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Supplementary Data from The MicroRNA Profile of Prostate Carcinoma Obtained by Deep Sequencing

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.

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.

Epstein-Barr virus (EBV) is a human tumour virus that efficiently growth-transforms primary human B-lymphocytes in vitro. The viral nuclear antigen 2 (EBNA2) is essential for immortalisation of B-cells and stimulates viral and cellular gene expression through interaction with DNA-bound transcription factors. Like its cellular homologue Notch, it associates with the DNA-bound repressor RBPJκ (CSL/CBF1) thereby converting RBPJκ into the active state. For instance, both EBNA2 and Notch activate the cellular HES1 promoter. In EBV-transformed lymphocytes, the RNA of the NP9 protein encoded by human endogenous retrovirus HERV-K(HML-2) Type 1 is strongly up-regulated. The NP9 protein is detectable both in EBV-positive Raji cells, a Burkitt's lymphoma cell line, and in IB4, an EBV-transformed human lymphoblastoid cell line. NP9 binds to LNX that forms a complex with the Notch regulator Numb. Therefore, the function of NP9 vis-à-vis Notch and EBNA2 was analysed. Here, we show that NP9 binds to EBNA2 and negatively affects the EBNA2-mediated activation of the viral C- and LMP2A promoters. In contrast, NP9 did neither interfere in the activation of the HES1 promoter by Notch nor the induction of the viral LMP1 promoter by EBNA2. In an electrophoretic mobility shift analysis, NP9 reduced the binding of EBNA2 to DNA-bound RBPJκ by about 50%. The down-regulation of EBNA2-activity by NP9 might represent a cellular defence mechanism against viral infection or could, alternatively, represent an adaptation of the virus to prevent excessive viral protein production that might otherwise be harmful for the infected cell.

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) growth-transforms B-lymphocytes. The virus-encoded nuclear antigen 2 (EBNA2) is essential for transformation and activates gene expression by association with DNA-bound transcription factors such as RBPJkappa (CSL/CBF1). We have previously shown that EBNA2 contains symmetrically dimethylated Arginine (sDMA) residues. Deletion of the RG-repeat results in a reduced ability of the virus to immortalise B-cells. We now show that the RG repeat also contains asymmetrically dimethylated Arginines (aDMA) but neither non-methylated (NMA) Arginines nor citrulline residues. We demonstrate that only aDMA-containing EBNA2 is found in a complex with DNA-bound RBPJkappa in vitro and preferentially associates with the EBNA2-responsive EBV C, LMP1 and LMP2A promoters in vivo. Inhibition of methylation in EBV-infected cells results in reduced expression of the EBNA2-regulated viral gene LMP1, providing additional evidence that methylation is a prerequisite for DNA-binding by EBNA2 via association with the transcription factor RBPJkappa.

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 upor 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. Copyright © 2009, American Society for Microbiology., link_to_OA_fulltext

PLoS one 7(8), e42106 (2012). doi:10.1371/journal.pone.0042106, Published by PLoS [u.a.], Lawrence, Kan.

The Epstein-Barr virus (EBV) is an oncogenic human Herpes virus involved in the pathogenesis of nasal NK/T-cell lymphoma. EBV encodes microRNAs (miRNAs) and induces changes in the host cellular miRNA profile. MiRNAs are short non-coding RNAs of about 19–25 nt length that regulate gene expression by post-transcriptional mechanisms and are frequently deregulated in human malignancies including cancer. The microRNA profiles of EBV-positive NK/T-cell lymphoma, non-infected T-cell lymphoma and normal thymus were established by deep sequencing of small RNA libraries. The comparison of the EBV-positive NK/T-cell vs. EBV-negative T-cell lymphoma revealed 15 up- und 16 down-regulated miRNAs. In contrast, the majority of miRNAs was repressed in the lymphomas compared to normal tissue. We also identified 10 novel miRNAs from known precursors and two so far unknown miRNAs. The sequencing results were confirmed for selected miRNAs by quantitative Real-Time PCR (qRT-PCR). We show that the proinflammatory cytokine interleukin 1 alpha (IL1A) is a target for miR-142-3p and the oncogenic BCL6 for miR-205. MiR-142-3p is down-regulated in the EBV-positive vs. EBV-negative lymphomas. MiR-205 was undetectable in EBV-negative lymphoma and strongly down-regulated in EBV-positive NK/T-cell lymphoma as compared to thymus. The targets were confirmed by reporter assays and by down-regulation of the proteins by ectopic expression of the cognate miRNAs. Taken together, our findings demonstrate the relevance of deregulated miRNAs for the post-transcriptional gene regulation in nasal NK/T-cell lymphomas.

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. ISSN:1362-4962 ISSN:0301-5610

Objective: We aimed at exploring the expression of neuropeptide Y (NPY), omentin and visfatin in adipose tissues of adults along with clinical parameters and hormones. Methods: We included 168 adult patients (31 surgical obese patients and 31 surgical controls, 76 non-surgical obese patients, 30 non-surgical controls). We measured plasma NPY (by radioimmunoassay), cortisol (with an electrochemiluminescence immunoassay) and urinary cortisol metabolites (by gas chromatography/ mass spectrometry). Expression of NPY, omentin and visfatin in subcutaneous and visceral adipose tissue specimens of the surgical patients was quantified using real-time PCR. Results: NPY was detectable in adipose tissue specimens and, like plasma NPY concentrations, comparable between groups. Omentin gene expression was higher in visceral than in subcutaneous adipose tissues (p < 0.0001). Visfatin expression was lower in the subcutaneous tissue of obese patients compared with controls (p < 0.05). Cortisol was lower in obese adults compared with controls (136.5 ± 74.1 vs. 162.2 ± 56.1 ng/ml; p < 0.05), cortisol metabolites were comparable between groups. Conclusion: In our obese adults, plasma NPY levels and the glucocorticoid measures were not elevated. Even though the expression of NPY, omentin and visfatin was comparable between obese individuals and controls, we have to consider differences in the total production rate of adipose tissue-derived factors.

MicroRNAs (miRNAs) are involved in sequence-specific cleavage, translational repression or deadenylation of specific target mRNAs resulting in post-transcriptional gene silencing. Epstein-Barr Virus (EBV) infection induces cellular non-coding (nc)RNAs e.g., the "vault" RNAs or miRNAs such as miR-21, miR-155 or miR-146a. MiR-146a is upregulated in various tumours and plays a role in innate immunity. We show that the EBV-encoded latent membrane protein 1 (LMP1) induces the expression of miR-146a via NFkappaB. LMP1 activates the miR-146a promoter but not a promoter with a mutation of the NFkappaB-response elements. Conversely, a LMP1-mutant deficient in NFkappaB-activation failed to activate the promoter. The "CAO"-LMP1 variant which has an increased potential to induce NFkappaB also showed a higher ability to activate the miR-146a promoter as compared to standard B95.8-LMP1. Northern blotting revealed high levels of miR-146a and miR-155 in the Burkitt's lymphoma cell line Jijoye which expresses LMP1 while the LMP1-deficient P3HR1 mutant derived from Jijoye expresses less miR-146a or miR-155. Likewise, EBV-latency type I Burkitt's lymphoma cells with low LMP1 levels also contain low levels of either miR-146a or miR-155 while their levels are increased in LMP1-expressing EBV-latency type III BL cells. Expression of LMP1 in P3HR1 cells upregulates miR-146a levels. Neither miR-146a nor miR-155 are detectable in BCBL-1 cells transformed by the Kaposi-Sarcoma Herpes virus (KSHV/HHV8). It is possible that the induction of miR-146a plays a role in the induction or maintenance of EBV latency by modulating innate immune responses to the virus infected host cell.

MicroRNAs (miRNAs) have been implicated in sequence-specific cleavage, translational repression or deadenylation of specific target mRNAs resulting in post-transcriptional gene silencing. Epstein-Barr virus (EBV) encodes 23 miRNAs of unknown function. Here we show that the EBV-encoded miRNA miR-BART2 down-regulates the viral DNA polymerase BALF5. MiR-BART2 guides cleavage within the 3'-untranslated region (3'UTR) of BALF5 by virtue of its complete complementarity to its target. Induction of the lytic viral replication cycle results in a reduction of the level of miR-BART2 with a strong concomitant decrease of cleavage of the BALF5 3'UTR. Expression of miR-BART2 down-regulates the activity of a luciferase reporter gene containing the BALF5 3'UTR. Forced expression of miR-BART2 during lytic replication resulted in a 40-50% reduction of the level of BALF5 protein and a 20% reduction of the amount of virus released from EBV-infected cells. Our results are compatible with the notion that EBV-miR-BART2 inhibits transition from latent to lytic viral replication.

Here we provide evidence that EBNA2 is methylated in vivo and that methylation of EBNA2 is a prerequisite for binding to SMN. We present SMN as a novel binding partner of EBNA2 by showing that EBNA2 colocalizes with SMN in nuclear gems and that both proteins can be coimmunoprecipitated from cellular extract. Furthermore, in vitro methylation of either wild-type EBNA2 or a glutathione S -transferase-EBNA2 fusion protein encompassing the arginine-glycine (RG) repeat element is necessary for in vitro binding to the Tudor domain of SMN. The recently shown functional cooperation of SMN and EBNA2 in transcriptional activation and the previous observation of a severely reduced transformation potential yet strongly enhanced transcriptional activity of an EBNA2 mutant lacking the RG repeat indicate that binding of SMN to EBNA2 is a critical step in B-cell transformation by Epstein-Barr virus.

The Epstein-Barr virus (EBV) causes infectious mononucleosis and is linked to the genesis of several human lymphoproliferative diseases (for a review, see reference 33). The EBV-encoded nuclear antigen 2 (EBNA2) is a viral transactivator essential for EBV-induced transformation of resting human B lymphocytes, by promoting the expression of the transforming latent membrane proteins LMP1 and 2, the nuclear EBV Cp promoter-driven EBNA proteins, and the cellular genes CD23 and c-fgr (for review, see reference 15). EBNA2 does not bind directly to DNA but exerts its function by interacting with the cellular proteins RBPJκ (CBF1) and, on the more complex LMP1 promoter, also Spi1 (PU.1), tethered to cognate response elements (12, 17, 20, 45, 46). Transcriptional activation is induced by binding of the C-terminal acidic domain (5) to components of the basal RNA polymerase II transcription machinery, such as RPA70, TAF40, TFIIB, and TFIIH (38, 39), and recruitment of the coactivators p300, CBP, and PCAF histone deacetylase (14, 41). In addition, by attracting the hSWI/SNF complex (42, 43) and targeting histone H1 (9, 34), EBNA2 likely promotes relief of nucleosome-mediated gene repression. We have recently shown that EBNA2 binds to DP103, a novel member of the DEAD-box family of putative RNA helicases (10). DP103 is a ubiquitously expressed 103-kDa phosphoprotein with an RNA-dependent ATPase activity; its other functions, in particular with regard to its interaction with EBNA2, remained unknown. While the work presented here was in progress, an interaction of DP103 (alternatively called Gemin3 [2]) and the survival motor neuron (SMN) protein, and their respective murine homologues, were described in two independent studies (1, 2). SMN is part of a multiprotein complex containing SIP1, DP103 (Gemin3), GIP1 (Gemin4), and several Sm proteins that is involved in the assembly and nuclear regeneration of snRNPs and spliceosomes (2, 7, 25, 31). Both SMN and DP103 are localized in the cytoplasm and distinct nuclear structures, described as coiled bodies and gems (gemini of coiled bodies) (2, 21, 31). Mutations in the SMN gene result in spinal muscular atrophy (SMA), a recessive genetic disease with loss of α-motor neurons in the spinal cord, leading to muscle weakness and subsequent death. The SMN gene exists in two inverted copies within the same chromosomal region on chromosome 5q13 (19). In most SMA patients, a mutated telomeric form of the SMN gene results in a nonfunctional exon 7-deleted SMN, unable to self-associate (22), which cannot be compensated by the low amounts of full-length SMN protein expressed from the centromeric allele (24). In a few cases of SMA, point mutations were described which exchange amino acid (aa) 272 (Y272C) (19) or aa 134 (E134K) (4), affecting a putative RNA binding tudor domain (26). Furthermore, knockout of the murine SMN gene or its yeast homologue Yab8p resulted in a lethal phenotype (11, 28, 35). Interestingly, a role for SMN in transcriptional regulation has been implicated, since SMN was shown to interact with the bovine papillomavirus E2 transactivator and to coactivate an E2-responsive viral promoter (1, 36). Furthermore, Ou et al. demonstrated that murine dp103 is also involved in transcriptional regulation by negatively modulating the expression of steroidogenic factor-1 (27). Finally, the SMN complex has recently been shown to associate with the C-terminal domain (CTD) of RNA polymerase II (30), although the functional consequences of this interaction have not yet been elucidated. Searching for DP103-associated cellular proteins by using the yeast two-hybrid system, we also identified SMN as an interaction partner of DP103. Here, we show that this interaction is also relevant in B cells and that SMN is able to coactivate the viral LMP1 promoter in the presence of EBNA2 in vitro and in vivo. Data obtained from analyzing different EBNA2, DP103, and SMN mutants regarding their binding domains, subcellular distribution, and influence on EBNA2-mediated transactivation suggest that SMN is a novel factor involved in EBNA2-mediated transactivation of the viral LMP1 promoter: by targeting of DP103 within spliceosomal complexes, EBNA2 subsequently releases transcriptionally active SMN, which functions as a coactivator, likely within the RNA polymerase II transcription complex.

Background: Prostate cancer is a leading cause of tumor mortality. In order to identify and characterize the underlying molecular mechanisms, we performed microRNA (miRNA) profiling of primary prostate cancers and non-cancer prostate tissue. Furthermore, using database analysis, we sought to identify regulatory targets of aberrantly expressed miRNAs. Material and Methods: We performed comparative miRNA expression profiling in ten prostate cancer specimens and ten non-cancer prostate tissue samples using deep sequencing of cDNA libraries. The deregulation of selected miRNAs was validated in established prostate cancer cell lines by Northern blotting as well as in 26 paired samples of prostate cancer tissue and adjacent normal tissue by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The regulative capabilities of selected miRNAs on potential target genes were analyzed in vitro using reporter gene constructs and transfection of miRNA expression vectors Results: Using deep sequencing we found that 16 miRNAs were up-regulated more than 1.5-fold and 17 miRNAs were down regulated more than 1.5-fold. The most pronounced deregulation was noted for miRNAs miR-375 and miR-200c which showed an up-regulation of more than 4.5-fold and miR-143 and miR-145 which were down regulated 4-fold each. The differential expression of these miRNAs could be confirmed by Northern blot analysis of established prostate cancer cell lines. By analyzing 26 primary prostate cancer samples by qRT-PCR we could detect a significant deregulation of the miRNAs miR-375, miR-143 and miR-145 (p Conclusion: Our findings indicate that specific miRNAs are reproducibly found to be deregulated in different cohorts of prostate cancer specimens using independent experimental methods. This is the first report to show that miRNAs miR-143 and miR-145 are capable of negatively regulating the protein expression of Myosin VI. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4036.

International audience; The mRNA targets of microRNAs (miRNAs) can be identified by immunoprecipitation of Argonaute (Ago) protein-containing RNA-induced silencing complexes (RISCs) followed by microarray analysis (RIP-Chip). Here we used Ago2-based RIP-Chip to identify transcripts targeted by Kaposi's sarcoma-associated herpesvirus (KSHV) miRNAs (n = 114), Epstein-Barr virus (EBV) miRNAs (n = 44), and cellular miRNAs (n = 2337) in six latently infected or stably transduced human B cell lines. Of the six KSHV miRNA targets chosen for validation, four showed regulation via their 3'UTR, while two showed regulation via binding sites within coding sequences. Two genes governing cellular transport processes (TOMM22 and IPO7) were confirmed to be targeted by EBV miRNAs. A significant number of viral miRNA targets were upregulated in infected cells, suggesting that viral miRNAs preferentially target cellular genes induced upon infection. Transcript half-life both of cellular and viral miRNA targets negatively correlated with recruitment to RISC complexes, indicating that RIP-Chip offers a quantitative estimate of miRNA function.