微小RNA在胰腺癌中的研究进展

目的总结微小RNA(microRNA,miRNA)与胰腺癌之间关系的研究现状,探讨其表达谱在胰腺癌诊断中的重要作用。方法应用PubMed及CNKI期刊全文数据库检索系统,以"microRNA、胰腺癌"等为关键词,检索2000～2012年的相关文献,共检索到英文文献60篇和中文文献15篇。纳入标准为:miRNA与胰腺癌的基础与临床研究以及miRNA在胰腺癌诊治中的前景。根据纳入标准,纳入31篇文献。结果研究发现,miRNA表达谱如miR-21、miR-34、miR-217、miR-196a、miR-10a、miR-155、miR-221、miR-222、miR-181a、miR-181b、miR-181d、miR-200、let-7等家族成员可作为肿瘤标志物用于胰腺癌与正常胰腺、慢性胰腺炎、胰腺内分泌肿瘤等的诊断和鉴别诊断,预测胰腺癌的预后等。结论胰腺癌中miRNA的表达谱不但与胰腺癌的诊断相关,也为胰腺癌的基因治疗提供了新的研究方向和方法。     

细胞外基底刚度调控宫颈癌细胞迁移的microRNA的筛选

目的通过microRNA芯片筛选不同基底刚度影响宫颈癌细胞迁移过程中的关键microRNA的候选者。方法用丙烯酰胺及双丙烯酰胺的不同比例配置基底刚度为1、20 k Pa的人工基底膜,在其上种植宫颈癌细胞系Siha,36 h后收取细胞提取总RNA,用microRNA芯片及qRT-PCR方法筛选2种基底刚度条件下的差异表达microRNA。结果在2种不同刚度条件下生长的宫颈癌细胞系Siha共有173个表达差异的microRNA,其中包括70个表达上调的microRNA和103个表达下调的microRNA。在宫颈癌细胞系Siha、Hela、Caski中,检测与宫颈癌细胞迁移相关的miR-21、miR-125a、miR-75b、miR-150、miR-595、miR-218、miR-200b、miR-107、miR-183等表达与microRNA芯片结果一致。结论不同基底刚度可引起宫颈癌细胞系Siha的microRNA表达谱不同,microRNA在基底刚度调控宫颈癌细胞迁移的过程中可能具有重要作用。     

microRNA-21通过上皮间质转化促进胆管癌细胞侵袭转移的研究

目的探讨microRNA-21（miR-21）对胆管癌RBE细胞侵袭转移能力的影响及其内在机制。 方法miR-21及其抑制基因片段（miR-21i）通过慢病毒载体感染胆管癌RBE细胞,RT-PCR用于检测RBE细胞中miR-21表达;Transwell体外侵袭实验和细胞划痕试验用于检测RBE细胞侵袭及转移能力;Western blotting用于检测PTEN蛋白和上皮间质转化（EMT）特征性蛋白E-cadherin、N-cadherin、Vimentin表达情况。 结果miR-21过表达促进RBE细胞侵袭和转移,miR-21低表达抑制RBE细胞的侵袭和转移。miR-21过表达能够降低PTEN蛋白和E-cadherin蛋白,增强N-cadherin和Vimentin蛋白的表达;转染miR-21i抑制基因组（miR-21i）组则有相反的效果,而转染空白基因组（对照组）相应蛋白表达无明显变化。 结论miR-21起到癌基因的作用,可增强胆管癌细胞侵袭和转移能力,其机制是通过调节抑癌基因PTEN及改变EMT相关蛋白的变化。这些发现为胆管癌侵袭转移的分子机制提供新的科学依据。     

IgA肾病外周血B淋巴细胞微核糖核酸差异表达及临床意义

目的通过检测人外周血B淋巴细胞微核糖核酸(microRNA)的差异表达,并分析其与IgA肾病临床病理特点、IgA1分子O-糖基化异常之间的关系,探索microRNA在IgA肾病发病中的可能机制。方法收集7例IgA肾病患者及4例正常人外周血5 ml,磁珠法分选出CD19~+B淋巴细胞,提取RNA,采用表达谱芯片筛选差异表达的microRNA。在另外29例IgA肾病患者和16例正常对照者中,对候选microRNAs用实时荧光定量聚合酶链反应(quantificational real-time polymerase chain reaction,QRT-PCR)进行验证,并分析它们与临床病理、IgA1分子半乳糖缺陷(galatose-defecient IgA1,Gd-IgA1)水平的关系。结果 IgA肾病患者外周血B淋巴细胞microRNA表达明显异常,其中85个明显上调,30个明显下调。对5个候选microRNA:miR-3189-5p、let-7g-5p、miR-4258-5p、miR-4695-3p及miR-99b-5p,进一步验证显示,其表达水平在IgA肾病患者与正常对照之间差异无统计学意义(P0.05)。且上述5个microRNA表达量与IgA肾病患者的临床病理指标没有明显的相关性。IgA肾病组血清IgA1、Gd-IgA1明显高于正常对照(P0.05或P0.01)。5个候选microRNA:miR-3189-5p、let-7g-5p、miR-4258-5p、miR-4695-3p及miR-99b-5p的水平与IgA肾病患者IgA1分子O-连接半乳糖缺陷之间没有明显相关性。结论 IgA肾病患者和正常人外周血CD19~+B淋巴细胞microRNA表达谱存在明显差异。初步验证显示miR-3189-5p、let-7g-5p、miR-4258-5p、miR-4695-3p及miR-99b-5p的表达水平在IgA肾病患者与正常对照之间差异无统计学意义,且与IgA肾病的临床病理特点和Gd-IgA1之间无明显相关性,提示这5个microRNA可能不是参与IgA肾病发病和进展的重要microRNA。     

下肢动脉硬化闭塞症的microRNA表达特点分析

目的探讨人下肢动脉硬化闭塞症(ASO)组织中microRNA的表达特征。方法采用microRNA芯片和real-time PCR芯片技术对正常人下肢动脉(3例)和ASO动脉(10例)中的microRNA的表达谱进行分析。结果正常动脉和ASO动脉的microRNA表达谱之间存在显著差异。MiR-21等microRNA表达上调明显(P0.05),miR-1298(P0.01)、miR-125b(P0.001)、miR-140-3p(P0.001)表达水平下降明显。进一步通过原位杂交染色发现,表达失调的microRNA主要定位于动脉壁中层平滑肌。结论与正常动脉相比,ASO组织中的microRNA表达谱显著不同,其在ASO发病中的作用有待于进一步研究。     

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。     

MicroRNA-133a-5p调控c-met表达对胆管癌细胞迁移和侵袭的影响

目的:探讨MicroRNA-133(miR-133)在胆管癌细胞系中的表达,研究miR-133基因表达在胆管癌细胞迁移和侵袭过程中的作用。方法:利用实时荧光定量PCR(RT-PCR)技术测定分析miR-133及其不同成熟体(miR-133b、miR-133a-3p、miR-133a-5p)在胆管癌QBC939细胞系中的表达。将胆管癌QBC939细胞系经转染后分为三组:自然生长组(空白对照组),细胞转染无关序列组(阴性对照组)以及细胞转染Anti-miR-133a-5p组(干扰组),利用RT-PCR检测各组胆管癌QBC939细胞系中miR-133a-5p、c-met的表达。然后利用Transwell小室法分别检测分析干扰miR-133a-5p基因表达对胆管癌QBC939细胞系迁移和侵袭能力的影响。结果:RT-PCR检测miR-133不同成熟体miR-133b(0.74±0.07)、miR-133a-3p(0.99±0.12)、miR-133a-5p(1.00±0.06)中的表达,其中miR-133a-5p在胆管癌QBC939细胞系中表达量最高(P0.05)。RT-PCR实验证实干扰组QBC939细胞系中miR-133a-5p(0.70±0.08)表达显著低于对照组(1.43±0.34);干扰组QBC939细胞系中c-met(0.09±0.02)表达也显著低于对照组(1.01±0.07),差异有统计学意义(P0.01)。Transwell小室实验证实干扰组中胆管癌QBC939细胞系迁移(69.5±8.3)和侵袭(19.3±3.3)能力明显低于对照组,差异均有统计学意义(P0.01)。结论:MicroRNA-133a-5p的低表达可以明显减弱c-met的表达,同时可以抑制胆管癌QBC939细胞系的迁移和侵袭能力,miR-133a-5p有可能成为胆管癌基因表达调控的新靶点。     

miR-124抑制结肠癌细胞增殖和侵袭与TET蛋白家族的关系

目的:探讨miR-124是否通过靶向调节TET蛋白家族的表达而抑制结肠癌细胞增殖与侵袭。方法:用双荧光素酶报告基因检测系统分别检测miR-124对TET家族(TET1、TET2、TET3)的3'UTR-荧光素酶活性的影响;用q RT-PCR与Western blot检测miR-124模拟物转染结肠癌HT29细胞后TET家族的m RNA与蛋白表达水平的变化;用MTS和Transwell实验观察HT29细胞转染miR-124模拟物及TET si RNA后增殖和侵袭能力的变化。结果:双荧光素酶报告基因检测结果显示,各TET m RNA的3'UTR均被miR-124特异性结合,其荧光素酶活性被明显抑制(均P0.05);转染miR-124模拟物后的HT29细胞TET的m RNA与蛋白表达水平明显减低(均P0.05);HT29细胞转染miR-124模拟物或TET si RNA后,增殖和侵袭能力均明显降低(均P0.05)。结论:miR-124可能通过直接靶向调控TET基因的表达,而抑制结肠癌细胞增殖和侵袭。     

微RNA在胰腺癌患者血浆中的表达及临床意义

目的 探讨微RNA(microRNA,miR)在胰腺癌诊断中的意义,为胰腺癌的早期诊断提供一种新的方法.方法 收集37例胰腺癌患者血浆标本及临床资料,10例正常人血浆标本作为对照.采用RT-PCR方法,以U6为内参,检测4种miR(miR-190、miR-196a、miR-221、miR-222)在胰腺癌患者及正常人中的相对表达量,并分析其与胰腺癌临床病理特征及CA199的关系.结果 胰腺痛患者血浆中4种miR的表达量均显著高于正常人.其相对表达量分别为miR 190(3.50±4.07)、miR-196a(4.21±4.13)、miR-221(4.00±4.11)、miR-222(2.57±2.55).其中miR-196a的高表达和胰腺癌临床分期呈正相关(P＜0.05),与其他病理特征无关.其余3种miR与临床病理特征均无关.4种miR的相对表达量与CA199浓度无相关性.结论 miR-190、miR-196a、miR-221、miR-222在胰腺癌患者血浆中显著高表达.血浆中miR-190、miR-196a、miR-221、miR-222的检测可能为胰腺癌的早期诊断提供一个新方法.     

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。     

Dysregulated microRNAs involved in contrast-induced acute kidney injury in rat and human

Abstract

Background: Contrast-induced nephropathy (CIN) is a complex syndrome of acute nephropathy that occurs following infusion of intravascular contrast agents, and is associated with an increased risk for adverse cardiovascular events. While there is no ideal biomarker for making an early diagnosis of CIN, we hypothesized that levels of specific circulating microRNA (miRNA) species might serve such a role. Methods: miRNA microarray assays were used to detect miRNAs in the kidney tissue of rats studied as an animal model of CIN. Real-time PCR was performed to validate results of the microarray assays. Kidney-enriched miRNAs detected in rat plasma were used as biomarkers to screen for CIN. Results obtained from the rat model of CIN were further validated in human patients with CIN. Results: Fifty-one miRNAs were aberrantly expressed in the kidney tissues between CIN and control rats; and among these, 17 miRNAs showed a >2-fold change of expression in the kidney tissues of CIN rats when compared with their expressions in non-CIN control rats. Among the 17 miRNAs aberrantly-expressed miRNAs screened from kidney tissue, only six also showed significantly different expression in the plasma of CIN rats. When compared with their levels in non-CIN control rats, the levels of three miR-30 family members (miR-30a, miR-30c, and miR-30e), as well as miR-320, were significantly increased in the plasma of CIN rats, while the plasma levels of miRNAs let-7a and miR-200a were significantly decreased. In a validation study of these results conducted with human plasma samples, only miR-30a, miR-30c, and miR-30e showed > 2-fold increases in CIN patients when compared with non-CIN patients. Receiver operating curves constructed to examine the abilities of miR-30a, miR-30c, and miR-30e to discriminate CIN patients from non-CIN patients showed AUCs of 0.954, 0.888, and 0.835, respectively. Conclusions: Our study provides the first evidence that plasma miRNAs, and especially three miR-30 family members (miR-30a, miR-30c, and miR-30e), might serve as early biomarkers and (or) target candidates for CIN.     

Suppression of microRNA-29 expression by TGF-β1 promotes collagen expression and renal fibrosis

Synthesis and deposition of extracellular matrix (ECM) within the glomerulus and interstitium characterizes renal fibrosis, but the mechanisms underlying this process are incompletely understood. The profibrotic cytokine TGF-β1 modulates the expression of certain microRNAs (miRNAs), suggesting that miRNAs may have a role in the pathogenesis of renal fibrosis. Here, we exposed proximal tubular cells, primary mesangial cells, and podocytes to TGF-β1 to examine its effect on miRNAs and subsequent collagen synthesis. TGF-β1 reduced expression of the miR-29a/b/c/family, which targets collagen gene expression, and increased expression of ECM proteins. In both resting and TGF-β1-treated cells, ectopic expression of miR-29 repressed the expression of collagens I and IV at both the mRNA and protein levels by targeting the 3'untranslated region of these genes. Furthermore, we observed low levels of miR-29 in three models of renal fibrosis representing early and advanced stages of disease. Administration of the Rho-associated kinase inhibitor fasudil prevented renal fibrosis and restored expression of miR-29. Taken together, these data suggest that TGF-β1 inhibits expression of the miR-29 family, thereby promoting expression of ECM components. Pharmacologic modulation of these miRNAs may have therapeutic potential for progressive renal fibrosis.     

MicroRNA-200a and -200b Mediated Hepatocellular Carcinoma Cell Migration Through the Epithelial to Mesenchymal Transition Markers

Background

MicroRNAs (miRNAs) play an essential role in mediating gene expression in both normal and malignant cells. However, little is known about specific miRNAs during the development of hepatocellular carcinoma (HCC) from well-differentiated to poorly differentiated cells.

Methods

We performed miRNA array analysis of three different HCC cell lines: HepG2, HepJ5, and skHep-1. The expression patterns of miR-200 family members were confirmed by real-time polymerase chain reaction (PCR). We overexpressed miR-200 family members by using a lentivirus system and selected for stably transduced cells using antibiotics. The migration ability of the cells was tested using the Transwell migration assay system.

Results

Our miRNA array and real-time PCR results indicated a decrease in the expression of miR-200 family members in poorly differentiated skHep-1 cells compared with well-differentiated HepG2 cells. We overexpressed miR-200a and miR-200b in both HepJ5 and skHep-1 cells and found that the overexpression of the miR-200 family members did not influence proliferation, although migration was decreased in these cells. We found that overexpression of miR-200 family members led to an upregulation of E-cadherin expression in both HepJ5 and skHep-1 cells. Furthermore, we silenced E-cadherin expression by shRNA in miR200a-HepJ5 cells and found that the migratory ability of these cells was enhanced upon the decrease in E-cadherin expression.

Conclusions

Members of the miR-200 family (miR-200a and miR-200b) play important roles in HCC migration by regulating E-cadherin expression.

     

Identification of key microRNAs in exosomes derived from patients with the severe acute pancreatitis

ObjectiveExosomes have been identified as important carriers of various genetic materials, including microRNAs (miRNAs). Increasing evidence indicates that the course of severe acute pancreatitis (SAP) is associated with miRNAs transported by exosomes. We aimed to identify the signature miRNAs as biomarkers of SAP.MethodsWe obtained exosomes from the SAP patients’ blood. After separation, purification, and identification, we performed high-throughput sequencing and screened the differentially expressed(DE) miRNAs in the exosomes. Bioinformatics analysis was performed to identified the target genes of the miRNAs and the pathways enriched based on Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses, and selected the key miRNAs related to SAP. Total RNA was extracted from patient serum exosomes to detect the expression levels of the selected miRNAs in exosomes of three experimental groups (mild -, moderately severe -, and severe AP) and a control group, using Real-time quantitative polymerase chain reaction (RT-qPCR).Results272 DE miRNAs were identified between SAP and control group. Using bioinformatics analysis, we determined that the functions of the target genes were enriched in six signaling pathways including focal adhesion. Based on this, seven candidate signature miRNAs were selected: miR-603, miR-548ad-5p, miR-122–5p, miR-4477a, miR-192–5p, miR-215–5p, and miR-583. The RT-qPCR results of the seven miRNAs in the SAP group were consistent with the sequencing results.ConclusionExosome-derived miR-603, miR-548ad-5p, miR-122–5p, miR-4477a, miR-192–5p, miR-215–5p, miR-583 are positively correlated with SAP, which might provide new insights into the pathogenesis of SAP and serve as the biomarkers of SAP.     

粪便miRNAs在胰腺导管腺癌筛查诊断中的应用价值

目的 检测胰腺导管腺癌（pancreatic ductal adenocarcinoma,PDAC）患者癌组织、配对癌旁组织及粪便miRNAs,评价粪便miRNAs筛查诊断PDAC的价值.方法 收集30例PDAC患者癌组织、癌旁组织及其粪便标本,10例慢性胰腺炎（CP）患者及15例健康志愿者粪便标本,抽提组织及粪便miRNAs,应用实时定量PCR法检测各组样本miR-21、miR-155、miR-196a、miR-216及miR-217的表达量.应用受试者工作曲线（receiver operating characteristic curve,ROC）及其曲线下面积（AUC）评估粪便miRNAs对PDAC的诊断价值.结果 目的miRNAs在癌组织、癌旁组织及粪便标本中均可被检测.粪便miRNAs抽提及检测方法具有可重复性.MiR-21、miR-155、miR-196a在PDAC组织中表达水平高于癌旁组织,而miR-216、miR-217在PDAC组织中表达水平低于癌旁组织（P＜0.01）.与慢性胰腺炎及健康对照组相比,PDAC患者粪便中miR-21及miR-155的相对表达水平都增加,miR-216的相对表达水平降低（P＜0.05）.PDAC组与对照组相比,miR-21、miR-155及miR-216两两组合后的AUC均比单个miRNA的AUC高,而三者联合后的AUC在所有组合中最高达到0.8667（95％CI：0.7722-0.9612）,诊断胰腺癌的敏感性和特异性分别为83.33％和83.33％,两组比较差异有统计学意义（P＜0.0001）.结论 粪便miRNAs的抽提和检测具有可重复性.粪便中miR-21、miR-155和miR-216有可能成为PDAC诊断的潜在分子标记物.     

Serum miRNAs as potential biomarkers for the bronchiolitis obliterans syndrome after lung transplantation

Lung transplantation (LTx) is the last treatment for patients suffering from end-stage lung diseases. Survival post-LTx is hampered by the development of the bronchiolitis obliterans syndrome (BOS) and diagnosis is often late. Given the urgent clinical need to recognize BOS patients at an early stage, we analyzed circulating miRNAs to identify possible stratification markers for BOS development post-transplantation. Therefore, pro-fibrotic (miR-21, miR-155), anti-fibrotic (miR-29a) and fibrosis-unrelated (miR-103, miR-191) miRNAs were analyzed in serum of end-stage lung disease patients and during LTx follow-up.Significant elevated levels of serum miRNAs were observed for all investigated miRNAs in both chronic obstructive pulmonary disease and interstitial lung disease patients compared to healthy controls. The same miRNAs were also significantly increased in the serum of BOS + vs. BOS − patients. Most importantly, miR-21, miR-29a, miR-103, and miR-191 levels were significantly higher in BOS + patients prior to clinical BOS diagnosis.We demonstrated that a selected group of miRNAs investigated is elevated in end-stage lung disease and BOS + patients, prior to clinical BOS diagnosis. Even if further research is expedient on the prognostic value of circulating miRNAs in BOS and lung conditions in general, these results strongly suggest that circulating miRNAs could be used as potential biomarkers for BOS development.     

Differentially expressed microRNA in testicular tissues of hyperuricaemia rats

This study is to identify the differentially expressed miRNAs in testicular tissues of rats with hyperuricaemia-induced male infertility. We found that the hyperuricaemia model group had significantly increased serum uric acid, while significantly decreased sperm concentration and motile sperm percentage than normal group (p < .05). A total of 39 differentially expressed miRNAs were identified in the testicular tissues of hyperuricaemia rats compared with the control rats, ten of which were validated by real-time PCR. The target mRNAs of 7 differentially expressed miRNAs (miR-10b-5p, miR-26a-5p, miR-136-5p, miR-151-3p, miR-183-5p, miR-362-3p and miR-509-5p) from 3’-untranslated region binding perspective were enriched in signalling pathways of Wnt, Jak-STAT, mTOR and MAPK. The target mRNAs of 6 differentially expressed miRNAs (miR-136-5p, miR-144-3p, miR-99a-5p, miR-509-5p, miR-451-5p and miR-362-3p) from coding sequence binding perspective were enriched in signalling pathways of Calcium, Notch and MAPK. The functions of miRNAs in testicular tissues of rats with hyperuricaemia were revealed by the differentially expressed miRNAs (miR-183-5p, miR-99a-5p, miR-10b-5p, miR-151-3p, miR-26a-5p, miR-451-5p, miR-362-3p, miR-136-5p, miR-144-3p and miR-509-5p)–mRNAs interaction network. The differentially expressed miRNAs in the testicular tissues of hyperuricaemia rats might shed light on the mechanism of hyperuricaemia-induced male infertility.     

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。