Hyun Yong Jin

MicroRNAs of the miR-17∼92 family are critical regulators of T(FH) differentiation.

Follicular helper T cells (TFH cells) provide critical help to B cells during humoral immune responses. Here we report that mice with T cell–specific deletion of the miR-17∼92 family of microRNAs (miRNAs) had substantially compromised TFH differentiation, germinal-center formation and antibody responses and failed to control chronic viral infection. Conversely, mice with T cell–specific expression of a transgene encoding miR-17∼92 spontaneously accumulated TFH cells and developed a fatal immunopathology. Mechanistically, the miR-17∼92 family controlled the migration of CD4+ T cells into B cell follicles by regulating signaling intensity from the inducible costimulator ICOS and kinase PI(3)K by suppressing expression of the phosphatase PHLPP2. Our findings demonstrate an essential role for the miR-17∼92 family in TFH differentiation and establish PHLPP2 as an important mediator of their function in this process.

MicroRNA-17∼92 plays a causative role in lymphomagenesis by coordinating multiple oncogenic pathways

MicroRNAs (miRNAs) have been broadly implicated in cancer, but their exact function and mechanism in carcinogenesis remain poorly understood. Elevated miR‐17∼92 expression is frequently found in human cancers, mainly due to gene amplification and Myc‐mediated transcriptional upregulation. Here we show that B cell‐specific miR‐17∼92 transgenic mice developed lymphomas with high penetrance and that, conversely, Myc‐driven lymphomagenesis stringently requires two intact alleles of miR‐17∼92. We experimentally identified miR‐17∼92 target genes by PAR‐CLIP and validated select target genes in miR‐17∼92 transgenic mice. These analyses demonstrate that miR‐17∼92 drives lymphomagenesis by suppressing the expression of multiple negative regulators of the PI3K and NFκB pathways and by inhibiting the mitochondrial apoptosis pathway. Accordingly, miR‐17∼92‐driven lymphoma cells exhibited constitutive activation of the PI3K and NFκB pathways and chemical inhibition of either pathway reduced tumour size and prolonged the survival of lymphoma‐bearing mice. These findings establish miR‐17∼92 as a powerful cancer driver that coordinates the activation of multiple oncogenic pathways, and demonstrate for the first time that chemical inhibition of miRNA downstream pathways has therapeutic value in treating cancers caused by miRNA dysregulation.

The MicroRNA-183-96-182 Cluster Promotes T Helper 17 Cell Pathogenicity by Negatively Regulating Transcription Factor Foxo1 Expression.

T helper 17 (Th17) cells are key players in autoimmune diseases. However, the roles of non-coding RNAs in Th17 cell development and function are largely unknown. We found that deletion of the endoribonuclease-encoding Dicer1 specifically in Th17 cells protected mice from experimental autoimmune encephalomyelitis. We found that the Dicer1-regulated microRNA (miR)-183-96-182 cluster (miR-183C) was highly expressed in Th17 cells and was induced by cytokine IL-6-STAT3 signaling. miR-183C expression enhanced pathogenic cytokine production from Th17 cells during their development and promoted autoimmunity. Mechanistically, miR-183C in Th17 cells directly repressed expression of the transcription factor Foxo1. Foxo1 negatively regulated the pathogenicity of Th17 cells in part by inhibiting expression of cytokine receptor IL-1R1. These findings indicate that the miR-183C drives Th17 pathogenicity in autoimmune diseases via inhibition of Foxo1 and present promising therapeutic targets.

Transfection of microRNA Mimics Should Be Used with Caution

Transient transfection of chemically synthesized microRNA (miRNA) mimics is being used extensively to study the functions and mechanisms of endogenous miRNAs. However, it remains unclear whether transfected miRNAs behave similarly to endogenous miRNAs. Here we show that transient transfection of miRNA mimics into HeLa cells by a commonly used method led to the accumulation of high molecular weight RNA species and a few hundred fold increase in mature miRNA levels. In contrast, expression of the same miRNAs through lentiviral infection or plasmid transfection of HeLa cells, transgenic expression in primary lymphocytes, and endogenous overexpression in lymphoma and leukemia cell lines did not lead to the appearance of high molecular weight RNA species. The increase of mature miRNA levels in these cells was below 10-fold, which was sufficient to suppress target gene expression and to drive lymphoma development in mice. Moreover, transient transfection of miRNA mimics at high concentrations caused non-specific alterations in gene expression, while at low concentrations achieved expression levels comparable to other methods but failed to efficiently suppress target gene expression. Small RNA deep sequencing analysis revealed that the guide strands of miRNA mimics were frequently mutated, while unnatural passenger strands of some miRNA mimics accumulated to high levels. The high molecular weight RNA species were a heterogeneous mixture of several classes of RNA species generated by concatemerization, 5′- and 3′-end tailing of miRNA mimics. We speculate that the supraphysiological levels of mature miRNAs and these artifactual RNA species led to non-specific changes in gene expression. Our results have important implications for the design and interpretation of experiments primarily employing transient transfection of miRNA mimics.

Regulation of B-cell development and tolerance by different members of the miR-17 ∼ 92 family microRNAs

The molecular mechanisms that regulate B-cell development and tolerance remain incompletely understood. In this study, we identify a critical role for the miR-17∼92 microRNA cluster in regulating B-cell central tolerance and demonstrate that these miRNAs control early B-cell development in a cell-intrinsic manner. While the cluster member miR-19 suppresses the expression of Pten and plays a key role in regulating B-cell tolerance, miR-17 controls early B-cell development through other molecular pathways. These findings demonstrate differential control of two closely linked B-cell developmental stages by different members of a single microRNA cluster through distinct molecular pathways.

A miR-155–Peli1–c-Rel pathway controls the generation and function of T follicular helper cells

Xiao and collaborators show that miR-155 regulates T follicular helper cell development and function by suppressing the E3 ubiquitin ligase Peli1.

MicroRNA Mechanisms of Action: What have We Learned from Mice?

MicroRNAs (miRNAs) are endogenously encoded single-stranded RNAs of about 22 nucleotides (nts) in length that play essential roles in a large variety of physiological processes in animals and plants (Ambros, 2004; Bushati and Cohen, 2007). Mature miRNAs are integrated into the RNA-induced silencing complex (RISC), whose core component is one of the Argonaute family proteins. MiRNAs then direct RISCs to target mRNAs, which are recognized through partial sequence complementarity. Bioinformatic prediction and experimental target gene identification have shown that a miRNA binds mRNAs of hundreds of protein coding genes, which often span a broad spectrum of functional categories (Bartel, 2009; Chi et al., 2009; Hafner et al., 2010). The functional consequence of miRNA-target mRNA interaction and the mechanism of miRNA action have been under intensive investigation and remain a matter of hot debate. It was initially thought that miRNAs repress the protein output of a small number of target genes without significantly affecting their mRNA levels in animals (Lee et al., 1993; Wightman et al., 1993). Subsequent genetic studies in C. elegans and zebrafish showed that miRNAs promote the degradation of their target mRNAs (Bagga et al., 2005; Giraldez et al., 2006). Later, a series of genome-wide studies of in vitro cultured mammalian cell lines transiently transfected with chemically synthesized miRNA mimics led to the conclusion that the predominant functional consequence of miRNA action is target mRNA degradation (Guo et al., 2010). A follow-up study employing temporal dissection of zebrafish development seems to reconcile these two opposite observations by revealing that translational repression precedes target mRNA decay, and suggesting that the immediate outcome of miRNA-target mRNA interaction is translation inhibition but mRNA degradation can follow (Bazzini et al., 2012). Similarly, re-analysis of the previous datasets from cultured cell lines transiently transfected with synthetic miRNA mimics also found that translation repression precedes mRNA degradation (Larsson and Nadon, 2013).

Differential Sensitivity of Target Genes to Translational Repression by miR-17~92

MicroRNAs (miRNAs) are thought to exert their functions by modulating the expression of hundreds of target genes and each to a small degree, but it remains unclear how small changes in hundreds of target genes are translated into the specific function of a miRNA. Here, we conducted an integrated analysis of transcriptome and translatome of primary B cells from mutant mice expressing miR-17~92 at three different levels to address this issue. We found that target genes exhibit differential sensitivity to miRNA suppression and that only a small fraction of target genes are actually suppressed by a given concentration of miRNA under physiological conditions. Transgenic expression and deletion of the same miRNA gene regulate largely distinct sets of target genes. miR-17~92 controls target gene expression mainly through translational repression and 5’UTR plays an important role in regulating target gene sensitivity to miRNA suppression. These findings provide molecular insights into a model in which miRNAs exert their specific functions through a small number of key target genes.

microRNA-17~92 is a powerful cancer driver and a therapeutic target

microRNAs (miRNAs) are a class of small non-coding RNAs of ~22 nucleotides in length that bind to their target mRNAs (mRNAs) and regulate their expression by translation repression and mRNA degradation. Over the past decade, several miRNA genes have been implicated in human cancers, but their exact function and underlying molecular mechanism in carcinogenesis remain poorly understood. This is a key obstacle in the development of miRNA-based cancer therapeutics. miR-17~92, a cluster of 6 miRNAs (miR-17, miR-18a, miR-19a, miR-20a, miR-19b, and miR-92), was the first miRNA gene implicated in cancer. Its overexpression occurs in a broad spectrum of human cancers, including lymphoma, leukemia, and solid tissue cancers. miR17~92 overexpression is mainly driven by gene amplification at the human chromosome 13q31 region, which harbors the miR-17~92 gene (termed MIR17HG), and by Myc-mediated transcriptional upregulation. A recent RNA sequencing analysis of patient biopsies showed that miR-17~92 expression was upregulated by 2~36-fold in 14 out of 79 (18%) cases of diffuse large B-cell lymphomas and in all the 28 cases of Burkitt lymphomas analyzed. As Myc overexpression is the defining feature of Burkitt lymphoma, these results confirmed that the Myc-miR-17~92 axis is operating in all patients with this malignancy. Previous studies showed that miR17~92 overexpression contributes to carcinogenesis. Thus, retroviral overexpression of miR-17~92 accelerated Myc-driven lymphomagenesis and Notch-driven leukemogenesis, while a miR-17~92 transgene promoted retinoblastoma initiated by inactivation of the Rb pathway. Another study showed that miR-17~92 played important roles in sustaining the optimal growth of Myc-driven lymphoma cell lines in tissue culture and in immunodeficient host. However, it was unclear whether elevated miR-17~92 expression, per se, is sufficient to drive carcinogenesis, and exactly what role miR-17~92 plays in Myc-driven carcinogenesis. A recent study from our lab provided some answers to these important questions. First, we created transgenic mice specifically overexpressing miR-17~92 in B cells. Those mice developed B-cell lymphomas with high penetrance, establishing miR-17~92 as a powerful cancer driver and validating its encoded miRNAs and downstream pathways as therapeutic targets (see below). Second, we used CD19-Cre to conditionally delete miR17~92 in B cells of λ-Myc mice, a commonly used model of Burkitt lymphoma. λ-Myc;CD19-Cre;miR-17~92 l mice exhibited a delay in lymphomagenesis. Strikingly, all lymphomas arising from those mice contained 2 intact miR-17~92 alleles. They escaped CD19-Cre-mediated deletion of miR-17~92 by restricting Myc-driven malignant transformation in CD19-negative early B-cell precursors or in a small fraction of CD19-positive cells with low Cre expression. These results show that Myc-driven lymphomagenesis stringently requires 2 intact alleles of miR17~92. This, together with a recent study demonstrating that miR-17~92 is essential for the development of retinoblastoma driven by the double deletion of Rb and p53, prompts us to hypothesize that the requirement of miR-17~92 is a common denominator of cancers. As miR-17~92 overexpression is found in many human cancers, future studies are warranted to investigate the effect of miR-17~92 deletion on carcinogenesis in other mouse models. We next explored the molecular mechanisms through which miR-17~92 drives lymphomagenesis. We performed PAR– CLIP analysis of human B cells, identified 868 protein coding genes containing miR17~92-binding sites that are conserved between human and mouse, and validated a select group of target genes in miR-17~92 transgenic B cells. Our analyses showed that miR-17~92 suppresses the expression of multiple inhibitors of the PI3K (Pten and Phlpp2) and NFκB (Cyld, A20, Itch, Rnf11, and Tax1BP1) pathways, and that both pathways are constitutively active in miR-17~92-driven lymphoma cells. Furthermore, chemical inhibition of either pathway significantly controlled tumor growth and prolonged survival of mice bearing miR-17~92-driven lymphomas. These results suggest that activation of the PI3K and NFκB pathways plays important roles in both the development and maintenance of miR-17~92-driven lymphomas, and that dual targeting of PI3K and NFκB pathways is a potential strategy to treat miR-17~92-driven lymphomas (Fig. 1). An alternative strategy to treat miR17~92-driven lymphomas is to sequester or knockdown miR-17~92 miRNAs directly. As miR-17~92 encodes for 6 distinct miRNAs, which may cooperate or even antagonize each other in driving lymphomagenesis, it will be essential to

Registered report: BET bromodomain inhibition as a therapeutic strategy to target c-Myc

The Reproducibility Project: Cancer Biology seeks to address growing concerns about reproducibility in scientific research by replicating selected results from a substantial number of high-profile papers in the field of cancer biology published between 2010 and 2012. This Registered report describes the proposed replication plan of key experiments from ‘BET bromodomain inhibition as a therapeutic strategy to target c-Myc’ by Delmore and colleagues, published in Cell in 2011 (Delmore et al., 2011). The key experiments that will be replicated are those reported in Figures 3B and 7C-E. Delmore and colleagues demonstrated that treatment with JQ1, a small molecular inhibitor targeting BET bromodomains, resulted in the transcriptional down-regulation of the c-Myc oncogene in vitro (Figure 3B; Delmore et al., 2011). To assess the therapeutic efficacy of JQ1 in vivo, mice bearing multiple myeloma (MM) lesions were treated with JQ1 before evaluation for tumor burden and overall survival. JQ1 treatment significantly reduced disease burden and increased survival time (Figure 7C-E; Delmore et al., 2011). The Reproducibility Project: Cancer Biology is a collaboration between the Center for Open Science and Science Exchange and the results of the replications will be published in eLife. DOI: http://dx.doi.org/10.7554/eLife.07072.001