miR-214、miR-503在上皮性卵巢癌组织及血清外泌小体中表达的研究

目的 检测正常卵巢、化疗敏感上皮性卵巢癌(EOC)及化疗耐药EOC组织及血清外泌小体中p53相关miR-214、miR-503的表达差异,初探p53相关miRNA与EOC耐药的关系.方法 采用免疫荧光法检测正常卵巢、化疗敏感EOC及化疗耐药EOC组织中p53的表达;RT-PCR法检测血清外泌小体及上述3种组织中p53相关miR-214、miR-503表达的差异.结果 免疫荧光法发现,与正常组织相比,p53在EOC组织中荧光较弱,其中化疗耐药EOC组织与化疗敏感EOC组织间比较差异无统计学意义(P﹥0.05).与敏感组相比,耐药组血清外泌小体中miR-214上调3倍,miR-503下调3倍,与组织中变化一致.结论 p53相关miR-214及miR-503由外泌小体携带进入血液循环,并可能在EOC化疗耐药中发挥重要作用.     

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.     

MicroRNA-regulated gene networks during mammary cell differentiation are associated with breast cancer

MicroRNAs (miRNAs) play pivotal roles in stem cell biology, differentiation and oncogenesis and are of high interest as potential breast cancer therapeutics. However, their expression and function during normal mammary differentiation and in breast cancer remain to be elucidated. In order to identify which miRNAs are involved in mammary differentiation, we thoroughly investigated miRNA expression during functional differentiation of undifferentiated, stem cell-like, murine mammary cells using two different large-scale approaches followed by qPCR. Significant changes in expression of 21 miRNAs were observed in repeated rounds of mammary cell differentiation. The majority, including the miR-200 family and known tumor suppressor miRNAs, was upregulated during differentiation. Only four miRNAs, including oncomiR miR-17, were downregulated. Pathway analysis indicated complex interactions between regulated miRNA clusters and major pathways involved in differentiation, proliferation and stem cell maintenance. Comparisons with human breast cancer tumors showed the gene profile from the undifferentiated, stem-like stage clustered with that of poor-prognosis breast cancer. A common nominator in these groups was the E2F pathway, which was overrepresented among genes targeted by the differentiation-induced miRNAs. A subset of miRNAs could further discriminate between human non-cancer and breast cancer cell lines, and miR-200a/miR-200b, miR-146b and miR-148a were specifically downregulated in triple-negative breast cancer cells. We show that miR-200a/miR-200b can inhibit epithelial-mesenchymal transition (EMT)-characteristic morphological changes in undifferentiated, non-tumorigenic mammary cells. Our studies propose EphA2 as a novel and important target gene for miR-200a. In conclusion, we present evidentiary data on how miRNAs are involved in mammary cell differentiation and indicate their related roles in breast cancer.     

宫颈癌及癌前病变组织中microRNAs的差异表达

目的 寻找与宫颈癌及宫颈癌前病变相关的microRNA。 方法 利用miRNA芯片,筛查宫颈 癌组织、宫颈上皮内瘤变及正常宫颈组织中差异表达的miRNA,并用实时定量RT-PCR在60份宫颈组 织标本中对4个miRNA进行验证。利用生物信息学对部分差异表达的miRNA的靶基因进行功能分析。 结果 与正常宫颈组织比较,宫颈癌及高级别宫颈病变（HSIL）中存差异表达的miRNAs,其中在宫 颈癌中下调最明显的是miR-218（下调倍数为0.175）,上调最明显的是miR-21（上调倍数为5.68）。 实时定量RT-PCR验证结果与miRNA芯片结果基本一致。功能分析显示预测的miR-218及miR-21的靶基 因与肿瘤的生长、侵袭转移有关。结论 宫颈癌及癌前病变中存在异常表达的miRNA,它们在宫颈癌 发生过程中可能起癌基因或抑癌基因的作用。     

MicroRNA gene expression deregulation in human breast cancer

MicroRNAs (miRNAs) are a class of small noncoding RNAs that control gene expression by targeting mRNAs and triggering either translation repression or RNA degradation. Their aberrant expression may be involved in human diseases, including cancer. Indeed, miRNA aberrant expression has been previously found in human chronic lymphocytic leukemias, where miRNA signatures were associated with specific clinicobiological features. Here, we show that, compared with normal breast tissue, miRNAs are also aberrantly expressed in human breast cancer. The overall miRNA expression could clearly separate normal versus cancer tissues, with the most significantly deregulated miRNAs being mir-125b, mir-145, mir-21, and mir-155. Results were confirmed by microarray and Northern blot analyses. We could identify miRNAs whose expression was correlated with specific breast cancer biopathologic features, such as estrogen and progesterone receptor expression, tumor stage, vascular invasion, or proliferation index.     

miR-483-5p在上皮性卵巢癌中的表达及其对顺铂敏感性的影响

背景与目的：顺铂是目前临床上治疗上皮性卵巢癌的一线化疗药物之一,但许多患者对铂类药物耐药。miR-483-5p在肺癌中过表达,然而目前尚未见miR-483-5p在上皮性卵巢癌中的研究。该研究检测miR-483-5p在上皮性卵巢癌组织和上皮性卵巢癌细胞系中的表达并探讨其对上皮性卵巢癌细胞对顺铂敏感性的影响。方法：采用实时荧光定量聚合酶链反应(real-time fluorescent quantitative polymerase chain reaction,RTFQ-PCR)检测43例上皮性卵巢癌患者的肿瘤组织、8例正常卵巢组织和5种上皮性卵巢癌细胞系中miR-483-5p的表达情况;通过慢病毒上调或敲低卵巢癌细胞miR-483-5p表达,应用CCK-8实验检测miR-483-5p对上皮性卵巢癌细胞系顺铂敏感性的影响。结果：上皮性卵巢癌组织中miR-483-5p表达明显高于正常卵巢组织(P<0.01)。此外,miR-483-5p在晚期上皮性卵巢癌组织中的表达水平显著高于早期肿瘤组织(P<0.05)。5种上皮性卵巢癌细胞系中SKOV3细胞表达miR-483-5p的量最低;miR-483-5p在上皮性卵巢癌顺铂耐药A2780/CP细胞中表达量最高。上调SKOV3细胞中miR-483-5p的表达能够降低上皮性卵巢癌细胞对顺铂的敏感性,并下调p21及Bcl-2的表达;下调A2780/CP细胞miR-483-5p的表达能够增加细胞对顺铂的敏感性,并上调p21及Bcl-2的表达。结论：miR-483-5p在上皮性卵巢癌组织中高表达并对顺铂耐药,可以作为临床预测上皮性卵巢癌对顺铂敏感性的生物标志物之一。     

microRNA Expression Profile in Patients with Stage II Colorectal Cancer: A Turkish Referral Center Study

Background: There are increasing data about microRNAs (miRNA) in the literature, providing abundantevidence that they play important roles in pathogenesis and development of colorectal cancer. In this study, weaimed to investigate the miRNA expression profiles in surgically resected specimens of patients with recurrentand non-recurrent colorectal cancer. Materials and Methods: The study population included 40 patients withstage II colorectal cancer (20 patients with recurrent tumors, and 20 sex and age matched patients withoutrecurrence), who underwent curative colectomy between 2004 and 2011 without adjuvant therapy. Expression of16 miRNAs (miRNA-9, 21, 30d, 31, 106a, 127, 133a, 133b, 135b, 143, 145, 155, 182, 200a, 200c, 362) was verifiedby quantitative real-time polymerase chain reaction (qRT-PCR) in all resected colon cancer tissue samples andin corresponding normal colonic tissues. Data analyses were carried out using SPSS 15 software. Values werestatistically significantly changed in 40 cancer tissues when compared to the corresponding 40 normal colonictissues (p<0.001). MiR-30d, miR-133a, miR-143, miR-145 and miR-362 expression was statistically significantlydownregulated in 40 resected colorectal cancer tissue samples (p<0.001). When we compared subgroups,miRNA expression profiles of 20 recurrent cancer tissues were similar to all 40 cancer tissues. However in 20non-recurrent cancer tissues, miR-133a expression was not significantly downregulated, moreover miR-133bexpression was significantly upregulated (p<0.05). Conclusions: Our study revealed dysregulation of expressionof ten miRNAs in Turkish colon cancer patients. These miRNAs may be used as potential biomarkers for earlydetection, screening and surveillance of colorectal cancer, with functional effects on tumor cell behavior.     

miRNA标志物在诊断云南省宣威地区早期非吸烟女性肺癌中的价值

目的初步探索与肿瘤发生相关的miRNA是否可以作为早期诊断云南省宣威地区非吸烟女性肺癌的小分子标志物。方法（1）应用miRNA微阵列芯片分析技术,检测37例宣威地区非吸烟女性肺癌患者和32例非宣威地区非吸烟女性肺癌患者癌组织中的miRNA表达谱,应用实时荧光定量PCR(Q-PCR)对生物芯片分析结果进行验证。（2）实时荧光定量PCR检测16例宣威地区Ⅰ期非吸烟女性肺癌患者和宣威肺癌高发区14例良性病变女性血清中miRNA表达水平。用受试者特征曲线（ROC）来评价血清中miRNA作为诊断宣威地区早期非吸烟女性肺癌患者的敏感度和特异性。结果发现与相邻对应非癌肺组织相比癌组织中有31个miRNA表达存在差异,癌组织中表达差异最显著的miRNA通过实时荧光定量PCR证实。其中miR-21、miR-10a、miR-494、miR-22、miR-141及miR-200b在两组癌组织中的表达水平差异有统计学意义（P<0.05）,且均在16例宣威地区Ⅰ期非吸烟女性肺癌患者和14例良性病变女性血清中稳定表达,其中miR-494、miR-22和miR-200b在两血清中的表达量显著不同（P<0.01）。用其作为分子标志物筛查早期肺癌患者,其敏感度和特异性为85.26%和94.45%。结论从表达的稳定性、敏感度和特异性上看,miR-494、miR-22和miR-200b可作为早期诊断宣威地区非吸烟女性肺癌患者有潜力的小分子标志物。     

Epithelial–mesenchymal transition-associated miRNAs in ovarian carcinoma,with highlight on the miR-200 family: Prognostic value and prospective role in ovarian cancer therapeutics

MicroRNAs (miRNAs) are a family of short ribonucleic acids found to play a pivotal role in cancer pathogenesis. MiRNAs are crucial in cellular differentiation, growth, stress response, cell death and other fundamental cellular processes, and their involvement in ovarian cancer has been recently shown. They can repress the expression of important cancer-related genes and they can also function both as oncogenes and tumour suppressor genes. During epithelial–mesenchymal transition (EMT), epithelial cells lose their cell polarity and cell–cell adhesion and gain migratory and invasive properties. In the ovarian surface epithelium, EMT is considered the key regulator of the post-ovulatory repair process and it can be triggered by a range of environmental stimuli. The aberrant expression of the miR-200 family (miR-200a, miR-200b, miR-200c, miR-141 and miR-429) in ovarian carcinoma and its involvement in ovarian cancer initiation and progression has been well-demonstrated. The miR-200 family members seem to be strongly associated with a pathologic EMT and to have a metastasis suppressive role. MiRNA signatures can accurately distinguish ovarian cancer from the normal ovary and can be used as diagnostic tools to predict the clinical response to chemotherapy. Recent evidence suggests a growing list of new miRNAs (miR-187, miR-34a, miR-506, miRNA-138, miR-30c, miR-30d, miR-30e-3p, miR-370 and miR-106a, among others) that are also implicated in ovarian carcinoma-associated EMT, either enhancing or suppressing it. MiRNA-based gene therapy provides a prospective anti-tumour approach for integrated cancer therapy. The aim of nanotechnology-based delivery approach for miRNA therapy is to overcome challenges in miRNA delivery and to effectively encourage the reprogramming of miRNA networks in cancer cells, which may lead to a clinically translatable miRNA-based therapy to benefit ovarian cancer patients.     

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.     

Identification of new aberrantly expressed miRNAs in intestinal-type gastric cancer and its clinical significance

miRNAs are small 19 to 22 nucleotide sequences of RNA that negatively regulate gene expression. miRNA expression profiles may become useful biomarkers for diagnostics, prognosis and prediction of response to treat, and it could be a powerful tool for cancer prevention and therapeutics. Several miRNA expression profiles of miRNAs in gastric cancer have been reported, but these studies screened only few miRNAs and samples used in experiments include several different subtypes of gastric cancers, which decrease the sensitivity to identify new aberrant miRNAs. In this study, a miRNA expression profile was identified by miRCURY LNA Array (v.14.0) between intestinal-type gastric cancers and normal tissues. Forty miRNA precursors were up-regulated and thirty-six miRNA were down-regulated in intestinal-type gastric cancers (p<0.01). Sixteen new miRNAs were found in intestinal-type gastric cancers. Seventeen new miRNAs were found in intestinal-type gastric cancers. miR-145, miR-27a, miR-494 are differently expressed between intestinal-type and diffuse-type gastric cancers. miR-32, miR-182 and miR-143 dysregulated expression levels are related with different pathological stages of intestinal-type gastric cancers (p<0.01). Taken together, aberrantly expressed miRNAs may offer new clues to tumorigenesis of gastric cancers. miR-32, miR-182 and miR-143 may be potential diagnostic biomarkers for intestinal-type gastric cancers.     

Identification of novel molecular targets regulated by tumor suppressive miR-1/miR-133a in maxillary sinus squamous cell carcinoma

Based on our microRNA (miRNA) expression signature analysis of maxillary sinus squamous cell carcinoma (MSSCC), we found that miR-1 and miR-133a were significantly reduced in tumor tissues. Quantitative real-time RT-PCR revealed that the expression levels of miR-1 and miR-133a were significantly downregulated in clinical MSSCC tumor tissues compared with normal tissues. We focused on the functional significance of miR-1 and miR-133a in cancer cells and identification of the novel cancer networks regulated by these miRNAs in MSSCC. Restoration of downregulated miRNAs (miR-1 or miR-133a) in cancer cells revealed that both miRNAs significantly inhibited cancer cell proliferation and induced cell apoptosis. Molecular target identification of these miRNAs showed that transgelin 2 (TAGLN2) and purine nucleoside phosphorylase (PNP) were regulated by miR-1 and miR-133a. Both TAGLN2 and PNP mRNA expression levels were significantly upregulated in clinical MSSCC tumor tissues. Silencing studies of target genes demonstrated that both genes inhibited cancer cell proliferation. The identification of novel miR-1/miR-133a-regulated cancer pathways could provide new insights into potential molecular mechanisms of MSSCC oncogenesis.     

Differential microRNA Expression by Solexa Sequencing in the Sera of Ovarian Cancer Patients

MicroRNAs are a class of small noncoding RNA which play important regulatory roles in a variety of cancers.MiRNA-specific expression profiles have been reported for several pathological conditions. In this study, wecombined large scale parallel Solexa sequencing to identify 11 up-regulated miRNAs and 19 down-regulatedmiRNAs with computational techniques in the sera of ovarian cancer patients while using healthy serum as thecontrol. Among the above, four miRNAs (miR-22, miR-93, miR-106b, miR-451) were validated by quantitativeRT-PCR and found to be significantly aberrantly expressed in the serum of ovarian cancer patients (P<0.05).There were no significant differences between samples from cancer stage Ⅰ/Ⅱ and Ⅲ/Ⅳ. However, the levelsof miR-106b (p=0.003) and miR-451 (p=0.007) were significantly different in those patients under and over 51yearsof age. MiR-451 and miR-93 were also specific when analyzed with reference to different levels of CA125.This study shows that Solexa sequencing provides a promising method for cancer-related miRNA profiling, andselectively expressed miRNAs could be used as potential serum-based biomarkers for ovarian cancer diagnosis.     

MicroRNA expression signature in gastric cancer

Objective: To identify the miRNA specific signature as novel diagnostic and prognostic tools for gastric cancer. Methods: miRNAs expression profiling of 3 normal gastric tissues, 24 malignant tissues, gastric cancer cell SGC7901 and normal gastric cell GES-1 were detected using microarray technology. The hierarchical clustering algorithm of the Cluster software was used to analyse the miRNAs expression of all samples. The expression levels of miR-433 and miR-9 which were significantly down-regulated in gastric cancer tissues and SGC7901 cells by microarray technology were validated by quantitative Real-time PCR (qRT-PCR).Results: Differential expressions of 26 individual miRNAs between normal samples (including 3 normal gastric tissues and GES-1 cells) and carcinomas (including 24 malignant tissues and SGC7901 cells) were discovered,19 of them showing down-regulation and 7 up-regulation in carcinoma samples. Hierarchical clustering of the cancer samples by their miRNA expression accurately separated the carcinomas from normal samples and further their histotypes of carcinomas. The expression levels of miR-433 and miR-9 were significantly down-regulated in gastric cancer tissues and SGC7901cells Conclusion: The differential expression of miR-433 and miR-9 may be used as a novel diagnostic tool for gastric cancer.