MicroRNAs (miRNAs) are short non-coding RNA molecules playing regulatory roles by repressing translation or cleaving RNA transcripts. Although the number of verified human miRNA is still expanding, only few have been functionally described. However, emerging evidences suggest the potential involvement of altered regulation of miRNA in pathogenesis of cancers and these genes are thought to function as both tumours suppressor and oncogenes.In our study, we examined by Real-Time PCR the expression of 156 mature miRNA in colorectal cancer. The analysis by several bioinformatics algorithms of colorectal tumours and adjacent nonneoplastic tissues from patients and colorectal cancer cell lines allowed identifying a group of 13 miRNA whose expression is significantly altered in this tumor. The most significantly deregulated miRNA being miR-31, miR-96, miR-133b, miR-135b, miR-145, and miR-183. In addition, the expression level of miR-31 was correlated with the stage of CRC tumor.Our results suggest that miRNA expression profile could have relevance to the biological and clinical behavior of colorectal neoplasia.

MicroRNAs (miRNAs) have been identified as promising prognostic markers in non-small-cell lung cancer (NSCLC) since they play an important role in oncogenesis. The miR-34 family is composed of three miRNAs (miR-34a, miR-34b and miR-34c) that are part of the p53 network and whose expression is directly induced by p53 in response to DNA damage or oncogenic stress. We have analyzed the impact of miR-34 expression on relapse and overall survival in surgically resected NSCLC patients. For this purpose, we used stem-loop reverse transcription-polymerase chain reaction to analyze the expression of the miR-34 family in paired tumor and normal tissue from 70 surgically resected NSCLC patients who received no postsurgical treatment until relapse. In addition, in patients with sufficient tumor tissue, we assessed p53 mutations and the methylation status of the MIRN34A gene promoter region and correlated these findings with miR-34a expression. Molecular findings were correlated with relapse and overall survival. The miR-34 family was downregulated in tumor compared with normal tissue, and low levels of miR-34a expression were correlated with a high probability of relapse (P = 0.04). A relation was also found between MIRN34A methylation and miR-34a expression (P = 0.008). Patients with both p53 mutations and low miR-34a levels had the highest probability of relapse (P = 0.001). In the multivariate analysis, miR-34a expression emerged as an independent prognostic marker for relapse. In summary, we have identified miR-34a as a novel prognostic marker in NSCLC patients, providing a potential mechanism for estimating a patient's risk of disease recurrence and a useful tool to help guide treatment decisions.

BackgroundThe p53 transcription factor is located at the core of a complex wiring of signaling pathways that are critical for the preservation of cellular homeostasis. Only recently it has become clear that p53 regulates the expression of several long intergenic noncoding RNAs (lincRNAs). However, relatively little is known about the role that lincRNAs play in this pathway.ResultsHere we characterize a lincRNA named Pint (p53 induced noncoding transcript). We show that Pint is a ubiquitously expressed lincRNA that is finely regulated by p53. In mouse cells, Pint promotes cell proliferation and survival by regulating the expression of genes of the TGF-β, MAPK and p53 pathways. Pint is a nuclear lincRNA that directly interacts with the Polycomb repressive complex 2 (PRC2), and is required for PRC2 targeting of specific genes for H3K27 tri-methylation and repression. Furthermore, Pint functional activity is highly dependent on PRC2 expression. We have also identified Pint human ortholog (PINT), which presents suggestive analogies with the murine lincRNA. PINT is similarly regulated by p53, and its expression significantly correlates with the same cellular pathways as the mouse ortholog, including the p53 pathway. Interestingly, PINT is downregulated in colon primary tumors, while its overexpression inhibits the proliferation of tumor cells, suggesting a possible role as tumor suppressor.ConclusionsOur results reveal a p53 autoregulatory negative mechanism where a lincRNA connects p53 activation with epigenetic silencing by PRC2. Additionally, we show analogies and differences between the murine and human orthologs, identifying a novel tumor suppressor candidate lincRNA.

IntroductionMature microRNAs (miRNAs) are naturally occurring small noncoding RNAs that act as negative regulators of gene expression through messenger RNA interference. These molecules were described for the first time in 1993 by Ambros and colleagues in Caenorhabditis elegans (Lee et al 1 ), and to date, hundreds of miRNAs have been identified in other species, including viruses. 2,3 miRNAs are encoded by intronic or intergenic DNA regions, primarily as large molecules that can exceed 1 Kb, and are cleaved by an RNase complex into fragments with characteristic stem-loop structures. In the cytoplasm, a RNase called Dicer further cleaves miRNA to generate a duplex molecule of 21 to 25 nucleotides in length. 4 One of the 2 chains is the mature miRNA that binds a protein complex called the RNA-induced silencing complex (RISC). When a miRNA and a messenger RNA exhibit total complementarities, RISC is capable of degrading target messenger RNA, 4 whereas if an incomplete base pairing complementarity takes place, translational silencing of the target occurs. Through these mechanisms, miRNAs decrease translation of human genes. 5,6 miRNAs play an important role in cellular proliferation and differentiation and embryonic development, and they also act as oncogenes or tumor suppressor genes. 7-10 Notably, the majority of miRNAs are found in cancer-associated genomic regions or in chromosome-fragile sites, 11 suggesting an important role for miRNAs in human tumorigenesis. There is also evidence that the influence of miRNAs in oncogenesis might be indirectly driven. For example, the presence of some viruses in a cell may change the host miRNA pattern. 12 Viruses may participate in the origin of some tumors, such as the Epstein-Barr virus (EBV) in Hodgkin lymphoma (HL).HL is a neoplasm characterized by the presence of relatively few tumoral cells (Hodgkin and Reed-Sternberg cells) in a nonneoplastic microenvironment. 13 Hodgkin and Reed-Sternberg cells arise from germinal center B cells. 14 Classic HL (cHL) is subclassified according to the morphology of Reed-Sternberg cells and the composition of the cellular background into nodular sclerosis, mixed cellularity, lymphocyte-rich, and lymphocyte depletion. 15 The 2 former subtypes are the most frequent forms of cHL and contain a variable proportion of neoplastic cells.EBV is present in the malignant cells of 40% to 60% of cHL patients. However, the precise role of the EBV in the pathogenesis of cHL is unknown. It has been reported that viruses have their own miRNA set, 16 and that there is an interaction between the host miRNAs and virus miRNAs. 17,18 The interaction between the virus and the malignant cells in cHL might be mediated in part by miRNAs.To investigate whether a specific expression signature of miRNAs is associated with cHL, we assessed the expression of 156 miRNAs, the majority of which are related to hematopoiesis or tumorigenesis, 7,8,11 in lymph nodes from patients with nodular sclerosis and mixed cellularity cHL and compared the expression patterns with tho...

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The behavior of classic Hodgkin lymphoma (cHL) is determined by both the intrinsic features of the tumor cells and the characteristics of the microenvironment, making the analysis of entire lymph nodes an effective approach to understanding the disease. We examined the influence of our previously reported 25-microRNA signature for cHL on clinical outcome in 89 homogeneously treated cHL patients with a median follow-up of 80 months. Patients with low miR-135a expression had a higher probability of relapse (P ‫؍‬ .04) and a shorter diseasefree survival (P ‫؍‬ .02). Functional analysis of cHL cell lines showed that mature miR-135a levels increased after pre-miR135a transfection, causing apoptosis and decreased cell growth. Target analysis showed a direct regulation by miR-135a of JAK2, a cytoplasmic tyrosine kinase involved in a specific subset of cytokine receptor signaling pathways. miR-135a-mediated JAK2 down-regulation led to decreased mRNA and protein levels of the antiapoptotic gene Bcl-xL, suggesting a role for Bcl-xL in miR-135a/JAK2-mediated apoptosis. Our findings confirm the critical role of miR-135a in the survival of cHL cells and in the prognosis of cHL patients, indicating that novel treatment approaches targeting miR-135a may potentially benefit these patients. (Blood. 2009;114:2945-2951)

BackgroundSeveral treatments in non-small cell lung cancer (NSCLC) are histology-dependent, and the need for histology-related markers is increasing. MicroRNAs (miRNAs) are promising molecular markers in multiple cancers and show differences in expression depending on histological subtype. The miRNA family miR-200 has been associated with the regulation of epithelial-mesenchymal (EMT)/mesenchymal-epithelial transition (MET). EMT involves profound phenotypic changes that include the loss of cell-cell adhesion, the loss of cell polarity, and the acquisition of migratory and invasive properties that facilitates metastasis. A dual role for the miR-200 family in the prognosis of several tumors has been related to tumor cell origin. However, the prognostic role and function of miR-200 family in early-stage NSCLC adenocarcinoma and squamous cell carcinoma (SCC) have not been well established.MethodsmiRNA expression was determined using TaqMan assays in 155 tumors from resected NSCLC patients. Functional studies were conducted in three NSCLC cell lines: H23, A-549 and HCC-44.ResultsHigh miR-200c expression was associated with shorter overall survival (OS) in the entire cohort (p = 0.024). High miR-200c (p = 0.0004) and miR-141 (p = 0.009) expression correlated with shorter OS in adenocarcinoma – but not in SCC. In the multivariate analysis, a risk score based on miR-141 and miR-200c expression emerged as an independent prognostic factor for OS in the entire cohort (OR, 2.787; p = 0.033) and in adenocarcinoma patients (OR, 10.649; p = 0.002). Functional analyses showed that miR-200c, was related to mesenchymal-epithelial transition (MET) and affected cell migration and E-cadherin levels, while overexpression of miR-141 reduced KLF6 protein levels and produced an increase of secretion of VEGFA in vitro (H23, p = 0.04; A-549, p = 0.03; HCC-44, p = 0.02) and was associated with higher blood microvessel density in patient tumor samples (p<0.001).ConclusionHigh miR-141 and miR-200c expression are associated with shorter OS in NSCLC patients with adenocarcinoma through MET and angiogenesis.

BackgroundCancer-derived exosomes are involved in metastasis. YKT6 is a SNARE protein that participates in the regulation of exosome production and release, but its role in non-small cell lung cancer (NSCLC) has not been examined.Materials and MethodsUltracentrifugation-purified exosomes from the A549 cell line were studied by CRYO-TEM, nanoparticle tracking analysis and western blot (TSG101 marker). YKT6 was inhibited using a DsiRNA and selected pre-microRNAs. MicroRNAs targeting YKT6 were validated by Renilla/Luciferase assay and western blot. YKT6 expression and its prognostic impact were analyzed in 98 tissue specimens from resected NSCLC patients.ResultsMembranous nanosized vesicles (mode size: 128nm) with TSG101 protein were purified from A549 cells. YKT6 inhibition reduced exosome release by 80.9%. We validated miR-134 and miR-135b as miRNAs targeting YKT6, and transfection with the pre-miRNAs also produced a significant reduction in exosome release. The analysis of YKT6 in tumor samples showed that patients with high levels had shorter disease-free and overall survival.ConclusionsYKT6 is a key molecule in the regulation of exosome release in lung cancer cells and is in turn precisely regulated by miR-134 and miR-135b. Moreover, YKT6 levels impact prognosis of resected NSCLC patients.