PM2.5染毒HBE细胞前后差异表达的microRNAs及其生物信息学分析

目的：采用microRNA（miRNA）测序和生物信息学方法，分析大气细颗粒物（PM_2.5）染毒人支气管上皮HBE细胞前后差异表达的miRNAs，预测差异miRNAs的生物学功能和信号转导途径。方法：用50 μg/mL的PM_2.5混悬液染毒HBE细胞，以未染毒组作为对照组，处理24 h后提取总RNA样品，Small RNA样品预处理试剂盒构建文库，并利用illumina HiSeq^TM 2500/MiSeq测序平台进行高通量测序。以调整后P < 0.05的筛选标准得到差异miRNAs，使用miRanda和RNAhybrid两个软件共同预测差异miRNAs的靶基因并进行GO和KEGG功能富集分析，利用STRING数据库和Cytoscape软件对miRNAs和靶基因及靶基因之间的相互作用关系进行可视化分析。结果：高通量测序方法共检测到PM_2.5染毒前后的包含1 137个miRNAs的表达谱，27个为差异表达的miRNAs，其中7个上调，20个下调。GO和KEGG富集分析发现，这些差异miRNAs主要参与细胞代谢、酶活性调节等生物学过程，聚集于胞内如细胞质、细胞器等，涉及代谢途径、PI3K-Akt信号通路、Ras信号通路、MAPK信号通路等与细胞增殖、分化、凋亡及癌症发生等相关的重要信号通路。此外，通过构建相互作用网络图，鉴定了miR-371b-3p、miR-371a-5p、miR-27a-3p、miR-7-5p、miR-372-3p等相互作用数量前11位的核心miRNAs，以及3个核心靶基因（VEGFA、MAPK3、CCR5）。结论：PM_2.5染毒HBE细胞后引起了27个miRNAs的差异表达，涉及PI3K-Akt信号通路、Ras信号通路及MAPK信号通路等与癌症发生发展相关的重要信号通路，其中筛选了11个核心miRNAs和3个核心基因，为深入研究PM_2.5致癌毒理作用机制提供了实验依据。     

PM2.5染毒HBE细胞前后差异表达的microRNAs及其生物信息学分析

目的：采用microRNA（miRNA）测序和生物信息学方法,分析大气细颗粒物（PM_2.5）染毒人支气管上皮HBE细胞前后差异表达的miRNAs,预测差异miRNAs的生物学功能和信号转导途径。方法：用50 μg/mL的PM_2.5混悬液染毒HBE细胞,以未染毒组作为对照组,处理24 h后提取总RNA样品,Small RNA样品预处理试剂盒构建文库,并利用illumina HiSeq^TM 2500/MiSeq测序平台进行高通量测序。以调整后P < 0.05的筛选标准得到差异miRNAs,使用miRanda和RNAhybrid两个软件共同预测差异miRNAs的靶基因并进行GO和KEGG功能富集分析,利用STRING数据库和Cytoscape软件对miRNAs和靶基因及靶基因之间的相互作用关系进行可视化分析。结果：高通量测序方法共检测到PM_2.5染毒前后的包含1 137个miRNAs的表达谱,27个为差异表达的miRNAs,其中7个上调,20个下调。GO和KEGG富集分析发现,这些差异miRNAs主要参与细胞代谢、酶活性调节等生物学过程,聚集于胞内如细胞质、细胞器等,涉及代谢途径、PI3K-Akt信号通路、Ras信号通路、MAPK信号通路等与细胞增殖、分化、凋亡及癌症发生等相关的重要信号通路。此外,通过构建相互作用网络图,鉴定了miR-371b-3p、miR-371a-5p、miR-27a-3p、miR-7-5p、miR-372-3p等相互作用数量前11位的核心miRNAs,以及3个核心靶基因（VEGFA、MAPK3、CCR5）。结论：PM_2.5染毒HBE细胞后引起了27个miRNAs的差异表达,涉及PI3K-Akt信号通路、Ras信号通路及MAPK信号通路等与癌症发生发展相关的重要信号通路,其中筛选了11个核心miRNAs和3个核心基因,为深入研究PM_2.5致癌毒理作用机制提供了实验依据。     

miR-10a-5p基因甲基化对卵巢癌铂类耐药的影响

目的 卵巢癌是死亡率最高的妇科肿瘤,较强的化疗耐药性是其预后差的主要原因之一,为了阐明卵巢癌对铂类药物的耐药机制,本研究探讨miRNA基因的甲基化水平对卵巢癌铂类耐药的影响.方法 将卵巢癌组织分为敏感组和耐药组,每组各3例;采用基因芯片技术,对比分析了两组微RNA(microRNA,miRNA)的表达差异;采用实时荧光定量PCR,分别在6例敏感和3例耐药组织、铂类药物敏感(CoC1)和耐药的卵巢癌细胞系(CoC1/DDP),检测了候选miRNA的表达差异;应用Massarray技术,检测敏感组织(15例)与耐药组织(6例)中miRNA基因启动子的甲基化差异;应用生物信息学分析,鉴定目标miRNA的潜在靶基因.结果 以铂类药物敏感的卵巢癌组织样本为对照,利用基因芯片筛选,鉴定了6条在耐药组织样本中出现表达上调的miRNA(miR-493-3p、miR-10a-5 p、miR-16-2-3p、miR-1248、miR-451a、miR-628-3p)和6条表达下调的miRNA(miR-509-3p、miR-1197、miR-376a-3p、miR-1273a、miR-550a-3p、miR-19b-3p).组织验证发现,miR-509-3p、miR-493-3p、miR-10a-5p、miR-16-2-3p和miR-451a,与芯片结果一致;培养细胞研究发现,4条miRNA的表达调控方式与组织芯片结果一致,miR-10a-5p、miR-16-2-3p、miR-1248和miR-628-3p在耐药细胞系中高表达.进一步研究发现,与敏感肿瘤组织相比,耐药组织中miR-10a-5p基因启动子的甲基化水平出现显著降低,P=0.04.结合生物信息学预测HOXA1和USF2为miR-10a-5p与耐药相关的靶基因.结论 与敏感组相比,耐药组miR-10a-5p基因启动子甲基化水平显著降低,miR-10a-5p表达升高,通过抑制 HOXA1和USF2,抑制细胞凋亡,导致铂类化疗药物耐受.     

miR-455-5p在上皮性卵巢癌中表达研究及其靶基因功能分析

目的 研究miR-455-5p在上皮性卵巢癌中的表达情况,分析其表达对上皮性卵巢癌发生发展的影响。方法 从GEO数据库中下载正常卵巢上皮组织与上皮性卵巢癌组织miRNA表达数据GSE83693,通过差异表达分析获得上皮性卵巢癌中miRNA差异表达数据,分析miR-455-5p在正常卵巢上皮与上皮性卵巢癌组织中的表达是否存在差异,应用qRT-PCR验证差异表达预测结果;应用生物信息学软件对miR-455-5p靶基因进行KEGG通路富集分析及GO基因功能注释,探究miR-455-5p表达失调在上皮性卵巢癌发生发展过程中的作用。结果 筛选出明显差异表达miRNA 101例,34例表达上调,67例表达下调;其中miR-455-5p呈明显下调(P＜0.01),差异倍数为-2.9019;qRT-PCR验证结果显示,上皮性卵巢癌细胞(SKOV-3,OVCAR-3,A2780)中miR-455-5p表达量明显低于正常卵巢上皮细胞(IOSE-80),差异均有统计学意义(P＜0.05);KEGG通路富集分析结果显示,miR-455-5p调控的靶基因主要参与的通路共5个,包括有TGF-β信号通路,Hippo信号通路,ECM-受体相互作用,癌症中的转录失调通路,慢性粒细胞白血病,这些通路均与肿瘤相关。GO功能注释分析结果显示上述通路中miR-455-5p调控的靶基因主要参与蛋白质磷酸化,促进细胞增殖、迁移,抑制细胞凋亡,促进上皮-间充质转化,转录调节及调控细胞周期等与肿瘤发生相关的功能。结论 miR-455-5p在上皮性卵巢癌中表达下调,miR-455-5p靶基因参与上皮性卵巢癌肿瘤相关通路与功能,与上皮性卵巢癌发生发展相关。     

肝细胞癌根治术后早期复发相关microRNAs的筛选

目的 筛选与肝细胞癌（hepatocellular carcinoma,HCC）患者根治术后早期复发相关的microRNAs。方法 收集10例HCC患者肝脏组织标本,其中早期复发组5例,非早期复发组5例,以及正常肝脏组织标本4例。利用microRNA基因芯片技术检测3组肝脏组织标本中microRNAs的表达谱,筛选出差异表达的microRNAs。结果 早期复发组与正常组相比,miR-21-3p、miR-21-5p、miR-222-3p、miR-3182、miR-3651、miR-3654、miR-4451、miR-4633-5p、miR-720的表达均上调,miR-4525的表达下调（P〈0.05）;非早期复发组与正常组相比,miR-106b-5p、miR-4451、miR-3651、miR-93-5p的表达均上调,miR-451a、miR-3692-5p的表达下调（P〈0.05）。结论 microRNAs的表达与HCC患者术后早期复发有关,检测患者肝脏组织中microRNAs的表达水平可能有助于监测HCC术后早期复发。     

KDM5A表达与卵巢浆液性囊腺癌临床病理因素的关系分析

摘 要：［目的］ 探讨卵巢浆液性囊腺癌中组蛋白赖氨酸去甲基化酶5A（KDM5A）的表达与临床病理因素的关系。［方法］ 利用R2平台的临床数据库对卵巢浆液性囊腺癌临床基因表达芯片数据进行详细分析，从生物信息学角度分析KDM5A表达与临床各参数的相关关系。［结果］ 生信分析结果显示，KDM5A表达与卵巢浆液性囊腺癌的年龄、病理分级、转移及耐药复发密切相关。［结论］ KDM5A的表达和卵巢浆液性囊腺癌的临床病理因素关系密切，其可作为卵巢浆液性囊腺癌病情评估及预后判断的临床参考指标。     

卵巢浆液性肿瘤中OCT4蛋白的表达及与化疗耐药的关系研究

目的:探讨OCT4蛋白在卵巢浆液性肿瘤组织中的表达与卵巢肿瘤发生发展及与化疗耐药的关系。方法:采用免疫组织化学链霉素抗生物素蛋白-过氧化酶连接法（SP法）检测OCT4蛋白在10例正常卵巢组织、10例卵巢良性浆液性囊腺瘤、10例卵巢交界性浆液性囊腺瘤和67例卵巢浆液性腺癌（其中化疗耐药30例,化疗敏感37例）的石蜡病理切片中的表达情况。结果:OCT4蛋白的阳性表达率在正常卵巢组织、卵巢良性浆液性囊腺瘤、卵巢交界性浆液性囊腺瘤和卵巢浆液性腺癌组织中分别为0、40.00%、50.00%和62.69%,差异有显著统计学意义（P=0.002）。在卵巢浆液性腺癌化疗耐药和化疗敏感组织中分别为83.33%和45.95%,差异有显著统计学意义（P=0.002）。OCT4蛋白的阳性表达率与卵巢浆液性腺癌的临床病理特征无明显相关性（P＞0.05）。结论:OCT4蛋白可能在卵巢浆液性肿瘤发生发展和卵巢癌化疗耐药中发挥重要作用,可能作为卵巢癌化疗耐药预测的一个重要参考指标。     

AKT1与卵巢浆液性肿瘤发生发展及化疗耐药的关系研究

[目的]探讨AKT1的表达与卵巢浆液性肿瘤发生发展及卵巢癌化疗耐药的关系。[方法]免疫组织化学SP法检测67例卵巢浆液性腺癌(其中化疗耐药30例、化疗敏感37例)、10例卵巢交界性浆液性囊腺瘤、10例卵巢良性浆液性囊腺瘤和10例正常卵巢组织中的AKT1表达情况。[结果]AKT1的阳性表达率在卵巢浆液性腺癌、卵巢交界性浆液性囊腺瘤、卵巢良性浆液性囊腺瘤和正常卵巢组织中分别为62.69%、70.00%、40.00%和0,有显著统计学差异(P=0.001),在化疗耐药卵巢浆液性腺癌中明显高于化疗敏感者(P=0.033)。AKT1的阳性表达率在Ⅰ～Ⅱ期和Ⅲ～Ⅳ期卵巢浆液性腺癌中分别为31.58%和75.00%,有显著统计学差异(P=0.001)。AKT1的阳性表达率与卵巢浆液性腺癌患者绝经状态、组织学分级和淋巴结转移情况无关(P>0.05)。[结论]AKT1与卵巢浆液性肿瘤发生发展和卵巢癌化疗耐药有关,可作为预后和化疗耐药性的间接预测指标之一。     

胰腺癌Panc-1细胞microRNA差异表达谱生物信息学的分析

目的:对胰腺癌细胞差异miRNAs表达谱进行生物信息学分析,以期从整体水平揭示microRNA在胰腺癌癌变和进展中的作用。方法:采用含有924条探针的microRNA微阵列检测胰腺癌Panc-1细胞,以3T3成纤维细胞为对照,筛选Panc-1细胞特异性microRNA表达谱;然后对上调和下调microRNA的靶基因进行Gene Ontology、Pathway和TFBS转录因子结合位点分析,以及构建microRNA和靶基因相互作用网络。结果:与3T3成纤维细胞的microRNA表达谱比较,筛选出9个Panc-1上调microR-NA,20个下调microRNA。TargetScan和mi-Randa软件预测出1 166个microRNA靶基因在Panc-1细胞中上调,212个靶基因下调。以上靶基因在DNA代谢、细胞间信号和胞质溶胶3种GO中富集显著;靶基因共涉及50条信号通路,其中富集度P<0.05的信号通路有6条;转录因子结合位点分析表明,CEBP-β、NF-κB和p53等对于上调以及下调的microRNA可能都有调节作用;microRNA和靶基因的相互作用网络分析表明,HIF-1A等基因连接度高。结论:利...     

萎缩性骨不连中miRNAs的异常表达与其靶基因的初步预测

目的筛选研究萎缩性骨不连断段修复组织中异常表达的microRNAs（miRNAs）,并对其可能的功能性靶基因进行预测分析。方法以萎缩性骨不连患者断端瘢痕组织（AN,3例）与正常骨折愈合后内固定钢板取出时钢板周围的骨痂组织（B组,3例）作为研究对象,采用Exiqon miRCURY^TM LNA microRNA芯片（11．0版）进行筛选分析,比较A组组织中是否存在异常表达的miRNAs,并采用浏览计算机预测数据库等生物信息学的方法预测其可能的靶基因,探寻miRNAs可能参与的萎缩性骨不连相关病理生理过程。结果A组相对B组有9种miRNAs发生1．5倍以上表达上调（hsa—miR-149,hsa—miR-221,hsa—miR-628—3p,hsa-miR-54—5p,以及5种hsa—miRPlus）,9种miRNAs发生1．5倍以上表达下调（hsa—let-7b＊,hsa—miR-220b,hsa—miR-513a-3p,hsa—miR-551a,hsa—miR-576-5p,hsa—miR-1236,kshv—miR—K12—6—5p,以及2种hsa—miRPlus）。生物信息学分析发现,表达异常的miRNAs与多数成骨基因存在作用靶点。结论萎缩性骨不连的断端修复组织存在miRNAs的异常表达,其靶基因包括BMP家族在内的众多成骨性基因。数种miRNAs,特别是4种上调的miRNAs（hsa-miR-149＊、hsa-miR-221、hsa-miR-628—3p和hsa—miR-654—5p）有可能在调控骨折向不愈合发展中具有重要作用。     

MiRNA expression signature for potentially predicting the prognosis of ovarian serous carcinoma

One of the best prognostic predictors for patients with epithelial ovarian cancer is the Federation of Obstetrics and Gynecology (FIGO) stage at diagnosis. Advanced-stage ovarian serous carcinoma (OSC) generally have poor prognosis. The goal of this study is to develop and validate a miRNA expression profile that can differentiate the OSC at early and advanced stages and study its correlation with the prognosis of OSC. To identify a unique microRNA (miRNA) pattern associated with the progression of OSC at early and advanced stages, a miRNA microarray was performed using Chinese tumor bank specimens of patients with OSC stage I or III in a retrospective analysis. The expression of four dysregulated miRNAs was validated using quantitative real-time polymerase chain reaction (qRT-PCR) in an external cohort of 51 cases of OSC samples at stages I and III. Kaplan–Meier analysis was performed to analyze the correlation between the expression of some miRNAs and prognosis. Of the 768 miRNAs analyzed in the microarray, 26 miRNAs were significantly either up- or downregulated, with at least a 2-fold difference, in OSC stage I compared with stage III. The qRT-PCR results showed that miR-510, miR-509-5p, and miR-508-3p were significantly downregulated and that miR-483-5p was upregulated in stage III OSC compared with stage I, which was consistent with the microarray results. Kaplan–Meier analysis showed low miR-510 expression, low miR-509-5p expression, and advanced FIGO stage, and chemotherapy resistance were significantly associated with poorer overall survival (P?<?0.05). Our results suggest that miRNAs may play a role in the progression of OSC, and miR-510 and miR-509-5p may be considered novel-candidate clinical biomarkers for predicting OSC outcome.     

Chemotherapy-induced modification of microRNA expression in esophageal cancer

Neoadjuvant chemotherapy is often used in the treatment of advanced esophageal cancer. In this study, we determined the impact of chemotherapy on microRNA (miRNA) expression in esophageal cancer cells, and whether identified changes might have biological relevance. Two esophageal carcinoma cell lines (one adenocarcinoma and one squamous cell carcinoma) were treated with cisplatin or 5-fluorouracil for 24 or 72 h. RNA was extracted from cells following 24-h treatment, and used for microarray studies. Promising miRNA candidates were selected for RT-PCR validation. Target prediction using TargetScan, combined with bioinformatic analysis (Ingenuity Pathway Analysis, IPA), was performed to evaluate the implications of the altered miRNA expression. Thirteen miRNAs (miR-199a-5p, miR-302f, miR-320a, miR-342-3p, miR-425, miR-455-3p, miR-486-3p, miR-519c-5p, miR-548d-5p, miR-617, miR-758, miR-766, miR-1286) were deregulated after 24- and/or 72-h treatment in both cell lines, and most miRNAs presented similar expression changes after short- or long-term exposure. IPA revealed that the major networks which incorporate the predicted targets, include functions such as 'Cell death', 'Cell cycle', 'Cellular growth and proliferation', 'DNA replication, recombination, and repair' and 'Drug metabolism'. Cisplatin or 5-fluorouracil alter miRNA expression in esophageal cancer cells. IPA suggests that these miRNAs may target molecular pathways involved in cell survival after chemotherapy.     

miR-142-5p对人上皮性卵巢癌细胞增殖和凋亡的影响

目的:探讨miR-142-5p靶向PTEN-PI3K/Akt信号调控人上皮性卵巢癌SKOV3细胞增殖和凋亡的具体机制。方法:Real-time PCR检测miR-142-5p在人卵巢癌组织及各卵巢癌细胞系中的表达情况。利用MTT、细胞克隆形成实验观察miR-142-5p对SKOV3细胞增殖活力的影响;Caspase-3活力检测miR-142-5p对SKOV3细胞凋亡情况;信号通路抑制剂处理及Western blot检测miR-142-5p、PTEN与PI3K/Akt信号通路的关系及PI3K/Akt信号下游基因Akt、FOXO1、cyclin D1等的表达情况;Luciferase实验验证miR-142-5p和PTEN 3' UTR的结合。结果:人上皮性卵巢癌组织及其细胞系中miR-142-5p表达显著增加（P<0.05)。与对照组相比,SKOV3细胞转染miR-142-5p mimics后活细胞数量显著增加,增殖能力显著上升,细胞凋亡下降（P<0.05);与之相反的,转染miR-142-5p inhibitor后SKOV3细胞增殖能力下调,凋亡增加。信号通路抑制剂处理显示miR-142-5p过表达激活PI3K/Akt信号通路。Luciferase实验显示miR-142-5p与PTEN 3' UTR直接结合。与对照组相比,miR-142-5p mimics组PTEN表达显著降低,p-Akt蛋白表达则显著上调（P<0.05）,同时过表达PTEN后p-Akt表达则显著降低（P<0.05）。结论:miR-142-5p通过抑制PTEN基因表达活化PI3K/Akt信号通路,促进人上皮性卵巢癌组织及SKOV3细胞增殖存活,抑制细胞凋亡。     

Extracellular microRNA profiling for prognostic prediction in patients with high-grade serous ovarian carcinoma

High-grade serous ovarian carcinoma is a leading cause of death in female patients worldwide. MicroRNAs (miRNAs) are stable noncoding RNAs in the peripheral blood that reflect a patient’s condition, and therefore, they have received substantial attention as noninvasive biomarkers in various diseases. We previously reported the usefulness of serum miRNAs as diagnostic biomarkers. Here, we investigated the prognostic impact of the serum miRNA profile. We used the GSE106817 dataset, which included preoperative miRNA profiles of patients with ovarian malignancies. Excluding patients with other malignancy or insufficient prognostic information, we included 175 patients with high-grade serous ovarian carcinoma. All patients except four underwent surgery and received chemotherapy as initial treatment. The median follow-up period was 54.6 months (range, 3.5-144.1 months). Univariate Cox regression analysis revealed that higher levels of miR-187-5p and miR-6870-5p were associated with both poorer progression-free survival (PFS) and overall survival (OS), and miR-1908-5p, miR-6727-5p, and miR-6850-5p were poor prognostic indicators of PFS. The OS and PFS prognostic indices were then calculated using the expression values of three prognostic miRNAs. Multivariate Cox regression analysis showed that both indices were significantly independent poor prognostic factors (hazard ratio for OS and PFS, 2.343 [P = .015] and 2.357 [P = .005], respectively). In conclusion, circulating miRNA profiles can potentially provide information to predict the prognosis of patients with high-grade serous ovarian carcinoma. Therefore, there is a strong demand for early clinical application of circulating miRNAs as noninvasive biomarkers.     

MicroRNA expression profiles in serous ovarian carcinoma.

PURPOSE: Although microRNAs have recently been recognized as riboregulators of gene expression, little is known about microRNA expression profiles in serous ovarian carcinoma. We assessed the expression of microRNA and the association between microRNA expression and the prognosis of serous ovarian carcinoma. EXPERIMENTAL DESIGN: Twenty patients diagnosed with serous ovarian carcinoma and eight patients treated for benign uterine disease between December 2000 and September 2003 were enrolled in this study. The microRNA expression profiles were examined using DNA microarray and Northern blot analyses. RESULTS: Several microRNAs were differentially expressed in serous ovarian carcinoma compared with normal ovarian tissues, including miR-21, miR-125a, miR-125b, miR-100, miR-145, miR-16, and miR-99a, which were each differentially expressed in >16 patients. In addition, the expression levels of some microRNAs were correlated with the survival in patients with serous ovarian carcinoma. Higher expression of miR-200, miR-141, miR-18a, miR-93, and miR-429, and lower expression of let-7b, and miR-199a were significantly correlated with a poor prognosis (P < 0.05). CONCLUSION: Our results indicate that dysregulation of microRNAs is involved in ovarian carcinogenesis and associated with the prognosis of serous ovarian carcinoma.     

MicroRNA expression profiling in human ovarian cancer: miR-214 induces cell survival and cisplatin resistance by targeting PTEN

MicroRNAs (miRNA) represent a novel class of genes that function as negative regulators of gene expression. Recently, miRNAs have been implicated in several cancers. However, aberrant miRNA expression and its clinicopathologic significance in human ovarian cancer have not been well documented. Here, we show that several miRNAs are altered in human ovarian cancer, with the most significantly deregulated miRNAs being miR-214, miR-199a*, miR-200a, miR-100, miR-125b, and let-7 cluster. Further, we show the frequent deregulation of miR-214, miR-199a*, miR-200a, and miR-100 in ovarian cancers. Significantly, miR-214 induces cell survival and cisplatin resistance through targeting the 3'-untranslated region (UTR) of the PTEN, which leads to down-regulation of PTEN protein and activation of Akt pathway. Inhibition of Akt using Akt inhibitor, API-2/triciribine, or introduction of PTEN cDNA lacking 3'-UTR largely abrogates miR-214-induced cell survival. These findings indicate that deregulation of miRNAs is a recurrent event in human ovarian cancer and that miR-214 induces cell survival and cisplatin resistance primarily through targeting the PTEN/Akt pathway.