藻蓝色素蛋白通过调控miR-642a-5p抑制LTEP-a2细胞的增殖和迁移

研究藻蓝色素蛋白抑制非小细胞肺癌LTEP-a2体外增殖迁移的机制.以LTEP-a2细胞为模型,采用高通量miRNA转录组学对藻蓝蛋白处理后细胞中差异表达的miRNA进行了筛选;对筛选出的差异miRNA,利用体外转染mimics的方法对细胞的增殖、迁移能力进行验证.研究结果显示,藻蓝蛋白处理LTEP-a2细胞后能够显著增加细胞内miR-642a-5p的表达水平;过表达miR-642a-5p能够显著抑制细胞的体外增殖、迁移能力;此外,藻蓝蛋白可以通过上调miR-642a-5p表达抑制核受体NF-κB信号通路,降低蛋白磷酸化水平,进而抑制LTEP-a2细胞的增殖迁移能力.研究能够为藻蓝色素蛋白的应用以及非小细胞肺癌的治疗提供一定的理论基础.      

全自动免疫组化筛查ALK基因融合非小细胞肺癌及其病理特征

目的探讨全自动免疫组化筛查间变性淋巴瘤激酶(ALK)基因融合非小细胞肺癌的临床特点及病理特征.方法选取经病理检查确诊的554例非小细胞肺癌组织,采用Ventana抗ALK试剂和全自动免疫组化(IHC)染色检测ALK状态,分析ALK基因融合非小细胞肺癌的临床特点和病理特征.结果本次研究的554例非小细胞肺癌患者组织中,共筛选出34例ALK阳性,占6.14%;年龄60岁的非小细胞肺癌患者ALK阳性率8.69%,明显高于年龄≥60岁的3.62%,差异具有显著统计学意义(P0.01);男性患者ALK阳性率6.71%高于女性5.21%,但差异无统计学意义(P0.05).组织形态学方面,34例ALK阳性非小细胞肺癌中28例为肺腺癌,6例为非肺腺癌.16例实体型为主腺癌合并黏液产生,7例腺泡型为主腺癌,1例为乳头型为主腺癌,4例为浸润性黏液腺癌,4例为鳞状细胞癌.EGFR基因突变检测显示:仅有1例合并该基因突变,其余均为野生型.9例IHC阳性样本,9例ALK基因融合非小细胞肺癌,9例IHC阴性样本经荧光原位杂交技术检测和RT-PCR检测均为阴性结果,6例IHC染色可能为阳性,经荧光原位杂交技术检测均显示为ALK融合阴性.结论 ALK基因融合肺癌是非小细胞肺癌一新的分子亚型,具有独特的临床表现和病理形态;Ventana抗ALK试剂和IHC染色是检测ALK阳性非小细胞肺癌首选方法,对提高该类型肺癌的检出率及个体化治疗具有重要意义.     

联合检测miRNA129与调节性T细胞对诊断结肠癌的临床意义

目的 探讨联合检测微小核糖核酸miRNA129与调节性T细胞(Treg)对诊断结肠癌的价值.方法 选取62例结肠癌患者为研究组,62例健康者为对照组,应用荧光定量PCR法检测miRNA129在两组患者中的相对表达量,同时应用流式细胞仪检测外周血中Treg细胞的百分数,评估miRNA129与Treg联合检测对诊断结肠癌的价值.结果 结肠癌组血清中miRNA 129的相对表达量高于健康组,差异具有统计学意义(P<0.05).同时,结肠癌组血清中Treg细胞百分数显著高于健康组,差异具有统计学意义(P<0.05).结论 miRNA129与Treg细胞在结肠癌患者血清中呈现高表达状态,miRNA129与Treg细胞可能成为结肠癌早期诊断的参考指标.     

105例肺癌患者外周血中miRNA-10b的表达

目的 探讨miRNA-10b在非小细胞肺癌(Non-small cell lung cancer,NSCLC)患者外周血的表达情况.方法 选取156例非小细胞肺癌患者,其中,105例列入实验组,51例排除入组,另选50例健康志愿人员为正常组.采用实时荧光定量PCR(RT-qPCR)检测两组外周血中miRNA-10b的表...     

miRNA-373在非小细胞型肺癌中的表达及其临床意义

目的:探讨非小细胞型肺癌石蜡包埋组织中miR-373的表达与临床病理因素之间的关系,及其在预测非小细胞型肺癌患者预后中的作用.方法:用微阵列芯片法和qRT-PCR检测非小细胞型肺癌石蜡包埋组织和癌旁组织中的miR-373表达.采用Kaplan-Meier法分析非小细胞型肺癌患者总生存期和无病生存期.结果:在原发非小细胞型肺癌中miR-373的表达下调,并用qRT-PCR进一步验证.另外,miR-373低表达与病理组织类型、分化程度、淋巴结转移、临床分期、血管浸润、复发时间和生存时间(OS)(P0.05)显著性相关.Kaplan-Meier分析结果表明,miR-373低表达与高表达患者OS和无病生存期(DFS)(P0.05)有显著性差异,miR-373低表达患者预后较差.多因素分析显示非小细胞型肺癌中miR-373的表达水平是预测OS和DFS(P0.05)的独立危险因素.结论:miR-373的表达与非小细胞型肺癌患者预后显著相关,miR-373可作为预测非小细胞型肺癌患者预后的独立标志物.     

儿童急性淋巴细胞性白血病复发期miRNA的表达特征研究

儿童急性淋巴细胞性白血病(Acute Lymphoblastic Leukemia,ALL)是儿童常见的恶性肿瘤.尽管已有研究报道miRNA在ALL发病机理中起重要调控作用,但其与ALL复发的关系目前还不完全清楚.本研究采用第二代高通量测序技术分别对一例B淋巴细胞ALL(B-ALL)和一例T淋巴细胞ALL(T-ALL)患儿初发和复发期的骨髓细胞进行miRNA检测,比较初发和复发期样本miRNA的表达差异.在T-ALL和B-ALL中分别鉴定出复发期较初发期表达发生明显变化的miRNA 168和123种.其中在T-ALL样本中,复发期表达上调的miRNA有115种,表达下调的有53种;而在B-ALL样本中,复发期表达上调的miRNA只有38种,表达下调的有85种.比较两种类型ALL,共有17种miRNA在两种类型中发生相同的改变.通过realtime RT-PCR验证,证实Hsa-miR-17在复发期ALL中明显升高,而Hsa-miR-10a则明显降低.     

CDK2在非小细胞肺癌组织中的表达

探讨CDK2在非小细胞肺癌组织中的表达与肺癌转移关系。将50例非小细胞肺癌组织分为转移组和非转移组,采用免疫组织化学和Western blot检测癌组织中CDK2蛋白的表达。结果表明:CDK2蛋白在肺癌细胞中主要位于细胞核。CDK2蛋白在肺癌组织中的表达水平显著高于癌旁组织(P0.05)。CDK2蛋白高水平表达与肺癌淋巴结转移呈正相关(P0.05),但与肿瘤类型无关(P0.05)。CDK2的过表达可能与肺癌的形成有关,并与淋巴转移有关。     

实时定量PCR检测B细胞恶性肿瘤中BCL11A基因的表达水平

目的:建立B细胞淋巴瘤/白血病BCL11A基因的定量检测方法,并分析其在B细胞恶性肿瘤中的表达水平。方法:利用实时定量RT-PCR分析B细胞淋巴瘤(18例)、B细胞性慢性淋巴细胞白血病(B-CLL,8例)、T细胞性急性淋巴细胞白血病(T-ALL,8例)和正常对照(15例)外周血单个核细胞(PBMC)中BCL11A基因的表达水平,以甘油醛-3-磷酸脱氢酶(GAPDH)基因作为内对照。结果:B-CLL组和B细胞淋巴瘤组患者PBMC中BCL11A表达水平均明显高于正常对照(P=0.000)和T-ALL组(P=0.000);T-ALL组和正常对照组BCL11A表达水平无显著差别(P=0.084);B-CLL组和B细胞淋巴瘤组BCL11A表达水平无显著差别(P=0.776)。在B细胞淋巴瘤不同的病例中,BCL11A表达水平有较大差异,其中最小值为0.04,最大值为9.70,中位值为1.00。结论:成功建立BCL11A基因的定量检测方法。     

Twist、E-cadherin和Vimentin在非小细胞肺癌中的表达及意义

目的:研究转录因子Twist、上皮性钙黏附素(E-Cadherin)和波形蛋白(Vimention)在非小细胞肺癌(NSCLC)中的表达,探讨其与非小细胞肺癌浸润转移及预后之间的关系.方法:采用SP免疫组织化学、Western blot和RT-PCR方法检测80例非小细胞肺癌组织Twist、E-Cadherin和vimention蛋白和mRNA,20例癌旁正常组织作对照.另采用免疫细胞化学法检测68例非小细胞肺癌所致胸腔积液中肿瘤细胞的表达.结果:Twist和Vimentin在NSCLC组织及NSCLC胸水标本中的表达显著高于癌旁正常肺组织(P<0.01);E-cadherin在NSCLC组织及NSCLC胸水标本中表达显著低于癌旁正常肺组织(P<0.01);NSCLC组织中Twist及E-cadherin表达均与组织分化,临床分期及淋巴结转移情况密切相关(P<0.01);并且Twist表达与E-cadherin表达降低(P<0.01)和Vimentin的表达上调(P<0.01)之间都存在相关性.结论:Twist蛋白在非小细胞肺癌组织及胸腔转移灶中过度表达,可能通过分别上调和下调Vimentin、E-ead-h...     

手术前后血管内皮生长因子、血小板活化因子水平与非小细胞肺癌的关系

目的：探讨非小细胞肺癌患者手术前、后血清血管内皮生长因子（VEGF）、外周血血小板活化标志物的动态变化规律及其与非小细胞肺癌（NSCLC）的关系。方法：用EUSA法检测120例非小细胞肺癌患者手术前、后血清VEGF水平和60例健康体检者VEGF水平；同时用流式细胞术（FCM）检测其外周血血小板活化标志物水平。结果：①NSCLC患者手术前、后VEGF水平和血小板活化标志物水平均显著高于正常对照组（P〈0．01）；②NSCLC患者手术后第7天血清VEGF水平显著高于术前和术后第1天水平（P〈0．01）；而血小板活化标志粉水平则相反；③NSCLC患者血清VEGF和外周血血小板活化标志物水平与有无淋巴结转移和TNM分期有密切关系（P〈0．05）。结论：NSCLC患者血清中VEGF和外周血血小板活化标志物水平都升高。且与NSCLC的分期有密切关系．它们可作为动态检测NSCLC患者病情进展、判断预后的拳考指标。     

CD4^＋CD25^＋调节性T细胞在肺癌患者外周血中的表达

目的探讨肺癌患者外周血中CD4^＋CD25^＋调节性T细胞的变化及其与临床病理因素的关系。方法用流式细胞术检测60例肺癌患者和60例健康对照者的CD4^＋CD25^＋调节性T细胞数量变化。结果肺癌患者外周血中CD4^＋CD25^＋调节性T细胞水平明显高于正常组（P〈0．05）,其变化与病理分期和是否有转移有关（P〈0．05）,而和性别、家族史、病理类型和肿瘤体积等无关。结论CD4^＋CD25^＋调节性T细胞可能在肿瘤免疫中发挥重要作用,导致和促进肺癌的发生和转移。     

联合检测血清中CYFRA21-1、NSE对肺癌诊断和预后的意义

目的 :评价肿瘤标记物CYFRA2 1- 1、NSE对肺癌诊断和预测预后的价值。方法 :将肺癌患者分为非小细胞性肺癌组 (NSCLC)和小细胞性肺癌组 (SCLC) ,良性疾病患者为对照组。采静脉血 ,按RIA方法检测CYFRA2 1- 1、NSE。采用STATA统计软件进行结果分析。结果 :CYFRA2 1- 1水平在NSCLC中明显高于SCLC和良性疾病患者 ,分别为 15 .45± 4.0 1、6.14± 3 .88、1.86± 0 .89。NSE则在SCLC中显著高于NSCLC和良性疾病组 ,分别为 3 .90± 1.47、2 .65± 1.47、2 .16± 0 .74。CYFRA2 1- 1、NSE联合检测与各单项指标相比 ,正确率增高 (P <0 .0 1) ,而且NSE是提示SCLC预后的有价值的指标 ,CYFRA2 1- 1则是提示NSCLC预后的有价值的指标。结论 :联合检测血清中CYFRA2 1- 1、NSE的含量诊断肺癌的阳性率最高 ,准确性达 87.41% ,同时显示 ,NSE、CYFRA2 1- 1分别是提示SCLC、NSCLC预后的有价值的独立指标 ,在临床上有重要的应用价值     

Cloning of aminopeptidase N promoter and its activity in hematopoietic cell and different tumor cell lines

Aminopeptidase N (APN) promoter region was cloned and sequenced from peripheral blood mononuclear cells. The recombinant reporter construct containing the promoter and luciferase gene, designated pXP1-APNLuc, was introduced into myeloblastic cell line, T lymphocyte cell line and various tumor cell lines. Luciferase assay showed that APN upstream promoter is myeloid-specific for high expression in myeloblastic cell line and much lower expression in T lymphocyte cell line. The promoter activity was relatively high in lung adenoma cell line compared with other tumor cell lines including hepatoma cell line, tong cancer cell line and esophageal cancer cell line in which the promoter activity significantly diminished or was almost undetectable. The characteristics of APN promoter may provide a new strategy for specific myeloprotection while tumor patients are being treated with chemotherapy and/or radiotherapy.     

AP-1及其相关基因uPA、uPAR在肺癌中的表达及其意义

研究AP-1及其相关基因uPA、uPAR在人肺癌中的表达及其与肺癌临床病理特征的关系.通过免疫组织化学方法检测101例肺癌组织及7例正常肺组织中AP-1、uPA、uPAR的表达情况,利用CMIAS2000型多功能真彩病理图像分析系统测量计算各病例中c-jun、c-fos、uPA、uPAR蛋白阳性细胞的平均光密度(DAO)和积分光密度(DIO).c-jun、c-fos在肺癌组织中表达的阳性率及其共表达率均显著高于正常肺组织;二者在NSCLC的表达显著强于SCLC;不同组织学类型肺癌中二者表达具有显著性差异,腺癌中表达最强,小细胞肺癌中表达最弱;c-jun、c-fos与肺癌分级、合并分期、淋巴结转移均呈显著正相关.uPA、uPAR与肺癌淋巴结转移密切相关,与合并分期呈显著正相关;uPAR与肺癌分级呈显著正相关;各指标之间均存在显著正相关关系.研究结果表明uPA、uPAR参与了肺癌侵袭转移,AP-1信号传导通路在该过程中起了重要调节作用.     

Isolation of rare circulating tumour cells in cancer patients by microchip technology

Viable tumour-derived epithelial cells (circulating tumour cells or CTCs) have been identified in peripheral blood from cancer patients and are probably the origin of intractable metastatic disease. Although extremely rare, CTCs represent a potential alternative to invasive biopsies as a source of tumour tissue for the detection, characterization and monitoring of non-haematologic cancers. The ability to identify, isolate, propagate and molecularly characterize CTC subpopulations could further the discovery of cancer stem cell biomarkers and expand the understanding of the biology of metastasis. Current strategies for isolating CTCs are limited to complex analytic approaches that generate very low yield and purity. Here we describe the development of a unique microfluidic platform (the 'CTC-chip') capable of efficient and selective separation of viable CTCs from peripheral whole blood samples, mediated by the interaction of target CTCs with antibody (EpCAM)-coated microposts under precisely controlled laminar flow conditions, and without requisite pre-labelling or processing of samples. The CTC-chip successfully identified CTCs in the peripheral blood of patients with metastatic lung, prostate, pancreatic, breast and colon cancer in 115 of 116 (99%) samples, with a range of 5-1,281 CTCs per ml and approximately 50% purity. In addition, CTCs were isolated in 7/7 patients with early-stage prostate cancer. Given the high sensitivity and specificity of the CTC-chip, we tested its potential utility in monitoring response to anti-cancer therapy. In a small cohort of patients with metastatic cancer undergoing systemic treatment, temporal changes in CTC numbers correlated reasonably well with the clinical course of disease as measured by standard radiographic methods. Thus, the CTC-chip provides a new and effective tool for accurate identification and measurement of CTCs in patients with cancer. It has broad implications in advancing both cancer biology research and clinical cancer management, including the detection, diagnosis and monitoring of cancer.     

A microRNA regulon that mediates endothelial recruitment and metastasis by cancer cells

Metastatic progression of cancer is a complex and clinically daunting process. We previously identified a set of human microRNAs (miRNAs) that robustly suppress breast cancer metastasis to lung and bone and which display expression levels that predict human metastasis. Although these findings revealed miRNAs as suppressors of cell-autonomous metastatic phenotypes, the roles of non-coding RNAs in non-cell-autonomous cancer progression processes remain unknown. Here we reveal that endogenous miR-126, an miRNA silenced in a variety of common human cancers, non-cell-autonomously regulates endothelial cell recruitment to metastatic breast cancer cells, in vitro and in vivo. It suppresses metastatic endothelial recruitment, metastatic angiogenesis and metastatic colonization through coordinate targeting of IGFBP2, PITPNC1 and MERTK--novel pro-angiogenic genes and biomarkers of human metastasis. Insulin-like growth factor binding protein 2 (IGFBP2) secreted by metastatic cells recruits endothelia by modulating IGF1-mediated activation of the IGF type-I receptor on endothelial cells; whereas c-Mer tyrosine kinase (MERTK) receptor cleaved from metastatic cells promotes endothelial recruitment by competitively antagonizing the binding of its ligand GAS6 to endothelial MERTK receptors. Co-injection of endothelial cells with breast cancer cells non-cell-autonomously rescues their miR-126-induced metastatic defect, revealing a novel and important role for endothelial interactions in metastatic initiation. Through loss-of-function and epistasis experiments, we delineate an miRNA regulatory network's individual components as novel and cell-extrinsic regulators of endothelial recruitment, angiogenesis and metastatic colonization. We also identify the IGFBP2/IGF1/IGF1R and GAS6/MERTK signalling pathways as regulators of cancer-mediated endothelial recruitment. Our work further reveals endothelial recruitment and endothelial interactions in the tumour microenvironment to be critical features of metastatic breast cancer.