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.     

Dual regulation by microRNA-200b-3p and microRNA-200b-5p in the inhibition of epithelial-to-mesenchymal transition in triple-negative breast cancer

Epithelial to mesenchymal transition (EMT) involves loss of an epithelial phenotype and activation of a mesenchymal one. Enhanced expression of genes associated with a mesenchymal transition includes ZEB1/2, TWIST, and FOXC1. miRNAs are known regulators of gene expression and altered miRNA expression is known to enhance EMT in breast cancer. Here we demonstrate that the tumor suppressive miRNA family, miR-200, is not expressed in triple negative breast cancer (TNBC) cell lines and that miR-200b-3p over-expression represses EMT, which is evident through decreased migration and increased CDH1 expression. Despite the loss of migratory capacity following re-expression of miR-200b-3p, no subsequent loss of the conventional miR-200 family targets and EMT markers ZEB1/2 was observed. Next generation RNA-sequencing analysis showed that enhanced expression of pri-miR-200b lead to ectopic expression of both miR-200b-3p and miR-200b-5p with multiple isomiRs expressed for each of these miRNAs. Furthermore, miR-200b-5p was expressed in the receptor positive, epithelial breast cancer cell lines but not in the TNBC (mesenchymal) cell lines. In addition, a compensatory mechanism for miR-200b-3p/200b-5p targeting, where both miRNAs target the RHOGDI pathway leading to non-canonical repression of EMT, was demonstrated. Collectively, these data are the first to demonstrate dual targeting by miR-200b-3p and miR-200b-5p and a previously undescribed role for microRNA processing and strand expression in EMT and TNBC, the most aggressive breast cancer subtype.     

The miRNA-200 family and miRNA-9 exhibit differential expression in primary versus corresponding metastatic tissue in breast cancer

Metastases are the major cause of cancer-related deaths, but the mechanisms of the metastatic process remain poorly understood. In recent years, the involvement of microRNAs (miRNAs) in cancer has become apparent, and the objective of this study was to identify miRNAs associated with breast cancer progression. Global miRNA expression profiling was performed on 47 tumor samples from 14 patients with paired samples from primary breast tumors and corresponding lymph node and distant metastases using LNA-enhanced miRNA microarrays. The identified miRNA expression alterations were validated by real-time PCR, and tissue distribution of the miRNAs was visualized by in situ hybridization. The patients, in which the miRNA profile of the primary tumor and corresponding distant metastasis clustered in the unsupervised cluster analysis, showed significantly shorter intervals between the diagnosis of the primary tumor and distant metastasis (median 1.6 years) compared to those that did not cluster (median 11.3 years) (p<0.003). Fifteen miRNAs were identified that were significantly differentially expressed between primary tumors and corresponding distant metastases, including miR-9, miR-219-5p and four of the five members of the miR-200 family involved in epithelial-mesenchymal transition. Tumor expression of miR-9 and miR-200b were confirmed using in situ hybridization, which also verified higher expression of these miRNAs in the distant metastases versus corresponding primary tumors. Our results demonstrate alterations in miRNA expression at different stages of disease progression in breast cancer, and suggest a direct involvement of the miR-200 family and miR-9 in the metastatic process.     

Epigenetic regulation of miR-34b and miR-129 expression in gastric cancer

MicroRNAs (miRNAs) are small noncoding RNAs that play fundamental roles in diverse biological and pathological processes by targeting the expression of specific genes. Here, we identified 38 methylation-associated miRNAs, the expression of which could be epigenetically restored by cotreatment with 5-aza-2'-deoxycytidine and trichostatin A. Among these 38 miRNAs, we further analyzed miR-34b, miR-127-3p, miR-129-3p and miR-409 because CpG islands are predicted adjacent to them. The methylation-silenced expression of these miRNAs could be reactivated in gastric cancer cells by treatment with demethylating drugs in a time-dependent manner. Analysis of the methylation status of these miRNAs showed that the upstream CpG-rich regions of mir-34b and mir-129-2 are frequently methylated in gastric cancer tissues compared to adjacent normal tissues, and their methylation status correlated inversely with their expression patterns. The expression of miR-34b and miR-129-3p was downregulated by DNA hypermethylation in primary gastric cancers, and the low expression was associated with poor clinicopathological features. In summary, our study shows that tumor-specific methylation silences miR-34b and miR-129 in gastric cancer cells.     

Regulation of Axl receptor tyrosine kinase expression by miR-34a and miR-199a/b in solid cancer

Axl is a receptor that induces proliferation, migration and invasion in cancer. In this study, we show that specific microRNAs (miRNAs) target the 3'-UTR of Axl. Luciferase-reporter assays with wild-type and deleted miR-34 and miR-199a/b seed sequences of Axl 3'-UTR confirmed the specificity of targeting. An inverse correlation between Axl protein and miR-34a expression in a panel of non-small cell lung cancer (NSCLC), colorectal cancer (CRC) and breast cancer (BRC) cell lines was observed, while miR-199a/b expression was completely suppressed. Pre-miR transfection inhibited in vitro migration and invasion and, in vivo, reduced the number of distant lung- or liver-metastases in a chorion-allantoic-membrane (CAM) assay. Moreover, methylation-specific PCR on bisulfite-converted DNA obtained from the cell lines showed that the miR-34a promoter methylation status was inversely correlated with its expression, and that miR-199a/b promoter regions were methylated in all cells tested. In a panel of NSCLC tissues (n=44), miR-34a and miR-199a/b were found to be downregulated and significantly co-expressed. A lower expression of all three miRs was significantly associated with squamous histotypes, and, in a preliminary series, NSCLC patients with miR-34a upregulation showed a positive association towards a longer survival. These results indicate that Axl receptor expression can be regulated by miR-34a and miR-199a/b, which are suppressed by promoter methylation in solid cancer cells.     

Methylation-associated silencing of microRNA-34b/c in gastric cancer and its involvement in an epigenetic field defect

Altered expression of microRNA (miRNA) is strongly implicated in cancer, and recent studies have shown that the silencing of some miRNAs is associated with CpG island hypermethylation. To identify epigenetically silenced miRNAs in gastric cancer (GC), we screened for miRNAs induced by treatment with 5-aza-2'-deoxycytidine and 4-phenylbutyrate. We found that miR-34b and miR-34c are epigenetically silenced in GC and that their downregulation is associated with hypermethylation of the neighboring CpG island. Methylation of the miR-34b/c CpG island was frequently observed in GC cell lines (13/13, 100%) but not in normal gastric mucosa from Helicobacter pylori-negative healthy individuals. Transfection of a precursor of miR-34b and miR-34c into GC cells induced growth suppression and dramatically changed the gene expression profile. Methylation of miR-34b/c was found in a majority of primary GC specimens (83/118, 70%). Notably, analysis of non-cancerous gastric mucosae from GC patients (n = 109) and healthy individuals (n = 85) revealed that methylation levels are higher in gastric mucosae from patients with multiple GC than in mucosae from patients with single GC (27.3 versus 20.8%; P < 0.001) or mucosae from H. pylori-positive healthy individuals (27.3 versus 20.7%; P < 0.001). These results suggest that miR-34b and miR-34c are novel tumor suppressors frequently silenced by DNA methylation in GC, that methylation of miR-34b/c is involved in an epigenetic field defect and that the methylation might be a predictive marker of GC risk.     

MicroRNA signatures in human ovarian cancer

Epithelial ovarian cancer (EOC) is the sixth most common cancer in women worldwide and, despite advances in detection and therapies, it still represents the most lethal gynecologic malignancy in the industrialized countries. Unfortunately, still relatively little is known about the molecular events that lead to the development of this highly aggressive disease. The relatively recent discovery of microRNAs (miRNA), a class of small noncoding RNAs targeting multiple mRNAs and triggering translation repression and/or RNA degradation, has revealed the existence of a new level of gene expression regulation. Multiple studies involving various types of human cancers proved that miRNAs have a causal role in tumorigenesis. Here we show that, in comparison to normal ovary, miRNAs are aberrantly expressed in human ovarian cancer. The overall miRNA expression could clearly separate normal versus cancer tissues. The most significantly overexpressed miRNAs were miR-200a, miR-141, miR-200c, and miR-200b, whereas miR-199a, miR-140, miR-145, and miR-125b1 were among the most down-modulated miRNAs. We could also identify miRNAs whose expression was correlated with specific ovarian cancer biopathologic features, such as histotype, lymphovascular and organ invasion, and involvement of ovarian surface. Moreover, the levels of miR-21, miR-203, and miR-205, up-modulated in ovarian carcinomas compared with normal tissues, were significantly increased after 5-aza-2'-deoxycytidine demethylating treatment of OVCAR3 cells, suggesting that the DNA hypomethylation could be the mechanism responsible for their overexpression. Our results indicate that miRNAs might play a role in the pathogenesis of human EOC and identify altered miRNA gene methylation as a possible epigenetic mechanism involved in their aberrant expression.     

MicroRNA-143 is downregulated in breast cancer and regulates DNA methyltransferases 3A in breast cancer cells

MicroRNAs (miRNAs) are small non-protein-coding RNAs that regulate expression of a wide variety of genes including those involved in cancer development. Here, we investigate the role of miR-143 in breast cancer. In this study, we showed that miR-143 was frequently downregulated in 80 % of breast carcinoma tissues compared to their adjacent noncancerous tissues. Ectopic expression of miR-143 inhibited proliferation and soft agar colony formation of breast cancer cells and also downregulated DNA methyltransferase 3A (DNMT3A) expression on both mRNA and protein levels. Restoration of miR-143 expression in breast cancer cells reduces PTEN hypermethylation and increases TNFRSF10C methylation. DNMT3A was demonstrated to be a direct target of miR-143 by luciferase reporter assay. Furthermore, miR-143 expression was observed to be inversely correlated with DNMT3A mRNA and protein expression in breast cancer tissues. Our findings suggest that miR-143 regulates DNMT3A in breast cancer cells. These findings elucidated a tumor-suppressive role of miR-143 in epigenetic aberration of breast cancer, providing a potential development of miRNA-based treatment for breast cancer.     

复发转移性乳腺癌患者血浆miRNA表达水平与临床病理特征、化疗疗效及预后关系的研究

目的：探讨血浆中miR-16、miR-34a、miR-200a、miR-375、miR-451化疗前、后表达水平的变化与乳腺癌临床病理特征、化疗疗效及预后之间的关系。方法选取复发转移性乳腺癌患者79例,采用GenEx ver-sion6软件、Normfinder软件和Genorm软件确定本实验的内对照,采用实时荧光定量PCR法检测患者化疗前及化疗2个周期时的血浆miRNA表达水平,并分析化疗前、后表达水平与患者临床病理特征、化疗疗效及预后的关系。结果 miR-34a为相对表达最稳定的内对照。血浆miR-451高表达的患者激素受体（hormone receptor, HR）阳性率显著高于低表达者（P=0.031）,分别为84.2%和62.5%。血浆miR-200a化疗后表达变化与客观缓解率（objective response rate,ORR）显著相关（P=0.032）,miR-200a化疗后表达升高者的ORR显著高于降低者,分别为57.9%和29.3%。血浆miR-200a化疗后表达水平升高组的无进展生存时间（progression-free survival,PFS）与降低组相比,组间差异具有统计学意义（P=0.009）,分别为10.3个月和5.2个月。miR-16、miR-200a、miR-375和miR-451表达水平及化疗后变化对总生存时间（overall survival,OS）均无显著影响。结论血浆miRNA化疗前、后表达水平对复发转移性乳腺癌患者临床病理特征、化疗疗效及预后有一定的预测价值。     

DNA甲基化调控miRNA-328的表达水平与乳腺癌预后的关系分析

目的 探讨DNA甲基化与微小RNA-328（miR-328）表达的关系及miR-328与浸润性乳腺癌临床病理特征和预后的关系。方法 回顾性分析1998年1月至2013年12月从TCGA乳腺癌芯片数据提取的1090例浸润性乳腺癌组织和104例癌旁组织的miR-328表达水平,分析从TCGA_BRCA_hMethyl450芯片数据中获取的775例浸润性乳腺癌组织和98例癌旁组织的miR-328启动子区CpG位点（cg16421621、cg04650403）甲基化率并计算启动子区甲基化与miR-328水平的相关性,从UCSC Genome Browser中的clinical_data_dictionary芯片数据中获取854例有完整临床病理资料及随访数据的乳腺癌标本,分析miR-328表达与临床病理特征及预后的关系,采用Cox回归模型分析影响预后的因素。结果 TCGA数据库1998年1月至2013年12月有miR-328表达量的1194例乳腺癌及癌旁组织中,1090例浸润性乳腺癌组织的miR-328水平为5.224±1.155,低于104例癌旁组织的6.246±0.923,差异有统计学意义（P＜0.01）;775例浸润性乳腺癌组织的miR-328基因启动子区域cg16421621和cg04650403的甲基化率分别为（0.415±0.201）%和（0.193±0.068）%,高于98例癌旁组织的（0.407±0.222）%和（0.094±0.079）%,差异有统计学意义(P＜0.01）;浸润性乳腺癌标本中miR-328的表达量与其启动子区域CG位点（cg16421621、cg04650403）甲基化率呈负相关（r=-0.127, P=0.005）。854例有完整信息的浸润性乳腺癌患者中,miR 328表达水平与淋巴结转移和肿瘤大小有关（P＜0.05）;miR-328低表达组（＜5.462）的中位总生存时间（OS）为7.25年,低于高表达组（≥5.462）的8.75年,差异有统计学意义（P<0.01）。Cox多因素回归分析显示,年龄、淋巴结转移与miR-328表达水平为影响OS的独立因素（P＜0.05）,miR-328高表达组的危险程度低于低表达组（P＜0.01）。结论 浸润性乳腺癌中miR-328表达受其基因启动子区甲基化调控;miRNA-328与浸润性乳腺癌的临床病理特征有关,并能作为影响浸润性乳腺癌预后的危险因素。     

DNA hypermethylation of microRNA-34b/c has prognostic value for stage Ⅰ non-small cell lung cancer

Lung cancer is the leading cause of cancer-related death in the world and approximately 30-40% of patients with stage Ⅰ non-small cell lung cancer (NSCLC) die of recurrent disease. miRNA expression profiles can be diagnostic and prognostic markers of lung cancer. Recently, miR-34 family has been shown to be part of the p53 pathway which is frequently involved in lung cancer, and the expression of miR-34 has been reported to be regulated by DNA methylation. In present study, we investigated the correlation between DNA methylation status of miR-34 family and recurrence of stage Ⅰ NSCLC patients. miR-34a and miR-34b/c promoter methylation status were determined by nested methylation-specific PCR in FFPE tumor tissues from 161 patients of stage Ⅰ NSCLC. Furthermore, mature miR-34b and miR-34c expression were analyzed by qRT-PCR in the same panel tissues. Our results revealed that aberrant DNA methylation of miR-34b/c was correlated with a high probability of recurrence (p = 0.026) and associated with poor overall survival (p = 0.010) and disease-free survival (p = 0.017). No significant association was found for miR-34a methylation. Multivariate analysis showed that promoter hypermethylation of miR-34b/c was an independent prognostic factor of stage Ⅰ NSCLC. Moreover, no significant association between mature miR-34b and miR-34c expression and DNA methylation status was found. In conclusion, we have identified promoter hypermethylation of miR-34b/c as a relatively common event in NSCLC and might be a potential prognostic factor for stage Ⅰ NSCLC.     

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.     

CpG island methylation status of miRNAs in esophageal squamous cell carcinoma

Previous studies on esophageal squamous cell carcinoma (ESCC) indicated that it contains much dysregulation of microRNAs (miRNAs). DNA hypermethylation in the miRNA 5' regulatory region is a mechanism that can account for the downregulation of miRNA in tumors (Esteller, N Engl J Med 2008;358:1148-59). Among those dysregulated miRNAs, miR-203, miR-34b/c, miR-424 and miR-129-2 are embedded in CpG islands, as is the promoter of miR-34a. We investigated their methylation status in ESCC by bisulfite sequencing PCR (BSP) and methylation specific PCR (MSP). The methylation frequency of miR-203 and miR-424 is the same in carcinoma and in the corresponding non-tumor tissues. The methylation ratio of miR-34a, miR-34b/c and miR-129-2 is 66.7% (36/54), 40.7% (22/54) and 96.3% (52/54), respectively in ESCC, which are significantly higher than that in the corresponding non-tumor tissues(p < 0.01). Quantitative RT-PCR analysis in clinical samples suggested that CpG island methylation is significantly correlated with their low expression in ESCC, 5-aza-2'-deoxycytidine (DAC) treatment partly recovered their expression in EC9706 cell line. We conclude that CpG island methylation of miR-34a, miR-34b/c and miR-129-2 are frequent events and important mechanism for their low expression in ESCC. DNA methylation changes have been reported to occur early in carcinogenesis and are potentially good early indicators of carcinoma (Laird, Nat Rev Cancer 2003;3:253-66). The high methylation ratio of miR-129-2 indicated its potential as a methylation biomarker in early diagnosis of ESCC.     

Genome structure-based screening identified epigenetically silenced microRNA associated with invasiveness in non-small-cell lung cancer

MicroRNA (miRNA) expression is frequently altered in human cancers. To search for epigenetically silenced miRNAs in non-small-cell lung cancer (NSCLC), we mapped human miRNAs on autosomal chromosomes and selected 55 miRNAs in silico. We treated six NSCLC cell lines with the DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-aza-CdR) and determined the expressions of the 55 miRNAs. Fourteen miRNAs were decreased in the cancer cell lines and were induced after 5-aza-CdR treatment. After a detailed DNA methylation analysis, we found that mir-34b and mir-126 were silenced by DNA methylation. Mir-34b was silenced by the DNA methylation of its own promoter, whereas mir-126 was silenced by the DNA methylation of its host gene, EGFL7. A chromatin immunoprecipitation assay revealed H3K9me2 and H3K9me3 in mir-34b and EGFL7, and H3K27me3 in EGFL7. The overexpression of mir-34b and mir-126 decreased the expression of c-Met and Crk, respectively. The 5-aza-CdR treatment of lung cancer cell line resulted in increased mir-34b expression and decreased c-Met protein. We next analyzed the DNA methylation status of these miRNAs using 99 primary NSCLCs. Mir-34b and mir-126 were methylated in 41 and 7% of all the cases, respectively. The DNA methylation of mir-34b was not associated with c-Met expression determined by immunohistochemistry, but both mir-34b methylation (p = 0.007) and c-Met expression (p = 0.005) were significantly associated with lymphatic invasion in a multivariate analysis. The DNA methylation of mir-34b can be used as a biomarker for an invasive phenotype of lung cancer.