MicroRNAs are noncoding RNAs of approximately 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.

p53 is a potent tumor suppressor, whose biological effects are largely due to its function as a transcriptional regulator. Here we report that, in addition to regulating the expression of hundreds of protein-coding genes, p53 also modulates the levels of microRNAs (miRNAs). Specifically, p53 can induce expression of microRNA-34a (miR-34a) in cultured cells as well as in irradiated mice, by binding to a perfect p53 binding site located within the gene that gives rise to miR-34a. Processing of the primary transcript into mature miR-34a involves the excision of a 30 kb intron. Notably, inactivation of miR-34a strongly attenuates p53-mediated apoptosis in cells exposed to genotoxic stress, whereas overexpression of miR-34a mildly increases apoptosis. Hence, miR-34a is a direct proapoptotic transcriptional target of p53 that can mediate some of p53's biological effects. Perturbation of miR-34a expression, as occurs in some human cancers, may thus contribute to tumorigenesis by attenuating p53-dependent apoptosis.

BackgroundCirculating 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 ResultsAfter 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 SignificanceConsidering 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.

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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.