XB130, a New Adaptor Protein,Regulates Expression of Tumor Suppressive MicroRNAs in Cancer Cells

XB130, a novel adaptor protein, promotes cell growth by controlling expression of many related genes. MicroRNAs (miRNAs), which are frequently mis-expressed in cancer cells, regulate expression of targeted genes. In this present study, we aimed to explore the oncogenic mechanism of XB130 through miRNAs regulation. We analyzed miRNA expression in XB130 short hairpin RNA (shRNA) stably transfected WRO thyroid cancer cells by a miRNA array assay, and 16 miRNAs were up-regulated and 22 miRNAs were down-regulated significantly in these cells, in comparison with non-transfected or negative control shRNA transfected cells. We chose three of the up-regulated miRNAs (miR-33a, miR-149 and miR-193a-3p) and validated them by real-time qRT-PCR. Ectopic overexpression of XB130 suppressed these 3 miRNAs in MRO cells, a cell line with very low expression of XB130. Furthermore, we transfected miR mimics of these 3 miRNAs into WRO cells. They negatively regulated expression of oncogenes (miR-33a: MYC, miR-149: FOSL1, miR-193a-3p: SLC7A5), by targeting their 3′ untranslated region, and reduced cell growth. Our results suggest that XB130 could promote growth of cancer cells by regulating expression of tumor suppressive miRNAs and their targeted genes.     

VDR regulation of microRNA differs across prostate cell models suggesting extremely flexible control of transcription

The Vitamin D Receptor (VDR) is a member of the nuclear receptor superfamily and is of therapeutic interest in cancer and other settings. Regulation of microRNA (miRNA) by the VDR appears to be important to mediate its actions, for example, to control cell growth. To identify if and to what extent VDR-regulated miRNA patterns change in prostate cancer progression, we undertook miRNA microarray analyses in 7 cell models representing non-malignant and malignant prostate cells (RWPE-1, RWPE-2, HPr1, HPr1AR, LNCaP, LNCaP-C4–2, and PC-3). To focus on primary VDR regulatory events, we undertook expression analyses after 30 minutes treatment with 1α,25(OH)₂D₃. Across all models, 111 miRNAs were significantly modulated by 1α,25(OH)₂D₃ treatment. Of these, only 5 miRNAs were modulated in more than one cell model, and of these, only 3 miRNAs were modulated in the same direction. The patterns of miRNA regulation, and the networks they targeted, significantly distinguished the different cell types. Integration of 1α,25(OH)₂D₃-regulated miRNAs with published VDR ChIP-seq data showed significant enrichment of VDR peaks in flanking regions of miRNAs. Furthermore, mRNA and miRNA expression analyses in non-malignant RWPE-1 cells revealed patterns of miRNA and mRNA co-regulation; specifically, 13 significant reciprocal patterns were identified and these patterns were also observed in TCGA prostate cancer data. Lastly, motif search analysis revealed differential motif enrichment within VDR peaks flanking mRNA compared to miRNA genes. Together, this study revealed that miRNAs are rapidly regulated in a highly cell-type specific manner, and are significantly co-integrated with mRNA regulation.     

Targeting Notch signalling by the conserved miR-8/200 microRNA family in development and cancer cells

Notch signalling is crucial for the correct development and growth of numerous organs and tissues, and when subverted it can cause cancer. Loss of miR-8/200 microRNAs (miRNAs) is commonly observed in advanced tumours and correlates with their invasion and acquisition of stem-like properties. Here, we show that this miRNA family controls Notch signalling activation in Drosophila and human cells. In an overexpression screen, we identified the Drosophila miR-8 as a potent inhibitor of Notch-induced overgrowth and tumour metastasis. Gain and loss of mir-8 provoked developmental defects reminiscent of impaired Notch signalling and we demonstrated that miR-8 directly inhibits Notch ligand Serrate. Likewise, miR-200c and miR-141 directly inhibited JAGGED1, impeding proliferation of human metastatic prostate cancer cells. It has been suggested that JAGGED1 may also be important for metastases. Although in metastatic cancer cells, JAGGED1 modestly regulated ZEB1, the miR-200c's target in invasion, studies in Drosophila revealed that only concurrent overexpression of Notch and Zfh1/ZEB1 induced tumour metastases. Together, these data define a new way to attenuate or boost Notch signalling that may have clinical interest.     

VDR regulation of microRNA differs across prostate cell models suggesting extremely flexible control of transcription

The Vitamin D Receptor (VDR) is a member of the nuclear receptor superfamily and is of therapeutic interest in cancer and other settings. Regulation of microRNA (miRNA) by the VDR appears to be important to mediate its actions, for example, to control cell growth. To identify if and to what extent VDR-regulated miRNA patterns change in prostate cancer progression, we undertook miRNA microarray analyses in 7 cell models representing non-malignant and malignant prostate cells (RWPE-1, RWPE-2, HPr1, HPr1AR, LNCaP, LNCaP-C4–2, and PC-3). To focus on primary VDR regulatory events, we undertook expression analyses after 30 minutes treatment with 1α,25(OH)₂D₃. Across all models, 111 miRNAs were significantly modulated by 1α,25(OH)₂D₃ treatment. Of these, only 5 miRNAs were modulated in more than one cell model, and of these, only 3 miRNAs were modulated in the same direction. The patterns of miRNA regulation, and the networks they targeted, significantly distinguished the different cell types. Integration of 1α,25(OH)₂D₃-regulated miRNAs with published VDR ChIP-seq data showed significant enrichment of VDR peaks in flanking regions of miRNAs. Furthermore, mRNA and miRNA expression analyses in non-malignant RWPE-1 cells revealed patterns of miRNA and mRNA co-regulation; specifically, 13 significant reciprocal patterns were identified and these patterns were also observed in TCGA prostate cancer data. Lastly, motif search analysis revealed differential motif enrichment within VDR peaks flanking mRNA compared to miRNA genes. Together, this study revealed that miRNAs are rapidly regulated in a highly cell-type specific manner, and are significantly co-integrated with mRNA regulation.     

Screening for possible miRNA–mRNA associations in a colon cancer cell line

MicroRNAs (miRNAs) are small non-coding RNAs mediating the regulation of gene expression in various biological contexts, including carcinogenesis. Here, we screened putative associations between 34, 45, and 103 miRNAs and 164, 391, and 81 mRNAs via Argonaute1 (Ago1) or Ago2 immunoprecipitation (IP) experiments in a colon cancer cell line. We used a combination of RIP Seq analysis. RNAs that were co-immunoprecipitated with Ago1 or Ago2 were used for massively parallel small RNA and mRNA sequencing. The detected miRNAs and mRNAs were further associated with one another based on in silico target predictions. Analysis of the putative associations indicated that, although Ago1 and Ago2 shared a similar repertory of miRNAs, the mRNAs possibly regulated by those miRNAs seemed different. The mRNAs detected with Ago1 IP were indicated to be frequently associated with genes having constitutive cellular functions, regulated by a smaller number of miRNAs, and appeared to receive more stringent translational regulation. In contrast, putative miRNA-mRNA associations detected with Ago2 IP appeared to be related to signal transduction genes, which had a larger number of possible miRNA binding sites. We then conducted a similar analysis using the colon cancer cells cultured under hypoxia and identified potential hypoxia-induced miRNA-mRNA associations, which included several well-characterized cancer-related genes as novel putative miRNA targets.     

The Porcine MicroRNA Transcriptome Response to Transmissible Gastroenteritis Virus Infection

Transmissible gastroenteritis virus (TGEV; Coronaviridae family) causes huge economic losses to the swine industry. MicroRNAs (miRNAs) play a regulatory role in viral infection and may be involved in the mammalian immune response. Here, we report a comprehensive analysis of host miRNA expression in TGEV-infected swine testis (ST) cells. Deep sequencing generated 3,704,353 and 2,763,665 reads from uninfected ST cells and infected ST cells, respectively. The reads were aligned to known Sus scrofa pre-miRNAs in miRBase 19, identifying 284 annotated miRNAs. Certain miRNAs were differentially regulated during TGEV infection. 59 unique miRNAs displayed significant differentially expression between the normal and TGEV-infected ST cell samples: 15 miRNAs were significantly up-regulated and 44 were significantly down-regulated. Stem-loop RT-PCR was carried out to determine the expression levels of specific miRNAs in the two samples, and the results were consistent with those of sequencing. Gene ontology enrichment analysis of host target genes demonstrated that the differentially expressed miRNAs are involved in regulatory networks, including cellular process, metabolic process, immune system process. This is the first report of the identification of ST cell miRNAs and the comprehensive analysis of the miRNA regulatory mechanism during TGEV infection, which revealed the miRNA molecular regulatory mechanisms for the viral infection, expression of viral genes and the expression of immune-related genes. The results presented here will aid research on the prevention and treatment of viral diseases.     

miRNAome analysis of the mammalian neuronal nicotinic acetylcholine receptor gene family

Nicotine binds to and activates a family of ligand-gated ion channels, neuronal nicotinic acetylcholine receptors (nAChRs). Chronic nicotine exposure alters the expression of various nAChR subtypes, which likely contributes to nicotine dependence; however, the underlying mechanisms regulating these changes remain unclear. A growing body of evidence indicates that microRNAs (miRNAs) may be involved in nAChR regulation. Using bioinformatics, miRNA library screening, site-directed mutagenesis, and gene expression analysis, we have identified a limited number of miRNAs that functionally interact with the 3′-untranslated regions (3′ UTRs) of mammalian neuronal nAChR subunit genes. In silico analyses revealed specific, evolutionarily conserved sites within the 3′ UTRs through which the miRNAs regulate gene expression. Mutating these sites disrupted miRNA regulation confirming the in silico predictions. In addition, the miRNAs that target nAChR 3′ UTRs are expressed in mouse brain and are regulated by chronic nicotine exposure. Furthermore, we show that expression of one of these miRNAs, miR-542-3p, is modulated by nicotine within the mesocorticolimbic reward pathway. Importantly, overexpression of miR-542-3p led to a decrease in the protein levels of its target, the nAChR β2 subunit. Bioinformatic analysis suggests that a number of the miRNAs play a general role in regulating cholinergic signaling. Our results provide evidence for a novel mode of nicotine-mediated regulation of the mammalian nAChR gene family.     

Epigenetic regulation of microRNAs in cancer: an integrated review of literature

MicroRNAs (miRNAs) belong to the heterogeneous class of non-coding RNAs (ncRNAs) that regulate the translation and degradation of target mRNAs, and control approximately 30% of human genes. MiRNA genes might be silenced in human tumors (oncomiRs) by aberrant hypermethylation of CpG islands that encompass or lie adjacent to miRNA genes and/or by histone modifications. We performed literature search for research articles describing epigenetically regulated miRNAs in cancer and identified 45 studies that were published between 2006 and 7/2010. The data from those papers are fragmented and methodologically heterogeneous and our work represents first systematic review towards to integration of diverse sets of information. We reviewed the methods used for detection of miRNA epigenetic regulation, which comprise bisulfite genomic sequencing PCR (BSP), bisulfite pyrosequencing, methylation specific PCR (MSP), combined bisulfite restriction analysis (COBRA), methylation sensitive single nucleotide primer extension (Ms-SNuPE), MassARRAY technique and some modifications of those methods. This integrative study revealed 122 miRNAs that were reported to be epigenetically regulated in 23 cancer types. Compared to protein coding genes, human oncomiRs showed an order of magnitude higher methylation frequency (11.6%; 122/1048 known miRNAs). Nearly half, (45%; 55/122) epigenetically regulated miRNAs were associated with different cancer types, but other 55% (67/122) miRNAs were present in only one cancer type and therefore representing cancer-specific biomarker potential. The data integration revealed miRNA epigenomic hot spots on the chromosomes 1q, 7q, 11q, 14q and 19q. CpG island analysis of corresponding miRNA precursors (pre-miRNAs) revealed that 20% (26/133) of epigenetically regulated miRNAs had a CpG island within the range of 5kb upstream, among them 14% (19/133) of miRNAs resided within the CpG island. Our integrative survey and analyses revealed candidate cancer-specific miRNA epigenetic signatures which provide the basis for new therapeutic strategies in cancer by targeting the epigenetic regulation of miRNAs.