DAB2IP 基因的研究进展

Human DAB2 interaction protein (DAB2IP) is a novel member of Ras GTPase-activating protein family. It interacts directly with disabled-2 protein (DAB2/DOC2) which suppresses growth of cancers derived from different tissues, including mammary, prostate and ovarian cancers. DAB2IP was identified as an immediate downstream effector mediated by DAB2/DOC2. DAB2IP and DAB2/DOC2 form a unique protein complex that has a negative regulatory effect on the Ras-mediated signal pathway. It is demonstrated that DAB2IP is a tumor suppressor gene inactivated by methylation in several cancers. This article reviews the structure and biological functions of DAB2IP gene as well as its potential roles in carcinogenesis and evolution.     

DAB2IP基因与肿瘤

DAB2IP是新发现的抑癌基因,是Ras-GTP酶激活蛋白家族的新成员。它通过介导多种肿瘤相关信号通路,如MAPK-ERK,PI3K-Akt,ASK1-JNK和GSK-3β-β-catenin等,影响细胞的增殖、存活和凋亡。研究发现DAB2IP在多种肿瘤中表达下调,包括前列腺癌、乳腺癌、肺癌、胃肠肿瘤、肝细胞癌以及成神经管细胞瘤等,并且与肿瘤的进展及预后密切相关。本文拟对当前关于DAB2IP与肿瘤相关性的研究文献进行汇总综述,讨论DAB2IP作为分子标志物判断肿瘤恶性进展以及治疗预后的可行性。     

辐射敏感性启动子调控CD基因表达联合125I照射对膀肤癌EJ细胞的杀伤作用

以脂质体介导的辐射敏感性基因联合胞嘧啶脱氨酶(cytosine deantinase,CD)基因转染膀胱癌EJ细胞,研究放射性核素125 I照射后5-氟胞嘧啶(5-fluorocytosine,5-FC)对转染膀胧癌EJ细胞的杀伤作用.人工合成辐射敏感性启动子E8,将启动子克隆至质粒pCD2的CD基因上游,构建以E8为...     

辐射敏感性启动子调控CD基因表达联合125I照射对膀肤癌EJ细胞的杀伤作用

以脂质体介导的辐射敏感性基因联合胞嘧啶脱氨酶(cytosine deantinase,CD)基因转染膀胱癌EJ细胞,研究放射性核素125 I照射后5-氟胞嘧啶(5-fluorocytosine,5-FC)对转染膀胧癌EJ细胞的杀伤作用.人工合成辐射敏感性启动子E8,将启动子克隆至质粒pCD2的CD基因上游,构建以E8为启动子、CD基因为目的基因的新质粒,并采用DNA测序法测定E8和CD基因的序列;脂质体Lipofectamine2000介导pE8-CD转染膀胧癌EJ细胞,用[3]I照射(吸收剂量为2舜)后,蛋白质免疫印迹分析(Western blot)测定CD蛋白表达;在转染EJ细胞中分别加人不同剂量125 I勺和5-FC,四哇盐比色法(MTT法)测定各组细胞存活率,并以未经125 I勺照射组、未加5-FC组和5-氟尿啼吮(5-FU)组(阳性对照组)进行对照.DNA测序显示构建的pE8-CD质粒含E8启动子及CD基因序列;Westem blot可检测到CD基因表达;125 I加5-FC组细胞存活率明显低于未经125 I照射组及未加5-FC组,与5-FU组相近.这表明放射性核素与基因治疗联合对肿瘤细胞具有协同杀伤作用.     

免疫相关基因在同一膀胱癌亚克隆细胞株中的差异表达

目的:筛选和鉴定同一膀胱癌亚克隆细胞株中差异表达的与BCG免疫相关的基因。方法:以已建立的两株同一来源、不同生物学表型的膀胱移行细胞癌细胞系BLX和BLS211为材料,采用抑制性削减杂交技术(SSH)筛选差异表达的基因片段。结果:在BLX细胞中获得9个高表达的基因,在BLS211细胞中获得15个高表达的基因。BLX细胞中有3个基因,即与卡介苗(BCG)免疫治疗相关的基因BCG诱导的膜蛋白(BIGM103)基因、纤维连接蛋白(FN)基因和补体因子B(BF)基因;BLS211细胞中有8个基因均为新的表达序列标签(EST),已被GenBank登录(登录号为DY50570813,DY2304478)。结论:SSH技术筛选表明,BIGM103等免疫相关基因的不同,可能与不同膀胱癌细胞对BCG免疫治疗的差异性有关;新的EST的获得,为进一步克隆全长、研究基因的功能创造了条件。     

FAK基因沉默增强白血病细胞对化疗药物敏感性

目的: 本实验研究黏着斑激酶(focal adhesion kinase, FAK)基因沉默对白血病细胞化疗药物敏感性的影响。方法: 构建FAK shRNA慢病毒载体,转染至BCR/ABL-BaF3白血病细胞株,通过蛋白质印迹方法检测FAK蛋白表达情况。 体外应用不同浓度的化疗药物伊马替尼处理BCR/ABL-BaF3细胞,予Annexin V标记检测细胞凋亡情况。建立白血病动物模型,联合应用FAK shRNA及伊马替尼进行处理,观察小鼠生存情况,分析白血病细胞在小鼠骨髓及脾脏的分布情况。结果: 成功建立FAK shRNA慢病毒载体,该载体能有效沉默FAK基因表达。体外实验发现5 μmol/L 伊马替尼处理时,空白对照组与FAK基因沉默组中Annexin V阳性细胞百分比分别为(9.76±1.97%和(21.90±3.20%,差异显著(P<0.05);予50 μmol/L 伊马替尼处理时,2组中Annexin V阳性细胞百分比分别为(56.10±6.00)%和(82.10±5.70)％,差异显著(P<0.05)。白血病动物实验中,与空白对照组相比,生存分析结果显示FAK基因沉默组小鼠的生存时间明显延长。白血病细胞输注后第60 d,与空白对照组相比,白血病细胞在FAK基因沉默组小鼠骨髓和脾脏的分布明显减少。结论: FAK基因沉默能明显增强白血病细胞对化疗药物敏感性,提示FAK基因沉默有望成为白血病治疗的新策略。     

CEA-HBD2重组基因在人结肠癌细胞中表达初步研究

目的　构建高表达人源化HBD2人结肠癌表达体系 .方法　构建人pLNCX2 -CEA信号肽—HBD2抗菌肽(成熟肽序列 )逆转录表达载体 ,DOTAP转染逆转录包装细胞 ,产生的含融合基因的侵袭性逆转录病毒 ,转染人结肠癌HCT116细胞 ,用免疫印迹法法鉴定转化细胞中HBD2蛋白的表达 .结果　正确构建了表达HBD2成熟肽的重组信号肽HBD2的人结肠癌表达体系 ,并出现正确的目的基因表达 .结论　此HBD2表达体系表达的特异性多肽经纯化后 ,可进行下一步检测等实验     

CEA-HBD2重组基因在人结肠癌细胞中表达初步研究

目的构建高表达人源化HBD2人结肠癌表达体系.方法构建人pLNCX2-CEA信号肽-HBD2抗菌肽(成熟肽序列)逆转录表达载体,DOTAP转染逆转录包装细胞,产生的含融合基因的侵袭性逆转录病毒,转染人结肠癌HCT116细胞,用免疫印迹法法鉴定转化细胞中HBD2蛋白的表达.结果正确构建了表达HBD2成熟肽的重组信号肽HBD2的人结肠癌表达体系,并出现正确的目的基因表达.结论此HBD2表达体系表达的特异性多肽经纯化后,可进行下一步检测等实验.     

Wip1基因沉默对结肠癌细胞化疗敏感性的影响

目的:观察靶向Wip1基因的特异性siRNA序列对结肠癌细胞Wip1基因的抑制效应,探讨Wip1基因沉默对结肠癌细胞化疗敏感性的影响。方法:将Wip1-811 siRNA转染入Wip1高表达的RKO结肠癌细胞株,采用real-time PCR法检测Wip1 mRNA的表达,采用Western blotting方法检测Wip1蛋白表达,采用MTS方法检测结肠癌细胞活力,流式细胞术检测细胞凋亡及细胞周期。结果:Wip1-811 siRNA明显抑制Wip1的表达。抑制Wip1基因表达后,转染组RKO结肠癌细胞对抗肿瘤药物的敏感性增加,5-氟尿嘧啶处理组RKO结肠癌细胞活力由(89.4±6.6)%降至(74.7±3.9)%(P0.05),奥沙利铂处理组细胞活力由(77.9±2.4)%降至(66.7±2.9)%(P0.05)。转染Wip1-811 siRNA后,5-氟尿嘧啶处理组细胞凋亡比例由(7.7±0.5)%升至(12.3±3.2)%(P0.05);奥沙利铂处理组细胞凋亡比例由(14.7±2.1)%升至(34.0±2.1)%(P0.05)。结论:沉默Wip1基因表达能增强结肠癌细胞的化疗敏感性。     

靶向调控Claudin-4基因表达介导DNA损伤修复增强膀胱癌细胞化疗敏感性的研究

目的 探究靶向下调紧密连接蛋白4(Claudin-4)基因表达对膀胱癌细胞DNA损伤修复及其化疗敏感性的影响。方法 采用si-Claudin-4及阴性对照(si-NC)转染人膀胱癌细胞系EJ下调Claudin-4的表达,采用qRT-PCR和Western blot检测细胞Claudin-4的表达,以确定转染效果。不同浓度顺铂（DDP）处理转染后的EJ细胞,CCK-8法检测细胞存活率。将EJ细胞随机分为对照组、si-Claudin-4组、DDP组、DDP+si-Claudin-4组。经转染处理与DDP处理后,CCK-8法检测各组细胞存活率;平板克隆形成实验检测各组细胞克隆形成能力;细胞免疫荧光染色观察各组细胞内γ-H2AX焦点生成情况;单细胞凝胶电泳法检测各组细胞DNA损伤情况;Western blot检测各组细胞内γ-H2AX、Ku70、Ku80以及DNA-PKcs的蛋白表达水平。结果 细胞转染后,si-Claudin-4组细胞内Claudin-4 mRNA和蛋白相对表达量均显著低于对照组和si-NC组（P<0.05）。经不同浓度DDP处理后,si-Claudin-4组EJ细胞存...     

DAB2IP with tumor-inhibiting activities exhibits frameshift mutations in gastrointestinal cancers

A scaffold protein DAB2 and its interaction partner DAB2IP have putative tumor suppressor gene (TSG) functions. Previous studies identified that both DAB2 and DAB2IP genes were inactivated by promoter hypermethylation in human cancers, but their mutational alterations in cancers remain largely unknown. The aim of our study was to find whether DAB2 and DAB2IP were mutated in gastric (GCs) and colorectal cancers (CRCs) by DNA sequencing. Both DAB2 and DAB2IP have mononucleotide repeats in their coding sequence that could be mutation targets in high microsatellite instability (MSI-H) cancers. We analyzed GC and CRC tissues and found that 8 of 34 GCs (23.5%) and 15 of 79 CRCs (20.0%) with MSI-H harbored DAB2IP frameshift mutations. DAB2 frameshift mutations were found in 2 of 79 CRCs (2.5%) with MSI-H. These mutations were not detected in microsatellite stable (MSS) cancers. We also found intratumoral heterogeneity (ITH) of DAB2IP frameshift mutations in 7 of 16 CRCs (43.8%). Loss of DAB2IP protein expression was found in approximately 20% of GCs and CRCs irrespective of MSI and DAB2IP frameshift mutation status. Our study shows that the TSG DAB2IP harbored frameshift mutations and ITH as well as expression loss. Together these tumor alterations might play a role in tumorigenesis of GC and CRC with MSI-H by down-regulating the tumor-inhibiting activities of DAB2IP.     

膀胱癌尿液差异蛋白鉴定及生物信息分析

目的对膀胱癌患者和正常对照尿液中的差异蛋白进行鉴定和生物信息分析。方法应用swiss-prot、ProtParam、Uniprot、Gene Ontology、KEGG等数据库对前期研究中筛选出的膀胱癌尿液差异蛋白分别从蛋白、基因、核酸水平进行基本理化性质、分子功能、参与生物进程、细胞组分、代谢途径等方面的综合分析。结果前期研究中筛选出的30个差异点鉴定出22个蛋白质,合并去重后共得到14个蛋白质,分别为:β2微球蛋白;脂肪细胞型脂肪酸结合蛋白;凝溶胶蛋白;凝溶胶蛋白亚型1;肌红蛋白;纤维蛋白原α链;载脂蛋白A-I;前列腺素合成酶;AMBP蛋白;转甲状腺素蛋白;角蛋白1;角蛋白8;推测蛋白白蛋白;推测蛋白甘露聚糖结合凝集素丝氨酸蛋白酶2。其中KRT8、KRT1、FGA、APOA1、FABP4、ALB、GSN、B2M、MASP2、TTR10个基因均具有蛋白结合的分子功能;PTGDS、ALB、TTR3个基因均具有转运蛋白活性的分子功能;ALB、MB2个基因具有氧结合功能,ALB还具有抗氧化活性。筛选的差异蛋白中大部分具有相似的本体注解,APOA1参与了膀胱癌分子通路途径中的PPAR信号通路。结论14个膀胱癌尿液差异蛋白具有各自不同的理化属性,其基因产物具有蛋白结合、转运蛋白活性、氧结合及抗氧化活性的分子功能;参与了转运和细胞骨架合成等生物进程。结合生物信息学分析和大量文献检索,为下一步的靶蛋白研究提供方向。     

非甾体类抗炎药对结肠癌细胞NAG-1 基因表达的诱导

研究非甾体类抗炎药(Non-steroidal anti-inflammatory drug,NSAID)对结肠癌细胞生长的影响及NSAID活化基因-1(NAG-1)的诱导作用。体外培养HT-29、SW480及LS174-T三种结肠癌细胞,分别加入不同浓度的aspirin、celecoxib及meloxicam作用于HT-29及SW480细胞,采用MTT法检测结肠癌细胞增殖;蛋白质印迹技术检测三种结肠癌细胞COX-2的表达;采用半定量RT-PCR技术分析NSAID对三种结肠癌细胞NAG-1基因表达的影响。aspirin、celecoxib及meloxicam均能有效抑制体外培养的HT-29、SW480结肠癌细胞生长,并具有良好的量-效关系。Western blot表明,HT-29细胞表达COX-2,而SW480细胞不表达COX-2。三种结肠癌细胞均表达NAG-1基因mRNA,其中LS174-T细胞NAG-1基础水平较低;NSAID能不同程度上调结肠癌细胞NAG-1基因表达。NSAID能有效抑制结肠癌细胞生长,这种作用可能部分通过诱导结肠癌细胞NAG-1基因表达实现,NAG-1基因表达不受肿瘤细胞是否表达COX-2的影响。     

血清miR-145、miR-29联合miR-217检测在膀胱癌的表达差异及价值分析

目的探讨血清微小RNA-145(microRNA-145,miR-145)、miR-29与miR-217检测在膀胱癌中的临床价值。方法选取2016年2月至2018年5月在我院经穿刺或术中病理切片确诊的膀胱癌患者80例,同期收取80例膀胱炎患者和80例健康体检者。应用实时荧光定量PCR(quantitative real-timePCR,RT-PCR)仪对血清miR-145、miR-29与miR-217的相对表达量进行检测。结果膀胱癌患者血清miR-145、miR-29和miR-217表达水平(检测结果经负对数转换后呈正态分布)显著低于膀胱炎患者和健康对照者(P<0.05),而膀胱炎患者和健康对照者间血清miR-145、miR-29和miR-217水平差异均无统计学意义(P>0.05)。血清miR-145、miR-29与miR-217表达水平与膀胱癌淋巴结转移及TNM分级呈线性关联(P<0.05)。ROC曲线分析显示,血清miR-145、miR-29和miR-217在区分膀胱癌与膀胱炎患者和健康对照的灵敏度/特异性分别为81.3%/82.9%、82.7%/89.7%和77.2%/83.3%;而联合三者后在区分膀胱癌与膀胱炎患者和健康对照的灵敏度显著提高,达到91.7%。二元Logistic回归分析显示,在校正了年龄、性别、BMI、吸烟、饮酒后,血清低miR-145、低miR-29和低miR-217是膀胱癌的独立危险因素。结论血清低miR-145、低miR-29和低miR-217水平对膀胱癌的诊断以及在评估膀胱癌患者发生肿瘤远处转移中均具有重要的临床意义。     

膀胱癌组织中MMP-2、MMP-9、Survivin、Livin和VEGF表达的临床意义分析

目的 探讨膀胱癌组织中MMP-2、MMP-9、Survivin、Livin和VEGF表达的临床意义.方法 应用RT-PCR方法检测51例膀胱癌手术切取标本中的MMP-2、MMP-9、Survivin、Livin和VEGF表达,并分析其临床意义.结果 膀胱癌组织中MMP-2、MMP-9、VEGF、Survivin、Livin表达水平均明显高于正常癌旁组织,组间比较差异有统计学意义,P＜0.05.随着膀胱癌分期及分级的增加,MMP-2、MMP-9、VEGF、Survivin、Livin表达水平均明显提高,且不同分级及分期间表达水平相比较差异有统计学意义(P＜0.05).结论 膀胱癌组织中存在多种基因的表达异常,其发生、发展以及转移与多基因的联合作用有着密切联系.     

Role of Cancer Stem-like Cells in the Process of Invasion and Mesenchymal Transformation by a Reconstituted Triple-negative Breast Cancer Cell Population Resistant to p53-induced Apoptosis

We investigated the role of cancer stem cells (CSCs) in a population of triple-negative breast cancer (TNBC) cells that are resistant to apoptosis. A human breast cancer cell population capable of inducing p53 expression with doxycycline (Dox) was created and used as an untreated control (UT). After the addition of Dox to UT for 5 days, the cell population reconstituted with cells showing resistance to apoptosis was named RE. Fluorescence-activated cell sorting (FACS) and immunostaining revealed that after the addition of Dox, the ratio of cells in the S and G2/M phases decreased in UT as apoptosis proceeded, but did not markedly change in apoptosis-resistant RE. CSC-like cells in RE exhibited a cell morphology with a larger ratio of the major/minor axis than UT. FACS showed that RE had a higher proportion of CSC-like cells and contained more CD44⁺CD24⁻ mesenchymal CSCs than ALDH1A3⁺ epithelial-like CSCs. In a Matrigel invasion assay, UT was more likely to form a three-dimensional cell population, whereas RE exhibited a planar population, higher migration ability, and the up-regulated expression of epithelial-mesenchymal transition-related genes. These results provide insights into the mechanisms by which TNBC cells acquire treatment resistance at the time of recurrence.     

Upregulation of MicroRNA-34a Sensitizes Ovarian Cancer Cells to Resveratrol by Targeting Bcl-2

PurposeResveratrol (REV), a natural compound found in red wine, exhibits antitumor activity in various cancers, including ovarian cancer (OC). However, its potential anti-tumor mechanisms in OC are not well characterized. Here, we tried to elucidate the underlying mechanisms of REV in OC cells.Materials and MethodsThe anti-proliferative effects of REV against OC cells were measured using CCK-8 assay. Apoptosis was measured using an Annexin V-FITC/PI apoptosis detection kit. The anti-metastasis effects of REV were evaluated by invasion assay and wound healing assay. The miRNA profiles in REV-treated cells were determined by microarray assay.ResultsOur results showed that REV treatment suppresses the proliferation, induces the apoptosis, and inhibits the invasion and migration of OV-90 and SKOV-3 cells. miR-34a was selected for further study due to its tumor suppressive roles in various human cancers. We found miR-34a overexpression enhanced the inhibitory effects of REV on OC cells, whereas miR-34a inhibition had the opposite effect in OC cells. In addition, we verified that BCL2, an anti-apoptotic gene, was found directly targeted by miR-34a. We also found that REV reduced the expression of Bcl-2 in OC cells. Further investigations revealed that overexpression of Bcl-2 significantly abolished the anti-tumor effects of REV on OC cells.ConclusionOverall, these results demonstrated that REV exerts anti-cancer effects on OC cells through an miR-34a/Bcl-2 axis, highlighting the therapeutic potential of REV for treatment of OC.     

Downregulation of AKT and MDM2, Melatonin Induces Apoptosis in AGS and MGC803 Cells

Melatonin, a neurohormone secreted by the pineal gland, has a variety of biological functions, such as circadian rhythms regulation, anti-oxidative activity, immunomodulatory effects, and anittumor, etc. At present, its antitumor effect has attracted people's attention due to its extensive tissue distribution, good tissue compatibility, and low toxic and side effects. In the gastrointestinal tract, there is high level of melatonin and many studies showed melatonin has effects of anti-gastric cancer. In this experiment, human gastric cancer cell lines AGS and MGC803 were used to investigate the intracellular molecular mechanism of melatonin against gastric cancer. After AGS and MGC803 have been treated with melatonin, the changes of cell morphology and cellular structure were observed under electron microscope. Flow cytometer and apoptosis detection kits were used to analyze the effect of apoptosis on AGS and MGC803. The alterations of apoptosis-related proteins Caspase 9, Caspase 3, and upstream regulators AKT, MDM2 including expression, phosphorylation, and activation were detected to analyze the intracellular molecular mechanism of melatonin inhibiting gastric cancer. In AGS and MGC803 cells with melatonin exposure, cleaved Caspase 9 was upregulated and Caspase 3 was activated; moreover, MDM2 and AKT expression and phosphorylation were downregulated. Melatonin promoted apoptosis of AGS and MGC803 cells by the downregulation of AKT and MDM2. Anat Rec, 302:1544–1551, 2019. © 2019 American Association for Anatomy