MicroRNA-31 controls G protein alpha-13 (GNA13) expression and cell invasion in breast cancer cells.

BACKGROUND: Gα13 (GNA13) is the α subunit of a heterotrimeric G protein that mediates signaling through specific G protein-coupled receptors (GPCRs). Our recent study showed that control of GNA13 expression by specific microRNAs (miRNAs or miRs) is important for prostate cancer cell invasion. However, little is known about the control of GNA13 expression in breast cancers. This project was carried out to determine (i) whether enhanced GNA13 expression is important for breast cancer cell invasion, and (ii) if so, the mechanism of deregulation of GNA13 expression in breast cancers. METHODS: To determine the probable miRNAs regulating GNA13, online miRNA target prediction tool Targetscan and Luciferase assays with GNA13-3'-UTR were used. Effect of miRNAs on GNA13 mRNA, protein and invasion was studied using RT-PCR, western blotting and in vitro Boyden chamber assay respectively. Cell proliferation was done using MTT assays. RESULTS: Overexpression of GNA13 in MCF-10a cells induced invasion, whereas knockdown of GNA13 expression in MDA-MB-231 cells inhibited invasion. Expression analysis of miRNAs predicted to bind the 3'-UTR of GNA13 revealed that miR-31 exhibited an inverse correlation to GNA13 protein expression in breast cancer cells. Ectopic expression of miR-31 in MDA-MB-231 cells significantly reduced GNA13 mRNA and protein levels, as well as GNA13-3'-UTR-reporter activity. Conversely, blocking miR-31 activity in MCF-10a cells induced GNA13 mRNA, protein and 3'-UTR reporter activity. Further, expression of miR-31 significantly inhibited MDA-MB-231 cell invasion, and this effect was partly rescued by ectopic expression of GNA13 in these cells. Examination of 48 human breast cancer tissues revealed that GNA13 mRNA levels were inversely correlated to miR-31 levels. CONCLUSIONS: These data provide strong evidence that GNA13 expression in breast cancer cells is regulated by post-transcriptional mechanisms involving miR-31. Additionally our data shows that miR-31 regulates breast cancer cell invasion partially via targeting GNA13 expression in breast cancer cells. Loss of miR-31 expression and increased GNA13 expression could be used as biomarkers of breast cancer progression.

Allele frequencies of variants in ultra conserved elements identify selective pressure on transcription factor binding.

Ultra-conserved genes or elements (UCGs/UCEs) in the human genome are extreme examples of conservation. We characterized natural variations in 2884 UCEs and UCGs in two distinct populations; Singaporean Chinese (n = 280) and Italian (n = 501) by using a pooled sample, targeted capture, sequencing approach. We identify, with high confidence, in these regions the abundance of rare SNVs (MAF<0.5%) of which 75% is not present in dbSNP137. UCEs association studies for complex human traits can use this information to model expected background variation and thus necessary power for association studies. By combining our data with 1000 Genome Project data, we show in three independent datasets that prevalent UCE variants (MAF>5%) are more often found in relatively less-conserved nucleotides within UCEs, compared to rare variants. Moreover, prevalent variants are less likely to overlap transcription factor binding site. Using SNPfold we found no significant influence of RNA secondary structure on UCE conservation. All together, these results suggest UCEs are not under selective pressure as a stretch of DNA but are under differential evolutionary pressure on the single nucleotide level.

Opposing activities of the Ras and Hippo pathways converge on regulation of YAP protein turnover.

Cancer genomes accumulate numerous genetic and epigenetic modifications. Yet, human cellular transformation can be accomplished by a few genetically defined elements. These elements activate key pathways required to support replicative immortality and anchorage independent growth, a predictor of tumorigenesis in vivo. Here, we provide evidence that the Hippo tumor suppressor pathway is a key barrier to Ras-mediated cellular transformation. The Hippo pathway targets YAP1 for degradation via the ßTrCP-SCF ubiquitin ligase complex. In contrast, the Ras pathway acts oppositely, to promote YAP1 stability through downregulation of the ubiquitin ligase complex substrate recognition factors SOCS5/6. Depletion of SOCS5/6 or upregulation of YAP1 can bypass the requirement for oncogenic Ras in anchorage independent growth in vitro and tumor formation in vivo. Through the YAP1 target, Amphiregulin, Ras activates the endogenous EGFR pathway, which is required for transformation. Thus, the oncogenic activity of Ras(V12) depends on its ability to counteract Hippo pathway activity, creating a positive feedback loop, which depends on stabilization of YAP1.

Viral small T oncoproteins transform cells by alleviating hippo-pathway-mediated inhibition of the YAP proto-oncogene.

Primary human cells can be transformed into tumor cells by a defined set of genetic alterations including telomerase, oncogenic Ras(V12), and the tumor suppressors p53 and pRb. SV40 small T (ST) is required for anchorage-independent growth in vitro and in vivo. Here, we identify the Hippo tumor suppressor pathway as a critical target of ST in cellular transformation. We report that ST uncouples YAP from the inhibitory activity of the Hippo pathway through PAK1-mediated inactivation of NF2. Membrane-tethered activated PAK is sufficient to bypass the requirement for ST in anchorage-independent growth. PAK acts via YAP to mediate the transforming effects of ST. Activation of endogenous YAP is required for ST-mediated transformation and is sufficient to bypass ST in anchorage-independent growth and xenograft tumor formation. Our findings uncover the Hippo tumor suppressor pathway as a final gatekeeper to transformation and tumorigenesis of primary cells.

High-content imaging of presynaptic assembly.

Presynaptic assembly involves the specialization of a patch of axonal membrane into a complex structure that supports synaptic vesicle exocytosis and neurotransmitter release. In mammalian neurons, presynaptic assembly is widely studied in a co-culture assay, where a synaptogenic cue expressed at the surface of a heterologous cell induces presynaptic differentiation in a contacting axon. This assay has led to the discovery of numerous synaptogenic proteins, but has not been used to probe neuronal mechanisms regulating presynaptic induction. The identification of regulatory pathways that fine-tune presynaptic assembly is hindered by the lack of adequate tools to quantitatively image this process. Here, we introduce an image-processing algorithm that identifies presynaptic clusters in mammalian co-cultures and extracts a range of synapse-specific parameters. Using this software, we assessed the intrinsic variability of this synaptic induction assay and probed the effect of eight neuronal microRNAs on presynaptic assembly. Our analysis revealed a novel role for miR-27b in augmenting the density of presynaptic clusters. Our software is applicable to a wide range of synaptic induction protocols (including spontaneous synaptogenesis observed in neuron cultures) and is a valuable tool to determine the subtle impact of disease-associated genes on presynaptic assembly.

New insights into lineage restriction of mammary gland epithelium using parity-identified mammary epithelial cells.

INTRODUCTION: Parity-identified mammary epithelial cells (PI-MECs) are an interesting cellular subset because they survive involution and are a presumptive target for transformation by human epidermal growth factor receptor 2 (HER2)/neu in mammary tumors. Depending on the type of assay, PI-MECs have been designated lobule-restricted progenitors or multipotent stem/progenitor cells. PI-MECs were reported to be part of the basal population of mammary epithelium based on flow cytometry. We investigated the cellular identity and lineage potential of PI-MECs in intact mammary glands. METHODS: We performed a quantitative and qualitative analysis of the contribution of PI-MECs to mammary epithelial cell lineages in pregnant and involuted mammary glands by immunohistochemistry, fluorescence-activated cells sorting (FACS), and quantitative polymerase chain reaction. PI-MECs were labeled by the activation of Whey Acidic Protein (WAP)-Cre during pregnancy that results in permanent expression of yellow fluorescent protein. RESULTS: After involution, PI-MECs are present exclusively in the luminal layer of mammary ducts. During pregnancy, PI-MECs contribute to the luminal layer but not the basal layer of alveolar lobules. Strikingly, whereas all luminal estrogen receptor (ER)-negative cells in an alveolus can be derived from PI-MECs, the alveolar ER-positive cells are unlabeled and reminiscent of Notch2-traced L cells. Notably, we observed a significant population of unlabeled alveolar progenitors that resemble PI-MECs based on transcriptional and histological analysis. CONCLUSIONS: Our demonstration that PI-MECs are luminal cells underscores that not only basal cells display multi-lineage potential in transplantation assays. However, the lineage potential of PI-MECs in unperturbed mammary glands is remarkably restricted to luminal ER-negative cells of the secretory alveolar lineage. The identification of an unlabeled but functionally similar population of luminal alveolar progenitor cells raises the question of whether PI-MECs are a unique population or the result of stochastic labeling. Interestingly, even when all luminal ER-negative cells of an alveolus are PI-MEC-derived, the basal cells and hormone-sensing cells are derived from a different source, indicating that cooperative outgrowth of cells from different lineages is common in alveologenesis.

Comparison of gene expression profiles in primary and immortalized human pterygium fibroblast cells.

PURPOSE: Pterygium is a fibrovascular growth on the ocular surface with corneal tissue destruction, matrix degradation and varying extents of chronic inflammation. To facilitate investigation of pterygium etiology, we immortalized pterygium fibroblast cells and profiled their global transcript levels compared to primary cultured cells. METHODS: Fibroblast cells were cultured from surgically excised pterygium tissue using the explant method and propagated to passage number 2-4. We hypothesized that intervention with 3 critical molecular intermediates may be necessary to propage these cells. Primary fibroblast cells were immortalized sequentially by a retroviral construct containing the human telomerase reverse transcriptase gene and another retroviral expression vector expressing p53/p16 shRNAs. Primary and immortalized fibroblast cells were evaluated for differences in global gene transcript levels using an Agilent Genechip microarray. RESULTS: Light microscopic morphology of immortalized cells was similar to primary pterygium fibroblast at passage 2-4. Telomerase reverse transcriptase was expressed, and p53 and p16 levels were reduced in immortalized pterygium fibroblast cells. There were 3308 significantly dysregulated genes showing at least 2 fold changes in transcript levels between immortalized and primary cultured cells (2005 genes were up-regulated and 1303 genes were down-regulated). Overall, 13.58% (95% CI: 13.08-14.10) of transcripts in immortalized cells were differentially expressed by at least 2 folds compared to primary cells. CONCLUSION: Pterygium primary fibroblast cells were successfully immortalized to at least passage 11. Although a variety of genes are differentially expressed between immortalized and primary cells, only genes related to cell cycle are significantly changed, suggesting that the immortalized cells may be used as an in vitro model for pterygium pathology.

Expression analysis of rare cellular subsets: direct RT-PCR on limited cell numbers obtained by FACS or soft agar assays.

Since tissues and tumors are heterogenous populations containing different cell types, their transcriptomes are blends of multiple mRNA expression profiles. Although fluorescence-activated cell sorting (FACS) allows isolation of individual cell types, RNA isolation and quantification remain problematic from rare subsets, such as tissue stem cells. Likewise, identification of transcriptional changes relevant to the tumorigenic potential of mammalian cells while they are actively growing as colonies in soft agar is also hampered by limited amounts of starting material. Here we describe a convenient method that fills the gap between single cell and whole tissue mRNA analysis, enabling mRNA quantification for individual colonies picked from soft agar. Our method involves direct lysis, reverse transcription and quantitative PCR (RT-qPCR) on 500 sorted cells or a single soft agar colony, thus allowing evaluation of up to 20 transcripts in functionally distinct subpopulations without the need for RNA isolation or amplification.

MicroRNA-182 and microRNA-200a control G-protein subunit α-13 (GNA13) expression and cell invasion synergistically in prostate cancer cells.

G protein-coupled receptors (GPCRs) and their ligands have been implicated in progression and metastasis of several cancers. GPCRs signal through heterotrimeric G proteins, and among the different types of G proteins, GNA12/13 have been most closely linked to tumor progression. In this study, we explored the role of GNA13 in prostate cancer cell invasion and the mechanism of up-regulation of GNA13 in these cells. An initial screen for GNA13 protein expression showed that GNA13 is highly expressed in the most aggressive cancer cell lines. Knockdown of GNA13 in highly invasive PC3 cells revealed that these cells depend on GNA13 expression for their invasion, migration, and Rho activation. As mRNA levels in these cells did not correlate with protein levels, we assessed the potential involvement of micro-RNAs (miRNAs) in post-transcriptional control of GNA13 expression. Expression analysis of miRNAs predicted to bind the 3'-UTR of GNA13 revealed that miR-182 and miR-141/200a showed an inverse correlation to the protein expression in LnCAP and PC3 cells. Ectopic expression of miR-182 and miR-141/200a in PC3 cells significantly reduced protein levels, GNA13-3'-UTR reporter activity and in vitro invasion of these cells. This effect was blocked by restoration of GNA13 expression in these cells. Importantly, inhibition of miR-182 and miR-141/200a in LnCAP cells using specific miRNA inhibitors elevated the expression of GNA13 and enhanced invasion of these cells. These data provide strong evidence that GNA13 is an important mediator of prostate cancer cell invasion, and that miR-182 and miR-200 family members regulate its expression post-transcriptionally.

p53-Independent regulation of p21Waf1/Cip1 expression and senescence by PRMT6.

p21 is a potent cyclin-dependent kinase inhibitor that plays a role in promoting G1 cell cycle arrest and cellular senescence. Consistent with this role, p21 is a downstream target of several tumour suppressors and oncogenes, and it is downregulated in the majority of tumours, including breast cancer. Here, we report that protein arginine methyltransferase 6 (PRMT6), a type I PRMT known to act as a transcriptional cofactor, directly represses the p21 promoter. PRMT6 knock-down (KD) results in a p21 derepression in breast cancer cells, which is p53-independent, and leads to cell cycle arrest, cellular senescence and reduced growth in soft agar assays and in severe combined immunodeficiency (SCID) mice for all the cancer lines examined. We finally show that bypassing the p21-mediated arrest rescues PRMT6 KD cells from senescence, and it restores their ability to grow on soft agar. We conclude that PRMT6 acts as an oncogene in breast cancer cells, promoting growth and preventing senescence, making it an attractive target for cancer therapy.

Oncogenic cooperation between SOCS family proteins and EGFR identified using a Drosophila epithelial transformation model.

MicroRNAs (miRNAs) are emerging as cooperating factors that promote the activity of oncogenes in tumor formation and disease progression. This poses the challenge of identifying the miRNA targets responsible for these interactions. In this study, we identify the growth regulatory miRNA bantam and its target, Socs36E, as cooperating factors in EGFR-driven tumorigenesis and metastasis in a Drosophila model of epithelial transformation. bantam promotes growth by limiting expression of Socs36E, which functions as a negative growth regulator. Socs36E has only a modest effect on growth on its own, but behaves as a tumor suppressor in combination with EGFR activation. The human ortholog of SOCS36E, SOCS5, behaves as a candidate tumor suppressor in cellular transformation in cooperation with EGFR/RAS pathway activation.

miR-Sens--a retroviral dual-luciferase reporter to detect microRNA activity in primary cells.

MicroRNA-mRNA interactions are commonly validated and deconstructed in cell lines transfected with luciferase reporters. However, due to cell type-specific variations in microRNA or RNA-binding protein abundance, such assays may not reliably reflect microRNA activity in other cell types that are less easily transfected. In order to measure miRNA activity in primary cells, we constructed miR-Sens, a MSCV-based retroviral vector that encodes both a Renilla luciferase reporter gene controlled by microRNA binding sites in its 3' UTR and a Firefly luciferase normalization gene. miR-Sens sensors can be efficiently transduced in primary cells such as human fibroblasts and mammary epithelial cells, and allow the detection of overexpressed and, more importantly, endogenous microRNAs. Notably, we find that the relative luciferase activity is correlated to the miRNA expression, allowing quantitative measurement of microRNA activity. We have subsequently validated the miR-Sens 3' UTR vectors with known human miRNA-372, miRNA-373, and miRNA-31 targets (LATS2 and TXNIP). Overall, we observe that miR-Sens-based assays are highly reproducible, allowing detection of the independent contribution of multiple microRNAs to 3' UTR-mediated translational control of LATS2. In conclusion, miR-Sens is a new tool for the efficient study of microRNA activity in primary cells or panels of cell lines. This vector will not only be useful for studies on microRNA biology, but also more broadly on other factors influencing the translation of mRNAs.

First somatic mutation of E2F1 in a critical DNA binding residue discovered in well-differentiated papillary mesothelioma of the peritoneum.

BACKGROUND: Well differentiated papillary mesothelioma of the peritoneum (WDPMP) is a rare variant of epithelial mesothelioma of low malignancy potential, usually found in women with no history of asbestos exposure. In this study, we perform the first exome sequencing of WDPMP. RESULTS: WDPMP exome sequencing reveals the first somatic mutation of E2F1, R166H, to be identified in human cancer. The location is in the evolutionarily conserved DNA binding domain and computationally predicted to be mutated in the critical contact point between E2F1 and its DNA target. We show that the R166H mutation abrogates E2F1's DNA binding ability and is associated with reduced activation of E2F1 downstream target genes. Mutant E2F1 proteins are also observed in higher quantities when compared with wild-type E2F1 protein levels and the mutant protein's resistance to degradation was found to be the cause of its accumulation within mutant over-expressing cells. Cells over-expressing wild-type E2F1 show decreased proliferation compared to mutant over-expressing cells, but cell proliferation rates of mutant over-expressing cells were comparable to cells over-expressing the empty vector. CONCLUSIONS: The R166H mutation in E2F1 is shown to have a deleterious effect on its DNA binding ability as well as increasing its stability and subsequent accumulation in R166H mutant cells. Based on the results, two compatible theories can be formed: R166H mutation appears to allow for protein over-expression while minimizing the apoptotic consequence and the R166H mutation may behave similarly to SV40 large T antigen, inhibiting tumor suppressive functions of retinoblastoma protein 1.

Genomic loss of miR-486 regulates tumor progression and the OLFM4 antiapoptotic factor in gastric cancer.

PURPOSE: MicroRNAs (miRNA) play pivotal oncogenic and tumor-suppressor roles in several human cancers. We sought to discover novel tumor-suppressor miRNAs in gastric cancer (GC). EXPERIMENTAL DESIGN: Using Agilent miRNA microarrays, we compared miRNA expression profiles of 40 primary gastric tumors and 40 gastric normal tissues, identifying miRNAs significantly downregulated in gastric tumors. RESULTS: Among the top 80 miRNAs differentially expressed between gastric tumors and normals (false discovery rate < 0.01), we identified hsa-miR-486 (miR-486) as a significantly downregulated miRNA in primary GCs and GC cell lines. Restoration of miR-486 expression in GC cell lines (YCC3, SCH and AGS) caused suppression of several pro-oncogenic traits, whereas conversely inhibiting miR-486 expression in YCC6 GC cells enhanced cellular proliferation. Array-CGH analysis of 106 primary GCs revealed genomic loss of the miR-486 locus in approximately 25% to 30% of GCs, including two tumors with focal genomic losses specifically deleting miR-486, consistent with miR-486 playing a tumor-suppressive role. Bioinformatic analysis identified the secreted antiapoptotic glycoprotein OLFM4 as a potential miR-486 target. Restoring miR-486 expression in GC cells decreased endogenous OLFM4 transcript and protein levels, and also inhibited expression of luciferase reporters containing an OLFM4 3' untranslated region with predicted miR-486 binding sites. Supporting the biological relevance of OLFM4 as a miR-486 target, proliferation in GC cells was also significantly reduced by OLFM4 silencing. CONCLUSIONS: miR-486 may function as a novel tumor-suppressor miRNA in GC. Its antioncogenic activity may involve the direct targeting and inhibition of OLFM4.

Cancer-related ectopic expression of the bone-related transcription factor RUNX2 in non-osseous metastatic tumor cells is linked to cell proliferation and motility.

INTRODUCTION: Metastatic breast cancer cells frequently and ectopically express the transcription factor RUNX2, which normally attenuates proliferation and promotes maturation of osteoblasts. RUNX2 expression is inversely regulated with respect to cell growth in osteoblasts and deregulated in osteosarcoma cells. METHODS: Here, we addressed whether the functional relationship between cell growth and RUNX2 gene expression is maintained in breast cancer cells. We also investigated whether the aberrant expression of RUNX2 is linked to phenotypic parameters that could provide a selective advantage to cells during breast cancer progression. RESULTS: We find that, similar to its regulation in osteoblasts, RUNX2 expression in MDA-MB-231 breast adenocarcinoma cells is enhanced upon growth factor deprivation, as well as upon deactivation of the mitogen-dependent MEK-Erk pathway or EGFR signaling. Reduction of RUNX2 levels by RNAi has only marginal effects on cell growth and expression of proliferation markers in MDA-MB-231 breast cancer cells. Thus, RUNX2 is not a critical regulator of cell proliferation in this cell type. However, siRNA depletion of RUNX2 in MDA-MB-231 cells reduces cell motility, while forced exogenous expression of RUNX2 in MCF7 cells increases cell motility. CONCLUSIONS: Our results support the emerging concept that the osteogenic transcription factor RUNX2 functions as a metastasis-related oncoprotein in non-osseous cancer cells.

Gene regulation and tumor suppression by the bromodomain-containing protein BRD7.

Oncogene-induced senescence (OIS) is a cellular defense mechanism against excessive mitogenic signaling and tumorigenesis. One of the major pathways required for OIS is the p53 tumor suppressor pathway. Consequently, many human tumors harbor p53 mutations while others show a dysfunctional p53 pathway, frequently by unknown mechanisms. We recently identified BRD7 as a potential tumor suppressor gene acting as a transcriptional cofactor for p53, affecting histone acetylation, p53 acetylation, and promoter activity on a subset of p53 target genes. We further found low BRD7 expression specifically in a subgroup of human breast tumors harboring wild-type, but not mutant, p53 and showed that one of the responsible mechanisms is deletion of the BRD7 gene locus. Here we further discuss the role of BRD7 as a cofactor in transcriptional regulation and highlight its role as a tumor suppressor via association with p53 and other tumor suppressor proteins.

BRD7 is a candidate tumour suppressor gene required for p53 function.

Oncogene-induced senescence is a p53-dependent defence mechanism against uncontrolled proliferation. Consequently, many human tumours harbour p53 mutations and others show a dysfunctional p53 pathway, frequently by unknown mechanisms. Here we identify BRD7 (bromodomain-containing 7) as a protein whose inhibition allows full neoplastic transformation in the presence of wild-type p53. In human breast tumours harbouring wild-type, but not mutant, p53 the BRD7 gene locus was frequently deleted and low BRD7 expression was found in a subgroup of tumours. Functionally, BRD7 is required for efficient p53-mediated transcription of a subset of target genes. BRD7 interacts with p53 and p300 and is recruited to target gene promoters, affecting histone acetylation, p53 acetylation and promoter activity. Thus, BRD7 suppresses tumorigenicity by serving as a p53 cofactor required for the efficient induction of p53-dependent oncogene-induced senescence.

MicroRNAs: Oncogenes, tumor suppressors or master regulators of cancer heterogeneity?

The realization that microRNAs are intimately linked to cancer pathogenesis has spawned an explosion of research activity in recent years. Their presence is not merely predictive of tumor origin and behavior, they are causally linked to the emergence and development of cancer by acting as oncogenes or tumor suppressors. The understanding of the functional consequences of altered microRNA expression in cancer is progressing rapidly, even though the prediction of microRNA targets is still a hit and miss process. MicroRNAs may not act primarily by strongly reducing the expression of a few prominent cancer-regulatory genes, but by influencing the properties of the network of which these regulators are a central part. By coordinately regulating many genes, microRNAs are exquisitely suited to act as stabilizers of networks and to prevent extreme variations in phenotype due to intrinsic and extrinsic disturbances. Many advanced tumors show defects in microRNA expression and processing, which could increase phenotypic variability within tumors. This allows small subsets of cells with altered characteristics to emerge, which can have grave consequences as typically a small fraction of tumor cells is responsible for metastasis and treatment resistance, and ultimately treatment failure. Investigating microRNAs from the perspective of master regulators of network stability in cancer calls for new experimental approaches and may help to understand causes of cancer heterogeneity and disease progression.

RNA-binding protein Dnd1 inhibits microRNA access to target mRNA.

MicroRNAs (miRNAs) are inhibitors of gene expression capable of controlling processes in normal development and cancer. In mammals, miRNAs use a seed sequence of 6-8 nucleotides (nt) to associate with 3' untranslated regions (3'UTRs) of mRNAs and inhibit their expression. Intriguingly, occasionally not only the miRNA-targeting site but also sequences in its vicinity are highly conserved throughout evolution. We therefore hypothesized that conserved regions in mRNAs may serve as docking platforms for modulators of miRNA activity. Here we demonstrate that the expression of dead end 1 (Dnd1), an evolutionary conserved RNA-binding protein (RBP), counteracts the function of several miRNAs in human cells and in primordial germ cells of zebrafish by binding mRNAs and prohibiting miRNAs from associating with their target sites. These effects of Dnd1 are mediated through uridine-rich regions present in the miRNA-targeted mRNAs. Thus, our data unravel a novel role of Dnd1 in protecting certain mRNAs from miRNA-mediated repression.

A genetic screen implicates miRNA-372 and miRNA-373 as oncogenes in testicular germ cell tumors.

Endogenous small RNAs (miRNAs) regulate gene expression by mechanisms conserved across metazoans. While the number of verified human miRNAs is still expanding, only few have been functionally annotated. To perform genetic screens for novel functions of miRNAs, we developed a library of vectors expressing the majority of cloned human miRNAs and created corresponding DNA barcode arrays. In a screen for miRNAs that cooperate with oncogenes in cellular transformation, we identified miR-372 and miR-373, each permitting proliferation and tumorigenesis of primary human cells that harbor both oncogenic RAS and active wild-type p53. These miRNAs neutralize p53-mediated CDK inhibition, possibly through direct inhibition of the expression of the tumorsuppressor LATS2. We provide evidence that these miRNAs are potential novel oncogenes participating in the development of human testicular germ cell tumors by numbing the p53 pathway, thus allowing tumorigenic growth in the presence of wild-type p53.