应用芯片技术筛查人结肠癌羟基喜树碱多药耐药相关微RNA的研究

目的 探讨新一类调控分子微RNA(miRNA)在人结肠癌羟基喜树碱多药耐药中的作用,为逆转肿瘤化学治疗的多药耐药性提供新的靶点.方法 运用miRCURYTM LNA Array V8.1版芯片检测人结肠癌细胞SW1116和人结肠癌羟基喜树碱多药耐药细胞SW1116/HCPT的miRNA表达谱,筛选出具有表达差异的miRNA(2倍以上).通过PiTcar,Targetscan,MIRanda等算法在线搜寻差异表达miRNA的靶基因.利用茎环特异miRNA引物反转录特异的miRNA,并采用荧光定量PCR法对个别差异表达的miRNA进行验证.结果 用于芯片分析的总RNA的吸光度比值分别为1.93(SW1116)和1.94(SW1116/HCPT).琼脂糖变性凝胶电泳进一步验证总RNA质量符合芯片要求.通过芯片检测后发现SW1116/HCPT相比SW1116表达下调的miRNA共有28条,表达上调的有36条.根据靶基因预测结果,我们选取其中2条表达下调hsa-miR-452,hsa-miR-373*和1条表达上调hsa-miR-506进行荧光定量PCR验证,hsa-miR-452和hsa-miR-506的结果与芯片相符合,但hsa-miR-373*表达量不足以由荧光定量PCR检测出.结论 miRNA表达量的改变可能在人结肠癌羟基喜树碱多药耐药中起有关键作用.     

miRNA在HBV从感染经由肝硬化到肝癌进程中表达谱的变化

目的:分析微小RNA(microRNA, miRNA)在HBV感染到肝硬化再到肝癌进程中表达谱的变化.方法:利用miRNA芯片技术检测人正常肝脏、乙型肝炎肝硬化、HBV相关性肝癌组织中miRNA表达谱的差异. 实时定量PCR检测上述3种肝脏组织中芯片结果差异性表达的miRNA, 验证芯片结果的可信性.结果:与正常肝脏组织相比, 乙型肝炎肝硬化和HBV相关性肝癌组织中表达上调超过2倍的microRNA有6个, 分别为:hsa-miR-602、hsa-miR-129-5p、has-miR-210、hsa-miR-671-5p、hsa-miR-30b*及hsa-miR-572; 表达下调超过2倍的miRNA有8个:hsa-miR-143、hsamiR-199a-5p、has-miR-195、hsa-miR-27a、hsa-miR-99a、hsa-miR-519e、has-miR-130a及hsa-miR-597. 这些正常肝脏组织相比有差异表达的miRNA, 在乙型肝炎肝硬化和HBV相关性肝癌组织中表达量无明显差别.结论:从HBV感染到肝硬化再到肝癌进程中伴有microRNA表达谱的变化, 且变化主要发生在进程早期.     

1型糖尿病大鼠心肌纤维化microRNA表达变化筛选

目的应用链脲佐菌素(STZ)诱导1型糖尿病大鼠心肌纤维化模型,检测心肌组织miRNA表达谱,对差异miRNA调控的靶基因进行初步预测。方法 STZ诱导大鼠心肌纤维化,微距阵基因芯片技术筛选大鼠心肌组织差异表达的miRNA,对差异表达的miRNA调控的靶基因生物信息学分析。结果与对照组比较,模型组大鼠心肌组织中差异倍数改变大于2倍的miRNA共有25个,其中hsa-miR-29a-3p、hsa-miR-551a、hsa-miR-34a-5p、hsa-miR-885-5p等表达上调,hsa-miR-208a、hsa-miR-150-5p等表达下调。生物信息学分析,差异miRNA调控的靶基因多与细胞增殖、凋亡、糖代谢及血管生成等生物学功能相关。结论 STZ诱导的1型糖尿病大鼠心肌纤维化模型心肌中,miRNA表达谱有明显差异,其中有些miRNA可能靶向与1型糖尿病心肌纤维化发生发展密切相关。     

结肠癌患者血清miRNA特异性表达谱及其靶基因筛选鉴定研究

目的探讨血清miRNA与结肠癌的预后及恶性程度的相关性,预测可能与结肠癌早期诊断及预后相关的标志物。方法收集结肠癌患者(结肠癌组,20例)及健康对照者(健康对照组,20名)的血清,进行实时荧光定量PCR检测,分析数据并筛选差异表达的miRNA。靶基因预测软件预测所筛选miRNA的靶基因并分析其与患者的性别、年龄、TNM分期、淋巴转移、预后、生存周期等的关系,依据生物信息学分析法,鉴定结肠癌患者血清中特异表达的miRNA的靶基因,分析其靶基因的生物学功能。结果靶基因预测软件预测显示,所筛选上调的hsa-miR-182和下调的hsa-miR-195与调控细胞周期、细胞死亡、细胞凋亡、细胞增殖等相关的信号通路有关。Western blotting检测结果显示,下调miRNA调控的靶基因IL-6在结肠癌患者中的表达上调,而上调miRNA调控的靶基因P53在结肠癌患者中的表达下调(P均0.05)。结论通过分析,我们筛选到了差异表达的miRNA,并预测了其作用的靶基因,通过实验验证了这些靶基因的可靠性,为后续的临床诊断和治疗提供了靶点。     

肝泡型包虫病特异microRNA的筛选及初步研究

目的肝泡型包虫病由多房棘球蚴感染造成,通过对肝泡型包虫病患者组织和血浆中差异表达的miRNA进行筛查,寻找肝泡型包虫病新的生物标志物。方法纳入2016年6月—2018年5月在青海大学附属医院确诊的肝泡型包虫病患者,选取2例肝泡型包虫病患者的病灶边缘组织和3例临近病灶边缘的正常组织,以及3例肝泡型包虫病患者和3例健康对照者的血浆样本,使用Agilent Human miRNA芯片检测组织和血浆的miRNA表达谱,根据差异倍数(FC>1.2)和P值(P<0.05)筛选差异表达的miRNA,根据差异miRNA的靶基因预测结果,结合文献报道,选择与肝脏疾病相关的血浆miRNA和组织miRNA进行实时荧光定量PCR(qRT-PCR)验证。计量资料2组间比较采用t检验,相关性分析采用Spearman相关分析。结果肝泡型包虫病患者中的microRNA表达谱与健康人相比有显著不同,qRT-PCR验证发现6个microRNA中有3个miRNA(hsa-miR-4644,hsa-miR-136-5p,hsa-miR-483-3p)在肝泡型包虫病患者中显著差异表达(P<0.05)。其中hsa-miR-4644和hsa-miR-483-3p在肝泡型包虫病患者中显著上调表达(P值均<0.05),hsa-miR-136-5p显著下调表达(P<0.05)。通过TargetScan,PITA,microRNAorg数据库对差异miRNA进行靶基因预测,对三个数据库预测到的靶基因取交集,共有137个基因和miRNA之间有靶向关系。差异的hsa-miR-483-3p靶向调控参与人体免疫反应及与肝脏疾病有关的基因(IL-17A,IL-5,CD40LG,TAP2,TNF)。通过GO与KEGG分析发现,hsa-miR-483-3p的靶基因在免疫缺陷(Primary immunodeficiency)信号通路,IL-17信号通路,TNF信号通路中起重要作用。结论肝泡型包虫病有独特的microRNA表达谱,其中hsa-miR-483-3p可作为肝泡型包虫病的一种新的生物学标志物,其调控的靶基因主要参与Primary immunodeficiency信号通路,IL-17信号通路,TNF信号通路。但这些miRNA与肝泡型包虫病之间的调控关系有待进一步验证。     

微小RNA作为单采血小板储存损伤标志物不同保存期的差异表达谱

目的:探讨单采血小板不同保存期微小RNA(mRNA)表达谱差异，发现由于储存过程中血小板生物学变化引起的新的敏感性表达miRNA。方法:通过新一代small RNA测序技术结合生物信息学分析方法，筛选出单采血小板在体外保存期间存在差异表达的miRNA,并采用实时荧光定量PCR(qRT-PCR)法验证。结果:miRNA表达谱芯片结果显示6组不同保存期单采血小板普遍高表达hsa-let-7家族，hsa-miR-26a-5p, hsa-miR-92a-3p, hsa-miR-199,hsa-miR-103a-3p。时间序列分析对944个差异表达miRNA进行趋势分类，共发现44个基因呈现14种趋势变化。以保存第1天为对照组，hsa-miR-4433b-5p, hsa-miR-22-3p, hsa-miR-30c-5p显著下降。结论:分析筛选出表达差异的miRNA-30c-5p, miRNA-22-3p, miRNA-4433b-5p为下一步研究单采血小板储存损伤标志物奠定基础。     

胃癌外周血微小核糖核酸表达谱的初步分析

目的 研究胃癌患者外周血微小核糖核酸(miRNA)的表达,初步建立胃癌特征性的循环miRNA表达谱,为深入研究miRNA与胃癌发生、发展并寻找新的分子标志物提供依据.方法 选取6例胃癌患者和6例健康体检者,提取外周血总RNA,进行miRNA表达谱检测和生物信息学分析,采用实时定量PCR技术对芯片检测结果进行验证,对筛选出的显著差异表达miRNA的靶基因进行预测.结果 胃癌组与对照组对比共有54个差异表达miRNA,其中表达上调的miRNA为35个(miRNA-504、miRNA-183、miRNA-938、miRNA-1285、miRNA-576-3p、miRNA-663 等),表达下调的miRNA为19个(miRNA-433、miRNA-193b、miRNA-329、miRNA-409-3p、miRNA-154等).实时定量PCR对其中2个上调miRNA(miRNA-504和miRNA-183)和2个下调miRNA(miRNA-433和miRNA-193b)的验证结果与芯片检测结果间具有较好的一致性.结论 胃癌患者外周血中具有特异性的miRNA表达谱,这些差异表达的miRNA有可能成为新的胃癌诊断分子标志物.     

结直肠腺瘤复发患者外周血miRNAs表达差异研究

背景:miRNAs是一类非编码小分子RNA,可在转录后水平调控基因表达,在肿瘤发生、发展中发挥重要作用。目的:以miRNA芯片筛选在结直肠腺瘤复发患者外周血中特异性表达的miRNAs,以期为复发预测提供敏感而特异的指标。方法:选取50例曾行结肠镜下结直肠腺瘤摘除术的患者,于2012年8月—2013年9月复查结肠镜,从中选取结直肠腺瘤复发者和未复发者各4例,采集外周血标本,以miRNA芯片检测miRNAs表达谱并筛选复发组与未复发组间的差异表达miRNAs。以real-time PCR对部分差异表达miRNAs进行验证。结果:与未复发组相比,复发组共筛选出11个差异表达miRNAs,其中7个表达上调,分别为hp_hsa-miR-548ai、hsa-miR-4446-3p、hsa-miR-139-3p、hsa-miR-27a-5p、hsa-miR-10a、hsa-miR-23a、hsa-miR-486-3p,4个表达下调,分别为hsa-miR-124、hsa-miR-3613-5p、hsa-miR-495、hsa-miR-485-5p。其中7个miRNAs经real-time PCR验证,表达变化趋势与芯片检测结果相一致。结论:部分差异表达miRNAs可能参与了结直肠腺瘤的复发机制,为进一步探索复发预测指标提供了线索。     

怀有先天性心脏病胎儿孕妇血浆microRNA的表达差异

目的检测血浆miRNA在怀有先天性心脏病(先心病)胎儿的孕妇和怀有正常心脏胎儿的孕妇之间的表达差异,筛选出表达显著差异的miRNAs,为产前筛查先心病提供生物标记物和为先心病的发生机制研究提供分子生物学依据。方法采集60例怀有先心病胎儿的孕妇及60例对照组的血样标本,用Solexa测序方法筛选出具有显著差异性的血浆miRNA。结果 Solexa测序结果显示,在病例组及对照组中分别检测出545个及580个miRNA的表达,以拷贝数≥30,病例组与对照组相比miRNA表达差异倍数≥9为标准,筛选出7个表达上调的miRNA:hsa-miR-137、hsa-miR-206、hsa-miR-224-3p、hsa-miR-34a-3p、hsa-miR-485-3p、hsa-miR-5094、hsa-miR-653-3p;4个表达下调的miRNA:hsa-miR-100-3p、hsa-miR-188-5p、hsa-miR-190a-3p、hsa-miR-216a-5p。结论怀有先心病胎儿的孕妇血浆中存在miRNA表达差异。     

先天性心脏病继发性肺动脉高压患者血浆中miRNA的研究

目的 应用微小RNA （microRNA,miRNA）芯片技术研究先天性心脏病合并重度肺动脉高压（pulmonary arterial hypertension,PAH）患者和不合并PAH患者血浆中miRNA表达谱的差异,并初步预测差异表达的miRNA调控的靶基因.方法 收集室间隔缺损（ventricular septal defect,VSD）合并重度PAH（PAH组）和不合并PAH患者（对照组）的血浆.分别提取总RNA,然后采用miRNA芯片进行miRNA表达谱差异分析,并对结果进行实时定量聚合酶链反应（polymerase chain reaction,PCR）验证.运用Targetscan、Pictar、Miranda软件预测可能调控的靶基因.结果 miRNA芯片结果提示：与对照组相比,左向右分流先天性心脏病继发重度PAH患者血浆中表达上调的miRNA有50个,表达下调的miRNA有36个.实时定量PCR验证miR-98与芯片结果一致,表达上调.靶基因预测软件显示内皮素（ET-1）为hsa-miR-98的重要靶基因.结论 miRNA在先天性心脏病继发重度PAH患者血浆中存在差异性表达,miRNA可能与PAH的发生、发展密切相关,血浆miR-98有可能成为先天性心脏病合并重度PAH的新的分子生物学标志物.     

miR-93 suppresses proliferation and colony formation of human colon cancer stem cells

AIM:To identify differentially expressed microRNAs(miRNAs) in human colon cancer stem cells(SW1116csc) and study their function in SW1116csc proliferation.METHODS:SW1116csc were isolated from the humancolon cancer cell line,SW1116 and cultured in serumfree medium. A miRNA microarray was used to detect differential expression profiles of miRNAs in SW1116cscand SW1116 cells. Real-time quantitative polymer asechain reaction(PCR) was performed to verify the differential expression of candidate miRNAs obtained f...     

人结直肠癌微小核糖核酸的差异表达及其临床意义

目的:研究人结直肠癌、腺瘤和癌旁正常组织中微小核糖核酸(microRNAs,miRNAs)的差异表达谱,并初步探讨其临床意义.方法:选取2008-01/07苏州大学附属第一医院和泰州市人民医院结直肠癌、对应癌旁正常组织以及结直肠腺瘤标本,提取组织总RNA,采用illumina microRNA芯片技术检测3种不同类型组织中miRNAs的表达,采用实时定量PCR技术对芯片检测结果进行验证.将结直肠癌组织中异常表达的miRNAs与结直肠癌患者的临床病理资料进行分析.结果:3种不同类型结直肠组织中miRNAs表达有明显差异,结直肠癌与癌旁正常组织相比,有65个miRNAs表达异常(P<0.001),其中35个上调,30个下调,而腺瘤与正常结直肠组织相比,有55个miRNAs表达差异(P<0.001),其中上调29个,下调26个.有25个miRNAs相对于正常组织同时在结直肠癌和腺瘤中异常表达,其中高表达12个,低表达有13个.与腺瘤相比,结直肠癌中有25个miRNAs表达异常(P<0.01),其中13个上调,12个下调.进一步定量PCR验证结果显示与正常结直肠黏膜组织相比,在癌组织中miR-552,miR-142-...     

结肠癌相关miRNA的转化机制

结肠癌的发生是多阶段、多步骤的病变过程,是从良性息肉-浸润性腺癌-癌远处转移的演变过程.这些病理变化与编码蛋白质的原癌基因和肿瘤抑制基因的异常激活或失活有关.但最近关于微小RNA(microRNAs,miRNAs)的研究改变了我们对非编码蛋白质基因在肿瘤发生中的作用的理解.在结肠癌组织中多种miRNAs的表达水平不同,...     

干细胞培养基成球培养法筛选结肠癌干细胞的生物学特性

目的探讨干细胞培养基成球培养法筛选结肠癌干细胞的相关蛋白表达及生物学特性。方法用干细胞培养基成球培养法筛选结肠癌干细胞,Western blot检测干细胞相关蛋白的表达,流式细胞仪检测细胞周期;同时检测干细胞增殖、黏附、耐药及侵袭能力,并摸索结肠癌干细胞最佳冻存条件。结果用干细胞培养基成球培养法成功培养出了结肠癌细胞球,检测发现干细胞相关蛋白表达增强,静止期细胞比例明显升高,细胞增殖速度慢,黏附性、耐药性、侵袭性均强于亲本细胞。结论干细胞培养基成球培养法筛选的细胞球中富集了肿瘤干细胞,为结肠癌干细胞的研究提供了良好的细胞模型。     

Proteome of human colon cancer stem cells: a comparative analysis

AIM: To isolate and identify the biological characteristics of human colon cancer stem cells (SW1116 cells) and further study their proteome. METHODS: SW1116 cells were isolated and cultured with a serum-free medium (SFM). Sphere formation was assayed to observe the formation of colon cancer stem cell spheres. SW1116 cells were inoculated into a serum-containing medium for observing their differentiation characteristics. Proliferation curve and cross-resistance of SW1116 cells to different drugs were detect...     

Gene profiles between non-invasive and invasive colon cancer using laser microdissection and polypeptide analysis

AIM： TO explore the expression of differential gene expression profiles of target cell between noninvasive submucosal and invasive advanced tumor in colon carcinoma using laser microdissection （LMD） in combination with polypeptide analysis. METHODS： Normal colon tissue samples from 20 healthy individuals and 30 cancer tissue samples from early non-invasive colon cancer cells were obtained. The cells from these samples were used LMD independently after P27-based amplification. aRNA from advanced colon cancer cells and metastatic cancer cells of 40 cases were applied to LMD and polypeptide analysis, semiquantitative reverse transcribed polymerase chain reaction （RT-PCR） and immunohistochemical assays were used to verify the results of microarray and further identify differentially expressed genes in non-invasive early stages of colon cancer. RESULTS： Five gene expressions were changed in colon carcinoma cells compared with that of controls. Of the five genes, three genes were downregulated and two were upregulated in invasive submucosal colon carcinoma compared with non-invasive cases. The results were confirmed at the level of aRNA and gene expression. Five genes were further identified as differentially expressed genes in the majority of cases （〉 50%, 25/40） in progression of colon cancer, and their expression patterns of which were similar to tumor suppressor genes or oncogenes. CONCLUSION： This study suggested that combined use of polypeptide analysis might identify early expression profiles of five differential genes associated with the invasion of colon cancer. These results reveal that this gene may be a marker of submucosal invasion in early colon cancer.     

Analysis of differential gene expression patterns in colon cancer and cancer stroma using microdissected tissues

BACKGROUND AND AIMS: The surrounding cancer stroma is increasingly recognized as playing an important role in cancer proliferation, invasion, and metastasis. Here, we analyzed patterns of gene expression in colon cancer cells, surrounding stroma, and normal mucosa and normal stroma using laser capture microdissection. METHODS: Tissues were fixed from 11 patients undergoing colorectal resection for cancer, and laser capture microdissection was performed. Samples (4 per patient) were extracted for RNA, which was then used for focused gene arrays. In addition, protein expression of selected genes was determined by Western blot or immunohistochemistry. RESULTS: We showed differential expression patterns in cancer cells and surrounding stroma compared with their normal counterparts. Genes contributing to angiogenesis, cell cycle regulation, and proliferation were significantly increased in cancer cells compared with the adjacent normal mucosa. Genes contributing to angiogenesis, cancer invasion, and metastasis were significantly increased in surrounding cancer stromal cells compared with normal stroma. CONCLUSIONS: Delineating the differential patterns of gene expression between colon cancers and the surrounding reactive stroma will better determine the role of the stromal components in the progression of colon cancers and may lead to chemopreventive therapy targeted specifically to the stroma.     

Expression of human intestinal mucin is modulated by the beta-galactoside binding protein galectin-3 in colon cancer

BACKGROUND & AIMS: Alterations in the production of the beta-galactoside binding protein galectin-3 and of MUC2 intestinal mucin have been independently correlated with the malignant behavior of human colon cancer cells. MUC2 mucin is a major ligand for galectin-3, and colon cancer cells that differ quantitatively in MUC2 expression may also vary in expression of galectin-3. The current study was designed to investigate the relationship between galectin-3 production and MUC2 mucin synthesis by human colon cancer cells. METHODS: The effect of galectin-3 on MUC2 mucin production was assessed by stable transfection of sense and antisense galectin-3 expression constructs under the control of constitutive or tetracycline-inducible promoters into human colon cancer cells. Galectin-3 and MUC2 expression were determined by fluorescence-activated cell sorter (cell surface galectin-3), Western and Northern analysis (galectin-3, MUC2), and gel filtration of secreted high-weight glycoprotein (MUC2). In vitro results were confirmed in vivo by analysis of cecal xenografts in athymic mice. RESULTS: Colon cancer cells with high levels of galectin-3 also had high levels of MUC2 mucin, whereas those with low galectin-3 levels had low MUC2 levels. Alterations in galectin-3 levels by expression of sense or antisense galectin-3 constructs resulted in parallel alterations of MUC2 protein and RNA. Induction of antisense to galectin-3 in vivo was associated with decreases in both galectin-3 and MUC2 protein in cecal xenografts. CONCLUSIONS: The beta-galactoside binding protein galectin-3 modulates the expression of its major ligand MUC2 mucin in human colon cancer cells. This may have important implications for understanding the role of galectin-3 in colon cancer metastasis.     

Gene expression patterns of human colon tops and basal crypts and BMP antagonists as intestinal stem cell niche factors

Human colonic epithelial cell renewal, proliferation, and differentiation are stringently controlled by numerous regulatory pathways. To identify genetic programs of human colonic epithelial cell differentiation in vivo as well as candidate marker genes that define colonic epithelial stem/progenitor cells and the stem cell niche, we applied gene expression analysis of normal human colon tops and basal crypts by using expression microarrays with 30,000 genes. Nine hundred and sixty-nine cDNA clones were found to be differentially expressed between human colon crypts and tops. Pathway analysis revealed the differential expression of genes involved in cell cycle maintenance and apoptosis, as well as genes in bone morphogenetic protein (BMP), Notch, Wnt, EPH, and MYC signaling pathways. BMP antagonists gremlin 1, gremlin 2, and chordin-like 1 were found to be expressed by colon crypts. In situ hybridization and RT-PCR confirmed that these BMP antagonists are expressed by intestinal cryptal myofibroblasts and smooth muscle cells at the colon crypt. In vitro analysis demonstrated that gremlin 1 partially inhibits Caco-2 cell differentiation upon confluence and activates Wnt signaling in normal rat intestinal epithelial cells. Collectively, the expression data set provides a comprehensive picture of human colonic epithelial cell differentiation. Our study also suggests that BMP antagonists are candidate signaling components that make up the intestinal epithelial stem cell niche.