。细胞实验中,利用miR-370的表达质粒转染细胞可以显著抑制FoxM1的表达,同时双荧光素酶报告基因检测表明FoxM1是miR-370的直接靶基因。幽门螺杆菌及CagA可显著下调miR-370的表达,并且miR-370介导了幽门螺杆菌及CagA对FoxM1的调控。利用细胞克隆形成实验表明miR-370在幽门螺杆菌诱发的细胞增殖中起负调控的作用。
4.体内分析miR-370-FoxM1在幽门螺杆菌感染所致慢性炎症中的作用：利用幽门螺杆菌标准菌株SS1进行小鼠灌胃造模,结果表明幽门螺杆菌的长期感染和定植可以触发慢性胃炎的发生,但是,单纯的幽门螺杆菌感染难以诱发小鼠胃癌的发生。在幽门螺杆菌所造成的慢性胃炎中,miR-370的表达是下调的,与之相对的是FoxM1表达的升高。
结论
1.组织标本检测中,FoxM1的表达升高不仅发生在胃部肿瘤组织中,也发生在胃炎标本中,FoxM1的异常表达是一个早期事件,提示其在恶性转化中的作用,是肿瘤发生的原因而非结果。
2.幽门螺杆菌感染胃癌皮细胞可以诱发FoxM1表达的上调,其中起主要作用的是其毒力因子CagA。
3.幽门螺杆及其毒力因子CagA参与胃癌细胞的增殖,机制在于通过上调FoxM1从而抑制P27kip1的表达和启动子活性。
4.miR-370直接靶向作用于FoxM1,并且miR-370-FoxM1信号途径参与到幽门螺杆菌诱导的细胞增殖。
5.动物实验表明幽门螺杆菌可以引起胃炎的发生,而miR-370调控FoxM1参与其中。
研究背景
吗啡是目前临床上常用的一种作用于中枢神经系统的强效镇痛药物,作为阿片类药物的一种,极易引起药物依赖性。不仅如此,相关研究发现,吗啡的滥用在引起神经系统器质性损害的同时,也能引起免疫系统的损伤。研究表明,吗啡能引起免疫细胞的凋亡,但具体的分子机制仍未十分明确。细胞凋亡主要由TNF家族所介导,作为其超家族成员之一的Fas,对于介导细胞凋亡,维持机体的免疫学功能有非常重要的作用。micro RNAs(miRN As)作为一类重要的转录后调控非编码小分子RNA,主要通过与靶基因3’UTR的完全或不完全配对,进而降解靶基因mRNA或抑制其翻译,从而参与调控个体发育、细胞凋亡、增殖及分化等生命活动。近期研究表明,miRNA广泛参与到机体的免疫反应中。本研究中,我们发现吗啡可以下调miR-146a的表达,而miR-146a通过负性靶向作用于Fas/FADD可消除吗啡所诱导的免疫抑制作用,并且miR-146a在解除吗啡的药物依赖中发挥重要作用,为吗啡诱导免疫细胞凋亡及药物成瘾提供了新的理论依据。
研究目的
本研究在于深入探讨miR-146a在阻断吗啡诱导的巨噬细胞凋亡及吗啡依赖中的作用及机制,为吗啡的戒断治疗及免疫抑制的改善提供了潜在的靶点。
方法和结果
1.体内及体外分析吗啡对miR-146a表达的影响：本研究中,首先用吗啡处理小鼠巨噬细胞系RAW264.7及分离获得的腹腔巨噬细胞,实时定量PCR检测证明miR-146a受吗啡调控而表达下调；体内实验中,以吗啡腹腔注射BALB/C小鼠,实时定量PCR检测相关组织中miR-146a的表达水平,得到与体外细胞实验相同结果。
2.预测及鉴定miR-146a的靶基因：利用生物信息学预测Fas及Fadd为miR-146a的潜在靶基因。用miR-146a的模拟类似物转染RAW264.7细胞系及小鼠腹腔巨噬细胞,实时定量PCR及免疫组织化学实验证实miR-146a可以抑制Fas及Fadd的mRNA及蛋白表达,用双荧光素酶报告基因分析证实二者为miR-146a的直接靶基因。
3.体内及体外分析miR-146a在吗啡诱导细胞凋亡中的作用：利用miR-146a的模拟物处理细胞,TUNEL检测分析提示miR-146a能够抑制吗啡诱导的巨噬细胞凋亡。体内实验中,利用尾静脉注射miR-146a模拟物,也得到同样的结果,提示niR-146a在吗啡诱导的凋亡中起抑制作用。同时,miR-146a能够抑制吗啡所引起的Fas及Fadd的表达升高,表明miR-146a的抗凋亡作用是通过靶向作用于凋亡相关蛋白Fas和Fadd实现的。
4.小鼠模型分析]miR-146a在吗啡的药物依赖中的作用：通过剂量递增法腹腔注射建立小鼠吗啡成瘾模型,期间辅以miR-146a类似物的注射,采用吗啡受体抑制剂纳洛酮催瘾造小鼠吗啡成瘾戒断模型,观察戒断模型小鼠的跳高、西狗样甩动、体重减轻指数、爪子震颤及腹泻等症状。结果表明,注射miR-146a的小鼠模型对吗啡的依赖性明显减轻。
结论
1.体内及体外实验均表明,吗啡能下调miR-146a的表达水平。
2.凋亡相关蛋白Fas及Fadd是miR-146a的直接靶基因,提示niR-146a在吗啡诱导的细胞凋亡中起作用。
3.miR-146a能够抑制吗啡诱导的细胞凋亡。
4.miR-146a能够阻抑吗啡所造成的药物依赖作用
Background
Gastric cancer is the fourth prevalent cancer and the second cause of cancer-related death worldwide, especially in developing countries. Gastric carcinogenesis is a complex multi-step and multi-factorial event. It has been extensively recognized that H.pylori infection is involved in the early stage of gastric cancer pathogenesis through induction of chronic gastritis, and that interaction of host-pathogen contributes to this carcinogenesis. H.pylori has been classified by the World Health Organization as a class1carcinogen in1994. It is well accepted that H.pylori and its key virulence factor CagA may be an initiating factor in the development process of gastric cancer, from chronic gastritis, peptic ulcer, intestinal metaplasia, atypical hyperplasia to gastric cancer. To date, however, the underlying molecular mechanism of carcinogensis relatived with H.pylori remains to be elucidated.
It has been proved that infection and inflammation was involved in carcinogenesis. Chronic inflammation elicited by H.pylori has been confirmed as a critical risk in the progression of gastric cancer, and this chronic inflammation can persist for decades and result in the nonresolving inflammation, a major driver to gastric cancer. Fork head Box M1(FoxM1), a member of the Fox transcription factor family, is a key positive cell-cycleregulator in cell proliferation. Aberrant expressionof FoxM1is involved in several tumor types, including hepatocellular carcinoma, basal cell carcinoma, breast cancer, lung cancer, prostate cancer, glioblastomas and GC, which implies an oncogene role in carcinogenesis. We previously reported that FoxM1is upregulated in GC, and its inhibition leads to cellular senescence, but therelevance of H.pylori infection and FoxMl expression associated with the pathogenesis of GC remains undefined. MicroRNAs (miRNAs), a family of small noncoding RNAs, are important negative regulators of post-transcriptional gene expression bydirectly targeting the3'untranslated regions (3'UTRs) of target mRNAs, eventually promoting the degradation or translation suppression of target mRNAs. MiRNAs are frequently deregulated in many types of human cancers and play critical roles in tumorigenesis, serving as tumor suppressors or oncogenes. However, the role and mechanisms of miRNAs in H.pylori-related gastric carcinogenesis is still unknown.
Aim
We previously reported that FoxM1is upregulated in GC, and its inhibition leads to cellular senescence, but therelevance of H.pylori infection and FoxM1expression associated with the pathogenesis of GC remains undefined. In this study, we attempted to explore the effect of H.pylori infection on FoxM1expression and possible mechanisms.
Methods and Results
1. The analysis of FoxM1expression in various gastric tissues. The expression of FoxM1was analyzed by Real-time PCR and IHC in various gastric specimens at different disease stages, including normal, superficial gastritis (SG), atrophic gastritis associated with intestinal metaplasia (AG/IM) and primary GC tissues. FoxM1expression was gradually increased in human gastric species from inflammation to cancer.
2. The effect of FoxM1on H.pylori-induced proliferation of gastric cancer cell in vitro. The gastric epithelial-derived cell lines infected with H.pylori showed up-regulation of FoxM1in the manner of time and concentration dependent. CagA, the key virulence factor of H.Pylori, showed the ability of increasing FoxMl expression.H.pylori and CagA inhibited expression of P27Kip1and subsequent promoted cells proliferation through up-regulating of FoxMl.
3. Prediction and identification of FoxMl as a direct target of miR-370, analysis the Correlation between miR-370and H. pylori-induced cell proliferation in gastric cancer cells. The expression of miR-370was declined in human species of gastritis and gastric cancer by Real-time PCR. We identified that FoxMl was directly down-regulated by hsa-miR-370in the gastric cell lines by western blot and Luciferase assay. H.pylori and CagA inhibited miR-370which led to overexpression of FoxM1and cell proliferation.
4. miR-370-FoxMl is involved in H.pylori-induced gastritis in vivo. The validation of mouse model of gastritis induced by H.pylori infection was confirmed by hematoxylin and eosin staining. After administration of H.pylori for8months,90%(18/20) of mice were validated as gastritis models in the H.pylori infection group as compared with13%(2/15) in the control group. FoxM1protein level was greater in the model than the control on IHC assay. In addition, the mRNA level of mmu-miR-370was reduced and that of FoxMl increased in mucosal epithelial samples of H.pylori-induced gastritis. The results in vivo revealed the involvement of FoxMl and miR-370inH.pylori-induced gastritis.
Conclusions
1. FoxM1expression was gradually increased in human gastric species from inflammation to cancer. Overexpression of FoxM1is an early event in H.pylori-relted gastric carcinogenesis.
2. H.pylori and its key virulence factor CagA promotes FoxM1expression in gastric epithelial cell lines.
3. H.pylori and CagA were involved in cell proliferation by suppressing p27kipl expression mediated by FoxMl.
4. FoxMl is a direct target of hsa-miR-370and miR-370-FoxM1pathway mediates H. pylori-induced cell proliferation.
5. FoxM1is involved in H. pylori-induced gastritis in vivo.
Backgroud
Morphine is one of the most potent pain reliever for centuries, but also a potent inducer of dependence.The uncontrolled use of opioids has been demonstrated to result in significant deterioration of the immune system.The mechanisms, however, remian to be identified. MicroRNAs (miRNAs) are a class of small non-coding regulatory RNAs that suppress protein expression mainly by binding to the3' untranlated region (UTR) of their target messenger RNAs. Here, we report that morphine can reduce the expression of microRNA-146a (miR-146a). MiR-146a blocks morphine-induced cell apoptosis by targeting the Fas/FADD pathway. Enforced expression of miR-146a diminishes morphine-induced cell apoptosis both in vitro and in vivo. Most interestingly, administration of miR-146a mimic in mice markedly attenuates morphine physical dependence.
Aim
Our discovery of modulation of miR-146a by opioids will enhance the understanding of the interactions between the immune and nervous systems. Current and further determinations of the function and mechanism of this novel signal will lead to our understanding of miRNA and apoptosis during the development of opioid dependence, providing potential molecular targets of novel therapeutics to treat opioid dependence and apoptosis.
Methods and Results
1. The effect of morphine administration on the expression of miR-146a.
To identify which miRNA was regulated by morphine, macrophage cell line RAW264.7andmurine peritoneal macrophages were treated with morphine and the expression of different miRNAs was determined by real-time quantitative RT-PCR (qRT-PCR). We found that morphine markedly decreased the levels of miR-146a. To determine the in vivo effect of morphine on miR-146a regulation, wild type (WT) mice were administered with morphine. Consistent with the results in vitro experiments, morphine reduced the expression of miR-146a in the spleen and liver.
2. Prediction and veryfiction of Fas and Fadd are direct targets of miR-146a.
We then applied the prediction program miRanda21to identify new possible miR-146targets. Cell deadth receptor Fas and Fas-associated death domain (FADD) have putative miR-146a binding elements in their3'-UTRs.Luciferase reporter assay was performed and the result suggested that Fas and FADD are direct targets of miR-146a. Consistent with that, miR-146a diminished the expression of Fas and FADD in both mRNAand protein levels.
3. The role and mechsnism of miR-146a in morphine-induced apoptosis in vivo and in vitro.
To assess the potential roles of miRNA-146a in morphine-induced apoptosis, we transfected RAW264.7cells with miR-146a mimic and identified apoptotic cells by the terminal deoxynucleotidy transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay. The RAW264.7cells forced miR-146a expressionand exposed to morphine led to asignificantlower number of apoptotic cells than the cells transfected with control mimic and morphine treatment. Then miRNA-146a mimic was administered in WT mice during chronic morphine administration, tissuesfrom the mice injected with both miR-146a mimic and morphine showed a dramatic decrease in the percentage of apoptotic cells. Taken together, these results suggest that miR-146a contributes to an anti-apoptotic effect and miR-146a inhibits apoptosis through the suppression of morphine-induced pro-apoptotic proteins.
4. Analysis the role of miR-146a on the main symptoms of morphine withdrawal.
Morphine dependence was evaluated by assessing withdrawal responses following administration of the opioid receptor antagonist naloxone. Notably, in administered mice with miR-146a mimic the main behavioral signs of morphine withdrawal were markedly attenuated, while the mice injected with control mimic still exhibited classic behavioral signs of withdrawal after administration of naloxone, suggesting that enforced expression of miR-146a in mice does not become dependent on morphine.
Conclusion
1. Morphine decreases the expression of miR-146a.
2. Fas and Fadd are direct targets of miR-146a.
3. miR-146a attenuates morphine-induced apoptosis.
4. miR-146a blocks the main aymptoms of morphine withdrawal.
胃癌是世界范围内最常见的恶性肿瘤之一,在与癌症相关的死亡中,胃癌致死率居于第二位。胃癌的发生是一个多因素交叉、多步骤渐进的过程。其中,幽门螺杆菌(Helicobacter pylori, H.pylori)感染与胃癌的发生之间关系密切,1994年世界卫生组织已将其正式列为类致癌因子。研究显示在慢性胃炎、萎缩性胃炎、肠上皮化生、异型增生至胃癌的演变过程中,幽门螺杆菌可能作为一个先导性的始动因素参与到胃癌的早期事件。幽门螺杆菌及其毒力因子CagA参与到幽门螺杆菌感染所导致的病理过程中,但涉及的分子机制尚未明了,仍需大量的科学研究来进一步探索。
近年来的研究证明,感染、炎症与一些肿瘤的发生有着密切的关系。胃癌发生的一个重要原因是基于幽门螺杆菌感染所诱发的慢性胃炎的形成。炎症恶性转化的调控网络是由众多基因、非编码RNA、蛋白质和代谢分子所构成的。其中,叉头蛋白M1(Forkhead Box M1, FoxM1)是细胞周期相关正调控转录因子,其促进肿瘤细胞的增殖和侵袭转移,调控下游细胞周期相关蛋白的表达,广泛参与到多种癌症的发生和发展过程中。microRNA(miRNA)是近年来发现的一类长度约为18—24个核苷酸的非编码小分子RNA。它主要通过与靶基因3’UTR的完全或不完全配对,进而降解靶基因信使RNA (mRNA)或抑制其翻译,从而参与调控个体发育、细胞凋亡、增殖及分化等生命活动。miRNA在肿瘤细胞和肿瘤组织中异常表达,可以作为原癌基因或者抑癌基因,与肿瘤的发生发展关系密切。已经证实FoxM1和miRNAs在癌细胞及癌组织中异常表达,影响癌症的发生、发展及预后等,然而FoxM1及miRNAs在幽门螺杆菌相关胃癌发生的具体作用机制仍有待于进一步研究。
研究目的
本课题组以往的研究发现,增殖相关细胞周期转录因子FoxM1通过调控下游细胞周期相关蛋白P27kip1在胃癌发生中发挥作用。在此基础上,本研究的目的是探讨FoxM1幽门螺杆菌感染所致的慢性炎症到癌症的恶性转化中相关分子机制,为寻求新的胃癌防治生物靶标提供理论依据。
方法和结果
1. FoxM1在幽门螺杆菌感染胃部组织标本中的表达分析：通过实时定量PCR (Real-time PCR)及免疫组织化学(IHC)检测,在幽门螺杆菌感染阳性的癌旁正常组织、浅表性胃炎、萎缩性胃炎伴肠化生及胃癌组织病例标本中,FoxM1在肿瘤早期的炎性阶段已经被显著活化,并随着病程的深入表达逐渐升高。
2.体外分析幽门螺杆菌及其毒力因子CagA调控FoxMl对胃癌细胞增殖的影响：通过Real-time PCR及western blot检测,在多种胃癌细胞株中均存在FoxM1的表达,并且幽门螺杆菌感染可以时间依赖性和剂量依赖性地活化FoxMl。当用幽门螺杆菌的不同组分处理胃癌细胞株时,毒力因子CagA在调控FoxM1的表达中发挥重要作用,同时,利用CagA质粒转染细胞也得到相似的结果。利用幽门螺杆菌感染或者CagA质粒转染胃癌细胞株均能够降低P27kiP1mRNA以及蛋白水平的表达,同时抑制P27kiP1启动子的活性,并且可以显著增强细胞的克隆形成能力,提示二者能够促进肿瘤细胞增殖。
3.预测鉴定FoxMl是miR-370的直接靶基因并检测FoxM与miR-370在不同胃癌细胞株和胃癌组织样本中的表达水平及相关性：分析miR-370调控FoxM1在幽门螺杆菌对胃癌细胞增殖中的影响,microRNA芯片筛查及生物信息学软件预测均提示miR-370可靶向作用于FoxM1。组织标本中,Real-time PCR检测表明在炎症向癌症转化的过程中miR-370表达呈下降趋势,与FoxM1的表达呈现负相关关系。细胞实验中,利用miR-370的表达质粒转染细胞可以显著抑制FoxM1的表达,同时双荧光素酶报告基因检测表明FoxM1是miR-370的直接靶基因。幽门螺杆菌及CagA可显著下调miR-370的表达,并且miR-370介导了幽门螺杆菌及CagA对FoxM1的调控。利用细胞克隆形成实验表明miR-370在幽门螺杆菌诱发的细胞增殖中起负调控的作用。
4.体内分析miR-370-FoxM1在幽门螺杆菌感染所致慢性炎症中的作用：利用幽门螺杆菌标准菌株SS1进行小鼠灌胃造模,结果表明幽门螺杆菌的长期感染和定植可以触发慢性胃炎的发生,但是,单纯的幽门螺杆菌感染难以诱发小鼠胃癌的发生。在幽门螺杆菌所造成的慢性胃炎中,miR-370的表达是下调的,与之相对的是FoxM1表达的升高。
结论
1.组织标本检测中,FoxM1的表达升高不仅发生在胃部肿瘤组织中,也发生在胃炎标本中,FoxM1的异常表达是一个早期事件,提示其在恶性转化中的作用,是肿瘤发生的原因而非结果。
2.幽门螺杆菌感染胃癌皮细胞可以诱发FoxM1表达的上调,其中起主要作用的是其毒力因子CagA。
3.幽门螺杆及其毒力因子CagA参与胃癌细胞的增殖,机制在于通过上调FoxM1从而抑制P27kip1的表达和启动子活性。
4.miR-370直接靶向作用于FoxM1,并且miR-370-FoxM1信号途径参与到幽门螺杆菌诱导的细胞增殖。
5.动物实验表明幽门螺杆菌可以引起胃炎的发生,而miR-370调控FoxM1参与其中。
研究背景
吗啡是目前临床上常用的一种作用于中枢神经系统的强效镇痛药物,作为阿片类药物的一种,极易引起药物依赖性。不仅如此,相关研究发现,吗啡的滥用在引起神经系统器质性损害的同时,也能引起免疫系统的损伤。研究表明,吗啡能引起免疫细胞的凋亡,但具体的分子机制仍未十分明确。细胞凋亡主要由TNF家族所介导,作为其超家族成员之一的Fas,对于介导细胞凋亡,维持机体的免疫学功能有非常重要的作用。micro RNAs(miRN As)作为一类重要的转录后调控非编码小分子RNA,主要通过与靶基因3’UTR的完全或不完全配对,进而降解靶基因mRNA或抑制其翻译,从而参与调控个体发育、细胞凋亡、增殖及分化等生命活动。近期研究表明,miRNA广泛参与到机体的免疫反应中。本研究中,我们发现吗啡可以下调miR-146a的表达,而miR-146a通过负性靶向作用于Fas/FADD可消除吗啡所诱导的免疫抑制作用,并且miR-146a在解除吗啡的药物依赖中发挥重要作用,为吗啡诱导免疫细胞凋亡及药物成瘾提供了新的理论依据。
研究目的
本研究在于深入探讨miR-146a在阻断吗啡诱导的巨噬细胞凋亡及吗啡依赖中的作用及机制,为吗啡的戒断治疗及免疫抑制的改善提供了潜在的靶点。
方法和结果
1.体内及体外分析吗啡对miR-146a表达的影响：本研究中,首先用吗啡处理小鼠巨噬细胞系RAW264.7及分离获得的腹腔巨噬细胞,实时定量PCR检测证明miR-146a受吗啡调控而表达下调；体内实验中,以吗啡腹腔注射BALB/C小鼠,实时定量PCR检测相关组织中miR-146a的表达水平,得到与体外细胞实验相同结果。
2.预测及鉴定miR-146a的靶基因：利用生物信息学预测Fas及Fadd为miR-146a的潜在靶基因。用miR-146a的模拟类似物转染RAW264.7细胞系及小鼠腹腔巨噬细胞,实时定量PCR及免疫组织化学实验证实miR-146a可以抑制Fas及Fadd的mRNA及蛋白表达,用双荧光素酶报告基因分析证实二者为miR-146a的直接靶基因。
3.体内及体外分析miR-146a在吗啡诱导细胞凋亡中的作用：利用miR-146a的模拟物处理细胞,TUNEL检测分析提示miR-146a能够抑制吗啡诱导的巨噬细胞凋亡。体内实验中,利用尾静脉注射miR-146a模拟物,也得到同样的结果,提示niR-146a在吗啡诱导的凋亡中起抑制作用。同时,miR-146a能够抑制吗啡所引起的Fas及Fadd的表达升高,表明miR-146a的抗凋亡作用是通过靶向作用于凋亡相关蛋白Fas和Fadd实现的。
4.小鼠模型分析]miR-146a在吗啡的药物依赖中的作用：通过剂量递增法腹腔注射建立小鼠吗啡成瘾模型,期间辅以miR-146a类似物的注射,采用吗啡受体抑制剂纳洛酮催瘾造小鼠吗啡成瘾戒断模型,观察戒断模型小鼠的跳高、西狗样甩动、体重减轻指数、爪子震颤及腹泻等症状。结果表明,注射miR-146a的小鼠模型对吗啡的依赖性明显减轻。
结论
1.体内及体外实验均表明,吗啡能下调miR-146a的表达水平。
2.凋亡相关蛋白Fas及Fadd是miR-146a的直接靶基因,提示niR-146a在吗啡诱导的细胞凋亡中起作用。
3.miR-146a能够抑制吗啡诱导的细胞凋亡。
4.miR-146a能够阻抑吗啡所造成的药物依赖作用
Background
Gastric cancer is the fourth prevalent cancer and the second cause of cancer-related death worldwide, especially in developing countries. Gastric carcinogenesis is a complex multi-step and multi-factorial event. It has been extensively recognized that H.pylori infection is involved in the early stage of gastric cancer pathogenesis through induction of chronic gastritis, and that interaction of host-pathogen contributes to this carcinogenesis. H.pylori has been classified by the World Health Organization as a class1carcinogen in1994. It is well accepted that H.pylori and its key virulence factor CagA may be an initiating factor in the development process of gastric cancer, from chronic gastritis, peptic ulcer, intestinal metaplasia, atypical hyperplasia to gastric cancer. To date, however, the underlying molecular mechanism of carcinogensis relatived with H.pylori remains to be elucidated.
It has been proved that infection and inflammation was involved in carcinogenesis. Chronic inflammation elicited by H.pylori has been confirmed as a critical risk in the progression of gastric cancer, and this chronic inflammation can persist for decades and result in the nonresolving inflammation, a major driver to gastric cancer. Fork head Box M1(FoxM1), a member of the Fox transcription factor family, is a key positive cell-cycleregulator in cell proliferation. Aberrant expressionof FoxM1is involved in several tumor types, including hepatocellular carcinoma, basal cell carcinoma, breast cancer, lung cancer, prostate cancer, glioblastomas and GC, which implies an oncogene role in carcinogenesis. We previously reported that FoxM1is upregulated in GC, and its inhibition leads to cellular senescence, but therelevance of H.pylori infection and FoxMl expression associated with the pathogenesis of GC remains undefined. MicroRNAs (miRNAs), a family of small noncoding RNAs, are important negative regulators of post-transcriptional gene expression bydirectly targeting the3'untranslated regions (3'UTRs) of target mRNAs, eventually promoting the degradation or translation suppression of target mRNAs. MiRNAs are frequently deregulated in many types of human cancers and play critical roles in tumorigenesis, serving as tumor suppressors or oncogenes. However, the role and mechanisms of miRNAs in H.pylori-related gastric carcinogenesis is still unknown.
Aim
We previously reported that FoxM1is upregulated in GC, and its inhibition leads to cellular senescence, but therelevance of H.pylori infection and FoxM1expression associated with the pathogenesis of GC remains undefined. In this study, we attempted to explore the effect of H.pylori infection on FoxM1expression and possible mechanisms.
Methods and Results
1. The analysis of FoxM1expression in various gastric tissues. The expression of FoxM1was analyzed by Real-time PCR and IHC in various gastric specimens at different disease stages, including normal, superficial gastritis (SG), atrophic gastritis associated with intestinal metaplasia (AG/IM) and primary GC tissues. FoxM1expression was gradually increased in human gastric species from inflammation to cancer.
2. The effect of FoxM1on H.pylori-induced proliferation of gastric cancer cell in vitro. The gastric epithelial-derived cell lines infected with H.pylori showed up-regulation of FoxM1in the manner of time and concentration dependent. CagA, the key virulence factor of H.Pylori, showed the ability of increasing FoxMl expression.H.pylori and CagA inhibited expression of P27Kip1and subsequent promoted cells proliferation through up-regulating of FoxMl.
3. Prediction and identification of FoxMl as a direct target of miR-370, analysis the Correlation between miR-370and H. pylori-induced cell proliferation in gastric cancer cells. The expression of miR-370was declined in human species of gastritis and gastric cancer by Real-time PCR. We identified that FoxMl was directly down-regulated by hsa-miR-370in the gastric cell lines by western blot and Luciferase assay. H.pylori and CagA inhibited miR-370which led to overexpression of FoxM1and cell proliferation.
4. miR-370-FoxMl is involved in H.pylori-induced gastritis in vivo. The validation of mouse model of gastritis induced by H.pylori infection was confirmed by hematoxylin and eosin staining. After administration of H.pylori for8months,90%(18/20) of mice were validated as gastritis models in the H.pylori infection group as compared with13%(2/15) in the control group. FoxM1protein level was greater in the model than the control on IHC assay. In addition, the mRNA level of mmu-miR-370was reduced and that of FoxMl increased in mucosal epithelial samples of H.pylori-induced gastritis. The results in vivo revealed the involvement of FoxMl and miR-370inH.pylori-induced gastritis.
Conclusions
1. FoxM1expression was gradually increased in human gastric species from inflammation to cancer. Overexpression of FoxM1is an early event in H.pylori-relted gastric carcinogenesis.
2. H.pylori and its key virulence factor CagA promotes FoxM1expression in gastric epithelial cell lines.
3. H.pylori and CagA were involved in cell proliferation by suppressing p27kipl expression mediated by FoxMl.
4. FoxMl is a direct target of hsa-miR-370and miR-370-FoxM1pathway mediates H. pylori-induced cell proliferation.
5. FoxM1is involved in H. pylori-induced gastritis in vivo.
Backgroud
Morphine is one of the most potent pain reliever for centuries, but also a potent inducer of dependence.The uncontrolled use of opioids has been demonstrated to result in significant deterioration of the immune system.The mechanisms, however, remian to be identified. MicroRNAs (miRNAs) are a class of small non-coding regulatory RNAs that suppress protein expression mainly by binding to the3' untranlated region (UTR) of their target messenger RNAs. Here, we report that morphine can reduce the expression of microRNA-146a (miR-146a). MiR-146a blocks morphine-induced cell apoptosis by targeting the Fas/FADD pathway. Enforced expression of miR-146a diminishes morphine-induced cell apoptosis both in vitro and in vivo. Most interestingly, administration of miR-146a mimic in mice markedly attenuates morphine physical dependence.
Aim
Our discovery of modulation of miR-146a by opioids will enhance the understanding of the interactions between the immune and nervous systems. Current and further determinations of the function and mechanism of this novel signal will lead to our understanding of miRNA and apoptosis during the development of opioid dependence, providing potential molecular targets of novel therapeutics to treat opioid dependence and apoptosis.
Methods and Results
1. The effect of morphine administration on the expression of miR-146a.
To identify which miRNA was regulated by morphine, macrophage cell line RAW264.7andmurine peritoneal macrophages were treated with morphine and the expression of different miRNAs was determined by real-time quantitative RT-PCR (qRT-PCR). We found that morphine markedly decreased the levels of miR-146a. To determine the in vivo effect of morphine on miR-146a regulation, wild type (WT) mice were administered with morphine. Consistent with the results in vitro experiments, morphine reduced the expression of miR-146a in the spleen and liver.
2. Prediction and veryfiction of Fas and Fadd are direct targets of miR-146a.
We then applied the prediction program miRanda21to identify new possible miR-146targets. Cell deadth receptor Fas and Fas-associated death domain (FADD) have putative miR-146a binding elements in their3'-UTRs.Luciferase reporter assay was performed and the result suggested that Fas and FADD are direct targets of miR-146a. Consistent with that, miR-146a diminished the expression of Fas and FADD in both mRNAand protein levels.
3. The role and mechsnism of miR-146a in morphine-induced apoptosis in vivo and in vitro.
To assess the potential roles of miRNA-146a in morphine-induced apoptosis, we transfected RAW264.7cells with miR-146a mimic and identified apoptotic cells by the terminal deoxynucleotidy transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay. The RAW264.7cells forced miR-146a expressionand exposed to morphine led to asignificantlower number of apoptotic cells than the cells transfected with control mimic and morphine treatment. Then miRNA-146a mimic was administered in WT mice during chronic morphine administration, tissuesfrom the mice injected with both miR-146a mimic and morphine showed a dramatic decrease in the percentage of apoptotic cells. Taken together, these results suggest that miR-146a contributes to an anti-apoptotic effect and miR-146a inhibits apoptosis through the suppression of morphine-induced pro-apoptotic proteins.
4. Analysis the role of miR-146a on the main symptoms of morphine withdrawal.
Morphine dependence was evaluated by assessing withdrawal responses following administration of the opioid receptor antagonist naloxone. Notably, in administered mice with miR-146a mimic the main behavioral signs of morphine withdrawal were markedly attenuated, while the mice injected with control mimic still exhibited classic behavioral signs of withdrawal after administration of naloxone, suggesting that enforced expression of miR-146a in mice does not become dependent on morphine.
Conclusion
1. Morphine decreases the expression of miR-146a.
2. Fas and Fadd are direct targets of miR-146a.
3. miR-146a attenuates morphine-induced apoptosis.
4. miR-146a blocks the main aymptoms of morphine withdrawal.
引文
1. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin 2011;61:69-90.
2. Catalano V, Labianca R, Beretta GD, Gatta G, de Braud F, Van Cutsem E. Gastric cancer. Crit Rev Oncol Hematol 2009;71:127-64.
3. Resende C, Thiel A, Machado JC, Ristimaki A. Gastric cancer:basic aspects. Helicobacter 2011;16:38-44.
4. Bornschein J, Malfertheiner P. Gastric carcinogenesis. Langenbecks Arch Surg 2011;396:729-42.
5. De Flora S, Bonanni P. The prevention of infection-associated cancers. Carcinogenesis 2011;32:787-95.
6. Carl Nathan. Nonresolving Inflammation. Cell 2010; 140:871-82.
7. Khan MK, Bemana M. Association of Helicobacter pylori infection and gastric carcinoma. Mymensingh Med J 2012;21:80-4.
8. Zhu Y, Zhong X, Zheng S, Du Q, Xu W. Transformed immortalized gastric epithelial cells by virulence factor CagA of Helicobacter pylori through Erk mitogen-activated protein kinase pathway. Oncogene 2005;24:3886-95.
9. Laoukili J, Stahl M, Medema RH. FoxM1:at the crossroads of ageing and cancer. Biochim Biophys Acta 2007; 1775:92-102.
10. Kalinichenko VV, Major ML, Wang X, Petrovic V, Kuechle J, Yoder HM, et al. Foxm1b transcription factor is essential for development of hepatocellular carcinomas and is negatively regulated by the p19ARF tumor suppressor. Genes Dev 2004;18:830-50.
11. Teh MT, Wong ST, Neill GW, Ghali LR, Philpott MP, Quinn AG. FOXM1 is a downstream target of Glil in basal cell carcinomas. Cancer Res 2002;62:4773-80.
12. Bektas N, Haaf A, Veeck J. Wild PJ, Liischer-Firzlaff J, Hartmann A, et al. Tight correlation between expression of the Forkhead transcription factor FOXM1 and HER2 in human breast cancer. BMC Cancer 2008;8:42.
13. Gialmanidis IP, Bravou V, Amanetopoulou SG, Varakis J, Kourea H, Papadaki H. Overexpression of hedgehog pathway molecules and F0XM1 in non-small cell lung carcinomas. Lung Cancer 2009;66:64-74.
14. Yang DK, Son CH, Lee SK, Choi PJ, Lee KE, Roh MS. Forkhead Box M1 expression in pulmonary squamous cell carcinoma:correlation with clinicopathologic features and its prognostic significance. Hum Pathol 2009;40:464-70.
15. Kalin TV, Wang IC, Ackerson TJ, Major ML, Detrisac CJ, Kalinichenko VV, et al. Increased levels of the FoxM1 transcription factor accelerate development and progression of prostate carcinomas in both TRAMP and LADY transgenic mice. Cancer Res 2006;66:1712-20.
16. Liu M, Dai B, Kang SH, Ban K, Huang FJ, Lang FF, et al. FoxM1B is overexpressed in human glioblastomas and critically regulates the tumorigenicity of glioma cells. Cancer Res 2006;66:3593-602.
17. Li Q, Zhang N, Jia Z, Le X, Dai B, Wei D, et al. Critical role and regulation of transcription factor FoxMl in human gastric cancer angiogenesis and progression. Cancer Res 2009;69:3501-09.
18. Zeng J, Wang L, Li Q, Li W, Bjorkholm M, Jia J, et al. FoxM1 is up-regulated in gastric cancer and its inhibition leads to cellular senescence, partially dependent on p27 kipl. J Pathol 2009;218:419-27.
19. Nelson P, Kiriakidou M, Sharma A, Maniataki E, Mourelatos Z. The microRNA world:Small is mighty. Trends Biochem Sci 2003;28:534-40.
20. Carthew RW, Sontheimer EJ. Origins and mechanisms of miRNAs and siRNAs. Cell 2009; 136:642-55.
21. Lee YS, Dutta A. The tumor suppressor microRNA let-7 represses the HMGA2 oncogene. Genes 2007;21:1025-30.
22. de Krijger I, Mekenkamp LJ, Punt CJ, Nagtegaal ID. MicroRNAs in colorectal cancer metastasis. J Pathol 2011;224:438-47.
23. Xiao B, Zhu ED, Li N, Lu DS, Li W, Li BS, et al. Increased miR-146a in gastric cancer directly targets SMAD4 and is involved in modulating cell proliferation and apoptosis. Oncol Rep 2012;27:559-66.
24. Oertli M, Engler DB, Kohler E, Koch M, Meyer TF, Miiller A. MicroRNA-155 is essential for the T cell-mediated control of Helicobacter pylori infection and for the induction of chronic Gastritis and Colitis. J Immunol 2011;187:3578-86.
25. Belair C, Darfeuille F, Staedel C. Helicobacter pylori and gastric cancer:possible role of microRNAs in this intimate relationship Clin Microbiol Infect 2009;15:806-12.
26. Yoshino H, Chiyomaru T, Enokida H, Kawakami K, Tatarano S, Nishiyama K, et al. The tumour-suppressive function of miR-1 and miR-133a targeting TAGLN2 in bladder cancer. Br J Cancer 2011; 104:808-18.
27. Meng F, Wehbe-Janek H, Henson R, Smith H, Patel T. Epigenetic regulation of microRNA-370 by interleukin-6 in malignant human cholangiocytes. Oncogene 2008;27:378-86.
28. Wang H, Sun Y, Liu S, Yu H, Li W, Zeng J, et al. Upregulation of progranulin by Helicobacter pylori in human gastric epithelial cells via p38MAPK and MEK1/2 signaling pathway:role in epithelial cell proliferation and migration. FEMS Immunol Med Microbiol 2011;63:82-92.
29. Houghton J, Wang TC. Helicobacter pylori and gastric cancer:a new paradigm for inflammation-associated epithelial cancers. Gastroenterology 2005;128:1567-78.
30. Schistosomes, liver flukes and Helicobacter pylori. IARC Working Group on the Evaluation of Carcinogenic Risks to Humans. Lyon,7-14 June 1994. IARC Monogr Eval Carcinog Risks Hum 1994;61:1-241.
31. Wroblewski LE, Shen L, Ogden S, Romero-Gallo J, Lapierre LA, Israel DA, et al. Helicobacter pylori dysregulation of gastric epithelial tight junctions by ureasemediated myosin Ⅱ activation. Gastroenterology 2009; 136:236-46.
32. Suzuki H, Nishizawa T, Tsugawa H, Mogami S, Hibi T. Roles of oxidative stress in stomach disorders. J Clin Biochem Nutr 2012;50:35-9.
33. Kim SS, Ruiz VE. Carroll JD, Moss SF. Helicobacter pylori in the pathogenesis of gastric cancer and gastric lymphoma. Cancer Letter 2011;305:228-38.
34. Hatakeyama M. Oncogenic mechanisms of the Helicobacter pylori CagA protein. Nat Rev Cancer 2004;4:688-94.
35. Yang ZM, Chen WW, Wang YF. Gene expression profiling in gastric mucosa from Helicobacter pylori-infected and uninfected patients undergoing chronic superficial gastritis. PLoS One 2012;7:e33030.
36. Myung N, Kim MR, Chung IP, Kim H, Jang JJ. Loss of p16 and p27 is associated with progression of human gastric cancer. Cancer Letter 2000; 153:129-36.
37. Lee KH, Lee HE, Cho SJ, Cho YJ, Lee HS, Kim JH, et al. Immunohistochemical analysis of cell cycle-related molecules in gastric carcinoma:prognostic significance, correlation with clinicopathological parameters, proliferation and apoptosis. Pathobiology 2008;75:364-72.
38. Alves MK, Lima VP, Andre AR, Ferreira MV, Barros MA, Rabenhorst SH. p27KIP1 expression in gastric cancer:differential pathways in the histological subtypes associated with Helicobacter pylori infection. Scand J Gastroenterol 2010;45:409-20.
39. Li SP, Chen XJ, Sun AH, Zhao JF, Yan J. CagA(+) H. pylori induces Aktl phosphorylation and inhibits transcription of p21WAF1/CIP1 and p27KIP1 via PI3K/Aktl pathway. Biomed Environ Sci 2010;23:273-8.
40. Byun SW, Chang YJ, Chung IS, Moss SF, Kim SS. Helicobacter pylori decreases p27 expression through the delta opioid receptor-mediated inhibition of histone acetylation within the p27 promoter. Cancer Letter 2012;326:96-104.
41. Wen S, So Y, Singh K, Slingerland JM, Resnick MB, Zhang S, et al. Promotion of cytoplasmic mislocalization of p27 by Helicobacter pylori in gastric cancer. Oncogene 2012;31:1771-80.
42. Zhu Y, Shu X, Chen J, Xie Y, Xu P, Huang DQ, et al. Effect of Helicobacter pylori eradication on oncogenes and cell proliferation. Eur J Clin Invest 2008;38:628-33.
43. Liu T, Tang H, Lang Y, Liu M, Li X. MicroRNA-27a functions as an oncogene in gastric adenocarcinoma by targeting prohibitin. Cancer Letter 2008;273:233-42.
44. Kim YK, Yu J, Han TS, Park SY, Namkoong B, Kim DH, et al. Functional links between clustered microRNAs:suppression of cell-cycle inhibitors by microRNA clusters in gastric cancer. Nucleic Acid Research 2009;37:1672-81.
45. Christoffersen NR, Silahtaroglu A, Orom UA, Kauppinen S, Lund AH. miR-200b mediates post-transcnptionai repression of ZFHXIB RNA 2007; 13:1172-8.
46. Gregory PA, Bert AG, Paterson EL, Barry SC, Tsykin A, Farshid G, et al. The miR-200 family and miR-205 regulate epithelial to mesenchymal transition by targeting ZEB1 and SIP1. Nat Cell Biol 2008;10:593-601.
47. Link A, Kupcinskas J, Wex T, Malfertheiner P. Macro-role of microRNA in gastric cancer. Dig Dis 2012;30:255-67.
1. Yin,D., Mufson,R.A., Wang,R. & Shi,Y. Fas-mediated cell death promoted by opioids. Nature397,218 (1999).
2. Nestler,E.J. & Aghajanian,GK. Molecular and cellular basis of addiction. Science278,58-63(1997).
3. O'Brien,C.P. A range of research-based pharmacotherapies for addiction. Science278,66-70(1997).
4. Rodriguez-Munoz,M., Sanchez-Blazquez,P., Vicente-Sanchez,A., Berrocoso,E. & Garzon,J. The mu-opioid receptor and the NMDA receptor associate in PAG neurons:implications in pain control. Neuropsychopharmacology37,338-349 (2012).
5. Li,Y. et al. Toll-like receptor 2 is required for opioids-induced neuronal apoptosis. Biochem. Biophys. Res. Commun.391,426-430 (2010).
6. Heinisch,S., Palma,J. & Kirby,L.G. Interactions between chemokine and mu-opioid receptors:anatomical findings and electrophysiological studies in the rat periaqueductal grey. Brain Behav. Immun.25,360-372 (2011).
7. Yin,D., Tuthill,D.. Mufson,R.A. & Shi,Y. Chronic restraint stress promotes lymphocyte apoptosis by modulating CD95 expression. J. Exp. Med.191, 1423-1428(2000).
8. Filipowicz,W., Bhattacharyya,S.N. & Sonenberg,N. Mechanisms of post-transcriptional regulation by microRNAs:are the answers in sight? Nat. Rev. Genet.9,102-114(2008).
9. Taganov,K.D.. Boldin.M.P., Chang,K.J. & Baltimore,D. NF-kappaB-dependent induction of microRNA miR-146, an inhibitor targeted to signaling proteins of innate immune responses. Proc. Natl. Acad. Sci. U. S. A103,12481-12486 (2006).
10. Bai,Y. et al. Integrin CD11b negatively regulates TLR9-triggered dendritic cell cross-priming by upregulating microRNA-146a. J. Immunol.188,5293-5302 (2012).
11. Lu,L.F. et al. Function of miR-146a in controlling Treg cell-mediated regulation of Thl responses. Celll42,914-929 (2010).
12. Du,C. et al. MicroRNA miR-326 regulates TH-17 differentiation and is associated with the pathogenesis of multiple sclerosis. Nat. Immunol.10, 1252-1259(2009).
13. Thum,T. et al. MicroRNA-21 contributes to myocardial disease by stimulating MAP kinase signalling in fibroblasts. Nature456,980-984 (2008).
14. Lu,J. et al. MicroRNA expression profiles classify human cancers. Nature435, 834-838 (2005).
15. Zhu,S. et al. The microRNA miR-23b suppresses IL-17-associated autoimmune inflammation by targeting TAB2, TAB3 and IKK-alpha. Nat. Med.18, 1077-1086(2012).
16. O'Connell,R.M. et al. MicroRNA-155 promotes autoimmune inflammation by enhancing inflammatory T cell development. Immunity.33,607-619 (2010).
17. Hou,J. et al. MicroRNA-146a feedback inhibits RIG-I-dependent Type I IFN production in macrophages by targeting TRAF6, IRAK1, and IRAK2. J. Immunol.183,2150-2158 (2009).
18. Medzhitov,R., Preston-Hurlburt,P. & Janeway.C.A., Jr. A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature388, 394-397(1997).
19. Rawlings,D.J., Schwartz,M.A., Jackson,S.W. & Meyer-Bahlburg,A. Integration of B cell responses through Toll-like receptors and antigen receptors. Nat. Rev. Immunol.12,282-294 (2012).
20. He,L-et al. Toll-like receptor 9 is required for opioid-induced microglia apoptosis. PLoS. One.6, e18190 (2011).
21. John,B. et al. Human MicroRNA targets. PLoS. Biol.2, e363 (2004).
22. Shaw-Lutchman,T.Z. et al. Regional and cellular mapping of cAMP response element-mediated transcription during naltrexone-precipitated morphine withdrawal. J. Neurosci.22,3663-3672 (2002).
23. Kielian,T. Toll-like receptors in central nervous system glial inflammation and homeostasis. J. Neurosci. Res.83,711-730 (2006).
24. Yang,H. et al. A critical cysteine is required for HMGB1 binding to Toll-like receptor 4 and activation of macrophage cytokine release. Proc. Natl. Acad. Sci. U. S. A107,11942-11947 (2010).
2. Catalano V, Labianca R, Beretta GD, Gatta G, de Braud F, Van Cutsem E. Gastric cancer. Crit Rev Oncol Hematol 2009;71:127-64.
3. Resende C, Thiel A, Machado JC, Ristimaki A. Gastric cancer:basic aspects. Helicobacter 2011;16:38-44.
4. Bornschein J, Malfertheiner P. Gastric carcinogenesis. Langenbecks Arch Surg 2011;396:729-42.
5. De Flora S, Bonanni P. The prevention of infection-associated cancers. Carcinogenesis 2011;32:787-95.
6. Carl Nathan. Nonresolving Inflammation. Cell 2010; 140:871-82.
7. Khan MK, Bemana M. Association of Helicobacter pylori infection and gastric carcinoma. Mymensingh Med J 2012;21:80-4.
8. Zhu Y, Zhong X, Zheng S, Du Q, Xu W. Transformed immortalized gastric epithelial cells by virulence factor CagA of Helicobacter pylori through Erk mitogen-activated protein kinase pathway. Oncogene 2005;24:3886-95.
9. Laoukili J, Stahl M, Medema RH. FoxM1:at the crossroads of ageing and cancer. Biochim Biophys Acta 2007; 1775:92-102.
10. Kalinichenko VV, Major ML, Wang X, Petrovic V, Kuechle J, Yoder HM, et al. Foxm1b transcription factor is essential for development of hepatocellular carcinomas and is negatively regulated by the p19ARF tumor suppressor. Genes Dev 2004;18:830-50.
11. Teh MT, Wong ST, Neill GW, Ghali LR, Philpott MP, Quinn AG. FOXM1 is a downstream target of Glil in basal cell carcinomas. Cancer Res 2002;62:4773-80.
12. Bektas N, Haaf A, Veeck J. Wild PJ, Liischer-Firzlaff J, Hartmann A, et al. Tight correlation between expression of the Forkhead transcription factor FOXM1 and HER2 in human breast cancer. BMC Cancer 2008;8:42.
13. Gialmanidis IP, Bravou V, Amanetopoulou SG, Varakis J, Kourea H, Papadaki H. Overexpression of hedgehog pathway molecules and F0XM1 in non-small cell lung carcinomas. Lung Cancer 2009;66:64-74.
14. Yang DK, Son CH, Lee SK, Choi PJ, Lee KE, Roh MS. Forkhead Box M1 expression in pulmonary squamous cell carcinoma:correlation with clinicopathologic features and its prognostic significance. Hum Pathol 2009;40:464-70.
15. Kalin TV, Wang IC, Ackerson TJ, Major ML, Detrisac CJ, Kalinichenko VV, et al. Increased levels of the FoxM1 transcription factor accelerate development and progression of prostate carcinomas in both TRAMP and LADY transgenic mice. Cancer Res 2006;66:1712-20.
16. Liu M, Dai B, Kang SH, Ban K, Huang FJ, Lang FF, et al. FoxM1B is overexpressed in human glioblastomas and critically regulates the tumorigenicity of glioma cells. Cancer Res 2006;66:3593-602.
17. Li Q, Zhang N, Jia Z, Le X, Dai B, Wei D, et al. Critical role and regulation of transcription factor FoxMl in human gastric cancer angiogenesis and progression. Cancer Res 2009;69:3501-09.
18. Zeng J, Wang L, Li Q, Li W, Bjorkholm M, Jia J, et al. FoxM1 is up-regulated in gastric cancer and its inhibition leads to cellular senescence, partially dependent on p27 kipl. J Pathol 2009;218:419-27.
19. Nelson P, Kiriakidou M, Sharma A, Maniataki E, Mourelatos Z. The microRNA world:Small is mighty. Trends Biochem Sci 2003;28:534-40.
20. Carthew RW, Sontheimer EJ. Origins and mechanisms of miRNAs and siRNAs. Cell 2009; 136:642-55.
21. Lee YS, Dutta A. The tumor suppressor microRNA let-7 represses the HMGA2 oncogene. Genes 2007;21:1025-30.
22. de Krijger I, Mekenkamp LJ, Punt CJ, Nagtegaal ID. MicroRNAs in colorectal cancer metastasis. J Pathol 2011;224:438-47.
23. Xiao B, Zhu ED, Li N, Lu DS, Li W, Li BS, et al. Increased miR-146a in gastric cancer directly targets SMAD4 and is involved in modulating cell proliferation and apoptosis. Oncol Rep 2012;27:559-66.
24. Oertli M, Engler DB, Kohler E, Koch M, Meyer TF, Miiller A. MicroRNA-155 is essential for the T cell-mediated control of Helicobacter pylori infection and for the induction of chronic Gastritis and Colitis. J Immunol 2011;187:3578-86.
25. Belair C, Darfeuille F, Staedel C. Helicobacter pylori and gastric cancer:possible role of microRNAs in this intimate relationship Clin Microbiol Infect 2009;15:806-12.
26. Yoshino H, Chiyomaru T, Enokida H, Kawakami K, Tatarano S, Nishiyama K, et al. The tumour-suppressive function of miR-1 and miR-133a targeting TAGLN2 in bladder cancer. Br J Cancer 2011; 104:808-18.
27. Meng F, Wehbe-Janek H, Henson R, Smith H, Patel T. Epigenetic regulation of microRNA-370 by interleukin-6 in malignant human cholangiocytes. Oncogene 2008;27:378-86.
28. Wang H, Sun Y, Liu S, Yu H, Li W, Zeng J, et al. Upregulation of progranulin by Helicobacter pylori in human gastric epithelial cells via p38MAPK and MEK1/2 signaling pathway:role in epithelial cell proliferation and migration. FEMS Immunol Med Microbiol 2011;63:82-92.
29. Houghton J, Wang TC. Helicobacter pylori and gastric cancer:a new paradigm for inflammation-associated epithelial cancers. Gastroenterology 2005;128:1567-78.
30. Schistosomes, liver flukes and Helicobacter pylori. IARC Working Group on the Evaluation of Carcinogenic Risks to Humans. Lyon,7-14 June 1994. IARC Monogr Eval Carcinog Risks Hum 1994;61:1-241.
31. Wroblewski LE, Shen L, Ogden S, Romero-Gallo J, Lapierre LA, Israel DA, et al. Helicobacter pylori dysregulation of gastric epithelial tight junctions by ureasemediated myosin Ⅱ activation. Gastroenterology 2009; 136:236-46.
32. Suzuki H, Nishizawa T, Tsugawa H, Mogami S, Hibi T. Roles of oxidative stress in stomach disorders. J Clin Biochem Nutr 2012;50:35-9.
33. Kim SS, Ruiz VE. Carroll JD, Moss SF. Helicobacter pylori in the pathogenesis of gastric cancer and gastric lymphoma. Cancer Letter 2011;305:228-38.
34. Hatakeyama M. Oncogenic mechanisms of the Helicobacter pylori CagA protein. Nat Rev Cancer 2004;4:688-94.
35. Yang ZM, Chen WW, Wang YF. Gene expression profiling in gastric mucosa from Helicobacter pylori-infected and uninfected patients undergoing chronic superficial gastritis. PLoS One 2012;7:e33030.
36. Myung N, Kim MR, Chung IP, Kim H, Jang JJ. Loss of p16 and p27 is associated with progression of human gastric cancer. Cancer Letter 2000; 153:129-36.
37. Lee KH, Lee HE, Cho SJ, Cho YJ, Lee HS, Kim JH, et al. Immunohistochemical analysis of cell cycle-related molecules in gastric carcinoma:prognostic significance, correlation with clinicopathological parameters, proliferation and apoptosis. Pathobiology 2008;75:364-72.
38. Alves MK, Lima VP, Andre AR, Ferreira MV, Barros MA, Rabenhorst SH. p27KIP1 expression in gastric cancer:differential pathways in the histological subtypes associated with Helicobacter pylori infection. Scand J Gastroenterol 2010;45:409-20.
39. Li SP, Chen XJ, Sun AH, Zhao JF, Yan J. CagA(+) H. pylori induces Aktl phosphorylation and inhibits transcription of p21WAF1/CIP1 and p27KIP1 via PI3K/Aktl pathway. Biomed Environ Sci 2010;23:273-8.
40. Byun SW, Chang YJ, Chung IS, Moss SF, Kim SS. Helicobacter pylori decreases p27 expression through the delta opioid receptor-mediated inhibition of histone acetylation within the p27 promoter. Cancer Letter 2012;326:96-104.
41. Wen S, So Y, Singh K, Slingerland JM, Resnick MB, Zhang S, et al. Promotion of cytoplasmic mislocalization of p27 by Helicobacter pylori in gastric cancer. Oncogene 2012;31:1771-80.
42. Zhu Y, Shu X, Chen J, Xie Y, Xu P, Huang DQ, et al. Effect of Helicobacter pylori eradication on oncogenes and cell proliferation. Eur J Clin Invest 2008;38:628-33.
43. Liu T, Tang H, Lang Y, Liu M, Li X. MicroRNA-27a functions as an oncogene in gastric adenocarcinoma by targeting prohibitin. Cancer Letter 2008;273:233-42.
44. Kim YK, Yu J, Han TS, Park SY, Namkoong B, Kim DH, et al. Functional links between clustered microRNAs:suppression of cell-cycle inhibitors by microRNA clusters in gastric cancer. Nucleic Acid Research 2009;37:1672-81.
45. Christoffersen NR, Silahtaroglu A, Orom UA, Kauppinen S, Lund AH. miR-200b mediates post-transcnptionai repression of ZFHXIB RNA 2007; 13:1172-8.
46. Gregory PA, Bert AG, Paterson EL, Barry SC, Tsykin A, Farshid G, et al. The miR-200 family and miR-205 regulate epithelial to mesenchymal transition by targeting ZEB1 and SIP1. Nat Cell Biol 2008;10:593-601.
47. Link A, Kupcinskas J, Wex T, Malfertheiner P. Macro-role of microRNA in gastric cancer. Dig Dis 2012;30:255-67.
1. Yin,D., Mufson,R.A., Wang,R. & Shi,Y. Fas-mediated cell death promoted by opioids. Nature397,218 (1999).
2. Nestler,E.J. & Aghajanian,GK. Molecular and cellular basis of addiction. Science278,58-63(1997).
3. O'Brien,C.P. A range of research-based pharmacotherapies for addiction. Science278,66-70(1997).
4. Rodriguez-Munoz,M., Sanchez-Blazquez,P., Vicente-Sanchez,A., Berrocoso,E. & Garzon,J. The mu-opioid receptor and the NMDA receptor associate in PAG neurons:implications in pain control. Neuropsychopharmacology37,338-349 (2012).
5. Li,Y. et al. Toll-like receptor 2 is required for opioids-induced neuronal apoptosis. Biochem. Biophys. Res. Commun.391,426-430 (2010).
6. Heinisch,S., Palma,J. & Kirby,L.G. Interactions between chemokine and mu-opioid receptors:anatomical findings and electrophysiological studies in the rat periaqueductal grey. Brain Behav. Immun.25,360-372 (2011).
7. Yin,D., Tuthill,D.. Mufson,R.A. & Shi,Y. Chronic restraint stress promotes lymphocyte apoptosis by modulating CD95 expression. J. Exp. Med.191, 1423-1428(2000).
8. Filipowicz,W., Bhattacharyya,S.N. & Sonenberg,N. Mechanisms of post-transcriptional regulation by microRNAs:are the answers in sight? Nat. Rev. Genet.9,102-114(2008).
9. Taganov,K.D.. Boldin.M.P., Chang,K.J. & Baltimore,D. NF-kappaB-dependent induction of microRNA miR-146, an inhibitor targeted to signaling proteins of innate immune responses. Proc. Natl. Acad. Sci. U. S. A103,12481-12486 (2006).
10. Bai,Y. et al. Integrin CD11b negatively regulates TLR9-triggered dendritic cell cross-priming by upregulating microRNA-146a. J. Immunol.188,5293-5302 (2012).
11. Lu,L.F. et al. Function of miR-146a in controlling Treg cell-mediated regulation of Thl responses. Celll42,914-929 (2010).
12. Du,C. et al. MicroRNA miR-326 regulates TH-17 differentiation and is associated with the pathogenesis of multiple sclerosis. Nat. Immunol.10, 1252-1259(2009).
13. Thum,T. et al. MicroRNA-21 contributes to myocardial disease by stimulating MAP kinase signalling in fibroblasts. Nature456,980-984 (2008).
14. Lu,J. et al. MicroRNA expression profiles classify human cancers. Nature435, 834-838 (2005).
15. Zhu,S. et al. The microRNA miR-23b suppresses IL-17-associated autoimmune inflammation by targeting TAB2, TAB3 and IKK-alpha. Nat. Med.18, 1077-1086(2012).
16. O'Connell,R.M. et al. MicroRNA-155 promotes autoimmune inflammation by enhancing inflammatory T cell development. Immunity.33,607-619 (2010).
17. Hou,J. et al. MicroRNA-146a feedback inhibits RIG-I-dependent Type I IFN production in macrophages by targeting TRAF6, IRAK1, and IRAK2. J. Immunol.183,2150-2158 (2009).
18. Medzhitov,R., Preston-Hurlburt,P. & Janeway.C.A., Jr. A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature388, 394-397(1997).
19. Rawlings,D.J., Schwartz,M.A., Jackson,S.W. & Meyer-Bahlburg,A. Integration of B cell responses through Toll-like receptors and antigen receptors. Nat. Rev. Immunol.12,282-294 (2012).
20. He,L-et al. Toll-like receptor 9 is required for opioid-induced microglia apoptosis. PLoS. One.6, e18190 (2011).
21. John,B. et al. Human MicroRNA targets. PLoS. Biol.2, e363 (2004).
22. Shaw-Lutchman,T.Z. et al. Regional and cellular mapping of cAMP response element-mediated transcription during naltrexone-precipitated morphine withdrawal. J. Neurosci.22,3663-3672 (2002).
23. Kielian,T. Toll-like receptors in central nervous system glial inflammation and homeostasis. J. Neurosci. Res.83,711-730 (2006).
24. Yang,H. et al. A critical cysteine is required for HMGB1 binding to Toll-like receptor 4 and activation of macrophage cytokine release. Proc. Natl. Acad. Sci. U. S. A107,11942-11947 (2010).