MicroRNAs (miRNAs) represent a conserved class of small noncoding RNAs that are found in all higher eukaryotes as well as some DNA viruses. miRNAs are 20 to 25 nucleotides in length and have important regulatory functions in biological processes such as embryonic development, cell differentiation, hormone secretion, and metabolism. Furthermore, miRNAs have been implicated in the pathology of various diseases, including cancer. miRNA expression profiles not only classify different types of cancer but also may even help to characterize distinct tumor stages, therefore constituting a valuable tool for prognosis. Here we report the miRNA profile of Epstein-Barr virus (EBV)-positive nasopharyngeal carcinoma (NPC) tissue samples characterized by cloning and sequencing. We found that all EBV miRNAs from the BART region are expressed in NPC tissues, whereas EBV miRNAs from the BHRF1 region are not found. Moreover, we identified two novel EBV miRNA genes originating from the BART region that have not been found in other tissues or cell lines before. We also identified three new human miRNAs which might be specific for nasopharyngeal tissues. We further show that a number of different cellular miRNAs, including miR-15a and miR-16, are up- or downregulated in NPC tissues compared to control tissues. We found that the tumor suppressor BRCA-1 is a target of miR-15a as well as miR-16, suggesting a miRNA role in NPC pathogenesis.

Prostate cancer is a leading cause of tumor mortality. To characterize the underlying molecular mechanisms, we have compared the microRNA (miRNA) profile of primary prostate cancers and noncancer prostate tissues using deep sequencing. MiRNAs are small noncoding RNAs of 21 to 25 nucleotides that regulate gene expression through the inhibition of protein synthesis. We find that 33 miRNAs were upregulated or downregulated >1.5-fold. The deregulation of selected miRNAs was confirmed by both Northern blotting and quantitative reverse transcription-PCR in established prostate cancer cell lines and clinical tissue samples. A computational search indicated the 3′-untranslated region (UTR) of the mRNA for myosin VI (MYO6) as a potential target for both miR-143 and miR-145, the expression of which was reduced in the tumor tissues. Upregulation of myosin VI in prostate cancer was previously shown by immunohistochemistry. The level of MYO6 mRNA was significantly induced in all primary tumor tissues compared with the nontumor tissue from the same patient. This finding was matched to the upregulation of myosin VI in established prostate cancer cell lines. In luciferase reporter analysis, we find a significant negative regulatory effect on the MYO6 3′UTR by both miR-143 and miR-145. Mutation of the potential binding sites for miR-143 and miR-145 in the MYO6 3′UTR resulted in a loss of responsiveness to the corresponding miRNA. Our data indicate that miR-143 and miR-145 are involved in the regulation of MYO6 expression and possibly in the development of prostate cancer. Mol Cancer Res; 8(4); 529–38. ©2010 AACR.

The Epstein–Barr virus (EBV) is an oncogenic human Herpes virus found in ∼15% of diffuse large B-cell lymphoma (DLBCL). EBV encodes miRNAs and induces changes in the cellular miRNA profile of infected cells. MiRNAs are small, non-coding RNAs of ∼19–26 nt which suppress protein synthesis by inducing translational arrest or mRNA degradation. Here, we report a comprehensive miRNA-profiling study and show that hsa-miR-424, -223, -199a-3p, -199a-5p, -27b, -378, -26b, -23a, -23b were upregulated and hsa-miR-155, -20b, -221, -151-3p, -222, -29b/c, -106a were downregulated more than 2-fold due to EBV-infection of DLBCL. All known EBV miRNAs with the exception of the BHRF1 cluster as well as EBV-miR-BART15 and -20 were present. A computational analysis indicated potential targets such as c-MYB, LATS2, c-SKI and SIAH1. We show that c-MYB is targeted by miR-155 and miR-424, that the tumor suppressor SIAH1 is targeted by miR-424, and that c-SKI is potentially regulated by miR-155. Downregulation of SIAH1 protein in DLBCL was demonstrated by immunohistochemistry. The inhibition of SIAH1 is in line with the notion that EBV impedes various pro-apoptotic pathways during tumorigenesis. The down-modulation of the oncogenic c-MYB protein, although counter-intuitive, might be explained by its tight regulation in developmental processes.

In primary prostate cancer (PCa), a major cause of cancer‐related death in men, the expression of various microRNAs (miRNAs) is deregulated. We previously detected several miRNAs, for example, miR‐24 and miR‐22, as significantly downregulated in PCa (Szczyrba et al., Mol Cancer Res 2010;8:529‐38). An in silico search predicted that zinc finger protein 217 (ZNF217) and importin 7 (IPO7) were potential target genes of these miRNAs. Additionally, for two genes that are deregulated in PCa (heterogeneous nuclear ribonucleoprotein K, hnRNP‐K, and vascular endothelial growth factor A, VEGF‐A), we identified two regulatory miRNAs, miR‐205 and miR‐29b. The regulation of the 3′‐untranslated regions of the four genes by their respective miRNAs was confirmed by luciferase assays. As expected, the upregulation of ZNF217, hnRNP‐K, VEGF‐A and IPO7 could be verified at the protein level in the PCa cell lines LNCaP and DU145. ZNF217 and IPO7, which had not yet been studied in PCa, were analyzed in more detail. ZNF217 mRNA is overexpressed in primary PCa samples, and this overexpression translates to an elevated protein level. However, IPO7 was upregulated at the protein level alone. The inhibition of ZNF217 and IPO7 by siRNA resulted in reduced proliferation of the PCa cell lines. ZNF217 could thus be identified as an oncogene that is overexpressed in PCa and affects the growth of PCa cell lines, whereas the function of IPO7 remains to be elucidated in greater detail.

MicroRNAs (miRNAs) are short 18–23 nucleotide long noncoding RNAs that posttranscriptionally regulate gene expression by binding to mRNA. Our previous miRNA profiling of diffuse large B‐cell lymphoma (DLBCL) revealed a mutation in the seed sequence of miR‐142‐3p. Further analysis now showed that miR‐142 was mutated in 11 (19.64%) of the 56 DLBCL cases. Of these, one case had a mutation in both alleles, with the remainder being heterozygous. Four mutations were found in the mature miR‐142‐5p, four in the mature miR‐142‐3p, and three mutations affected the miR‐142 precursor. Two mutations in the seed sequence redirected miR‐142‐3p to the mRNA of the transcriptional repressor ZEB2 and one of them also targeted the ZEB1 mRNA. However, the other mutations in the mature miR‐142‐3p did not influence either the ZEB1 or ZEB2 3′ untranslated region (3′ UTR). On the other hand, the mutations affecting the seed sequence of miR‐142‐3p resulted in a loss of responsiveness in the 3′ UTR of the known miR‐142‐3p targets RAC1 and ADCY9. In contrast to the mouse p300 gene, the human p300 gene was not found to be a target for miR‐142‐5p. In one case with a mutation of the precursor, we observed aberrant processing of the miR‐142‐5p. Our data suggest that the mutations in miR‐142 probably lead to a loss rather than a gain of function. This is the first report describing mutations of a miRNA gene in a large percentage of a distinct lymphoma subtype.

MicroRNAs (miRNA) posttranscriptionally regulate gene expression and are important in tumorigenesis. Previous deep sequencing identified the miRNA profile of prostate carcinoma versus nonmalignant prostate tissue. Here, we generated miRNA expression profiles of prostate carcinoma by deep sequencing, with increasing tumor stage relative to corresponding nonmalignant and healthy prostate tissue, and detected clearly changed miRNA expression patterns. The miRNA profiles of the healthy and nonmalignant tissues were consistent with our previous findings, indicating a high fidelity of the method employed. In the tumors, quantitative real-time PCR (qRT-PCR) analysis of 40 paired samples of prostate carcinoma versus normal tissue revealed significant upregulation of miR-20a, miR-148a, miR-200b, and miR-375 and downregulation of miR-143 and miR-145. Hereby, miR-375 increased from normal to organ-confined tumors (pT2 pN0), slightly decreased in tumors with extracapsular growth (pT3 pN0), but was then expressed again at higher levels in lymph node metastasizing (pN1) tumors. The sequencing data for miR-375 were confirmed by Northern blotting and qRT-PCR. The regulation for other selected miRNAs could, however, not be confirmed by qRT-PCR in individual tumor stages. MiR-200b, in addition to miR-200c and miR-375 reduced the expression of SEC23A. Interestingly, miR-375, found by sequencing in pT2 upregulated by us and others in tumor versus normal tissue, and miR-15a, found by sequencing in pT2 and pT3 and in the metastasizing tumors, target the phosphatases PHLPP1 and PHLPP2, respectively. PHLPP1 and PHLPP2 dephosphorylate members of the AKT family of signal transducers, thereby inhibiting cell growth. Coexpression of miR-15a and miR-375 resulted in downregulation of PHLPP1/2 and strongly increased prostate carcinoma cell growth. Implications: These genomic data reveal relevant miRNAs in prostate cancer that may have biomarker and therapeutic potential. Mol Cancer Res; 12(2); 250–63. ©2013 AACR.