Zvi Bentwich

Identification of hundreds of conserved and nonconserved human microRNAs

MicroRNAs are noncoding RNAs of ∼22 nucleotides that suppress translation of target genes by binding to their mRNA and thus have a central role in gene regulation in health and disease. To date, 222 human microRNAs have been identified, 86 by random cloning and sequencing, 43 by computational approaches and the rest as putative microRNAs homologous to microRNAs in other species. To prove our hypothesis that the total number of microRNAs may be much larger and that several have emerged only in primates, we developed an integrative approach combining bioinformatic predictions with microarray analysis and sequence-directed cloning. Here we report the use of this approach to clone and sequence 89 new human microRNAs (nearly doubling the current number of sequenced human microRNAs), 53 of which are not conserved beyond primates. These findings suggest that the total number of human microRNAs is at least 800.

Serum MicroRNAs Are Promising Novel Biomarkers

Background Circulating nucleic acids (CNAs) offer unique opportunities for early diagnosis of clinical conditions. Here we show that microRNAs, a family of small non-coding regulatory RNAs involved in human development and pathology, are present in bodily fluids and represent new effective biomarkers. Methods and Results After developing protocols for extracting and quantifying microRNAs in serum and other body fluids, the serum microRNA profiles of several healthy individuals were determined and found to be similar, validating the robustness of our methods. To address the possibility that the abundance of specific microRNAs might change during physiological or pathological conditions, serum microRNA levels in pregnant and non pregnant women were compared. In sera from pregnant women, microRNAs associated with human placenta were significantly elevated and their levels correlated with pregnancy stage. Conclusions and Significance Considering the central role of microRNAs in development and disease, our results highlight the medically relevant potential of determining microRNA levels in serum and other body fluids. Thus, microRNAs are a new class of CNAs that promise to serve as useful clinical biomarkers.

MicroRNAs accurately identify cancer tissue origin

MicroRNAs (miRNAs) belong to a class of noncoding, regulatory RNAs that is involved in oncogenesis and shows remarkable tissue specificity. Their potential for tumor classification suggests they may be used in identifying the tissue in which cancers of unknown primary origin arose, a major clinical problem. We measured miRNA expression levels in 400 paraffin-embedded and fresh-frozen samples from 22 different tumor tissues and metastases. We used miRNA microarray data of 253 samples to construct a transparent classifier based on 48 miRNAs. Two-thirds of samples were classified with high confidence, with accuracy >90%. In an independent blinded test-set of 83 samples, overall high-confidence accuracy reached 89%. Classification accuracy reached 100% for most tissue classes, including 131 metastatic samples. We further validated the utility of the miRNA biomarkers by quantitative RT-PCR using 65 additional blinded test samples. Our findings demonstrate the effectiveness of miRNAs as biomarkers for tracing the tissue of origin of cancers of unknown primary origin.

MiR-24-mediated downregulation of H2AX suppresses DNA repair in terminally differentiated blood cells

Terminally differentiated cells have a reduced capacity to repair double-stranded breaks, but the molecular mechanism behind this downregulation is unclear. Here we find that miR-24 is upregulated during postmitotic differentiation of hematopoietic cell lines and regulates the histone variant H2AX, a protein that has a key role in the double-stranded break response. We show that the H2AX 3′ untranslated region contains conserved miR-24 binding sites that are indeed regulated by miR-24. During terminal differentiation, both H2AX mRNA and protein levels are substantially reduced by miR-24 upregulation in in vitro differentiated cells; similar diminished levels are found in primary human blood cells. miR-24–mediated suppression of H2AX renders cells hypersensitive to γ-irradiation and genotoxic drugs, a phenotype that is fully rescued by overexpression of miR-24–insensitive H2AX. Therefore, miR-24 upregulation in postreplicative cells reduces H2AX and makes them vulnerable to DNA damage.

MiR‐92b and miR‐9/9* Are Specifically Expressed in Brain Primary Tumors and Can Be Used to Differentiate Primary from Metastatic Brain Tumors

A recurring challenge for brain pathologists is to diagnose whether a brain malignancy is a primary tumor or a metastasis from some other tissue. The accurate diagnosis of brain malignancies is essential for selection of proper treatment. MicroRNAs are a class of small non‐coding RNA species that regulate gene expression; many exhibit tissue‐specific expression and are misregulated in cancer. Using microRNA expression profiling, we found that hsa‐miR‐92b and hsa‐miR‐9/hsa‐miR‐9* are over‐expressed, specifically in brain primary tumors, as compared to primary tumors from other tissues and their metastases to the brain. By considering the expression of only these two microRNAs, it is possible to distinguish between primary and metastatic brain tumors with very high accuracy. These microRNAs thus represent excellent biomarkers for brain primary tumors. Previous reports have found that hsa‐miR‐92b and hsa‐miR‐9/hsa‐miR‐9* are expressed more strongly in developing neurons and brain than in adult brain. Thus, their specific over‐expression in brain primary tumors supports a functional role for these microRNAs or a link between neuronal stem cells and brain tumorigenesis.

Epithelial microRNAs regulate gut mucosal immunity via epithelium-T cell crosstalk

Colonic homeostasis entails epithelium-lymphocyte cooperation, yet many participants in this process are unknown. We show here that epithelial microRNAs mediate the mucosa–immune system crosstalk necessary for mounting protective T helper type 2 (TH2) responses. Abolishing the induction of microRNA by gut-specific deletion of Dicer1 (Dicer1Δgut), which encodes an enzyme involved in microRNA biogenesis, deprived goblet cells of RELMβ, a key TH2 antiparasitic cytokine; this predisposed the host to parasite infection. Infection of Dicer1Δgut mice with helminths favored a futile TH1 response with hallmarks of inflammatory bowel disease. Interleukin 13 (IL-13) induced the microRNA miR-375, which regulates the expression of TSLP, a TH2-facilitating epithelial cytokine; this indicated a TH2-amplification loop. We found that miR-375 was required for RELMβ expression in vivo; miR-375-deficient mice had significantly less intestinal RELMβ, which possibly explains the greater susceptibility of Dicer1Δgut mice to parasites. Our findings indicate that epithelial microRNAs are key regulators of gut homeostasis and mucosal immunity.

Treatment of intestinal worms is associated with decreased HIV plasma viral load

Background: We have previously suggested that helminthic infections make the host more susceptible to HIV infection and enhance its progression due to the chronic immune activation they cause. Objective: To study the effect of antihelminthic treatment on HIV plasma viral load (VL) in HIV‐ and helminth‐infected individuals living in Ethiopia. Methods: Fifty‐six clinically asymptomatic HIV‐1‐infected individuals, 31 (55%) of whom were also infected with helminths, were studied. All participants received antihelminthic treatment at baseline and at 3 and 6 months. Worm egg excretion, HIV plasma VL, and T‐cell subsets were determined at baseline and 6 months after treatment. Results: The mean age, number of CD4 T cells, and gender distribution were similar in the helminth‐infected and ‐noninfected groups. At baseline, HIV plasma VL was strongly correlated to the number of eggs excreted (p < .001) and was higher in individuals infected with more than one helminth (5.28 ± 0.35 versus 4.30 ± 1.13 log10 RNA copies/mL, respectively; p = .16). After treatment of helminths, the 6‐month change in HIV plasma VL was significantly different between the successfully treated group and the persistently helminth‐positive group (p = .04) Conclusions: Helminth “load” is correlated to HIV plasma VL, and successful deworming is associated with a significant decrease in HIV plasma VL. The results of the current study, if confirmed in a larger study, may have important implications for slowing disease progression and reducing risks of transmission.

miR-34a contributes to megakaryocytic differentiation of K562 cells independently of p53

The role of miRNAs in regulating megakaryocyte differentiation was examined using bipotent K562 human leukemia cells. miR-34a is strongly up-regulated during phorbol ester-induced megakaryocyte differentiation, but not during hemin-induced erythrocyte differentiation. Enforced expression of miR-34a in K562 cells inhibits cell proliferation, induces cell-cycle arrest in G(1) phase, and promotes megakaryocyte differentiation as measured by CD41 induction. miR-34a expression is also up-regulated during thrombopoietin-induced differentiation of CD34(+) hematopoietic precursors, and its enforced expression in these cells significantly increases the number of megakaryocyte colonies. miR-34a directly regulates expression of MYB, facilitating megakaryocyte differentiation, and of CDK4 and CDK6, to inhibit the G(1)/S transition. However, these miR-34a target genes are down-regulated rapidly after inducing megakaryocyte differentiation before miR-34a is induced. This suggests that miR-34a is not responsible for the initial down-regulation but may contribute to maintaining their suppression later on. Previous studies have implicated miR-34a as a tumor suppressor gene whose transcription is activated by p53. However, in p53-null K562 cells, phorbol esters induce miR-34a expression independently of p53 by activating an alternative phorbol ester-responsive promoter to produce a longer pri-miR-34a transcript.

MicroRNA Expression Patterns and Function in Endodermal Differentiation of Human Embryonic Stem Cells

Background/Aims microRNAs (miRNAs) are small noncoding RNAs that regulate cognate mRNAs post-transcriptionally. Human embryonic stem cells (hESC), which exhibit the characteristics of pluripotency and self-renewal, may serve as a model to study the role of miRNAs in early human development. We aimed to determine whether endodermally-differentiated hESC demonstrate a unique miRNA expression pattern, and whether overexpression of endoderm-specific miRNA may affect hESC differentiation. Methods miRNA expression was profiled in undifferentiated and NaButyrate-induced differentiated hESC of two lines, using microarray and quantitative RT-PCR. Then, the effect of lentiviral-based overexpression of liver-specific miR-122 on hESC differentiation was analyzed, using genomewide gene microarrays. Results The miRNA profiling revealed expression of three novel miRNAs in undifferentiated and differentiated hESC. Upon NaButyrate induction, two of the most upregulated miRNAs common to both cell lines were miR-24 and miR-10a, whose target genes have been shown to inhibit endodermal differentiation. Furthermore, induction of several liver-enriched miRNAs, including miR-122 and miR-192, was observed in parallel to induction of endodermal gene expression. Stable overexpression of miR-122 in hESC was unable to direct spontaneous differentiation towards a clear endodermal fate, but rather, delayed general differentiation of these cells. Conclusions Our results demonstrate that expression of specific miRNAs correlates with that of specific genes upon differentiation, and highlight the potential role of miRNAs in endodermal differentiation of hESC.

Comprehensive Gene and microRNA Expression Profiling Reveals a Role for microRNAs in Human Liver Development

Background and Aims microRNAs (miRNAs) are small noncoding RNAs that regulate cognate mRNAs post-transcriptionally. miRNAs have been implicated in regulating gene expression in embryonic developmental processes, including proliferation and differentiation. The liver is a multifunctional organ, which undergoes rapid changes during the developmental period and relies on tightly-regulated gene expression. Little is known regarding the complex expression patterns of both mRNAs and miRNAs during the early stages of human liver development, and the role of miRNAs in the regulation of this process has not been studied. The aim of this work was to study the impact of miRNAs on gene expression during early human liver development. Methods Global gene and miRNA expression were profiled in adult and in 9–12w human embryonic livers, using high-density microarrays and quantitative RT-PCR. Results Embryonic liver samples exhibited a gene expression profile that differentiated upon progression in the developmental process, and revealed multiple regulated genes. miRNA expression profiling revealed four major expression patterns that correlated with the known function of regulated miRNAs. Comparison of the expression of the most regulated miRNAs to that of their putative targets using a novel algorithm revealed a significant anti-correlation for several miRNAs, and identified the most active miRNAs in embryonic and in adult liver. Furthermore, our algorithm facilitated the identification of TGFβ-R1 as a novel target gene of let-7. Conclusions Our results uncover multiple regulated miRNAs and genes throughout human liver development, and our algorithm assists in identification of novel miRNA targets with potential roles in liver development.