Sachi Inukai

Novel microRNAs differentially expressed during aging in the mouse brain.

MicroRNAs (miRNAs) are endogenous small RNA molecules that regulate gene expression post-transcriptionally. Work in Caenorhabditis elegans has shown that specific miRNAs function in lifespan regulation and in a variety of age-associated pathways, but the roles of miRNAs in the aging of vertebrates are not well understood. We examined the expression of small RNAs in whole brains of young and old mice by deep sequencing and report here on the expression of 558 known miRNAs and identification of 41 novel miRNAs. Of these miRNAs, 75 known and 18 novel miRNAs exhibit greater than 2.0-fold expression changes. The majority of expressed miRNAs in our study decline in relative abundance in the aged brain, in agreement with trends observed in other miRNA studies in aging tissues and organisms. Target prediction analysis suggests that many of our novel aging-associated miRNAs target genes in the insulin signaling pathway, a central node of aging-associated genetic networks. These novel miRNAs may thereby regulate aging-related functions in the brain. Since many mouse miRNAs are conserved in humans, the aging-affected brain miRNAs we report here may represent novel regulatory genes that also function during aging in the human brain.

Age-associated changes in expression of small, noncoding RNAs, including microRNAs, in C. elegans

Small, noncoding RNAs (sncRNAs), including microRNAs (miRNAs), impact diverse biological events through the control of gene expression and genome stability. However, the role of these sncRNAs in aging remains largely unknown. To understand the contribution of sncRNAs to the aging process, we performed small RNA profiling by deep-sequencing over the course of Caenorhabditis elegans (C. elegans) aging. Many small RNAs, including a significant number of miRNAs, change their expression during aging in C. elegans. Further studies of miRNA expression changes under conditions that modify lifespan demonstrate the tight control of their expression during aging. Adult-specific loss of argonaute-like gene-1 (alg-1) activity, which is necessary for miRNA maturation and function, resulted in an abnormal lifespan, suggesting that miRNAs are, indeed, required in adulthood for normal aging. miRNA target prediction algorithms combined with transcriptome data and pathway enrichment analysis revealed likely targets of these age-associated miRNAs with known roles in aging, such as mitochondrial metabolism. Furthermore, a computational analysis of our deep-sequencing data identified additional age-associated sncRNAs, including miRNA star strands, novel miRNA candidates, and endo-siRNA sequences. We also show an increase of specific transfer RNA (tRNA) fragments during aging, which are known to be induced in response to stress in several organisms. This study suggests that sncRNAs including miRNAs contribute to lifespan regulation in C. elegans, and indicates new connections between aging, stress responses, and the small RNA world.

miR-34a Silences c-SRC to Attenuate Tumor Growth in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is an aggressive subtype with no clinically proven biologically targeted treatment options. The molecular heterogeneity of TNBC and lack of high frequency driver mutations other than TP53 have hindered the development of new and effective therapies that significantly improve patient outcomes. miRNAs, global regulators of survival and proliferation pathways important in tumor development and maintenance, are becoming promising therapeutic agents. We performed miRNA-profiling studies in different TNBC subtypes to identify miRNAs that significantly contribute to disease progression. We found that miR-34a was lost in TNBC, specifically within mesenchymal and mesenchymal stem cell-like subtypes, whereas expression of miR-34a targets was significantly enriched. Furthermore, restoration of miR-34a in cell lines representing these subtypes inhibited proliferation and invasion, activated senescence, and promoted sensitivity to dasatinib by targeting the proto-oncogene c-SRC. Notably, SRC depletion in TNBC cell lines phenocopied the effects of miR-34a reintroduction, whereas SRC overexpression rescued the antitumorigenic properties mediated by miR-34a. miR-34a levels also increased when cells were treated with c-SRC inhibitors, suggesting a negative feedback exists between miR-34a and c-SRC. Moreover, miR-34a administration significantly delayed tumor growth of subcutaneously and orthotopically implanted tumors in nude mice, and was accompanied by c-SRC downregulation. Finally, we found that miR-34a and SRC levels were inversely correlated in human tumor specimens. Together, our results demonstrate that miR-34a exerts potent antitumorigenic effects in vitro and in vivo and suggests that miR-34a replacement therapy, which is currently being tested in human clinical trials, represents a promising therapeutic strategy for TNBC.

MicroRNAs mediate dietary-restriction-induced longevity through PHA-4/FOXA and SKN-1/Nrf transcription factors

BACKGROUND Dietary restriction (DR) has been shown to prolong longevity across diverse taxa, yet the mechanistic relationship between DR and longevity remains unclear. MicroRNAs (miRNAs) control aging-related functions such as metabolism and lifespan through regulation of genes in insulin signaling, mitochondrial respiration, and protein homeostasis. RESULTS We have conducted a network analysis of aging-associated miRNAs connected to transcription factors PHA-4/FOXA and SKN-1/Nrf, which are both necessary for DR-induced lifespan extension in Caenorhabditis elegans. Our network analysis has revealed extensive regulatory interactions between PHA-4, SKN-1, and miRNAs and points to two aging-associated miRNAs, miR-71 and miR-228, as key nodes of this network. We show that miR-71 and miR-228 are critical for the response to DR in C. elegans. DR induces the expression of miR-71 and miR-228, and the regulation of these miRNAs depends on PHA-4 and SKN-1. In turn, we show that PHA-4 and SKN-1 are negatively regulated by miR-228, whereas miR-71 represses PHA-4. CONCLUSIONS Based on our findings, we have discovered new links in an important pathway connecting DR to aging. By interacting with PHA-4 and SKN-1, miRNAs transduce the effect of dietary-restriction-mediated lifespan extension in C. elegans. Given the conservation of miRNAs, PHA-4, and SKN-1 across phylogeny, these interactions are likely to be conserved in more-complex species.

MiR-33 connects cholesterol to the cell cycle.

Comment on: Cirera-Salinas D, et al. Cell Cycle 2012; 11:922–33

A microRNA feedback loop regulates global microRNA abundance during aging

Expression levels of many microRNAs (miRNAs) change during aging, notably declining globally in a number of organisms and tissues across taxa. However, little is known about the mechanisms or the biological relevance for this change. We investigated the network of genes that controls miRNA transcription and processing during C. elegans aging. We found that miRNA biogenesis genes are highly networked with transcription factors and aging-associated miRNAs. In particular, miR-71, known to influence life span and itself up-regulated during aging, represses alg-1/Argonaute expression post-transcriptionally during aging. Increased ALG-1 abundance in mir-71 loss-of-function mutants led to globally increased miRNA expression. Interestingly, these mutants demonstrated widespread mRNA expression dysregulation and diminished levels of variability both in gene expression and in overall life span. Thus, the progressive molecular decline often thought to be the result of accumulated damage over an organisms life may be partially explained by a miRNA-directed mechanism of age-associated decline.

Abstract LB-300: Reintroduction of tumor-suppressor miR-34a shows therapeutic efficacy in triple-negative breast cancer

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Triple-negative breast cancer (TNBC) accounts for a disproportionate share of the total breast cancer morbidity because of its aggressive behavior and lack of effective targeted therapies to treat the disease. MicroRNAs, global regulators of survival and proliferation pathways important in tumor development and maintenance, are highly dysregulated in cancer. We identified miR-34a to be aberrantly lost in TNBC lines when compared to both a luminal cancer subtype as well as normal breast cells. Re-introduction of miR-34a in TNBC lines results in inhibition of cell proliferation and invasion, reactivation of senescence, and enhanced sensitivity to apoptosis inducing agents. Furthermore, intratumoral delivery of miR-34a into subcutaneous tumors in nude mice, as well as systemic delivery of poly(amine-co-ester) PACE-loaded miR-34a in an orthotopic setting, delayed tumor growth. In conclusion, re-introduction of miR-34a in TNBC promotes potent anti-tumorigenic phenotypes in vitro and in vivo, and could be a promising targeted therapeutic agent to treat the disease. Citation Format: Brian D. Adams, Vikram Wali, Chris Cheng, Sachi Inukai, David Rimm, Lajos Pusztai, Mark Saltzman, Frank Slack. Reintroduction of tumor-suppressor miR-34a shows therapeutic efficacy in triple-negative breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-300. doi:10.1158/1538-7445.AM2015-LB-300