STAT3反义寡核苷酸对人肺腺癌放射治疗的增效作用

目的:评价STAT3反义寡核苷酸对人肺腺癌细胞A549增殖抑制作用及辐射增敏作用,探讨新的肺癌分子靶向治疗药物。方法:2003-10/2004-11于解放军第二军医大学放射医学教研室,应用阳离子脂质体介导STAT3反义寡核苷酸转染人肺腺癌A549细胞,westernblot检测STAT3总蛋白和p-STAT3蛋白的表达;MTT法检测细胞增殖状态;流式细胞术检测细胞周期;用克隆形成分析和SF2研究人肺腺癌细胞A549辐射敏感性的变化。结果:STAT3反义寡核苷酸转染人肺腺癌A549后STAT3蛋白的表达和磷酸化水平下降,细胞增殖受抑制,出现G1阻滞;照射后12d,反义寡核苷酸组测定SF2值是(46.78%±3.25%),较对照组(61.52%±2.74%)明显降低(P<0.01)。结论:STAT3反义寡核苷酸可以抑制STAT3蛋白表达和细胞增殖,对人肺腺癌细胞A549有辐射增敏作用。     

过表达SOCS5对非小细胞肺癌增殖、迁移和侵袭的作用及机制研究

摘要：目的?分析细胞因子信号传导抑制因子5(SOCS5)对非小细胞肺癌(NSCLC)细胞增殖、迁移和侵袭能力的影响，并探讨其可能的作用机制。方法?应用癌症基因组图谱(TCGA)数据库分析SOCS5在肺癌组织中的表达情况； western blot检测SOCS5在NSCLC细胞系PC-9、A549和SPC-A-1及正常支气管上皮细胞系16HBE中的表达水平；选择SOCS5表达水平最低的NSCLC细胞系进行SOCS5过表达质粒转染，实验设对照(NC)组和SOCS5过表达质粒转染(oe-SOCS5)组。采用MTS试验检测各组细胞增殖能力；划痕试验检测各组细胞迁移能力；Boyden试验检测各组细胞侵袭能力；western blot检测各组细胞中PI3K/Akt/mTOR信号通路活性。结果?TCGA数据库分析结果显示，SOCS5?mRNA在肺腺癌和肺鳞癌组织中的表达水平显著低于正常肺组织(P<0.05)。western blot结果显示，SOCS5蛋白在PC-9、A549和SPC-A-1细胞中的表达水平均低于其在16HBE细胞中的表达水平(P<0.05)；选择表达水平最低的PC-9细胞进行SOCS5过表达质粒转染，结果发现与NC组相比，oe-SOCS5组PC-9细胞增殖、迁移和侵袭能力均降低(P<0.05)；western blot结果显示，与NC组相比，oe-SOCS5组细胞pPI3K、pAkt和pmTOR蛋白的表达水平均降低(P<0.05)。结论?SOSC5在NSCLC中呈异常低表达，过表达SOCS5可抑制NSCLC细胞的增殖、迁移和侵袭能力，抑制PI3K/Akt/mTOR信号通路的激活可能是作用机制之一。     

SATB2基因siRNA对口腔鳞状细胞癌生长及STAT3信号的抑制作用

目的:探讨抑制SATB2基因表达对口腔鳞状细胞癌(oral squamous cell carcinoma,OSCC)细胞增殖、凋亡、侵袭和迁移的影响及机制。方法:以LipofectaminTM2000为载体,将阴性对照siRNA(阴性组)与SATB2特异性siRNA(抑制组)转染人OSCC细胞株CAL-27,设置空白组,CCK-8法检测siRNA转染4 d的细胞增殖;siRNA转染CAL-27细胞48 h,Western印迹法检测E-cadherin,STAT3,p-STAT3和cleaved caspase-3蛋白表达。克隆形成实验、Transwell小室及流式细胞术分别检测细胞克隆形成率、细胞侵袭和迁移能力及细胞凋亡率。结果:转染siRNA的CAL-27细胞SATB2蛋白表达明显低于空白组(P0.05)。与空白组相比,抑制组细胞增殖、侵袭和迁移能力均明显降低,细胞凋亡率明显升高,E-cadherin和cleaved caspase-3蛋白表达明显升高,p-STAT3蛋白表达明显降低(P0.05)。结论:RNA干扰抑制SATB2基因表达可抑制OSCC细胞生长,机制与下调STAT3信号有关。     

长链非编码RNA HMMR-AS1促进肺腺癌的恶性进展

目的探讨长链非编码RNA(long noncoding RNA, lncRNA)HMMR-AS1对肺腺癌(lung adenocarcinoma, LUAD)增殖转移的影响。方法采用实时荧光定量聚合酶链反应技术(RT-qPCR)检测LUAD细胞系中HMMR-AS1及其正义链HMMR的表达水平;通过小干扰RNA敲减HMMR-AS1的水平,并利用RT-qPCR检测转染效率及其对HMMR水平的影响;采用CCK-8法、克隆形成实验、细胞凋亡实验、划痕实验、Transwell侵袭实验等表型实验检测干扰HMMR-AS1的表达对A549和H1299细胞生物学功能的影响;western blot检测该两种细胞中HMMR-AS1水平降低对HMMR蛋白表达水平的影响。结果与正常肺上皮细胞BEAS-2A相比,LUAD细胞系A549和H1299中HMMR-AS1的表达水平分别上调了3.06倍和5.02倍(P0.05);转染小干扰RNA后A549和H1299细胞中HMMR-AS1的表达水平明显下降(P0.05),并且明显抑制HMMR的转录和蛋白质表达水平(P0.05);表型实验结果显示,与阴性对照组比较,敲减HMMR-AS1能够抑制LUAD细胞的生长、迁移和侵袭能力,并促进凋亡。结论 LncRNA HMMR-AS1能够促进LUAD细胞的生长、迁移和侵袭能力,影响肺腺癌的恶性进展。     

STAT3反义寡核苷酸对人肺腺癌放射治疗的增效作用

目的：评价STAT3反义寡核苷酸对人肺腺癌细胞A549增殖抑制作用及辐射增敏作用，探讨新的肺癌分子靶向治疗药物。方法：2003-10／2004-11于解放军第二军医大学放射医学教研室，应用阳离子脂质体介导STAT3反义寡核苷酸转染人肺腺癌A549细胞，westem blot检测STAT3总蛋白和p—STAT3蛋白的表达；MTT法检测细胞增殖状态；流式细胞术检测细胞周期；用克隆形成分析和SF2研究人肺腺癌细胞A549辐射敏感性的变化。结果：STAT3反义寡核苷酸转染人肺腺癌A549后STAT3蛋白的表达和磷酸化水平下降，细胞增殖受抑制，出现Gl阻滞；照射后12d．反义寡核苷酸组测定SF2值是(46．78％&;#177;3．25％)．较对照组(61．52％&;#177;2．74％)明显降低(P&;lt;0．01)。结论：STAT3反义寡核苷酸可以抑制STAT3蛋白表达和细胞增殖，对人肺腺癌细胞A549有辐射增敏作用。     

长链非编码RNA LINC00222对肺腺癌细胞增殖、迁移、侵袭和凋亡的影响及作用机制的研究

目的　检测肺腺癌组织及细胞系中LncRNA LINC00222表达,探究其对肺腺癌细胞增殖、迁移、侵袭和凋亡的影响和相关作用机制。方法　采用实时荧光定量PCR（qRT-PCR）法和Western blot 实验检测肺腺癌组织及细胞中LINC00222表达;构建LINC00222过表达载体,验证其转染效率;采用CCK-8法、Hoechst 33342/PI 染色法、划痕实验及Transwell实验分别检测过表达LINC00222对肺腺癌细胞增殖、凋亡、迁移和侵袭的影响;采用Western blot 法检测肺腺癌细胞中P-GSK-3β,GSK-3β蛋白及β-catenin核蛋白表达;采用荧光霉素基因实验及RIP实验验证探究LINC00222影响肺腺癌生物学行为的相关作用机制。结果　肺腺癌组织中LINC00222 mRNA和蛋白表达明显低于癌旁正常组织,差异有统计学意义（t=7.388,15.100,均P<0.001）;肺腺癌细胞系中LINC00222表达明显低于人正常肺胚细胞（F=21.926,P<0.001）。过表达LINC00222后,肺腺癌细胞增殖、迁移、侵袭能力明显抑制,细胞凋亡数目明显增多（P<0.01）。过表达 LINC00222后,GSK-3β磷酸化明显降低, GSK-3β催化活性明显增强,β-catenin 核转位受到抑制（P<0.01）。LINC00222靶向调控结合GSK-3β和GSK-3β蛋白共沉淀中LINC00222表达显著高于IgG蛋白共沉淀（P<0.05）。结论　肺腺癌中LncRNA LINC00222低表达,其过表达可抑制肺腺癌细胞的增殖、迁移及侵袭,促进细胞凋亡,可能与其调控GSK-3β催化活性,抑制β-catenin 核转位有关。     

miRNA-181c对人肺腺癌细胞系SPC-A1增殖和侵袭的影响

目的探讨miRNA-181c(miR-181c)在人肺腺癌组织中的表达及其对人肺腺癌细胞系SPC-A1增殖和侵袭的影响。方法收集37例肺腺癌患者组织标本,实时荧光定量PCR(qRT-PCR)检测肺腺癌组织及相应癌旁组织中miR-181c的表达水平。脂质体法将miR-181c模拟物和miR-181c抑制剂分别转染至SPC-A1细胞中,CCK-8增殖实验检测转染后SPC-A1细胞的增殖能力,Transwell法检测转染后SPC-A1细胞的侵袭能力。结果肺腺癌组织中miR-181c的表达水平[3.259(1.637,4.920)×10-3]高于癌旁组织[2.008(1.200,3.292)×10-3],差异有统计学意义(U=-641.0,P0.05)。miR-181c的表达水平与淋巴结转移密切相关(P0.05)。转染SPC-A1细胞120 h后,miR-181c模拟物转染组的细胞增殖能力高于于阴性对照组,差异有统计学意义(2.029±0.034 vs 1.476±0.071;t=7.044,P0.01);而miR-181c抑制剂转染组细胞的增殖能力弱于阴性对照组,差异有统计学意义(0.998±0.050 vs 1.414±0.058;t=5.461,P0.01)。Transwell结果显示,转染miR-181c模拟物后SPC-A1侵袭能力显著增强(432.00±12.22 vs 219.50±9.31;t=14.11,P0.01),转染抑制剂后侵袭能力显著减弱(73.60±5.32 vs 227.30±11.27;t=11.52,P0.01)。结论 miR-181c在肺腺癌组织中高表达,并能促进SPC-A1细胞的增殖与侵袭。     

羧肽酶A4活化STAT3促进肝癌细胞增殖、迁移和侵袭

目的探究肿瘤相关基因羧肽酶A4(CPA4)对肝癌细胞增殖、迁移和侵袭的影响及其相关分子机制。方法慢病毒载体构建MHCC97L-CPA4过表达细胞系和MHCC97H-shCPA4敲减细胞系,分别以MHCC97L-vector和MHCC97H-shNC细胞系作为对照。采用实时荧光定量PCR(qRT-PCR)和western blot检测各组细胞中CPA4 mRNA和蛋白质的表达;CCK8试验和克隆形成试验检测各组细胞的体外增殖能力;Transwell试验检测细胞的体外迁移、侵袭能力;western blot检测各组细胞STAT3/p-STAT3/c-myc的表达差异。结果与vector组相比,CPA4组MHCC97L细胞的增殖、迁移、侵袭能力均升高(P<0.05);与shNC组相比,shCPA4组MHCC97H细胞的增殖、迁移、侵袭能力均降低(P<0.05)。western blot结果显示,与vector组相比,CPA4组MHCC97L细胞p-STAT3、c-myc蛋白的表达水平升高(P<0.05);与shNC组相比,shCPA4组MHCC97H细胞p-STAT3、c-myc蛋白的表达水平降低(P<0.05)。结论CPA4可能通过激活STAT3信号通路促进肝癌细胞的增殖、迁移和侵袭。     

血小板源性生长因子-DD对肾小球系膜细胞JAK/STAT信号途径的影响

目的 探讨血小板源性生长因子-DD(PDGF-DD)对肾小球系膜细胞JAK/STAT信号途径的影响.方法 将实验用人肾小球系膜细胞分成对照组、PDGF-DD组(20μg/L)和PDGF-DD+AG490组(20 μg/L PDGF-DD+10 μmol/L AG490).采用免疫组化、蛋白印迹法、反转录聚合酶链反应等方法观察PDGF-DD对肾小球系膜细胞p-JAK2、STAT3、p-STAT3、SOCS1和SOCS3的蛋白及mRNA表达的影响.结果 PDGF-DD刺激后肾小球系膜细胞p-JAK2、STAT3、p-STAT3、SOCS1和SOCS3表达增强,AG490抑制上述蛋白的表达(P＜0.05或＜0.01).结论 PDGF-DD能够激活肾小球系膜细胞JAK/STAT信号途径,促进系膜细胞增殖.     

TIGAR调节肺癌细胞的增殖和侵袭能力研究

目的探讨P53下游基因TIGAR在肺癌细胞A549增殖、迁移及侵袭中的作用。方法采用siRNA技术在A549细胞中干扰TIGAR的表达,细胞计数试剂盒(CCK-8)检测细胞增殖,小室法检测细胞迁移,肿瘤细胞侵袭实验检测细胞侵袭,免疫印迹杂交检测相关蛋白水平变化。结果在A549细胞中成功干扰TIGAR后,细胞增殖显著降低(P0.05),细胞迁移和侵袭能力显著减弱,侵袭相关蛋白基质金属蛋白酶2(MMP-2)和基质金属蛋白酶9(MMP-9)的表达量均下调。结论 TIGAR促进肺癌细胞A549的增殖,并促进细胞的迁移和侵袭能力。     

Selective repression of v-abl-encoded protein by N-methylisatin-beta-4',4'-diethylthiosemicarbazone and N-allylisatin-beta-4',4'-diallylthiosemicarbazone.

N-Methylisatin-beta-4',4'-diethylthiosemicarbazone (M-IBDET) and N-allylisatin-beta-4',4'-diallylthiosemicarbazone (A-IBDAT) selectively inhibited v-abl protein (P120), an oncogene product associated with tyrosine kinase activity. Concentrations of M-IBDET ranging between 0.17 and 0.64 microM and concentrations of A-IBDAT from 1.45 to 2.9 microM reduced tyrosine kinase activity significantly, whereas 0.64 microM M-IBDET and 2.9 microM A-IBDAT blocked P120 production. Cellular growth rate, protein production, and synthesis of p45 actin and p53 nuclear oncogene were not affected at these conditions. M-IBDET and A-IBDAT selectively suppress the v-abl oncogene as well as Moloney murine leukemia virus production.     

ATP-dependent transport of leukotrienes B4 and C4 by the multidrug resistance protein ABCC4 (MRP4)

The proinflammatory mediators leukotriene (LT) B(4) and LTC(4) must be transported out of cells before they can interact with LT receptors. Previously, we identified the multidrug resistance protein ABCC1 (MRP1) as an efflux pump for LTC(4). However, the molecular basis for the efflux of LTB(4) was unknown. Here, we demonstrate that human ABCC4 mediates the ATP-dependent efflux of LTB(4) in the presence of reduced glutathione (GSH), whereby the latter can be replaced by S-methyl GSH. Transport studies were performed with inside-out membrane vesicles from V79 fibroblasts and Sf9 insect cells that contained recombinant ABCC4, with vesicles from human platelets and myelomonocytic U937 cells, which were rich in endogenous ABCC4, but ABCC1 was below detectability. Moreover, human polymorphonuclear leukocytes contained ABCC4. K(m) values for LTB(4) were 5.2 muM with vesicles from fibroblasts and 5.6 muM with vesicles from platelets. ABCC4, with its broad substrate specificity, also functioned as an ATP-dependent efflux pump for LTC(4) with a K(m) of 0.13 muM in vesicles from fibroblasts and 0.32 muM in vesicles from platelets. However, GSH was not required for the transport of this glutathionylated leukotriene. The transport of LTC(4) by ABCC4 explains its release from platelets during transcellular synthesis. ATP-dependent transport of LTB(4) and LTC(4) by ABCC4 was inhibited by several organic anions, including S-decyl GSH, sulindac sulfide, and by the LTD(4) receptor antagonists montelukast and 3-(((3-(2-(7-chloro-2-quinolinyl)ethenyl)phenyl)-((3-dimethyl-amino-3-oxopropyl)-thio)-methyl)thio)propanoic acid (MK571). Thus, as an efflux pump for the proinflammatory mediators LTB(4) and LTC(4), ABCC4 may represent a novel target for anti-inflammatory therapies.     

Metabolism of leukotriene C4, D4 and E4 in allergic inflammation in rats

Using an allergic inflammation model of air pouch type in rats, levels of peptide-leukotriene (LT) C4, D4 and E4 in the pouch fluid were measured chromatographically, and peptide-LT metabolizing activities in the pouch fluid in the anaphylactic phase were examined. 10 min after injection of an antigen (azobenzene arsonate-conjugated acetyl bovine serum albumin) solution into a preformed air pouch on the back of the immunized rats, LTC4 level in the pouch fluid was the highest, followed by LTD4 and LTE4. At 30 min, the order of the level was reversed to LTE4 greater than LTD4 greater than LTC4, and total amount of peptide-LTs (LTC4 + LTD4 + LTE4) was the highest. Supernatant fraction of the pouch fluid collected 30 min after the antigenic challenge, converted [3H]LTC4 into [3H]LTD4, and [3H]LTD4 into [3H]LTE4 in time- and concentration-dependent manner. [3H]LTE4 was not metabolized under these conditions. Heat denaturation of the pouch fluid diminished the conversion of [3H]LTC4 into [3H]LTD4, and [3H]LTD4 into [3H]LTE4. In the granule fraction of purified mast cells, no metabolic activity of [3H]LTs was found. In intact mast cells as well as degranulating mast cells, a small but significant amount of [3H]LTC4 was metabolized into [3H]LTD4 and [3H]LTE4. In contrast, rat serum showed potent metabolizing activities of peptide-LTs. Since plasma exudation into the pouch is very prominent in the anaphylactic phase in this model, peptide-LT metabolizing activities in the pouch fluid are suggested to be attributable to plasma leaked into the pouch during the anaphylactic phase.