白细胞介素-1通过JNK信号通路调控Schlafen2基因的表达

目的：在多效生长因子（Ptn）基因稳定沉默的小鼠胚胎成纤维细胞Ptn-siRNA B/MEF241中,研究白细胞介素-1（IL-1）调控Schlafen2（Slfn2）基因表达的机制。方法：应用Northernblot检测Ptn沉默细胞Ptn-siRNAB/MEF241处于不同生长密度时Slfn2基因的表达变化,以确定Ptn沉默细胞中Slfn2基因的表达是否受到某种分泌性细胞因子的调控;用不同浓度的IL-1α中和抗体及IL-1受体拮抗剂处理Ptn沉默细胞,通过Northern blot检测细胞内Slfn2表达的抑制情况;用不同浓度的IL-1α中和抗体及IL-1受体拮抗剂处理Ptn沉默细胞不同时间,通过Western blot检测细胞中JNK磷酸化水平;Northern blot检测SP600125（JNK/MAPK通路抑制剂）对Ptn沉默细胞中Slfn2基因表达的影响。结果：Ptn沉默细胞中Slfn2基因的表达水平同细胞密度相关;用中和抗体和受体拮抗剂阻断IL-1通路,Slfn2表达受到显著抑制;IL-1受到抑制会影响JNK通路的活化;阻断JNK通路,Slfn2的表达受到显著抑制。结论：IL-1可以通过JNK通路诱导Slfn2的表达。     

多效生长因子通过JNK信号通路负调控Schlafen2基因表达

实验室前期研究发现,多效生长因子（pleiotrophin,PTN）基因稳定沉默的小鼠胚胎成纤维细胞中Schlafen2 (Slfn2)基因高表达.为了探讨Ptn沉默诱导Slfn2基因表达可能涉及的信号通路,应用Western印迹检测外源性PTN因子（终浓度50 ng/μl）对Ptn沉默细胞JNK磷酸化水平的影响;应用Northern 印迹分别检测JNK和p38通路特异性抑制剂对Ptn沉默细胞Slfn2基因转录水平的影响.结果发现,Ptn沉默细胞内JNK磷酸化水平高于对照细胞,外源性PTN处理后沉默细胞内JNK磷酸化水平下调;阻断JNK通路呈时间依赖性抑制Ptn沉默细胞中Slfn2基因转录,阻断p38通路对Ptn沉默细胞中Slfn2转录水平没有明显影响结果提示,Ptn可能通过抑制其下游JNK/MAPK通路来负调控Slfn2的表达.     

血管外膜肌成纤维细胞分化的基因表达谱

我们以往的研究表明,TGF-β1可以诱导血管外膜成纤维细胞（adventitial fibroblasts,AFs）向肌成纤维细胞（myofibroblasts,MFs）分化。为寻找可能涉及MF分化的基因,本实验采用寡核苷酸芯片技术动态检测细胞表型转化过程中基因表达的变化,实时定量RT-PCR验证芯片结果。在芯片上的15866条总探针组中,2121个探针组在TGF-β1刺激后至少一个时间点的表达发生2倍以上变化,其中l318个基因表达上调,761个基因表达下调,还有少数基因（42个）在不同的时间点既有上调又有下调表达。在1231个已知功能基因中,分泌磷蛋白l（secreted phosphoprotein1．APP1）、Rhoassociated coiled-coil forming kinase2（ROCK2）的表达趋势与标志基因α-平滑肌肌动蛋白（α-SM-actin）的表达趋势相同,TGF-D1诱导MF分化过程中上调了电压门控性钾通道Shal家族成员2（potassium voltage-gated channel,Shal-related family and member2,KCND2）的表达,这些基因参与了MF的分化;此外,还发现内皮素1（endothelin 1,EDN1）、补体成分、NADPH氧化酶4（NADPH oxidase 4,NOX4）和NAD（P）H dehydrogenase,quinone 1 （NQO1）可能参与了MF分化。本实验用寡核苷酸芯片技术验证了通过其它技术证实的同MF分化相关的基因,并发现了新的涉及该过程的基因,基因表达谱研究有利于鉴定参与细胞分化的基因和通路。     

脂氧合酶OsRCI-1正调控水稻对二化螟的抗性

本文克隆了虫害诱导的水稻脂氧合酶基因OsRCI-1全编码序列, 实时定量QRT-PCR 检测结果表明二化螟Chilo suppressalia Walker取食能快速且持续地诱导OsRCI-1基因的表达。利用农杆菌介导的水稻遗传转化获得了两株OsRCI-1基因的RNAi沉默突变体,突变体株系ir-rci-1和ir-rci-2中目的基因OsRCI-1的表达被明显的抑制,其表达水平分别仅为对照(秀水11)的4773%和3233%。生测结果表明OsRCI-1基因沉默导致水稻对二化螟抗性显著地降低,取食RNAi突变体株系的二化螟体重分别是取食秀水11的158倍和215倍。因此,水稻OsRCI-1基因正调控对二化螟的抗性,该基因介导的防御途径可能在水稻虫害诱导防御反应中起重要的作用。     

人同源盒基因NKX3.1对前列腺癌细胞的诱导凋亡作用

构建人同源盒基因NKX3.1 cDNA真核表达载体,研究其在前列腺癌细胞PC-3、LNCaP 中的表达及对细胞的促凋亡作用.以人前列腺癌细胞LNCaP细胞中的总RNA为模板,RT-PCR扩增NKX3.1基因全长编码片段,将NKX3.1 cDNA重组到真核表达载体pcDNA3.1（+）中; 将pcDNA3.1-NKX3.1表达载体瞬时转染前列腺癌细胞PC-3和LNCaP 细胞,用RT-PCR和Western印迹检测NKX3.1 cDNA在转录水平和蛋白水平的表达;绘制细胞生长曲线,观察NKX3.1对前列腺癌细胞增殖的抑制作用;用DNA/ladder和流式细胞术检测NKX3.1对前列腺癌细胞凋亡的影响,进一步用RT PCR检测凋亡相关基因caspase3、caspase8、caspase9、Apaf1、survivin和Bcl2表达的变化.人同源盒基因NKX3.1 cDNA真核表达载体pcDNA3.1-NKX3.1经酶切及测序鉴定正确. pcDNA3.1-NKX3.1转染PC-3和LNCaP细胞后,经RT-PCR和Western印迹证明能有效表达NKX3.1.生长曲线显示,前列腺癌细胞转染NKX3.1 cDNA后细胞增殖受到抑制;前列腺癌细胞转染NKX3.1 cDNA 48 h后,DNA电泳呈现具有凋亡特征的DNA ladder;流式细胞术检测出现明显凋亡峰;RT-PCR检测凋亡相关基因.结果显示,caspase3、caspase8、caspase9基因表达明显增加,Bcl2基因表达明显减少.本研究成功构建了真核表达载体pcDNA3.1 NKX3.1, 转染PC3和LNCaP细胞后能有效表达,并对细胞具有诱导凋亡作用     

db/db小鼠糖尿病肾病相关基因的分析和克隆

用GM-U74A基因芯片分别检测了正常对照组（db/m小鼠）、糖尿病肾病组（db/db小鼠）、大黄酸治疗组（大黄酸150 mg/kg治疗12周）肾脏基因表达谱.发现在12 437个基因（包括表达序列标签）中,与正常对照组相比,糖尿病肾病组有1 085个基因表达下调,37个基因表达上调,其中变化幅度大于2倍,表达下调的有166个和表达上调的有29个.与糖尿病肾病组相比,大黄酸治疗组有384个基因表达下调,155个表达上调,其中变化幅度大于2倍,表达下调的有47个和表达上调的有30个.在此基础上,对其中的一个差异表达的表达序列标签(EST)进行了详细的生物信息学分析,发现它是一个未知功能基因——“REKEN cDNA 0610006H10”基因的一部分.在用RT-PCR进一步验证了其与糖尿病肾病的相关性后,对“REKEN cDNA 0610006H10”基因进行了克隆.     

p53、bax和bcl-2基因在SO 2染毒大鼠肝中的表达

为了进一步探讨SO₂的毒理学作用机制,运用荧光实时定量RT-PCR和免疫组化技术研究SO2吸入对大鼠肝细胞中p53、bax和bcl-2三种细胞凋亡相关基因mRNA和蛋白表达的影响.结果显示,肝中p53和bax mRNA水平呈剂量依赖性增加,在SO ₂浓度为28.00和56.00 mg/m3时显著增加（p53 mRNA在28 mg/m ³为1.30倍,在56 mg/m^3为3.43倍;bax mRNA在28 mg/m3为1.63倍,在56 mg/m ³为2.17倍）;而bcl-2 mRNA水平显著降低（在28 mg/m3为0.63倍,在56 mg/m ³为0.45倍）.免疫组化实验结果表明,吸入SO ₂后,大鼠肝中p53和bax蛋白表达水平呈剂量依赖性增加,而bcl-2蛋白表达水平呈剂量依赖性降低.结果表明,SO2可以改变凋亡相关基因的表达,诱导大鼠肝组织细胞凋亡,这可能与一些凋亡相关疾病的发生有关.     

LCB1基因表达对酵母神经酰胺合成的影响

酿酒酵母(Saccharomyces cerevisiae)LCB1（Long chain base）基因被克隆到酵母诱导表达载体pYES2中,并转入到FY2中,用半乳糖诱导表达。与对照相比,质粒所含LCB1基因的表达,使酵母细胞干重略有下降,而神经酰胺的含量提高为对照的1.9倍。     

Twist对小鼠乳腺癌细胞基因表达谱的调控研究

摘要 Twist是一个bHLH（basic Helix-loop-Helix）类型的转录因子,近年来研究发现,Twist在乳腺癌中的表达显著升高,并能促进乳腺癌的转移。为了探索Twist促进乳腺癌转移的分子机制,本文采用RNA干扰技术在小鼠乳腺癌细胞株4T1中沉默Twist的表达,通过全基因组基因芯片技术检测了Twist沉默前后4T1细胞基因表达谱的差异性。体内实验结果证明Twist表达被沉默后4T1细胞的肺转移能力明显被抑制。芯片结果表明：表达差异显著的基因有167条,其中与肿瘤相关的基因有26条,包括15条上调基因和11条下调基因。这些基因中可能存在能被Twist调控并与肿瘤转移相关的基因,为以后研究Twist影响乳腺癌转移的分子机制提供了帮助。     

乏氧诱导因子-1α基因沉默对肺癌细胞乏氧应答相关基因表达的影响

乏氧诱导因子-1α (HIF-1α)是肿瘤细胞适应乏氧微环境的关键调控因子,具有作为治疗靶基因的潜力,以克服乏氧诱导的治疗抗拒等效应.下调其表达可能影响肿瘤细胞内一系列乏氧应答相关基因的表达.本研究采用已构建的HIF-1α RNAi慢病毒载体转导肺腺癌A549细胞,经杀稻瘟素（blasticidin）筛选建立HIF-1α基因稳定沉默的A549细胞株.应用cDNA微阵列技术检测并比较HIF-1α基因沉默A549细胞株和其亲本细胞株在常氧和乏氧状态下的基因表达谱改变. 应用定量RT PCR方法验证部分cDNA芯片差异表达基因的表达改变.HIF-1α基因稳定沉默细胞株A549/HIF-1α,在常氧和乏氧条件下HIF-1αmRNA水平分别较A549细胞下降89.2％和88.1％,HIF-1α蛋白水平分别下降97.2％和88.4％. 在乏氧条件下,cDNA微阵列检测的1 280个基因中,52个基因表达上调,15个基因表达下调. HIF-1α基因沉默显著影响其中27个基因的乏氧诱导效应.定量RT-PCR验证其中ENO2、BCL-2、CXCR4和MMP11的表达水平,与cDNA芯片结果相符合.结果提示,HIF-1α基因沉默能够在一定程度上阻断肺癌细胞的乏氧应答,在克服乏氧导致的肺癌治疗抗拒方面具有潜力.     

PTEN deletion leads to up-regulation of a secreted growth factor pleiotrophin

Tumor suppressor gene PTEN is highly mutated in a wide variety of human tumors. To identify unknown targets or signal transduction pathways that are regulated by PTEN, microarray analysis was performed to compare the gene expression profiles of Pten null mouse embryonic fibroblasts (MEFs) cell lines and their isogenic counterparts. Expression of a heparin binding growth factor, pleiotrophin (Ptn), was found to be up-regulated in Pten-/- MEFs as well as Pten null mammary tumors. Further experiments revealed that Ptn expression is regulated by the PTEN-PI3K-AKT pathway. Knocking down the expression of Ptn by small interfering RNA resulted in the reduction of Akt and GSK-3beta phosphorylation and suppression of the growth and the tumorigenicity of Pten null MEFs. Our results suggest that PTN participates in tumorigenesis caused by PTEN loss and PTN may be a potential target for anticancer therapy, especially for those tumors with PTEN deficiencies.     

Macrophage activation and differentiation signals regulate schlafen-4 gene expression: evidence for Schlafen-4 as a modulator of myelopoiesis

Background

The ten mouse and six human members of the Schlafen (Slfn) gene family all contain an AAA domain. Little is known of their function, but previous studies suggest roles in immune cell development. In this report, we assessed Slfn regulation and function in macrophages, which are key cellular regulators of innate immunity.

Methodology/Principal Findings

Multiple members of the Slfn family were up-regulated in mouse bone marrow-derived macrophages (BMM) by the Toll-like Receptor (TLR)4 agonist lipopolysaccharide (LPS), the TLR3 agonist Poly(I∶C), and in disease-affected joints in the collagen-induced model of rheumatoid arthritis. Of these, the most inducible was Slfn4. TLR agonists that signal exclusively through the MyD88 adaptor protein had more modest effects on Slfn4 mRNA levels, thus implicating MyD88-independent signalling and autocrine interferon (IFN)-β in inducible expression. This was supported by the substantial reduction in basal and LPS-induced Slfn4 mRNA expression in IFNAR-1^−/− BMM. LPS causes growth arrest in macrophages, and other Slfn family genes have been implicated in growth control. Slfn4 mRNA levels were repressed during macrophage colony-stimulating factor (CSF-1)-mediated differentiation of bone marrow progenitors into BMM. To determine the role of Slfn4 in vivo, we over-expressed the gene specifically in macrophages in mice using a csf1r promoter-driven binary expression system. Transgenic over-expression of Slfn4 in myeloid cells did not alter macrophage colony formation or proliferation in vitro. Monocyte numbers, as well as inflammatory macrophages recruited to the peritoneal cavity, were reduced in transgenic mice that specifically over-expressed Slfn4, while macrophage numbers and hematopoietic activity were increased in the livers and spleens.

Conclusions

Slfn4 mRNA levels were up-regulated during macrophage activation but down-regulated during differentiation. Constitutive Slfn4 expression in the myeloid lineage in vivo perturbs myelopoiesis. We hypothesise that the down-regulation of Slfn4 gene expression during macrophage differentiation is a necessary step in development of this lineage.     

Midkine, a newly discovered regulator of the renin-angiotensin pathway in mouse aorta: significance of the pleiotrophin/midkine developmental gene family in angiotensin II signaling

We previously demonstrated that pleiotrophin (PTN the protein, Ptn the gene) highly regulates the levels of expression of the genes encoding the proteins of the renin-angiotensin pathway in mouse aorta. We now demonstrate that the levels of expression of these same genes are significantly regulated in mouse aorta by the PTN family member midkine (MK the protein, Mk the gene); a 3-fold increase in expression of renin, an 82-fold increase in angiotensinogen, a 6-fold decrease in the angiotensin converting enzyme, and a 6.5-fold increase in the angiotensin II type 1 and a 9-fold increase in the angiotensin II type 2 receptor mRNAs were found in Mk-/- mouse aorta in comparison with the wild type (WT, +/+). The results in Mk-/- mice are remarkably similar to those previously reported in Ptn-/- mouse aorta, with the single exception of that the levels of the angiotensinogen gene expression in Ptn-/- mice are equal to those in WT+/+ mouse aorta, and thus, in contrast to Mk gene expression unaffected by levels of Ptn gene expression. The data indicate that MK and PTN share striking but not complete functional redundancy. These data support potentially high levels importance of MK and the MK/PTN developmental gene family in downstream signals initiated by angiotensin II either in development or in the many pathological conditions in which MK expression levels are increased, such as atherosclerosis and many human neoplasms that acquire constitutive endogenous Mk gene expression by mutation during tumor progression and potentially provide a target through the renin-angiotensin pathway to treat advanced malignancies.     

Expression and Regulatory Effects of Murine Schlafen (Slfn) Genes in Malignant Melanoma and Renal Cell Carcinoma

There is emerging evidence that the IFN-inducible family of Slfn genes and proteins play important roles in cell cycle progression and control of cellular proliferation, but the precise functional roles of different Slfn members in the regulation of tumorigenesis remain unclear. In the present study, we undertook a systematic analysis on the expression and functional relevance of different mouse Slfn genes in malignant melanoma and renal cell carcinoma cells. Our studies demonstrate that several mouse Slfn genes are up-regulated in response to IFN treatment of mouse melanoma and renal cell carcinoma cells, including Slfn1, Slfn2, Slfn4, Slfn5, and Slfn8. Our data show that Slfn2 and Slfn3 play essential roles in the control of mouse malignant melanoma cell proliferation and/or anchorage-independent growth, suggesting key and non-overlapping roles for these genes in the control of malignant melanoma tumorigenesis. In renal cell carcinoma cells, in addition to Slfn2 and Slfn3, Slfn5 also exhibits important antineoplastic effects. Altogether, our findings indicate important functions for distinct mouse Slfn genes in the control of tumorigenesis and provide evidence for differential involvement of distinct members of this gene family in controlling tumorigenesis. They also raise the potential of future therapeutic approaches involving modulation of expression of members of this family of genes in malignant melanoma and renal cell carcinoma.     

Identification of the angiogenesis signaling domain in pleiotrophin defines a mechanism of the angiogenic switch

Neoplasms progress through genetic and epigenetic mutations that deregulate pathways in the malignant cell that stimulate more aggressive growth of the malignant cell itself and/or remodel the tumor microenvironment to support the developing tumor mass. The appearance of new blood vessels in malignant tumors is known as the "angiogenic switch." The angiogenic switch triggers a stage of rapid tumor growth supported by extensive tumor angiogenesis and a more aggressive tumor phenotype and its onset is a poor prognostic indicator for host survival. Identification of the factors that stimulate the angiogenic switch thus is of high importance. Pleiotrophin (PTN the protein, Ptn the gene) is an angiogenic factor and the Ptn gene has been found to be constitutively expressed in many human tumors of different cell types. These studies use a nude mouse model to test if Ptn constitutively expressed in premalignant cells is sufficient to trigger an angiogenic switch in vivo. We introduced an ectopic Ptn gene into "premalignant" SW-13 cells and analyzed the phenotype of SW-13 Ptn cell tumor implants in the flanks of nude mice. SW-13 Ptn cell subcutaneous tumor implants grew very rapidly and had a striking increase in the density of new blood vessels compared to the SW-13 cell tumor implants, suggesting that constitutive PTN signaling in the premalignant SW-13 cell implants in the nude mouse recapitulates fully the angiogenic switch. It was found also that ectopic expression of the C-terminal domain of PTN in SW-13 cell implants was equally effective in initiating an angiogenic switch as the full-length PTN whereas implants of SW-13 cells in nude mice that express the N-terminal domain of PTN grew rapidly but failed to develop tumor angiogenesis. The data suggest the possibility that mutations that activate Ptn in premalignant cells are sufficient to stimulate an angiogenic switch in vivo and, since these mutations are frequently found in human malignancies, that constitutive PTN signaling may be an important contributor to progression of human tumors. The data also suggest that the C-terminal and the N-terminal domains of PTN equally initiate switches in premalignant cells to cells of a more aggressive tumor phenotype but the separate domains of PTN signal different mechanisms and perhaps signal through activation of a separate receptor-like protein.     

Pleiotrophin: a cytokine with diverse functions and a novel signaling pathway.

Pleiotrophin (PTN the protein, Ptn the gene) is a 136 amino acid secreted heparin-binding cytokine that signals diverse functions, including lineage-specific differentiation of glial progenitor cells, neurite outgrowth, and angiogenesis. Pleiotrophin gene expression is found in cells in early differentiation during different development periods and upregulated in cells with an early differentiation phenotype in wound repair. The Ptn gene is a protooncogene. It is strongly expressed in different human tumor cells and expression of the Ptn gene in tumor cells in vivo accelerates growth and stimulates tumor angiogenesis. Separate independent domains have been identified in PTN to signal transformation and tumor angiogenesis. Pleiotrophin is the first ligand of any of the known transmembrane tyrosine phosphatases. Pleiotrophin inactivates the receptor protein tyrosine phosphatase (RPTP) beta/zeta. The interaction of PTN and RPTP beta/zeta increases steady-state tyrosine phosphorylation of beta-catenin. Pleiotrophin thus regulates both normal cell functions and different pathological conditions at many levels. It signals these functions through a transmembrane tyrosine phosphatase.     

Identification of MrgX2 as a human G-protein-coupled receptor for proadrenomedullin N-terminal peptides

Pleiotrophin (PTN) is a heparin-binding growth/differentiation inducing cytokine that shares 50% amino acid sequence identity and striking domain homology with Midkine (MK), the only other member of the Ptn/Mk developmental gene family. The Ptn gene is expressed in sites of early vascular development in embryos and in healing wounds and its constitutive expression in many human tumors is associated with an angiogenic phenotype, suggesting that PTN has an important role in angiogenesis during development and in wound repair and advanced malignancies. To directly test whether PTN is angiogenic in vivo, we injected a plasmid to express PTN into ischemic myocardium in rats. Pleiotrophin stimulated statistically significant increases in both normal appearing new capillaries and arterioles each of which had readily detectable levels of the arteriole marker, smooth muscle cell alpha-actin. Furthermore, the newly formed blood vessels were shown to interconnect with the existent coronary vascular system. The results of these studies demonstrate directly that PTN is an effective angiogenic agent in vivo able to initiate new vessel formation that is both normal in appearance and function. The data suggest that PTN signals the more "complete" new blood vessel formation through its ability to stimulate different functions in different cell types not limited to the endothelial cell.