微小RNA let-7基因表达在膀胱癌生物学行为中的作用

目的 探讨膀胱癌组织中微小RNA(miRNA)表达在膀胱癌发生发展中的作用. 方法 Trizol法抽提9例膀胱癌患者癌组织及癌旁正常膀胱组织标本总RNA,分离与富集miRNA,随机选取2组miRNA标本与miRNA表达谱芯片杂交,采用LuxScan 3.0和SAM 2.1软件对芯片结果 进行图像数据分析.在差异性表达的miRNA中筛选出感兴趣的let-7基因,采用印迹杂交技术对9组miRNA标本中let-7基因进行验证. 结果 膀胱癌组织和正常膀胱组织标本中差异性表达有显著性意义的miRNA 71个,其中表达上调33个,表达下调38个;差异性表达有极显著性意义的miRNA(上调或下调≥4倍)26个,其中显著上调12个,显著下调14个.let-7基因验证结果 与芯片表达结果 一致,癌组织let-7基因表达吸光度值为479.6±228.2,正常膀胱组织为694.6±152.9,组间差异有统计学意义(P<0.05). 结论 膀胱癌组织和正常膀胱组织中存在差异表达的miRNA,let-7基因在膀胱癌的发生中可能起着抑癌基因的作用.     

微小RNA在结肠慢传输型便秘病变组织中的差异表达

目的 观察微小RNA(miRNA)在结肠慢传输型便秘(STC)病变结肠组织和正常结肠组织中的差异表达.方法 收集6例STC病变结肠组织和正常结肠组织标本,应用miRNA芯片检测miRNA在STC病变结肠组织和正常结肠组织中的表达.结果 5种miRNA在STC病变结肠组织正常结肠组织中的表达差异有统计学意义(P＜0.05).与正常结肠组织比较,miR-20b、miR-27b、miR-30b、miR-128及miR-129-3p在STC结肠组织中表达下调,统计学组间差异倍数均在1.2倍以上,其中miR-128及miR-129-3p下调明显,统计学组间差异倍数大于2倍.结论 部分miRNA在STC结肠组织和正常结肠组织中存在差异表达,可能参与了STC发生的分子机制.     

Preliminary study of microRNA expression profiles in human pancreatic cancer

目的:探讨微小RNA(miRNA)在人胰腺癌中的表达谱,为进一步研究miRNA在胰腺癌发生发展中的分子机制奠定基础。方法:利用miRNA芯片技术比较胰腺癌组织和癌旁正常胰腺组织的miRNA表达差异;挑选其中两个差异表达的miRNA运用荧光定量实时聚合酶链反应(qRT-PCR)验证。结果:与正常胰腺组织相比,胰腺癌组织中表达上调超过1.5倍的miRNA有15个,表达下调超过1.5倍的miRNA有11个;qRT-PCR检测8例胰腺癌组织和相应癌旁正常胰腺组织中miR-10b的相对表达量分别为0.0743±0.0222和0.0287±0.0129;miR-21相对表达量分别为0.3062±0.1117和0.0240±0.0137(均P<0.05),与miRNA芯片结果相符合。结论:miRNA芯片筛选出的26种差异表达的miRNA可能与胰腺癌的发生发展相关,其各自在胰腺癌中的作用值得进一步研究。     

miRNA在皮肤瘢痕组织中的表达研究

目的研究正常皮肤组织、非增生性瘢痕和瘢痕疙瘩组织中miRNA的表达,筛选对瘢痕异常增生可能发挥作用的miRNA。方法使用miRNA芯片检测正常皮肤组织(C)、非增生性瘢痕(T_1)和瘢痕疙瘩(T2)中miRNA的表达谱,并对各组miRNA进行对比,筛选共同表达但表达有差异的miRNA部分,再选取其中表达差异较为明显的miRNA,对其进行Real-Time quantitative PCR验证实验。结果共计测得miRNA 2539条,差异表达结果为:T_1/C,上调2/下调17;T_2/C,上调3/下调94;T2/T_1,上调9/下调87。选取差异表达显著的miRNA,从C至T_2,miR-100、miR-29a表达呈现下调趋势,而miR-181a、miR-198表达呈现上调趋势。结论非增生性瘢痕和瘢痕疙瘩的miRNA表达均有别于正常皮肤组织,而miR-100、miR-29a、miR-181a、miR-198对瘢痕组织内成纤维细胞增殖和胶原合成过程中发挥作用的重要信号通路均有影响。瘢痕增生是多基因复合性表达作用的结果 。     

miR-193b对胃癌细胞浸润和转移的影响

目的:探讨miR-193b转染对胃癌细胞浸润、转移的影响及其作用机制。 方法:应用microRNA芯片检测TGF-β1处理胃癌细胞BGC823前后miRNA的差异表达谱;应用划痕实验,transwell 迁移浸润及裸鼠成瘤等实验分别检测瞬时转染miR-193b抑制剂前后胃癌细胞株生物学行为的变化。 结果:TGF-β1处理胃癌细胞BGC823前后miRNA的表达谱存在6个差异表达,包括3个（miR-27a, miR-29b-1和 miR-194）表达上调,3个（miR-193b, miR-574-3p和miR-130b）表达下调。转染miR-193b 抑制剂后,胃癌细胞株BGC823迁移、浸润和转移的能力明显增强。 结论:TGF-β1可能通过下调miR-193b的表达负向调控胃癌细胞的迁移、浸润和转移。     

早期胃癌特征性miRNA筛选

目的检测早期胃癌组织中微小RNA（miRNA）表达谱,筛选出早期胃癌的特征性miRNA。方法应用中通量基因芯片技术来检测5例早期胃癌组织及其癌旁组织标本中miRNA的表达。结果相对于癌旁组织,早期胃癌组织中共有36个miRNA表达下调,如miR-9．1、miR-103、miR-141等;12个miRNA表达上调,如miR．196a、miR．142．3p、miR-25等。结论在早期胃癌组织中表达异常的miRNA可能与胃癌发生发展有着一定的相关性。     

肝内及肝外胆管癌组织表达上调microRNAs表达谱的鉴定

目的 筛选、鉴定肝内及肝外胆管癌组织表达上调的miRNAs,并探讨其在胆管癌细胞增殖中的作用.方法 利用miRNA-基因芯片方法筛选肝内、肝外胆管癌组织 特异表达上调或共同表达上调的miRNAs;利用实时PCR方法对其进行验证;选择特异miRNA抑制剂转染胆管癌细胞系QBC939并利用MTT检测方法,对上述表达上调明显的miRNAs进行初步的功能研究,探讨miRNAs表达上调与胆管细胞癌细胞增殖的关系.结果 12个miRNAs在肝外及肝内胆管癌组织共同表达上调,肝外胆管癌组织特异表达上调的miRNAs有28个,其中miR-125b与miR-19a分别表达上调3.7倍与3.6倍(P＜0.05);肝内胆管癌有12个miRNAs特异表达上调,其中miR-92a与miR-205分别表达上调约4.5倍与3.5倍(P＜0.05);在胆管癌细胞系QBC939中抑制miR-125b、miR-19a、miR 21以及miR-378*的内源性表达,可以明显抑制QBC939细胞的增殖,其抑制效率分别为71％、72％、69％与76％(P＜0.05,36 h),61％、63％、60％与59％ (P＜0.01,48 h),61％、56％、60％与59％ (P＜0.05,60 h).结论 肝内及肝外胆管癌具有不同的miRNAs上调表达谱,敲低表达上调的miRNAs可以明显抑制胆管癌细胞增殖.     

胃癌中微小RNA表达谱及miR-429表达水平的研究

目的 检测胃癌组织及胃癌细胞中微小RNA (miRNA)表达谱,并测定胃癌组织及胃癌细胞中miR-429的表达水平.方法 采用基因芯片技术检测胃癌组织、癌旁正常胃组织标本及胃癌细胞株( SGC-7901)、正常人胃上皮细胞(GES-1)中miRNAs的表达.挑选基因芯片结果中胃癌组织表达显著下调的miR-429,并采用实时定量聚合酶链反应(qRT-PCR)检测7例正常胃组织、52例胃癌组织、胃癌细胞SGC-7901、GES-1中miR-429的表达.结果 基因芯片结果表明,胃癌组织中有23个miRNA上调,如miR-21、miR-30b等,31个下调如miR-429、miR-200等.qRT-PCR检测显示,miR-429在胃癌组织中下降约63.4％ (P＜0.01),在胃癌细胞株SGC7901中下降约76.2％(P＜0.01).结论 miR-429在胃癌组织中表达显著下调,可能作为胃癌特异性miRNA,在胃癌的发生发展过程中有重要作用.     

直肠癌中差异表达的microRNA及其与预后因子的关系

目的：检测直肠癌组织中差异表达的microRNA（miRNA）,并分析其与预后因子的关系。 方法：选20例直肠癌患者手术所得癌组织标本与癌旁组织,用q-PCR方法筛选在癌组织与癌旁组织中有差异表达的miRNA以及相关的预后因子。 结果：筛选得到26个在直肠癌组织中有差异表达的miRNA,其中15个miRNA表达上调,11个表达下调。相关分析显示,下调的miR-4770、miR-4790-5p与CK20表达呈负相关;上调的miR-182、miR-125a-5p、miR-126与p53表达呈正相关、miR-143和k-ras呈负相关、miR-9与vilin基因呈负相关（均P<0.05）。 结论：直肠癌组织中差异表达的miRNA与预后因子密切相关,可作为直肠癌诊断或预后判断的生物标记。     

应用液相芯片分析肝细胞癌及癌旁组织小分子RNA表达谱差异

目的运用液相芯片技术研究肝细胞癌（HCC）及毗邻癌旁组织中小分子RNA（miRNA）表达谱差异。方法常规抽提20例HCC及对照癌旁组织中总RNA，采用含有114种miRNA的液相芯片及配套的Luminex 100检测系统进行miRNA差异表达谱分析；选取部分筛选出的差异表达miRNA，以半定量逆转录-聚合酶链反应（RT—PCR）和Western blot分析其相对应的靶mRNA和目的蛋白表达状况。结果HCC的miRNA表达谱中差异表达miRNA共有28个，其中上调6个，下调22个，20例标本中共存性差异表达miRNA有9个（上调2个，下调7个，P〈0．01）；选取表达明显上调的miR-222作为进一步研究对象，半定量RT—PCR和Western blot分别证实其调节的靶目标-结缔组织生长因子（CTGF）在mRNA水平HCC和癌旁组织中差异无统计学意义。而蛋白质水平在HCC中表达明显下降。结论液相芯片筛选差异表达miRNA为HCC发病机制和诊断治疗的研究提供了新的思路；miR-18可能通过转录后基因沉默机制抑制CTGF mRNA翻译，从而促进HCC浸润转移。     

miR-490-5p和miR-363作为结直肠癌诊断标记的研究

目的筛查可作为结直肠癌诊断标记的微小RNA（miRNA）。方法采用实时荧光定量PCR分析miRNA在结直肠癌患者肿瘤和瘤旁组织之间的表达谱．运用非配对t检验的方法,筛查出表达水平具有统计学差异的miRNA。并通过受试者特征曲线（ROC）分析miR-363和miR-490．5p作为诊断标记筛查结直肠癌患者的特异性和敏感性。结果在男性和女性样本中分别发现73和42个表达具有显著性差异的miRNA。而33个miRNA同时在男、女样本中都具有显著性的异常表达,其中10个miRNA的异常表达水平超过5倍,这10个miRNA在男女混合样本中同样具有显著性的异常表达。结论男、女结直肠癌患者中的部分miRNA的表达水平具有显著差异：miR-363和miR-490-5p具有作为结直肠癌的临床筛查指标的潜能。     

Micro-RNA profiling in kidney and bladder cancers

OBJECTIVES: Micro-RNAs are a group of small noncoding RNAs with modulator activity of gene expression. Recently, micro-RNA genes were found abnormally expressed in several types of cancers. To study the role of the micro-RNAs in human kidney and bladder cancer, we analyzed the expression profile of 245 micro-RNAs in kidney and bladder primary tumors. METHODS AND MATERIALS: A total of 27 kidney specimens (20 carcinomas, 4 benign renal tumors, and 3 normal parenchyma) and 27 bladder specimens (25 urothelial carcinomas and 2 normal mucosa) were included in the study. Total RNA was used for hybridization on an oligonucleotide microchip for micro-RNA profiling developed in our laboratories. This microchip contains 368 probes in triplicate, corresponding to 245 human and mouse micro-RNA genes. RESULTS: A set of 4 human micro-RNAs (miR-28, miR-185, miR-27, and let-7f-2) were found significantly up-regulated in renal cell carcinoma (P < 0.05) compared to normal kidney. Human micro-RNAs miR-223, miR-26b, miR-221, miR-103-1, miR-185, miR-23b, miR-203, miR-17-5p, miR-23a, and miR-205 were significantly up-regulated in bladder cancers (P < 0.05) compared to normal bladder mucosa. Of the kidney cancers studied, there was no differential micro-RNA expression across various stages, whereas with increasing tumor-nodes-metastasis staging in bladder cancer, miR-26b showed a moderate decreasing trend (P = 0.082). CONCLUSIONS: Our results show that different micro-RNAs are deregulated in kidney and bladder cancer, suggesting the involvement of these genes in the development and progression of these malignancies. Further studies are needed to clarify the role of micro-RNAs in neoplastic transformation and to test the potential clinical usefulness of micro-RNAs microarrays as diagnostic and prognostic tool.     

Up-regulation of microRNA in bladder tumor tissue is not common

MicroRNAs (miRNAs) have recently been shown to down-regulate gene expression by targeting mRNA translation and to play a critical role in tumorigenesis; how they regulate bladder tumor development, particularly in patients, is, however, poorly understood. The difference in miRNA expression in a bladder tumor compared with healthy tissue from the same patients was examined using microRNA arrays in seven patients. Here, we showed that up-regulation of miRNA was not commonly found in this limited number of patients, and four miRNAs (miR-26a, miR-29c, miR-30c, miR-30e-5p) were down-regulated as a common marker in patients with a 1–3 grade of disease. Our data suggest that instead of up-regulation of carcinogenic miRNAs, loss of regulation of these miRNA may be critical for bladder tumor development in patients.     

miR-18a-3p在肝癌中的表达及其调控ADCY1表达对肝癌细胞侵袭及增殖的影响

背景与目的 研究表明多种microRNA（miRNA）可能在肝癌的发生发展中发挥重要作用,其作用机制仍值得进一步研究和探讨。因此,本研究从已报道的肝癌差异表达miRNA中进一步筛选关键miRNA,并验证和探讨其作用机制。方法 从已发表的研究中筛选出肝癌组织及肝癌患者血清/血浆中与正常肝组织及正常血清/血浆中共同的差异表达miRNA;用qRT-PCR在正常肝细胞与肝癌细胞中对筛选出的目标miRNA表达情况进行验证;用过表达和抑制的方法观察目标miRNA对肝癌细胞侵袭能力（Transwell实验）与增殖能力（MTT实验）的影响,以及在30例临床标本中检测目标miRNA的表达并通过KM plotter网站分析其对肝癌患者生存的影响;通过miRDB和GEPIA数据库预测和分析目标miRNA的靶基因,并用逆转实验和双荧光素酶报告实验进一步验证。结果 在肝癌组织（vs.正常肝组织）及肝癌患者血清/血浆（vs.正常人血清/血浆）中共同高表达的miRNA有4个（miR-18a-3p、miR-221-3p、miR-222-3p、miR-224-3p）,共同低表达的miRNA有2个（miR-26a-3p、miR-125b-3p）。qRT-PCR实验证实,与正常肝细胞比较,miR-18a在肝癌细胞中高表达,miR-26a在肝癌细胞中低表达（均P<0.05）。过表达/抑制miR-18a-3p表达能促进/降低肝癌细胞的侵袭及生长能力（均P<0.05）,而过表达/抑制miR-26a-3p对肝癌细胞的侵袭及生长能力影响无不法确定。分析结果显示,ADCY1是miR-18a-3p的靶基因,过表达ADCY1能部分逆转miR-18a-3p对肝癌细胞的上述作用,同时,表达上调的miR-18a-3p能通过结合到ADCY1 mRNA 3''UTR抑制ADCY1的表达。结论 miR-18a-3p可能在肝癌的发生发展中起了关键作用,其在肝癌中表达上调,并能通过抑制下游靶基因ADCY1的表达增强进肝癌细胞的侵袭和增殖能力。     

Microarray analysis identifies distinct gene expression profiles associated with histological subtype in human osteosarcoma

Osteosarcoma is the most common primary malignant bone tumour. Currently osteosarcoma classification is based on histological appearance. It was the aim of this study to use a more systematic approach to osteosarcoma classification based on gene expression analysis and to identify subtype specific differentially expressed genes. We analysed the global gene expression profiles of ten osteosarcoma samples using Affymetrix U133A arrays (five osteoblastic and five non-osteoblastic osteosarcoma patients). Differential gene expression analysis yielded 75 genes up-regulated and 97 genes down-regulated in osteoblastic versus non-osteoblastic osteosarcoma samples, respectively. These included genes involved in cell growth, chemotherapy resistance, angiogenesis, steroid- and neuropeptide hormone receptor activity, acute-phase response and serotonin receptor activity and members of the Wnt/ß-catenin pathway and many others. Furthermore, we validated the highly differential expression of six genes including angiopoietin 1, IGFBP3, ferredoxin 1, BMP, decorin, and fibulin 1 in osteoblastic osteosarcoma relative to non-osteoblastic osteosarcoma. Our results show the utility of gene expression analysis to study osteosarcoma subtypes, and we identified several genes that may play a role as potential therapeutic targets in the future.     

MicroRNA-148b influences high glucose-induced endoplasmic reticulum stress in rat mesangial cell by targeting AMPKα1

Objective To observe the expression of microRNA-148b (miR-148b) induced by high glucose in rat mesangial cells, and to explore its effect on its target gene AMP-activated protein kinase α1 (AMPKα1) and extracellular matrix excretion. Methods Rat mesangial cells were divided into 3 groups: normal glucose (NG, 5.5 mmol/L glucose) group, hypertonic (MA, 5.5 mmol/L glucose+ 19.5 mmol/L mannitol) group and high-glucose (HG, 25.0 mmol/L glucose) group. MiR-148b expression was detected by real time PCR. Then miR-148b inhibitor was transfected to rat mesangial cells. Their protein expressions of AMPKα1, glucose regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), fibronectin (FN) and collagen Ⅳ were detected by Western blotting. The expression of AMPKα1 mRNA was detected by real time PCR. The expression of collagen Ⅳ was also detected by immunofluorescence. Results Compared with NG group, HG group showed up-regulated miR-148b expression, down-regulated AMPKα1 mRNA and protein expressions, and up-regulated CHOP, GRP78, collagen Ⅳ and FN expressions (all P＜0.05). HG-induced mesangial cells with miR-148b inhibitor had up-regulated AMPKα1 mRNA and protein expressions, and down-regulated CHOP, GRP78, collagen Ⅳ, FN expressions as compared with HG-induced cells without miR-148b inhibitor (all P＜0.05). Conclusions HG can up-regulate miR-148b expression and down-regulate AMPKα1 expression in rat mesangial cells, then activate endoplasmic reticulum stress to induce extracellular matrix excretion. MiR-148b inhibitor up-regulates AMPKα1 expression, inhibits endoplasmic reticulum stress and reduces extracellular matrix excretion.     

Expression of miR-200a in peritoneal fibrosis associated with peritoneal dialysis

Objective To find the key miRNA that relative to peritoneal fibrosis associated with peritoneal dialysis (PD) by microarray technology, and verify its expression in vitro and in vivo. Methods The peritoneal fibrosis mouse model associated with PD were established by intraperitoneal injection of lipopolysaccharide (LPS) + 4.25% peritoneal dialysate. The expression of miRNA was detected by microarray in peritoneal tissues. The expression of miRNA profiles between fibrotic and normal peritoneal tissues was compared. The differentially expressed miRNA (miR-200a) was validated by real-time PCR in lager sample size cohorts. The expressions of miR-200a were also detected in the epithelial-mesenchymal transition (EMT) process of human peritoneal mesothelium cells. Results In mice model of PD, peritoneal tissue was markedly thickened and with a massive extracellular matrix accumulation. In contrast with control, the expression level of epithelial marker E - cadherin was significantly decreased, α - SMA, Col - I and FN were remarkably increased (P ﹤ 0.05). By miRNA microarray analysis, miR - 200a was significantly down - regulated (3.31 folds change, P ﹤ 0.05) in fibrotic peritoneal tissues. The down-regulated expression level of miR-200a was also validated by real- time PCR in larger cohorts (P ﹤ 0.05). Then, the expression level of miR-200a was detected in the EMT process of human peritoneal mesothelium cells. During the process of TGF-β1 induced EMT, miR -200a was significantly down-regulated compared with the control (P﹤0.05). Conclusions Down- regulated expression of miR-200a was observed both during peritoneal fibrosis and TGF-β1 induced EMT in vivo and in vitro, suggesting that miR - 200a may be involved in the peritoneum fibrosis by regulating the target genes of EMT.