T follicular helper (T FH ) cells provide critical help to B cells during humoral immune responses.Here we report that mice with T cell-specific deletion of miR-17~92 family miRNAs (tKO mice) exhibited severely compromised T FH differentiation, germinal center formation, antibody responses, and failed to control chronic virus infection. Conversely, T cell-specific miR-17~92 transgenic mice spontaneously accumulated T FH cells and developed fatal immunopathology. Mechanistically, miR-17~92 family miRNAs control CD4 + T cell migration into B cell follicles by regulating ICOS-PI3K signaling intensity through suppressing the expression of the Akt phosphatase Phlpp2. These findings demonstrate that miR-17~92 family microRNAs play an essential role in T FH differentiation and establish Phlpp2 as an important mediator of their function in this process.MicroRNAs (miRNAs) are endogenously encoded small RNAs of ~22 nucleotides in length that play important roles in a large diversity of biological processes 1,2,3 .
Cancer cells develop mechanisms to escape immunosurveillance, among which modulating the expression of immune suppressive messenger RNAs is most well-documented. However, how this is molecularly achieved remains largely unresolved. Here, we develop an in vivo mouse model of liver cancer to study oncogene cooperation in immunosurveillance. We show that MYC overexpression (MYCTg) synergizes with KRASG12D to induce an aggressive liver tumor leading to metastasis formation and reduced mouse survival compared with KRASG12D alone. Genome-wide ribosomal footprinting of MYCTg;KRASG12 tumors compared with KRASG12D revealed potential alterations in translation of mRNAs, including programmed-death-ligand 1 (PD-L1). Further analysis revealed that PD-L1 translation is repressed in KRASG12D tumors by functional, non-canonical upstream open reading frames in its 5′ untranslated region, which is bypassed in MYCTg;KRASG12D tumors to evade immune attack. We show that this mechanism of PD-L1 translational upregulation was effectively targeted by a potent, clinical compound that inhibits eIF4E phosphorylation, eFT508, which reverses the aggressive and metastatic characteristics of MYCT9;KRASG12D tumors. Together, these studies reveal how immune-checkpoint proteins are manipulated by distinct oncogenes at the level of mRNA translation, which can be exploited for new immunotherapies.
Summary 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 Dicer1 gene 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 cytokine receptor IL-1R1 expression. These findings indicate that the miR-183C drives Th17 pathogenicity in autoimmune diseases via inhibition of Foxo1 and present promising therapeutic targets.
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.
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.
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.
Xiao and collaborators show that miR-155 regulates T follicular helper cell development and function by suppressing the E3 ubiquitin ligase Peli1.
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