A Seven-microRNA Expression Signature Predicts Survival in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is the fifth common cancer. The differential expression of microRNAs (miRNAs) has been associated with the prognosis of various cancers. However, limited information is available regarding genome-wide miRNA expression profiles in HCC to generate a tumor-specific miRNA signature of prognostic values. In this study, the miRNA profiles in 327 HCC patients, including 327 tumor and 43 adjacent non-tumor tissues, from The Cancer Genome Atlas (TCGA) Liver hepatocellular carcinoma (LIHC) were analyzed. The associations of the differentially expressed miRNAs with patient survival and other clinical characteristics were examined with t-test and Cox proportional regression model. Finally, a tumor-specific miRNA signature was generated and examined with Kaplan–Meier survival, univariate\multivariate Cox regression analyses and KEGG pathway analysis. Results showed that a total of 207 miRNAs were found differentially expressed between tumor and adjacent non-tumor HCC tissues. 78 of them were also discriminatively expressed with gender, race, tumor grade and AJCC tumor stage. Seven miRNAs were significantly associated with survival (P value <0.001). Among the seven significant miRNAs, six (hsa-mir-326, hsa-mir-3677, hsa-mir-511-1, hsa-mir-511-2, hsa-mir-9-1, and hsa-mir-9-2) were negatively associated with overall survival (OS), while the remaining one (hsa-mir-30d) was positively correlated. A tumor-specific 7-miRNAs signature was generated and validated as an independent prognostic predictor. Collectively, we have identified and validated an independent prognostic model based on the expression of seven miRNAs, which can be used to assess patients’ survival. Additional work is needed to translate our model into clinical practice.     

Identification of miRNAs Expression Profile in Gastric Cancer Using Self-Organizing Maps (SOM)

In this paper, an unsupervised artificial neural network was implemented to identify the patters of specific signatures. The network was based on the differential expression of miRNAs (under or over expression) found in healthy or cancerous gastric tissues. Among the tissues analyzes, the neural network evaluated 514 miRNAs of gastric tissue that exhibited significant differential expression. The result suggested a specific expression signature nine miRNAs (hsa-mir-21, hsa-mir-29a, hsa-mir-29c, hsa-mir-148a, hsa-mir-141, hsa-let-7b, hsa-mir-31, hsa-mir-451, and hsa-mir-192), all with significant values (p-value < 0.01 and fold change > 5) that clustered the samples into two groups: healthy tissue and gastric cancer tissue. The results obtained “in silico” must be validated in a molecular biology laboratory; if confirmed, this method may be used in the future as a risk marker for gastric cancer development.     

Patterns of MiRNA Expression in Arctic Charr Development

Micro-RNAs (miRNAs) are now recognized as a major class of developmental regulators. Sequences of many miRNAs are highly conserved, yet they often exhibit temporal and spatial heterogeneity in expression among species and have been proposed as an important reservoir for adaptive evolution and divergence. With this in mind we studied miRNA expression during embryonic development of offspring from two contrasting morphs of the highly polymorphic salmonid Arctic charr (Salvelinus alpinus), a small benthic morph from Lake Thingvallavatn (SB) and an aquaculture stock (AC). These morphs differ extensively in morphology and adult body size. We established offspring groups of the two morphs and sampled at several time points during development. Four time points (3 embryonic and one just before first feeding) were selected for high-throughput small-RNA sequencing. We identified a total of 326 conserved and 427 novel miRNA candidates in Arctic charr, of which 51 conserved and 6 novel miRNA candidates were differentially expressed among developmental stages. Furthermore, 53 known and 19 novel miRNAs showed significantly different levels of expression in the two contrasting morphs. Hierarchical clustering of the 53 conserved miRNAs revealed that the expression differences are confined to the embryonic stages, where miRNAs such as sal-miR-130, 30, 451, 133, 26 and 199a were highly expressed in AC, whereas sal-miR-146, 183, 206 and 196a were highly expressed in SB embryos. The majority of these miRNAs have previously been found to be involved in key developmental processes in other species such as development of brain and sensory epithelia, skeletogenesis and myogenesis. Four of the novel miRNA candidates were only detected in either AC or SB. miRNA candidates identified in this study will be combined with available mRNA expression data to identify potential targets and involvement in developmental regulation.     

肺腺癌预后相关miRNA生物信息学筛选及其临床意义

目的：探讨肺腺癌预后相关miRNA组学特征及其临床意义。方法：应用癌症基因组图谱（TCGA）数据库,检索人肺腺癌miRNA表达谱数据,进行差异分析,再利用Cox风险回归模型筛选预后相关miRNA;利用mirwalk分析平台,对筛选出的miRNA进行靶向调控基因预测、KEGG功能富集分析,最后,预测出预后相关miRNA的功能。结果：共筛选肺腺癌差异miRNA46个,其中,上调19个、下调27个;通过Cox生存分析筛选到预后相关miRNA有6个,即hsa-mir-21、hsa-mir-142、hsa-mir-200a高表达,hsa-mir-101、hsa-let-7c、hsa-mir-378e低表达,其中hsa-mir-21、hsa-mir-378e与肺腺癌患者不良预后有关,生存期显著缩短（P<0.05,AUC=0.618）。KEGG分析上述预后相关miRNA靶向调控基因与免疫反应通路、miRNA与癌症通路、代谢通路等有关。结论：hsa-mir-21、hsa-mir-378e与肺腺癌预后不良有关,未来经进一步临床验证有可能作为肺腺癌预后相关的分子标记物。     

The Lin28/Let-7 System in Early Human Embryonic Tissue and Ectopic Pregnancy

Our objective was to determine the expression of the elements of the Lin28/Let-7 system, and related microRNAs (miRNAs), in early stages of human placentation and ectopic pregnancy, as a means to assess the potential role of this molecular hub in the pathogenesis of ectopic gestation. Seventeen patients suffering from tubal ectopic pregnancy (cases) and forty-three women with normal on-going gestation that desired voluntary termination of pregnancy (VTOP; controls) were recruited for the study. Embryonic tissues were subjected to RNA extraction and quantitative PCR analyses for LIN28B, Let-7a, miR-132, miR-145 and mir-323-3p were performed. Our results demonstrate that the expression of LIN28B mRNA was barely detectable in embryonic tissue from early stages of gestation and sharply increased thereafter to plateau between gestational weeks 7–9. In contrast, expression levels of Let-7, mir-132 and mir-145 were high in embryonic tissue from early gestations (≤6-weeks) and abruptly declined thereafter, especially for Let-7. Opposite trends were detected for mir-323-3p. Embryonic expression of LIN28B mRNA was higher in early stages (≤6-weeks) of ectopic pregnancy than in normal gestation. In contrast, Let-7a expression was significantly lower in early ectopic pregnancies, while miR-132 and miR-145 levels were not altered. Expression of mir-323-3p was also suppressed in ectopic embryonic tissue. We are the first to document reciprocal changes in the expression profiles of the gene encoding the RNA-binding protein, LIN28B, and the related miRNAs, Let-7a, mir-132 and mir-145, in early stages of human placentation. This finding suggests the potential involvement of LIN28B/Let-7 (de)regulated pathways in the pathophysiology of ectopic pregnancy in humans.     

Differential expression of microRNA and predicted targets in pulmonary sarcoidosis

BackgroundRecent studies show that various inflammatory diseases are regulated at the level of RNA translation by small non-coding RNAs, termed microRNAs (miRNAs). We sought to determine whether sarcoidosis tissues harbor a distinct pattern of miRNA expression and then considered their potential molecular targets.Methods and resultsGenome-wide microarray analysis of miRNA expression in lung tissue and peripheral blood mononuclear cells (PBMCs) was performed and differentially expressed (DE)-miRNAs were then validated by real-time PCR. A distinct pattern of DE-miRNA expression was identified in both lung tissue and PBMCs of sarcoidosis patients. A subgroup of DE-miRNAs common to lung and lymph node tissues were predicted to target transforming growth factor (TGFβ)-regulated pathways. Likewise, the DE-miRNAs identified in PBMCs of sarcoidosis patients were predicted to target the TGFβ-regulated “wingless and integrase-1” (WNT) pathway.ConclusionsThis study is the first to profile miRNAs in sarcoidosis tissues and to consider their possible roles in disease pathogenesis. Our results suggest that miRNA regulate TGFβ and related WNT pathways in sarcoidosis tissues, pathways previously incriminated in the pathogenesis of sarcoidosis.     

MicroRNA-21 targets the vitamin D-dependent antimicrobial pathway in leprosy

Leprosy provides a model to investigate mechanisms of immune regulation in humans, given that the disease forms a spectrum of clinical presentations that correlate with host immune responses. Here we identified 13 miRNAs that were differentially expressed in the lesions of subjects with progressive lepromatous (L-lep) versus the self-limited tuberculoid (T-lep) disease. Bioinformatic analysis revealed a significant enrichment of L-lep-specific miRNAs that preferentially target key immune genes downregulated in L-lep versus T-lep lesions. The most differentially expressed miRNA in L-lep lesions, hsa-mir-21, was upregulated in Mycobacterium leprae-infected monocytes. By directly downregulating Toll-like receptor 2/1 heterodimer (TLR2/1)-induced CYP27B1 and IL1B expression as well as indirectly upregulating interleukin-10 (IL-10), hsa-mir-21 inhibited expression of the genes encoding two vitamin D-dependent antimicrobial peptides, CAMP and DEFB4A. Conversely, knockdown of hsa-mir-21 in M. leprae-infected monocytes enhanced expression of CAMP and DEFB4A and restored TLR2/1-mediated antimicrobial activity against M. leprae. Therefore, the ability of M. leprae to upregulate hsa-mir-21 targets multiple genes associated with the immunologically localized disease form, providing an effective mechanism to escape from the vitamin D-dependent antimicrobial pathway.     

Identification of novel MiRNAs and MiRNA expression profiling during grain development in indica rice

ABSTRACT: BACKGROUND: MicroRNAs (miRNAs) modulate gene expression in different tissues and at diverse developmental stages, including grain development in japonica rice. To identify novel miRNAs in indica rice and to study their expression patterns during the entire grain filling process, small RNAs from all stages of grain development were sequenced and their expression patterns were studied using customized miRNA chips. RESULTS: A total of 21 conserved and 91 non-conserved miRNA families were found in developing indica grains. We also discovered 11 potential novel miRNAs based on the presence of their miRNA*s. Expression patterns of these identified miRNAs were analyzed using customized miRNA chips. The results showed that during the filling phase about half of the detected miRNAs were up-regulated, whereas the remainder were down-regulated. Predicted targets of differentially expressed miRNAs may participate in carbohydrate metabolism, hormone signaling and pathways associated with seed maturity, suggesting potentially important roles in rice grain development. CONCLUSIONS: This study is the first genome-wide investigation of miRNAs during the grain-filling phase of an indica variety of rice. The novel miRNAs identified might be involved in new miRNA regulatory pathways for grain development. The complexity of these miRNAs and their targets and interactions require further study to obtain a better understanding of the molecular mechanisms underlying grain development. Key words: miRNA, grain filling, indica rice.     

Identification of a 4-miRNA signature as a potential prognostic biomarker for pancreatic adenocarcinoma

An microRNA (miRNA) signature to predict the clinical outcome of pancreatic adenocarcinoma (PAAD) is still lacking. In the current study, we aimed at identifying and evaluating a prognostic miRNA signature for patients with PAAD. The miRNA expression profile and the clinical information regarding patients with PAAD were recruited from The Cancer Genome Atlas database. Differentially expressed miRNAs were identified between normal and tumor samples. By means of survival analysis, a 4-miRNA signature for predicting patients' with PAAD overall survival (OS) was constructed. Receiver operating characteristic (ROC) analysis was applied to determine the efficiency of survival prediction. Furthermore, the biological function of the predicted miRNAs was evaluated using a bioinformatics approach. Four (hsa-mir-126, hsa-mir-3613, hsa-mir-424, and hsa-mir-4772) out of 17 differentially expressed miRNAs were associated to the OS of patients with PAAD. Moreover, the area under the curve (AUC) of the constructed 4-miRNA signature associated to patients' with PAAD 2-year survival was 0.789. The multivariate Cox's proportional hazards regression model suggested that this 4-miRNA signature was an independent prognostic factor of other clinical parameters in patients with PAAD. Further pathway enrichment analyses revealed that the miRNAs in the 4-miRNA signature might regulate genes that affect focal adhesion, Wnt signaling pathway, and PI3K-Akt signaling pathway. Thus, these findings indicated that the 4-miRNA signature might be an effective independent prognostic biomarker in the prediction of PAAD patients' survival.     

Identification of Differential MicroRNAs in Cerebrospinal Fluid and Serum of Patients with Major Depressive Disorder

Major depression is a debilitating disease. To date, the development of biomarkers of major depressive disorder (MDD) remains a challenge. Recently, alterations in the expression of microRNAs (miRNAs) from post-mortem brain tissue and peripheral blood have been linked to MDD. The goals of this study were to detect the differential miRNAs in cerebrospinal fluid (CSF) and serum of MDD patients. First, the relative expression levels of 179 miRNAs (relative high levels in serum) were analyzed by miRNA PCR Panel in the CSF of MDD patients. Then, the differentially altered miRNAs from CSF were further assessed by qRT-PCR in the serum of the same patients. Finally, the serum differentially altered miRNAs were further validated by qRT-PCR in the serum of another MDD patients. The CSF-results indicated that 11 miRNAs in MDD patients were significantly higher than these in control subjects, and 5 miRNAs were significantly lower than these in control subjects. The serum-results from the same patients showed that 3 miRNAs (miR-221-3p, miR-34a-5p, and let-7d-3p) of the 11 miRNAs were significantly higher than these in control subjects, and 1 miRNA (miR-451a) of 5 miRNAs was significantly lower than these in control subjects. The up-regulation of miR-221-3p, miR-34a-5p, let-7d-3p and down-regulation of miR-451a was further validated in another 32 MDD patients. ROC analysis showed that the area under curve of let-7d-3p, miR-34a-5p, miR-221-3p and miR-451a was 0.94, 0.98, 0.97 and 0.94, with specificity of 90.48%, 95.24%, 90.48% and 90.48%, and sensitivity of 93.75%, 96.88%, 90.63% and 84.85%, respectively. In addition, target gene prediction found that the altered miRNAs are involved in affecting some important genes and pathway related to MDD. Our results suggested that differentially altered miRNAs in CSF might be involved in MDD, and serum miR-221-3p, miR-34a-5p, let-7d-3p, and miR-451a might be able to serve as biomarkers for MDD.     

Genome-wide microRNA profiling of bovine milk-derived exosomes infected with <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

Bovine milk is rich in exosomes, which contain abundant miRNAs and play important roles in the regulation of neonatal growth and development of adaptive immunity. Here, we analyzed miRNA expression profiles of bovine milk exosomes from three healthy and three mastitic cows, and then six miRNA libraries were constructed. Interestingly, we detected no scRNAs and few snRNAs in milk exosomes; this result indicated a potential preference for RNA packaging in milk exosomes. A total of 492 known and 980 novel exosomal miRNAs were detected, and the 10 most expressed miRNAs in the six samples accounted for 80–90% of total miRNA-associated reads. Expression analyses identified 18 miRNAs with significantly different expression between healthy and infected animals; the predicted target genes of differentially expressed miRNAs were significantly enriched in immune system process, response to stimulus, growth, etc. Moreover, target genes were significantly enriched in several Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways including inflammatory, immune, and cancer pathways. Our survey provided comprehensive information about milk exosomes and exosomal miRNAs involved in mastitis. Moreover, the differentially expressed miRNAs, especially miR-223 and miR-142-5p, could be considered as potential candidates for mastitis.     

MicroRNA-196a Is a Putative Diagnostic Biomarker and Therapeutic Target for Laryngeal Cancer

Background

MicroRNA (miRNA) is an emerging subclass of small non-coding RNAs that regulates gene expression and has a pivotal role for many physiological processes including cancer development. Recent reports revealed the role of miRNAs as ideal biomarkers and therapeutic targets due to their tissue- or disease-specific nature. Head and neck cancer (HNC) is a major cause of cancer-related mortality and morbidity, and laryngeal cancer has the highest incidence in it. However, the molecular mechanisms involved in laryngeal cancer development remain to be known and highly sensitive biomarkers and novel promising therapy is necessary.

Methodology/Principal Findings

To explore laryngeal cancer-specific miRNAs, RNA from 5 laryngeal surgical specimens including cancer and non-cancer tissues were hybridized to microarray carrying 723 human miRNAs. The resultant differentially expressed miRNAs were further tested by using quantitative real time PCR (qRT-PCR) on 43 laryngeal tissue samples including cancers, noncancerous counterparts, benign diseases and precancerous dysplasias. Significant expressional differences between matched pairs were reproduced in miR-133b, miR-455-5p, and miR-196a, among which miR-196a being the most promising cancer biomarker as validated by qRT-PCR analyses on additional 84 tissue samples. Deep sequencing analysis revealed both quantitative and qualitative deviation of miR-196a isomiR expression in laryngeal cancer. In situ hybridization confirmed laryngeal cancer-specific expression of miR-196a in both cancer and cancer stroma cells. Finally, inhibition of miR-196a counteracted cancer cell proliferation in both laryngeal cancer-derived cells and mouse xenograft model.

Conclusions/Significance

Our study provided the possibilities that miR-196a might be very useful in diagnosing and treating laryngeal cancer.