大规模平行测序技术筛选人肾细胞癌组织中差异表达microRNAs及其验证

目的:以大规模平行测序技术(massively parallel signature sequencing,MPSS)筛选人肾细胞癌(renal cell carcinoma,RCC)和癌旁组织中差异表达的microRNAs(miRNAs),并验证差异表达miRNAs之一的miR-660在人RCC中的表达。方法:MPSS检测10例RCC组织及相应癌旁组织中miRNAs的表达,筛选RCC组织中差异表达miRNAs。RT-PCR检测5例RCC与癌旁组织中miR-660的表达,qPCR检测40例RCC与癌旁组织中miR-660的表达。结果:MPSS结果显示,RCC组织中283个miRNAs表达下调,187个表达上调,其中miR-660在RCC组织中表达量是癌旁组织的17.5%。RT-PCR初步验证了此结果;qPCR验证结果显示,40例RCC组织中33例miR-660表达显著低于癌旁组织,RCC组织中miR-660平均表达量是癌旁的19.5%。结论:人RCC组织中283个miRNAs表达下调、187个上调。miR-660在RCC组织中低表达,有可能成为RCC诊断及治疗的新靶点。     

miR-30通过调节CD90表达抑制肝细胞肝癌

  目的  miRNA能通过转录后调节靶基因的表达量,并在致癌过程中发挥抑癌或促癌作用。研究发现miR-30家族在人类肿瘤中起着重要作用,并参与维持干细胞特性的上皮细胞间质化过程,本研究主要识别miR-30家族与肝细胞肝癌的关系。  方法  应用qRT-PCR实验方法检测93例肝细胞肝癌患者癌组织和癌旁正常组织中miR-30c,miR-30b和miR-30e表达水平,另对121例肝细胞肝癌患者癌组织进行miR-30家族的表达及CD90免疫组织化学表达检测,分析miR-30家族与CD90在肝细胞肝癌中的相关性。  结果  本研究发现miR-30c(P < 0.001)、miR-30b(P=0.004)和miR-30e(P < 0.001)与癌旁正常组织相比,癌组织中表达显著下调。CD90蛋白在癌组织中的表达水平显著高于癌旁正常组织(P=0.007)。MiR-30c(P=0.032)和miR-30e(P=0.015)在CD90阴性表达的患者中表达水平显著高于CD90阳性表达的患者。  结论  miR-30家族在肝细胞肝癌中可作为抑癌miRNA,并通过调节CD90蛋白来发挥这一作用。      

食管癌组织中miRNA表达差异研究

目的探讨食管癌组织microRNA（miRNA）表达的差异,为研究miRNA在食管癌发生、发展中的作用提供新线索。方法 Trizol法抽提食管癌及癌旁组织总RNA,分离miRNA,采用基因芯片技术,将组织中miRNA与哺乳动物miRNA芯片杂交,采用图像软件和SAM version 2.1进行数据分析。结果与正常癌旁组织相比,有23个miRNAs在食管癌组织中有显著差异,包括18个上调和5个下凋。结论部分差异表达miRNAs可能参与食管癌癌变分子机制,为进一步探索提供思路。     

MicroRNA与DNA甲基化相互调节机制在肝细胞癌中的研究进展

肝细胞癌是原发性肝癌的主要类型,也是人类恶性程度较高的肿瘤之一,其发病机制至今尚未完全阐明.表观遗传学机制在肿瘤的发生、发展中起重要作用,DNA甲基化和微小RNA (microRNA,miRNA)的调控机制属于表观遗传学的研究范畴.研究表明,DNA甲基化及miRNA在肝细胞癌的形成中分别或协同发挥着重要作用,miRNA是一类在转录后水平调节基因表达的非编码短链RNA.研究表明,DNA甲基化和组蛋白修饰不仅可以调节蛋白编码基因的表达,而且可以调节miRNA的表达.在肝细胞癌中,一些异常表达的miRNAs(如miR-125b、miR-1-1、miR-124、miR-203和miR-191)是通过表观遗传学机制调控的.另外,在肝细胞癌中还发现了一类miRNAs通过调控表观遗传学通路中关键分子来改变整个基因组的表观遗传学状态.本文就DNA甲基化和miRNA之间复杂的相互调节机制在肝细胞癌发生和发展中的研究进展进行综述.     

肝细胞癌中Hep Par 1、p53、HBsAg表达差异与细胞形态计量学变化的关系

目的 探讨原发性肝癌组织、癌旁组织和正常肝组织中肝细胞抗原（Hep Par1)，p53，乙型肝炎表面抗原（HBsAg)的表达差异及与细胞形态学变化的相关性。方法 用免疫组织化学方法（两步法－EnVision System-HRP)检测10对肝癌和癌旁组织，3例正常肝组织中Hep Par1,p53,HBsAg等蛋白的表达差异；用HPIAS－1000图像分析系统测量实质细胞的总面积（以细胞直径代替）和核质比。结果 1）Hep Par 1:正常肝组织中肝细胞中度表达，癌旁肝组织中肝细胞为弥漫性高表达，肝癌细胞中阳性率为24．53％（13／53）；2）p53：正常肝组织和癌旁肝组织中均呈阴性；肝癌组织，在被检的10例样品中肝癌细胞均为阳性；3）HBsAg:正常肝组织中肝细胞呈阴性，癌旁肝组织中肝细胞为片状弥漫性或散在性高表达，肝癌组织，在被检的10例样品中肝癌细胞仅有1例呈阳性；4）各种组织中实质细胞的平均直径为癌旁肝细胞＞正常肝细胞＞肝细胞癌细胞；5）正常肝细胞和癌旁肝细胞的核质比无差别（P＞0．05），但均明显＜肝细胞癌细胞（P＜0．01和P＜0．01）。结论 肝细胞抗原（Hep Par 1)在分化较高的肝细胞癌细胞中有不同程度的表达，可作为鉴别原发性肝细胞癌细胞来源的标志；原发性肝细胞癌细胞核质比明显＞正常肝细胞和癌旁肝细胞（P＜0.01和P＜0.01)。     

miR-96与miR-103在肝癌组织中的表达及其与预后的关系

目的 探讨miR-96与miR-103在肝癌组织中的表达及其与预后的关系。方法 选取122例肝细胞癌患者作为本研究对象。聚合酶链式反应检测miR-96和miR-103表达水平,采用单因素和多因素Cox回归模型,探讨肝癌患者中位生存时间的危险因素并建立预测模型。结果 肝癌组织中miR-96和miR-103相对表达水平均显著高于癌旁正常组织;肝癌组织中miR-96和miR-103的表达与肿瘤直径、淋巴结转移、临床分期及分化程度均有关（均P<0.05）,而与性别、年龄及合并肝硬化等无关（P>0.05）;所有患者随访60月,中位生存时间为37.8月。单因素分析结果显示：肝细胞癌患者中位生存时间与肿瘤直径、淋巴结转移、TNM分期、病理分化程度、miR-96和miR-103表达水平差异均有统计学意义（均P<0.05）。多因素分析结果显示：TNM分期为Ⅲ~Ⅳ、低分化、淋巴结转移、miR-96和miR-103高表达是肝癌患者中位生存时间独立危险因素。结论 肝癌组织中miR-96和miR-103的高表达是患者预后差的独立影响因素,可作为原发性肝癌患者预后评估指标。     

实时荧光定量PCR检测结直肠癌与癌旁正常组织中miR-145的表达

目的:比较结直肠癌组织与正常癌旁组织中microRNA-145(miR-145)的表达变化。方法:收集30例手术切除的结直肠癌组织和远离癌组织的正常结直肠黏膜组织。提取总RNA,microRNA的引物反转录后,用miR-145特异性引物做荧光定量PCR反应。结果:荧光定量PCR显示结直肠癌组织miR-145的表达量明显降低,癌组织中的表达量为正常癌旁组织的0.353倍,差异有统计学意义(P<0.05)。结论:结直肠癌组织中miR-145的表达低于正常组织,在结直肠癌中发挥抑癌基因的作用。     

miR-122在肝癌细胞中的表达及其与细胞周期调控的关系

目的 探讨miR-122在肝癌细胞中的表达及其与细胞周期调控间的关系。方法 应用荧光定量PCR和原位杂交法分别检测50例肝癌及相应癌旁组织中miR-122的表达水平;用流式细胞术检测肝癌细胞的细胞周期;分析miR-122的表达水平与细胞周期的关系。结果 50例肝癌组织中miR-122的相对表达量为癌旁正常组织的（17.74±9.20）％。其中,40例高中分化肝癌组织中miR-122的相对表达量为癌旁组织的（19.89±8.80）％,而在10例低分化肝癌中仅为癌旁组织的（9.16±4.77）％。肝癌组织中miR-122的阳性表达率为30.0％（15／50）,癌旁组织为80.0％（40／50）,组间差异有统计学意义（P＜0.05）。与癌旁组织相比,肝癌细胞G1／G0期比例降低（P＜0.05）,S期比例增加;与miR-122阳性表达组比较,miR-122阴性表达组的肝癌细胞S期比例增加,而G1／G0期细胞比例降低（P＜0.05）。结论 miR-122在肝癌组织中低表达,且表达水平随着肿瘤分化程度的降低而下调,这种改变促进肝癌细胞的分裂和增殖。     

人醛氧化酶蛋白在肝癌中的表达及其临床意义

目的:探讨人醛氧化酶(human aldehyde oxidase, hAOX)蛋白在肝癌细胞系、肝癌组织、癌旁肝组织、肝硬化及正常肝组织中的表达及其临床意义.方法:采用Western印迹法和免疫组织化学法检测hAOX蛋白在7个肝癌细胞系、2个永生化肝细胞系、10例正常肝、12例肝硬化、57例肝癌及癌旁肝组织中的表达差异,分析其表达差异的临床意义.结果:Western印迹法检测结果表明,hAOX蛋白在MHCC-LM3、Hep3B、Huh-7和BEL-7402肝癌细胞系以及人永生化肝细胞系L-02和Chang's liver等6个细胞系中均有较高表达,在MHCC- 97L、 HepG2和SMMC-7721细胞中则为低表达.免疫组织化学检测发现,hAOX在正常肝、肝硬化组织和癌旁肝组织中高表达,在癌旁肝组织中的表达较匹配肝癌组织的高(P<0.001).Western印迹法检测结果提示癌旁肝组织中hAOX表达明显高于肝癌组织.hAOX表达与肝癌患者性别、年龄、血清甲胎蛋白(alpha-fetoprotein,AFP)水平、乙肝表面抗原、肿瘤大小、病理学分级以及是否伴有肝硬化均无相关性(P>0.05),而与肿瘤血管侵犯和肿瘤生长方式有一定的相关性(P=0.053, P=0.011).结论:hAOX在正常肝、肝硬化、癌旁肝组织中高表达,而在肝癌组织中表达明显降低,其表达可能会影响肝癌的血管侵犯和肿瘤生长方式.     

肝细胞癌组织microRNA-187-3p表达预后意义

目的 微小RNA (microRNA,miRNA)差异表达与多种肿瘤的发生发展密切相关,但微小RNA-187-3p(miR-187-3p)在肝细胞癌(hepatocellular carcinoma,HCC)中的表达及预后意义研究较少.本研究检测HCC组织中microRNA的表达谱,分析miR-187-3p在HCC组织中的表达及其与临床病理特征之间的关系,探讨miR-187-3p表达在其发生、发展及预后中的作用.方法 利用microRNA芯片杂交技术筛选5例HCC组织及相应癌旁组织中差异表达的microRNA,然后通过实时荧光定量PCR(real-time fuorescence quantitative-PCR,RFQ-PCR)法验证86例HCC组织及相应癌旁组织中miR-187-3p表达水平,并统计临床病理资料,随访生存期.结果 148个microRNAs在HCC组织中差异表达,其中102个microRNAs在HCC组织较相应癌旁组织表达下调,46个microRNAs在HCC组织较相应癌旁组织表达上调.RFQ-PCR结果显示,miR-187-3p在HCC组织中表达水平明显低于相应癌旁组织,差异有统计学意义,Z=-2.244,P=0.025.HCC中miR-187-3p表达状态与肿瘤大小(x2=5.031,P=0.025)相关,而与性别(x2=3.648,P=0.056)、年龄(x2=0.003,P=0.956)、是否转移(x2=0.005,P=0.943)、TNM分期(x2=0.129,P=0.719)、肿瘤数目(x2 =0.126,P=0.722)、乙肝表面抗原(x2=0.019,P=0.890)、AFP水平(x2=0.187,P=0.665)和Child分级(x2=1.665,P=0.197)无关.miR-187-3p低表达的HCC患者总生存期较高表达者明显缩短,x2=7.684,P=0.006.且多因素分析发现,miR-187-3p表达水平和TNM分期是影响HCC患者生存预后的独立危险因素.结论 miR-187-3p在一定程度上参与了HCC的发生发展,其有望成为HCC新的潜在的治疗靶点以及诊断、判断病情和预测预后的分子标志物.     

Upregulation of miR-23a approximately 27a approximately 24 decreases transforming growth factor-beta-induced tumor-suppressive activities in human hepatocellular carcinoma cells

Transforming growth factor-beta (TGF-beta) plays a dual and complex role in human cancer. In this report, we observe a specific set of MicroRNAs (miRNAs) changed in response to TGF-beta in human hepatocellular carcinoma (HCC) cells by miRNA microarray screening. A cluster of miRNA, miR-23a approximately 27a approximately 24, is induced in an early stage by TGF-beta in Huh-7 cells. Knockdown of Smad4, Smad2 or Smad3 expression by RNA interference can attenuate the response of miR-23a approximately 27a approximately 24 to TGF-beta addition, indicating that this induction is dependent on Smad pathway. We also explore that miR-23a approximately 27a approximately 24 can function as an antiapoptotic and proliferation-promoting factor in liver cancer cells. In addition, expression of this miRNA cluster is found to be remarkably upregulated in HCC tissues versus normal liver tissues. These findings suggest a novel, alternative mechanism through which TGF-beta could induce specific miRNA expression to escape from tumor-suppressive response in HCC cells.     

Reanalysis of microRNA expression profiles identifies novel biomarkers for hepatocellular carcinoma prognosis

The aim of our study is to identify microRNAs (miRNAs) that have significance in the prognosis and pathogenesis of hepatocellular carcinoma (HCC). The miRNAs differentially expressed in HCC were examined by using a human miRNA microarray dataset, and then the acquired candidates were screened by another microarray dataset. As a result, we got 25 miRNAs which were aberrantly expressed in cancer and meanwhile predicated distinct prognosis. Among them, miR-139-5p was down-regulated in HCC and its low expression in cancer tissue meant poor prognosis. Additionally, we demonstrated that its low expression was also related to several clinicopathologic characteristics such as vein invasion, BCLC stage, p-AKT expression, and pIGFR1 expression. In vitro, it has been discovered that treatment of HCC cells with a miR-139-5p mimic lead to inhibition of cell growth and migration. Moreover, luciferase assay showed that KPNA4 was not the direct target of miR-139-5p. Ectopic expression of miR-139-5p has not repressed the expression of KPNA4, but inhibited the nuclear import of NF-κB and phosphorylation of Akt. In conclusion, for the first time, we identify 25 deregulated miRNAs that are associated with prognosis and prove that miR-139-5p functions as a tumor suppressor in HCC and its low expression predicts poor prognosis.

     

Hepatocellular carcinoma associated microRNA expression signature: integrated bioinformatics analysis,experimental validation and clinical significance

microRNA (miRNA) expression profiles varied greatly among current studies due to different technological platforms and small sample size. Systematic and integrative analysis of published datesets that compared the miRNA expression profiles between hepatocellular carcinoma (HCC) tissue and paired adjacent noncancerous liver tissue was performed to determine candidate HCC associated miRNAs. Moreover, we further validated the confirmed miRNAs in a clinical setting using qRT-PCR and Tumor Cancer Genome Atlas (TCGA) dataset. A miRNA integrated-signature of 5 upregulated and 8 downregulated miRNAs was identified from 26 published datesets in HCC using robust rank aggregation method. qRT-PCR demonstrated that miR-93-5p, miR-224-5p, miR-221-3p and miR-21-5p was increased, whereas the expression of miR-214-3p, miR-199a-3p, miR-195-5p, miR-150-5p and miR-145-5p was decreased in the HCC tissues, which was also validated on TCGA dataset. A miRNA based score using LASSO regression model provided a high accuracy for identifying HCC tissue (AUC = 0.982): HCC risk score = 0.180E_miR-221 + 0.0262E_miR-21 - 0.007E_miR-223 - 0.185E_miR-130a. E_miR-n = Log 2 (expression of microRNA n). Furthermore, expression of 5 miRNAs (miR-222, miR-221, miR-21 miR-214 and miR-130a) correlated with pathological tumor grade. Cox regression analysis showed that miR-21 was related with 3-year survival (hazard ratio [HR]: 1.509, 95%CI: 1.079–2.112, P = 0.016) and 5-year survival (HR: 1.416, 95%CI: 1.057–1.897, P = 0.020). However, none of the deregulated miRNAs was related with microscopic vascular invasion. This study provides a basis for further clinical application of miRNAs in HCC.     

miR-93-5p和miR-27a-3p在直肠癌组织中的表达及其意义

目的 探讨在直肠癌组织中呈现出特异性表达的miRNA与临床病理分期、肿瘤浸润深度、淋巴结转移等参数之间的关系及其可能的意义。方法 用微阵列基因芯片技术分析未经任何术前放化疗的71例直肠癌患者癌组织与癌旁组织间miRNA表达的区别,筛选出上调的miR-93-5p和下调的miR-27a-3p,扩大样本量后进行qRT-PCR验证,随后进行多种临床病理参数分析并探讨其可能存在的意义。结果 miR-27a-3p的表达在芯片检测中呈现出低表达,但在PCR验证时呈现出了高表达,且数据较为离散;miR-93-5p在两种检测方法中均显示出高表达的特性(癌组织表达量是癌旁组织的3.165倍,P=0.006),并与肿瘤体积(P=0.004)、治疗前癌胚抗原水平(P=0.001)及淋巴结转移数目(r=0.534,P=0.005)具有相关性,其中与治疗前癌胚抗原水平及淋巴结转移阳性组受侵数目存在正相关。结论 在直肠癌组织中存在特异性表达的miRNA。根据miR-93-5p和miR-27a-3p在直肠癌组织中的表达特点,miR-93-5p有望作为新型生物标记物,为直肠癌的临床诊疗提供参考价值。     

Changes in microRNA (miRNA) expression during pancreatic cancer development and progression in a genetically engineered KrasG12D;Pdx1-Cre mouse (KC) model

Differential expression of microRNAs (miRNAs) has been demonstrated in various cancers, including pancreatic cancer (PC). Due to the lack of tissue samples from early-stages of PC, the stage-specific alteration of miRNAs during PC initiation and progression is largely unknown. In this study, we investigated the global miRNA expression profile and their processing machinery during PC progression using the Kras^G12D;Pdx1-Cre (KC) mouse model. At 25 weeks, the miRNA microarray analysis revealed significant downregulation of miR-150, miR-494, miR-138, miR-148a, miR-216a, and miR-217 and upregulation of miR-146b, miR-205, miR-31, miR-192, and miR-21 in KC mice compared to controls. Further, expression of miRNA biosynthetic machinery including Dicer, Exportin-5, TRKRA, and TARBP2 were downregulated, while DGCR8 and Ago2 were upregulated in KC mice. In addition, from 10 to 50 weeks of age, stage-specific expression profiling of miRNA in KC mice revealed downregulation of miR-216, miR-217, miR-100, miR-345, miR-141, miR-483-3p, miR-26b, miR-150, miR-195, Let-7b and Let-96 and upregulation of miR-21, miR-205, miR-146b, miR-34c, miR-1273, miR-223 and miR-195 compared to control mice. Interestingly, the differential expression of miRNA in mice also corroborated with the miRNA expression in human PC cell lines and tissue samples; ectopic expression of Let-7b in CD18/HPAF and Capan1 cells resulted in downregulation of KRAS and MSST1 expression. Overall, the present study aids an understanding of miRNA expression patterns during PC pathogenesis and helps to facilitate the identification of promising and novel early diagnostic/prognostic markers and therapeutic targets.     

MiR-542-5p is a negative prognostic factor and promotes osteosarcoma tumorigenesis by targeting HUWE1

Recent evidence has demonstrated that microRNAs (miRNAs) are involved in the proliferation and metastasis of osteosarcoma. Using miRNA microarray and functional screening methods to compare miRNA expression profiles in osteosarcoma cell lines treated with Trichostatin A (TSA), overexpression of miR-542-5p was determined to be involved in the proliferation of osteosarcoma. We used isobaric tags for relative and absolute quantitation (iTRAQ) and nanoscale liquid chromatography-mass spectrometry (NanoLC−MS/MS) to identify differentially expressed proteins in MNNG/HOS and U2OS osteosarcoma cell lines transfected with miR-542-5p; in both cell lines, seven proteins were downregulated, and nine were upregulated. HUWE1 was found to be a direct target of miR-542-5p in both osteosarcoma cell lines, and was negatively correlated with miR-542-5p levels in human osteosarcoma tissues. Moreover, the expression of miR-542-5p was upregulated in human osteosarcoma tissue compared with non-tumor adjacent tissue. Kaplan-Meier analysis revealed that overexpression of miR-542-5p predicted poor prognosis for osteosarcoma patients. Taken together, our results indicated that miR-542-5p plays a critical role in the proliferation of osteosarcoma and targets HUWE1.     

milk-145通过下调OCT4基因仰制肺腺癌干细胞增殖

背景与目的 miR-145是通过miRNA芯片及qPCR验证筛选出的一种潜在肺癌保护性miRNA。本研究旨在探讨miR-145与肺癌干细胞之间的关系及分子机制。方法 miRNA芯片对肺腺癌患者瘤旁和正常组织进行表达谱分析;生物信息学软件预测miR-145潜在的靶基因;脂质体2000介导转染miR-145模拟物和阻遏物进入A549细胞株;实时定量PCR检测miR-145表达水平;Western blot检测OCT4蛋白水平;双荧光素酶报告基因验证miR-145是否作用于OCT4mRNA的3'UTR区预测靶位;细胞增殖实验检测miR-145对于A549细胞生长的作用;流式细胞术检测干细胞表型CD133+的表达。结果在肺腺癌组织中miR-145表达明显低于瘤旁正常组织;miRanda软件预测OCT4是miR-145潜在靶基因;与对照组相比,miR-145模拟物组和阻遏物组miR-145表达分别明显上调和下调;miR-145对A549细胞的生长有双向调节作用,过表达miR-145抑制细胞生长;过表达miR-145可明显降低OCT4蛋白水平及干细胞表型CD133百分比,而抑制miR-145表达则明显增加OCT4蛋白水平...     

Identification of recurrence-related microRNAs in hepatocellular carcinoma following liver transplantation

Tumor recurrence-related microRNAs (miRNAs) in hepatocellular carcinoma (HCC) following orthotopic liver transplantation (OLT) are not clear yet. This study was designed to determine whether altered miRNA expression is associated with HCC recurrence and prognosis following OLT. 18 miRNAs, including 6 up-regulated and 12 down-regulated miRNAs were identified by microarray in primary HCC samples of patients who had developed HCC recurrence (n = 5) compared to those with non-recurrence (n = 5) following OLT by using p < 0.05 as cutoff value. The six most significantly altered miRNAs (fold change ≥ 2: miR-19a, miR-886-5p, miR-126, miR-223, miR-24 and miR-147) were further confirmed by qRT-PCR in the remaining 105 HCC samples. In receiver-operating characteristic curve analysis, this six miRNAs were of high sensitivity and specificity in predicting HCC recurrence. Using Cox regression and risk score analysis, we built a six-miRNA signature based on their qRT-PCR readings for the prediction of outcome of HCC following OLT. Kaplan-Meier and Cox proportional regression revealed this six-miRNA signature was a significant independent predictor of overall survival (log-rank p = 0.020) and recurrence-free survival (log-rank p < 0.001). Finally, the data were further reconfirmed in an independent cohort of 50 patients from another transplant center. In addition, bioinformatics Gene Ontology and pathway analysis were also performed to better understand the critical roles of these miRNAs in HCC recurrence. Our study, in addition to suggesting a different miRNA expression pattern between HCC samples of patients with recurrence and those with non-recurrence, proposes that this six-miRNA signature may serve as biomarker for prognosis of HCC patients following OLT.