Virally encoded microRNAs (miRNAs) have recently been discovered in herpesviruses. However, their biological roles are mostly unknown. We developed an algorithm for the prediction of miRNA targets and applied it to human cytomegalovirus miRNAs, resulting in the identification of the major histocompatibility complex class I-related chain B (MICB) gene as a top candidate target of hcmv-miR-UL112. MICB is a stress-induced ligand of the natural killer (NK) cell activating receptor NKG2D and is critical for the NK cell killing of virus-infected cells and tumor cells. We show that hcmv-miR-UL112 specifically down-regulates MICB expression during viral infection, leading to decreased binding of NKG2D and reduced killing by NK cells. Our results reveal a miRNA-based immunoevasion mechanism that appears to be exploited by human cytomegalovirus.MiRNAs constitute a large family of small noncoding RNAs that regulate gene expression posttranscriptionally, affecting mRNA degradation and translation by base-pairing with the 3′ untranslated regions (3′UTRs) (1). The recent discovery of virally encoded miRNAs, mostly in herpesviruses, intriguingly suggests that miRNAs may function in interspecies Copyright © 2007 We applied RepTar and subsequently cRepTar to all human 3′UTRs, searching for potential binding sites of the 11 HCMV miRNAs listed in miRBase 7.0 (9). MICB, an immunorelated gene, was among the highest ranking predicted targets and the top prediction for hcmv-miR-UL112 (Fig. 1A). MICB is a stress-induced ligand of NKG2D, a natural killer (NK) activating receptor expressed on almost all human NK cells and activated cytotoxic T lymphocytes (CTLs) (10). The importance of MICB in the immune response against HCMV infection is substantiated by the specific down-regulation of MICB surface expression via the UL16 protein of HCMV (11,12). MICA, another stress-induced ligand of NKG2D, was also ranked among the top predicted targets of hcmv-miR-UL112 (Fig. 1A). The hcmv-miR-UL112 putative binding sites of both genes are almost identical and are located within a highly similar but not evolutionarily conserved (7) 150-nucleotide (nt) region of their 3′UTRs.To assess the function of hcmv-miR-UL112, we expressed this miRNA in various human tumor cell lines that endogenously express MICA and MICB with the use of recombinant lentiviral vectors: hcmv-miR-UL112 and two control vectors, a non-miRNA sequence (miRcontrol) and hcmv-miR-US5-1. The expression of hcmv-miR-UL112 was confirmed by quantative real-time polymerase chain reaction (qPCR) ( fig. S1). The vectors contained green fluorescent protein (GFP) for monitoring the infection efficiency (7). No difference in the transduction efficiency of the different lentiviral vectors was measured ( fig. S2). Analysis of the various tumor cells transduced with hcmv-miR-UL112 revealed a specific and extensive reduction of MICB and little or no reduction of MICA (Fig. 1B). The downregulation was specific to MICB and to hcmv-miR-UL112, because no change in the level of major histocompati...

Highlights d Cancer-associated fibroblasts contribute to pancreatic cancer heterogeneity d Cancer cells can have a double-positive phenotype: proliferation and invasion d High CAF abundance linked with DP cells enriched for MAPK and STAT3 co-signaling d Intra-tumoral gland types provide tissue heterogeneity linked with clinical outcome

MICA and MICB are stress-induced ligands recognized by the activating receptor NKG2D. A microRNA encoded by human cytomegalovirus downregulates MICB expression by targeting a specific site in the MICB 3' untranslated region. As this site is conserved among different MICB alleles and a similar site exists in the MICA 3' untranslated region, we speculated that these sites are targeted by cellular microRNAs. Here we identified microRNAs that bound to these MICA and MICB 3' untranslated region sequences and obtained data suggesting that these microRNAs maintain expression of MICA and MICB protein under a certain threshold and facilitate acute upregulation of MICA and MICB during cellular stress. These microRNAs were overexpressed in various tumors and we demonstrate here that they aided tumor avoidance of immune recognition.

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Elevated concentrations of tumor necrosis factor-α (TNF-α) are detected in pathologies characterized by chronic inflammation. Whether TNF-α plays a role in manipulating the host's immune system toward generating an immunosuppressive milieu, typical of ongoing chronic inflammation, is unclear. Here we showed that TNF-α exhibited a dual function during chronic inflammation: arresting differentiation of immature myeloid-derived suppressor cells (MDSCs) primarily via the S100A8 and S100A9 inflammatory proteins and their corresponding receptor (RAGE) and augmenting MDSC suppressive activity. These functions led to in vivo T and NK cell dysfunction accompanied by T cell antigen receptor ζ chain downregulation. Furthermore, administration of etanercept (TNF-α antagonist) during early chronic inflammatory stages reduced MDSCs' suppressive activity and enhanced their maturation into dendritic cells and macrophages, resulting in the restoration of in vivo immune functions and recovery of ζ chain expression. Thus, TNF has a fundamental role in promoting an immunosuppressive environment generated during chronic inflammation.

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Cellular senescence is thought to contribute to age-associated deterioration of tissue physiology. The senescence effector p16Ink4a is expressed in pancreatic beta cells during aging and limits their proliferative potential; however, its effects on beta cell function are poorly characterized. We found that beta cell-specific activation of p16Ink4a in transgenic mice enhances glucose-stimulated insulin secretion (GSIS). In mice with diabetes, this leads to improved glucose homeostasis, providing an unexpected functional benefit. Expression of p16Ink4a in beta cells induces hallmarks of senescence—including cell enlargement, and greater glucose uptake and mitochondrial activity—which promote increased insulin secretion. GSIS increases during the normal aging of mice and is driven by elevated p16Ink4a activity. We found that islets from human adults contain p16Ink4a-expressing senescent beta cells and that senescence induced by p16Ink4a in a human beta cell line increases insulin secretion in a manner dependent, in part, on the activity of the mechanistic target of rapamycin (mTOR) and the peroxisome proliferator-activated receptor (PPAR)-γ proteins. Our findings reveal a novel role for p16Ink4a and cellular senescence in promoting insulin secretion by beta cells and in regulating normal functional tissue maturation with age.

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