Germline Genetic Variants Disturbing the Let-7/LIN28 Double-Negative Feedback Loop Alter Breast Cancer Susceptibility

Previous studies have shown that let-7 can repress the post-transcriptional translation of LIN28, and LIN28 in turn could block the maturation of let-7, forming a double-negative feedback loop. In this study, we investigated the effect of germline genetic variants on regulation of the homeostasis of the let-7/LIN28 loop and breast cancer risk. We initially demonstrated that the T/C variants of rs3811463, a single nucleotide polymorphism (SNP) located near the let-7 binding site in LIN28, could lead to differential regulation of LIN28 by let-7. Specifically, the C allele of rs3811463 weakened let-7–induced repression of LIN28 mRNA, resulting in increased production of LIN28 protein, which could in turn down-regulate the level of mature let-7. This effect was then validated at the tissue level in that the normal breast tissue of individuals with the rs3811463-TC genotype expressed significantly lower levels of let-7 and higher levels of LIN28 protein than those individuals with the rs3811463-TT genotype. Because previous in vitro and ex vivo experiments have consistently suggested that LIN28 could promote cellular transformation, we then systematically evaluated the relationship between rs3811463 as well as other common LIN28 SNPs and the risk of breast cancer in a stepwise manner. The first hospital-based association study (n = 2,300) demonstrated that two SNPs were significantly associated with breast cancer risk, one of which was rs3811463, while the other was rs6697410. The C allele of the rs3811463 SNP corresponded to an increased risk of breast cancer with an odds ratio (OR) of 1.25 (P = 0.0091), which was successfully replicated in a second independent study (n = 1,156) with community-based controls. The combined P-value of the two studies was 8.0×10−5. Taken together, our study demonstrates that host genetic variants could disturb the regulation of the let-7/LIN28 double-negative feedback loop and alter breast cancer risk

Circadian Consequence of Socio-Sexual Interactions in Fruit Flies Drosophila melanogaster

In fruit flies Drosophila melanogaster, courtship is an elaborate ritual comprising chasing, dancing and singing by males to lure females for mating. Courtship interactions peak in the night and heterosexual couples display enhanced nighttime activity. What we do not know is if such socio-sexual interactions (SSI) leave long-lasting after-effects on circadian clock(s). Here we report the results of our study aimed at examining the after-effects of SSI (as a result of co-habitation of males and females in groups) between males and females on their circadian locomotor activity rhythm. Males undergo reduction in the evening activity peak and lengthening of circadian period, while females show a decrease in overall activity. Such after-effects, at least in males, require functional circadian clocks during SSI as loss-of-function clock mutants and wild type flies interacting under continuous light (LL), do not display them. Interestingly, males with electrically silenced Pigment Dispersing Factor (PDF)-positive ventral lateral (LNv) clock neurons continue to show SSI mediated reduction in evening activity peak, suggesting that the LNv clock neurons are dispensable for SSI mediated after-effects on locomotor activity rhythm. Such after-effects in females may not be clock-dependent because clock manipulated females with prior exposure to males show decrease in overall activity, more or less similar to rhythmic wild type females. The expression of SSI mediated after-effects requires a functional olfactory system in males because males with compromised olfactory ability do not display them. These results suggest that SSI causes male-specific, long-lasting changes in the circadian clocks of Drosophila, which requires the presence of functional clocks and intact olfactory ability in males

MicroRNA let-7c Is Downregulated in Prostate Cancer and Suppresses Prostate Cancer Growth

Prostate cancer (PCa) is characterized by deregulated expression of several tumor suppressor or oncogenic miRNAs. The objective of this study was the identification and characterization of miR-let-7c as a potential tumor suppressor in PCa.Levels of expression of miR-let-7c were examined in human PCa cell lines and tissues using qRT-PCR and in situ hybridization. Let-7c was overexpressed or suppressed to assess the effects on the growth of human PCa cell lines. Lentiviral-mediated re-expression of let-7c was utilized to assess the effects on human PCa xenografts.We identified miR-let-7c as a potential tumor suppressor in PCa. Expression of let-7c is downregulated in castration-resistant prostate cancer (CRPC) cells. Overexpression of let-7c decreased while downregulation of let-7c increased cell proliferation, clonogenicity and anchorage-independent growth of PCa cells in vitro. Suppression of let-7c expression enhanced the ability of androgen-sensitive PCa cells to grow in androgen-deprived conditions in vitro. Reconstitution of Let-7c by lentiviral-mediated intratumoral delivery significantly reduced tumor burden in xenografts of human PCa cells. Furthermore, let-7c expression is downregulated in clinical PCa specimens compared to their matched benign tissues, while the expression of Lin28, a master regulator of let-7 miRNA processing, is upregulated in clinical PCa specimens.These results demonstrate that microRNA let-7c is downregulated in PCa and functions as a tumor suppressor, and is a potential therapeutic target for PCa

Involving people with diabetes and the wider community in diabetes research: a realist review protocol.

BACKGROUND: Patient and public involvement in diabetes research is now actively encouraged in different countries because it is believed that involving people with experience of the condition will improve the quality and relevance of the research. However, reviews of patient involvement have noted that inadequate resources, patients' and communities' lack of research knowledge, and researchers' lack of skills to involve patients and communities in research may present significant contextual barriers. Little is known about the extent of patient/community involvement in designing or delivering interventions for people with diabetes. A realist review of involvement will contribute to assessing when, how and why involvement works, or does not work, to produce better diabetes interventions. METHODS/DESIGN: This protocol outlines the process for conducting a realist review to map how patients and the public have been involved in diabetes research to date. The review questions ask the following: How have people with diabetes and the wider community been involved in diabetes research? What are the characteristics of the process that appear to explain the relative success or failure of involvement? How has involvement (or lack of involvement) in diabetes research influenced the development and conduct of diabetes research? The degree of support in the surrounding context will be assessed alongside the ways in which people interact in different settings to identify patterns of interaction between context, mechanisms and outcomes in different research projects. The level and extent of the involvement will be described for each stage of the research project. The descriptions will be critically reviewed by the people with diabetes on our review team. In addition, researchers and patients in diabetes research will be asked to comment. Information from researcher-patient experiences and documents will be compared to theories of involvement across a range of disciplines to create a mid-range theory describing how involvement (or lack of involvement) in diabetes research influences the development and conduct of diabetes research

Phenotypic Variation and Bistable Switching in Bacteria

Microbial research generally focuses on clonal populations. However, bacterial cells with identical genotypes frequently display different phenotypes under identical conditions. This microbial cell individuality is receiving increasing attention in the literature because of its impact on cellular differentiation, survival under selective conditions, and the interaction of pathogens with their hosts. It is becoming clear that stochasticity in gene expression in conjunction with the architecture of the gene network that underlies the cellular processes can generate phenotypic variation. An important regulatory mechanism is the so-called positive feedback, in which a system reinforces its own response, for instance by stimulating the production of an activator. Bistability is an interesting and relevant phenomenon, in which two distinct subpopulations of cells showing discrete levels of gene expression coexist in a single culture. In this chapter, we address techniques and approaches used to establish phenotypic variation, and relate three well-characterized examples of bistability to the molecular mechanisms that govern these processes, with a focus on positive feedback.

Widespread translational control of fibrosis in the human heart by RNA-binding proteins

BACKGROUND: Fibrosis is a common pathology in many cardiac disorders and is driven by the activation of resident fibroblasts. The global post-transcriptional mechanisms underlying fibroblast-to-myofibroblast conversion in the heart have not been explored. METHODS: Genome-wide changes of RNA transcription and translation during human cardiac fibroblast activation were monitored with RNA sequencing and ribosome profiling. We then used an RNA-binding protein-based analyses to identify translational regulators of fibrogenic genes. The integration with cardiac ribosome occupancy levels of 30 dilated cardiomyopathy patients demonstrates that these post-transcriptional mechanisms are also active in the diseased fibrotic human heart. RESULTS: We generated nucleotide-resolution translatome data during the TGFβ1-driven cellular transition of human cardiac fibroblasts to myofibroblasts. This identified dynamic changes of RNA transcription and translation at several time points during the fibrotic response, revealing transient and early-responder genes. Remarkably, about one-third of all changes in gene expression in activated fibroblasts are subject to translational regulation and dynamic variation in ribosome occupancy affects protein abundance independent of RNA levels. Targets of RNA-binding proteins were strongly enriched in post-transcriptionally regulated genes, suggesting genes such as MBNL2 can act as translational activators or repressors. Ribosome occupancy in the hearts of patients with dilated cardiomyopathy suggested the same post-transcriptional regulatory network was underlying cardiac fibrosis. Key network hubs include RNA-binding proteins such as PUM2 and QKI that work in concert to regulate the translation of target transcripts in human diseased hearts. Furthermore, silencing of both PUM2 and QKI inhibits the transition of fibroblasts toward pro-fibrotic myofibroblasts in response to TGFβ1. CONCLUSIONS: We reveal widespread translational effects of TGFβ1 and define novel post-transcriptional regulatory networks that control the fibroblast-to-myofibroblast transition. These networks are active in human heart disease and silencing of hub genes limits fibroblast activation. Our findings show the central importance of translational control in fibrosis and highlight novel pathogenic mechanisms in heart failure

Multiple mechanisms disrupt the let-7 microRNA family in neuroblastoma

Poor prognosis in neuroblastoma is associated with genetic amplification of MYCN. MYCN is itself a target of let-7, a tumour suppressor family of microRNAs implicated in numerous cancers. LIN28B, an inhibitor of let-7 biogenesis, is overexpressed in neuroblastoma and has been reported to regulate MYCN. Here we show, however, that LIN28B is dispensable in MYCN-amplified neuroblastoma cell lines, despite de-repression of let-7. We further demonstrate that MYCN messenger RNA levels in amplified disease are exceptionally high and sufficient to sponge let-7, which reconciles the dispensability of LIN28B. We found that genetic loss of let-7 is common in neuroblastoma, inversely associated with MYCN amplification, and independently associated with poor outcomes, providing a rationale for chromosomal loss patterns in neuroblastoma. We propose that let-7 disruption by LIN28B, MYCN sponging, or genetic loss is a unifying mechanism of neuroblastoma development with broad implications for cancer pathogenesis.United States. National Institutes of Health (R01GM107536)Alex's Lemonade Stand FoundationHoward Hughes Medical InstituteBoston Children's Hospital. Manton Center for Orphan Disease ResearchNational Institute of General Medical Sciences (U.S.) (T32GM007753

DAZL Relieves miRNA-Mediated Repression of Germline mRNAs by Controlling Poly(A) Tail Length in Zebrafish

BACKGROUND:During zebrafish embryogenesis, microRNA (miRNA) miR-430 contributes to restrict Nanos1 and TDRD7 to primordial germ cells (PGCs) by inducing mRNA deadenylation, mRNA degradation, and translational repression of nanos1 and tdrd7 mRNAs in somatic cells. The nanos1 and tdrd7 3'UTRs include cis-acting elements that allow activity in PGCs even in the presence of miRNA-mediated repression. METHODOLOGY/PRINCIPAL FINDINGS:Using a GFP reporter mRNA that was fused with tdrd7 3'UTR, we show that a germline-specific RNA-binding protein DAZ-like (DAZL) can relieve the miR-430-mediated repression of tdrd7 mRNA by inducing poly(A) tail elongation (polyadenylation) in zebrafish. We also show that DAZL enhances protein synthesis via the 3'UTR of dazl mRNA, another germline mRNA targeted by miR-430. CONCLUSIONS/SIGNIFICANCE:Our present study indicated that DAZL acts as an "anti-miRNA factor" during vertebrate germ cell development. Our data also suggested that miRNA-mediated regulation can be modulated on specific target mRNAs through the poly(A) tail control

lin-28 Controls the Succession of Cell Fate Choices via Two Distinct Activities

lin-28 is a conserved regulator of cell fate succession in animals. In Caenorhabditis elegans, it is a component of the heterochronic gene pathway that governs larval developmental timing, while its vertebrate homologs promote pluripotency and control differentiation in diverse tissues. The RNA binding protein encoded by lin-28 can directly inhibit let-7 microRNA processing by a novel mechanism that is conserved from worms to humans. We found that C. elegans LIN-28 protein can interact with four distinct let-7 family pre-microRNAs, but in vivo inhibits the premature accumulation of only let-7. Surprisingly, however, lin-28 does not require let-7 or its relatives for its characteristic promotion of second larval stage cell fates. In other words, we find that the premature accumulation of mature let-7 does not account for lin-28's precocious phenotype. To explain let-7's role in lin-28 activity, we provide evidence that lin-28 acts in two steps: first, the let-7–independent positive regulation of hbl-1 through its 3′UTR to control L2 stage-specific cell fates; and second, a let-7–dependent step that controls subsequent fates via repression of lin-41. Our evidence also indicates that let-7 functions one stage earlier in C. elegans development than previously thought. Importantly, lin-28's two-step mechanism resembles that of the heterochronic gene lin-14, and the overlap of their activities suggests a clockwork mechanism for developmental timing. Furthermore, this model explains the previous observation that mammalian Lin28 has two genetically separable activities. Thus, lin-28's two-step mechanism may be an essential feature of its evolutionarily conserved role in cell fate succession