Wnt/β-catenin信号通路对靶基因转录的调控

Wnt/β-catenin信号通路又被称为经典Wnt信号通路,在早期胚胎发育、成体组织稳态维持、干细胞干性调控和肿瘤发生等过程中均发挥重要作用.经典Wnt信号通路的核心信号转导因子β-catenin与核内转录因子TCF/LEF家族成员结合后,通过募集或替换一系列协同作用因子,诱导染色质结构变化,调控Wnt信号靶基因的转录.本文将从Wnt信号靶基因转录调控的基本模式、分子机制、表观遗传学调控和意义等方面,总结近年来有关Wnt信号靶基因转录调控的研究成果,方便读者更好地理解Wnt信号通路靶基因的转录调控.     

Nodal/Smad2靶基因的系统鉴定及其在早期胚胎发育中的功能机制

Nodal/Smad2信号通路在脊椎动物胚胎中内胚层诱导及其背腹分化中发挥着主导作用,但是在胚胎早期发育中,Nodal/Smad2信号调控哪些靶基因表达,这些靶基因如何在Nodal/Smad2信号下游发挥作用,人们仍然所知甚少。以国家自然科学基金委员会"细胞编程与重编程的表观遗传机制"重大研究计划为依托,王强实验室在全基因组水平上对斑马鱼胚胎原肠早期Nodal/Smad2信号通路的靶基因进行了系统鉴定,并通过分析Smad2结合区域的其他转录因子保守的结合序列的出现频率,鉴定了一批潜在的Smad2的协同转录因子。研究发现,Nodal/Smad2的靶基因主要由转录因子、发育相关基因及重要信号通路的调控因子组成,其中F-actin捆绑蛋白Fascin1a和鸟核苷酸交换因子Net1分别通过调控受体内吞与Smad2转录活性反馈调控Nodal信号转导和中内胚层形成,而BPTF做为Smad2协同转录因子,通过调节核小体滑动来调控wnt8a表达,在中枢神经系统后部化过程中发挥重要作用。相关研究工作构建了Nodal/Smad2信号在斑马鱼中内胚层诱导及体轴建立中的分子网络,为理解脊椎动物早期胚胎发育过程中的基因表达调控机制提供了有意义的线索。     

新型抗癌药物分子靶标STAT3的研究进展

信号转导与转录激活因子(STATs)是一类发挥信号转导和转录因子调节作用的蛋白质家族,它们可以作为信号转导分子和转录调节因子参与到细胞因子和生长因子对于正常细胞的调控作用中。STATs的异常激活,特别是STAT3激活,和多种人类恶性肿瘤相关联。相关的分子生物学和药理学模型的研究也已确认STAT3在肿瘤发生中的重要作用,这些工作为抗癌药物研发和治疗癌症提供了新的靶标。此外,结构性活化的STAT3突变体就足以诱导瘤原细胞的转化,并且进一步在体内形成肿瘤。结构性激活的STAT3信号通路常常伴随着一些基因如cyclinD1,c-Myc和Bcl-x的上调,同时也会破坏正常细胞生长与生存的调控机制。体外和体内的实验研究结果也证明,对于STAT3信号通路结构性的阻断可以导致STAT3高表达肿瘤类型中的细胞生长抑制和凋亡。这种已被证实了的肿瘤细胞内的结构性激活和生长存活之间的相互联系,为癌症治疗提供了广阔的应用前景。近年来针对STAT3抑制剂的研究逐渐成为热点,本文就此作一综述。     

不同牛种STAT5B基因启动子多态性分析

目的:试验旨在筛选STAT5B基因启动子区SNP及研究其对启动子功能元件的影响,为地方牛种选种选育提供一定理论依据.方法:选择品种差异较大的贵州荷斯坦奶牛和务川黑牛构建不同DNA池,直接测序筛选SNP位点.结果:STAT5B基因5'调控区及第1外显子存在3个SNPs位点,分别为:T-181C、C-31G、T+119C.生物信息学软件预测得到STAT5B基因核心启动子区和转录因子结合位点,SNP位点导致5个转录因子结合位点消失,而产生8个新的转录因子结合位点.软件未发现可能的CpG岛范围,但STAT5B基因RNA二级结构和最小自由能在突变后显著改变.结论:DNA池结合测序技术可快速筛选SNP位点,STAT5B启动子区存在功能性SNP位点.     

STAT3基因沉默降低小鼠乳腺癌细胞的体外迁移能力

信号转导子与转录活化子3(STAT3)是一个具有信号转导和转录调控双重功能的转录因子,有文献报道STAT3在乳腺癌中的表达显著升高,并能促进乳腺癌的转移。为了深入探索STAT3在肿瘤发生发展中的作用和影响乳腺癌转移的分子机制,采用RNA干扰技术在小鼠乳腺癌细胞株4T1中沉默STAT3的表达。MTT实验结果显示STAT3沉默对4T1细胞的增殖能力没有影响;细胞迁移实验结果表明STAT3表达被沉默后4T1细胞的迁移能力明显被抑制;定量PCR结果显示,STAT3基因沉默后4T1细胞中VEGF和IL-6的mRNA水平下降,E-cadherin表达上升,mosin表达下降;信号通路检测显示STAT3基因表达沉默后MAPK的活化明显降低。研究表明STAT3在小鼠乳腺癌细胞的迁移过程中发挥重要作用,为以STAT3基因为靶向的治疗提供了一定的实验依据。     

EB病毒潜伏膜蛋白1在鼻咽癌细胞中通过STAT3促进VEGF表达

探讨了EB病毒编码的潜伏膜蛋白1(LMP1)是否通过STAT3调控诱导血管内皮细胞生长因子(VEGF)的表达.利用蛋白质印迹的方法对HNE2、HNE2-LMP1以及瞬时转染STAT3显性负性突变体STAT3β的HNE2-LMP1细胞中VEGF含量进行检测,发现LMP1可以上调VEGF的表达,而STAT3β可以抑制VEGF的上调;利用LMP1可控表达细胞系tet-on-LMP1-HNE2进行LMP1时间和剂量诱导表达研究,发现VEGF可以随LMP1的动态表达而表达;将VEGF野生型报告基因和VEGF潜在的STAT3转录因子突变体报告基因与LMP1表达载体分别共转染研究发现,LMP1可以激活VEGF的转录,这种转录通过VEGF启动子区STAT3转录因子的结合位点发挥作用;电泳迁移率变动分析(EMSA)确证了STAT3的这种DNA位点的特异性活性.结果表明：EB病毒编码的LMP1 在鼻咽癌细胞中可以增加VEGF的转录和表达,并能通过VEGF启动子区STAT3转录因子结合位点发挥作用.     

TFIID在配子发生和早期胚胎发育过程中的作用

配子发生以及胚胎早期发育过程受严格且有序的基因表达调控。多种转录因子与靶基因结合,激活基因的时空特异性表达,实现受精卵全能性的获得,完成母型基因组转录调控向合子基因组转录调控的转变以及随后胚胎细胞的分化调节。研究表明,TFIID转录因子家族在这些关键阶段起重要作用,在基因转录调节的起始阶段,TFIID转录因子家族成员作为通用转录因子被招募到靶基因的启动子上,与其他转录因子共同形成转录前起始复合物,起始转录。该文总结了TFIID转录因子的结构、作用方式,以及在配子发生和早期胚胎发育中的调控作用。     

基于高覆盖(磷酸化)蛋白质组对人胚胎干细胞干性维持调控网络的解析

目的:对人胚胎干细胞H9的(磷酸化)蛋白质组进行鉴定和深入分析,探讨维持人胚胎干细胞干性的核心调控网络。方法:利用新近发表的TAFT磷酸化肽段富集策略和高精度质谱鉴定技术,采用无标定量方法对H9细胞(磷酸化)蛋白进行定量分析;结合MOTIFX、iGPS、IPA等多种生物信息学分析软件,分析H9细胞的磷酸化基序-激酶、转录因子-靶基因调控网络。结果:获得了目前高度覆盖的H9细胞(磷酸化)蛋白质组数据集,鉴定到8674种蛋白质,其中3898种能够发生磷酸化修饰,包括13 676条磷酸化肽段,高可信度(class 1)磷酸化位点有11 870种。与已有的H9细胞磷酸化蛋白质组数据相比,其中2247种磷酸化蛋白质和10 025种磷酸化位点是本研究新鉴定到的。基于基序和激酶预测分析,推导出与干性维持相关的已知转录调节因子的新的磷酸化修饰基序以及调控其发生磷酸化的激酶。结合多能性转录因子对靶基因的调控信息,构建了多能性调控分子磷酸化修饰及其转录调控的核心网络。此外,还对特定位点的磷酸化修饰水平及其对应蛋白的总体丰度水平进行了比较及功能分析。基于各自的百分位数,比较磷酸化修饰水平与其对应蛋白的总体丰度,发现具有高丰度但低磷酸化修饰水平的蛋白倾向于参与物质转换或物质运输等生物过程,而低丰度但高磷酸化水平的蛋白质倾向于参与信息转导或调控过程。结论:提供了人胚胎干细胞H9高覆盖的(磷酸化)蛋白质组数据,这些数据有助于深入了解蛋白质磷酸化修饰在胚胎干细胞干性维持中所发挥的重要作用,为胚胎干细胞基础和应用研究提供了宝贵的数据资源。     

转录因子Ets-1与β1,3 N-乙酰氨基葡萄糖基转移酶基因启动子结合活性分析

β1,3-N-乙酰氨基葡萄糖转移酶-2,-8（β3GnT-2, β3GnT-8）共同参与多聚N-乙酰氨基乳糖(［Galβ1→4GlcNAcβ1→3］n)的合成,从而使得细胞表面的相应糖链结 构延长进而影响细胞的恶性转化.已有研究表明,在全反式维甲酸诱导人白血病细胞株 HL-60分化过程中β3GnT-2,-8的表达上调,但其分子机制不明.本文旨在探讨ATRA诱导 HL-60分化过程中,转录因子Ets-1对β3GnT-2,-8表达调控的分子机制.采用10-6 mol/L ATRA 诱导人白血病细胞株HL-60向粒系分化,RT-PCR检测到细胞中Ets-1的表达 明显增加;进一步采用染色质免疫沉淀（ChIP）结合电泳迁移率变动实验（EMSA）检测 证实,有活化的Ets-1结合至β3GnT-2/-8基因调控区. 以上结果表明,转录因子Ets-1对 人白血病细胞株HL-60分化过程中β3GnT-2,-8基因有表达调控作用.     

Increased unbound retinol-binding protein 4 concentration induces apoptosis through receptor-mediated signaling

The increase of apo-/holo-retinol-binding protein 4 (RBP4) concentrations has been found in subjects with renal dysfunction and even in diabetic patients with microalbuminuria. Holo-RBP4 is recognized to possess cytoprotective function. Therefore, we supposed that the relative increase in apo-RBP4 might induce cell damage. In this study, we investigated the signal transduction that activated apoptosis in response to the increase of apo-/holo-RBP4 concentration. We found that increase of apo-/holo-RBP4 concentration ratio delayed the displacement of RBP4 with "stimulated by retinoic acid 6" (STRA6), enhanced Janus kinase 2 (JAK2)/STAT5 cascade, up-regulated adenylate cyclase 6 (AC6), increased cAMP, enhanced JNK1/p38 cascade, suppressed CRBP-I/RARα (cellular retinol-binding protein/retinoic acid receptor α) expression, and led to apoptosis in HK-2 and human umbilical vein endothelial cells. Furthermore, STRA6, JAK2, STAT5, JNK1, or p38 siRNA and cAMP-PKA inhibitor reversed the repression of CRBP-I/RARα and apoptosis in apo-RBP4 stimulation. In conclusion, this study indicates that the increase of apo-/holo-RBP4 concentration may influence STRA6 signaling, finally causing apoptosis.     

Testing potential developmental toxicants with a cytotoxicity assay based on human embryonic stem cells

Since the differentiation of embryonic stem cells mimics early development, these cells could potentially permit the detection of embryotoxicants which interfere with this process. Although reliable tests based on murine embryonic stem cells exist, no such methods are available for human embryonic stem (hES) cells. Nonetheless, to avoid the false classification of substances due to inter-species differences, human-relevant toxicity tests are needed. We therefore developed an assay based on three human cell types, representing different degrees of developmental maturation, namely, human foreskin fibroblasts, hES cell-derived progenitor cells, and pluripotent hES cells. A set of embryotoxicants for which existing in vivo data were available, namely, all-trans retinoic acid (ATRA), 13-cis retinoic acid (13CRA), valproic acid (VPA) and dimethyl sulphoxide (DMSO), were tested. 5-fluorouracil (5-FU) was used as a positive control, and saccharin as a negative control. Two methods were compared for the assessment of cell viability -- the determination of intracellular ATP content and of resazurin reduction. In addition, the protective capacity of basic fibroblast growth factor (bFGF) against retinoid-induced toxicity was investigated. This novel assay system reliably detected the embryotoxic potentials of the test substances, 5-FU, ATRA, 13-CRA (a substance that displays inter-species differences in its effects) and VPA. This was possible due to the apparent differences in the sensitivities of the human cell types used in the assay system. Thus, our results clearly indicate the advantages and relevance of using hES cells in in vitro developmental toxicity testing.     

LINC01433 promotes hepatocellular carcinoma progression via modulating the miR-1301/STAT3 axis

Long noncoding RNAs (lncRNAs) have been demonstrated to play significant roles in hepatocellular carcinoma (HCC) tumor progression. LINC01433 has been implicated in the progression of lung cancer. However, its biological role in HCC remains poorly understood. In our current study, we focused on the detailed mechanism of LINC01433 in HCC development. First, it was exhibited that LINC01433 was remarkably elevated in HCC cells, which indicated that LINC01433 was involved in HCC. Then, knockdown of LINC01433 was able to restrain HCC cell proliferation and cell colony formation and greatly induced cell apoptosis. On the contrary, overexpression of LINC01433 promoted HCC cell proliferation, increased cell colony formation, and enhanced cell invasion capacity. Subsequently, we found that miR-1301 was remarkably decreased in HCC cells, and it can serve as a target of LINC01433 according to bioinformatics analysis. In addition, the binding correlation between them was validated by performing RNA pull-down experiments and RIP assay. Moreover, STAT3 was predicted and validated as a target of miR-1301, and it was shown that miR-1301 mimics significantly suppressed STAT3 in HCC cells. Finally, in vivo models were established, and the results demonstrated that silencing of LINC01433 could repress HCC development through modulating miR-1301 and STAT3. Taken together, these results indicated in our study that LINC01433 participated in HCC progression through modulating the miR-1301/STAT3 axis and it might act as a novel biomarker in HCC diagnosis and treatment.     

Deregulation in STAT signaling is important for cutaneous T-cell lymphoma (CTCL) pathogenesis and cancer progression

Deregulation of STAT signaling has been implicated in the pathogenesis for a variety of cancers, including CTCL. Constitutive activation of STAT5 and STAT3 was observed in early and late stages of CTCL, respectively. In early stages, IL-2, IL-7 and IL-15 signaling via JAK1 and JAK3 kinases is believed to be responsible for activating STAT5, while in advanced stages development of IL-21 autocrine signaling is thought to be important for STAT3 activation. Recent molecular evidence further suggests that upregulation of STAT5 in early disease stages results in increased expression of oncogenic miR-155 microRNA that subsequently targets STAT4 expression on mRNA level. STAT4 signaling is known to be critical for T helper (Th) 1 phenotype differentiation and its loss results in a switch from Th1 to Th2 phenotype in malignant T cells. During this switch the expression of STAT6 is often upregulated in CTCL. In advanced stages, activation of STAT3 and STAT5 may become completely cytokine-independent and be driven only via constitutively active JAK1 and JAK3 kinases. Further research into the molecular pathogenesis of JAK/STAT signaling in this cancer may enable us to develop effective therapies for our patients.     

Intact JAK-STAT signaling pathway is a prerequisite for STAT1 to reinforce the expression of RIG-G gene

We previously reported that IRF-9/STAT2 functional interaction could drive the expression of retinoic acid-induced gene G (RIG-G), independently of STAT1 and the classical JAK–STAT pathway, providing a novel alternative pathway for interferons (IFN) to mediate their multiple biological properties. In addition, we also found that IRF-1 could regulate RIG-G induction as well as the expression of IRF-9 and STAT2 in some cases. But the mechanisms by which IRF-1 exerted its action remained to be elucidated. Here, we showed that STAT1 could significantly enhance the effects of the IRF-9/STAT2 complex or IRF-1 on RIG-G induction through an activated JAK–STAT pathway, though it was not essential for RIG-G expression. In STAT1-deficient U3A cells, IRF-1 could induce RIG-G expression via the IFN-stimulated response elements in the RIG-G gene promoter, but it failed to upregulate IRF-9 and STAT2 unless the U3A cells were reconstituted by exogenous STAT1. In STAT1-expressing cells, IRF-1 indirectly activated RIG-G expression through an IRF-9/STAT2-dependent manner. Taken together, we concluded that the expression of RIG-G was independent on the classical JAK–STAT pathway, but could be greatly increased by it. This work will be of great benefit to us for a better understanding of the mechanisms on RIG-G gene expression regulation.     

白血病抑制因子与胚胎干细胞

白血病抑制因子对细胞的生长和分化有多种作用,通过与其受体结合传导信号,gp130与LIF受体β链的结合激活JAK激酶(JAK1和JAK2),JAK激酶磷酸化STAT信号转录子,STAT3的磷酸化对于阻止体外培养的干细胞的分化具有十分重要的作用。     

Analysis of STAT4 expression in cutaneous T-cell lymphoma (CTCL) patients and patient-derived cell lines

Deregulation of STAT signaling has been implicated in the pathogenesis for a variety of cancers, including CTCL. Recent reports indicate that loss of STAT4 expression is an important prognostic marker for CTCL progression and is associated with the acquisition of T helper 2 cell phenotype by malignant cells. However, little is known about the molecular mechanism behind the downregulation of STAT4 in this cancer. In the current work we test the expression of STAT4 and STAT6 via RT-PCR and/or Western Blot in CTCL lesional skin samples and in immortalized patient-derived cell lines. In these malignant cell lines we correlate the expression of STAT4 and STAT6 with the T helper (Th) phenotype markers and test the effect of Histone Deacetylase (HDAC) inhibitors and siRNA-mediated knock down of miR-155 on STAT4 expression. Our findings demonstrate that STAT4 expression correlates with Th1 phenotype, while STAT6 is associated with the Th2 phenotype. Our results further document that STAT4 and STAT6 genes are inversely regulated in CTCL. Treatment with HDAC inhibitors upregulates STAT4 expression, while at the same time decreases STAT6 expression in MyLa cells. Also, siRNA-mediated knock down of miR-155 leads to upregulation in STAT4 expression in MyLa cells. In summary, our results suggest that loss of STAT4 expression and associated switch to Th2 phenotype during Mycosis Fungoides progression may be driven via aberrant histone acetylation and/or upregulation of oncogenic miR-155 microRNA.