In this study, we performed small RNA library sequencing using human placental tissues to identify placenta-specific miRNAs. We also tested the hypothesis that human chorionic villi could secrete miRNAs extracellularly via exosomes, which in turn enter into maternal circulation. By small RNA library sequencing, most placenta-specific miRNAs (e.g., MIR517A) were linked to a miRNA cluster on chromosome 19. The miRNA cluster genes were differentially expressed in placental development. Subsequent validation by real-time PCR and in situ hybridization revealed that villous trophoblasts express placenta-specific miRNAs. The analysis of small RNA libraries from the blood plasma showed that the placenta-specific miRNAs are abundant in the plasma of pregnant women. By real-time PCR, we confirmed the rapid clearance of the placenta-specific miRNAs from the plasma after delivery, indicating that such miRNAs enter into maternal circulation. By using the trophoblast cell line BeWo in culture, we demonstrated that miRNAs are indeed extracellularly released via exosomes. Taken together, our findings suggest that miRNAs are exported from the human placental syncytiotrophoblast into maternal circulation, where they could target maternal tissues. Finally, to address the biological functions of placenta-specific miRNAs, we performed a proteome analysis of BeWo cells transfected with MIR517A. Bioinformatic analysis suggests that this miRNA is possibly involved in tumor necrosis factor-mediated signaling. Our data provide important insights into miRNA biology of the human placenta.

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MicroRNAs (miRNAs) are endogenous non-coding small RNAs that can regulate the expression of complementary mRNA targets. Identifying tissue-specific miRNAs is the first step toward understanding the biological functions of miRNAs, which include the regulation of tissue differentiation and the maintenance of tissue identity. In this study, we performed small RNA library sequencing in adult mouse testis and ovary to reveal their characteristic organ-and gender-specific profiles and to elucidate the characteristics of the miRNAs expressed in the reproductive system. We obtained 10 852 and 11 744 small RNA clones from mouse testis and ovary respectively (greater than 10 000 clones per organ), which included 6630 (159 genes) and 10 192 (154 genes) known miRNAs. A high level of efficiency of miRNA library sequencing was achieved: 61% (6630 miRNA clones/10 852 small RNA clones) and 87% (10 192/11 744) for adult mouse testis and ovary respectively. We obtained characteristic miRNA signatures in testis and ovary; 55 miRNAs were detected highly, exclusively, or predominantly in adult mouse testis and ovary, and discovered two novel miRNAs. Male-biased expression of miRNAs occurred on the X-chromosome. Our data provide important information on sex differences in miRNA expression that should facilitate studies of the reproductive organ-specific roles of miRNAs. Reproduction (2008) 136 811-822

Abstract-In this study, to search for novel preeclampsia (PE) biomarkers, we focused on microRNA expression and function in the human placenta complicated with PE. By comprehensive analyses of microRNA expression, we identified 22 microRNAs significantly upregulated in preeclamptic placentas, 5 of which were predicted in silico to commonly target the mRNA encoding hydroxysteroid (17-␤) dehydrogenase 1 (HSD17B1), a steroidogenetic enzyme expressed predominantly in the placenta. In vivo HSD17B1 expression, at both the mRNA and protein levels, was significantly decreased in preeclamptic placentas. Of these microRNAs, miR-210 and miR-518c were experimentally validated to target HSD17B1 by luciferase assay, real-time PCR, and ELISA. Furthermore, we found that plasma HSD17B1 protein levels in preeclamptic pregnant women reflected the decrease of its placental expression. Moreover, a prospective cohort study of plasma HSD17B1 revealed a significant reduction of plasma HSD17B1 levels in pregnant women at 20 to 23 and 27 to 30 weeks of gestation before PE onset compared with those with normal pregnancies. The sensitivities/specificities for predicting PE at 20 to 23 and 27 to 30 weeks of gestation were 0.75/0.67 (cutoff valueϭ21.9 ng/mL) and 0.88/0.51 (cutoff valueϭ30.5 ng/mL), and the odds ratios were 6.09 (95% CI: 2.35-15.77) and 7.83 (95% CI: 1.70 -36.14), respectively. We conclude that HSD17B1 is dysregulated by miR-210 and miR-518c that are aberrantly expressed in preeclamptic placenta and that reducing plasma level of HSD17B1 precedes the onset of PE and is a potential prognostic factor for PE. (Hypertension. 2012;59:265-273.) • Online Data Supplement Key Words: preeclampsia Ⅲ microRNA Ⅲ biomarker Ⅲ placenta Ⅲ prospective cohort study T he pathophysiology and etiology of preeclampsia (PE) remain largely unknown, and its final diagnosis can only be made when symptoms have regressed after delivery. 1 Thus, it is of clinical significance to predict PE before its onset. Dysregulation of the serum levels of angiogenic/ antiangiogenic factors has been demonstrated previously; examples include placental growth factor (PlGF), soluble fms-like tyrosine kinase 1 (sFlt-1), and soluble endoglin. [2][3][4] However, these proteins may not sufficiently characterize the clinical features and pathophysiological mechanisms of PE onset. 5 If there are any other parameters of which serum levels change in PE, they may illustrate the pathophysiology of PE in a manner different from the previous studies.MicroRNAs (miRNAs), small noncoding RNAs of Ϸ22 nucleotides in length, play a critical role in posttranscriptional gene regulation. 6,7 Although many miRNAs are ubiquitously expressed in mammals, some miRNAs exhibit specific expression patterns in an organ-or cell-type-dependent manner. 8 For instance, miRNAs derived from the miRNA cluster in human chromosome 19, a primate-specific miRNA cluster encompassing 46 miRNAs in the human genome, 9 have been demonstrated to exhibit a placenta-specific expression pattern. 10 Although a few studi...

Interaction domains located in the NH2 terminus of IRS-1 mediate its recognition by the insulin receptor. Alignment of IRS-1 and IRS-2 reveals two homology regions: the IH1(PH) contains a pleckstrin homology (PH) domain, and the IH2(PTB) contains a phosphotyrosine binding (PTB) domain. A third region in IRS-1 called SAIN was proposed to contain another functional PTB domain. Peptide competition experiments demonstrated that the IH2(PTB) in IRS-2, like the corresponding domain in IRS-1, binds directly to peptides containing NPXY motifs. In contrast, these peptides do not bind to IH1(PH) or the SAIN regions. In 32D cells the IH1(PH) was essential for insulin-stimulated tyrosine phosphorylation of IRS-1 and insulin-stimulated phosphatidylinositol 3-kinase activity and p70(s6k) phosphorylation. In contrast, the IH2(PTB) and the SAIN regions were not required for these insulin actions; however, the IH2(PTB) improved the coupling between IRS-1 and the insulin receptor. Overexpression of the insulin receptor in 32DIR cells increased IRS-1 tyrosine phosphorylation and mediated insulin-stimulated DNA synthesis. The sensitivity of these responses was partially reduced by deletion of either the IH1(PH) or the IH2(PTB) and significantly reduced when both regions were deleted together. Thus, the PH and PTB domains equally couple IRS-1 to high levels of insulin receptor normally expressed in most cells, whereas at low levels of insulin receptors the PTB domain is inefficient and the PH domain is essential for a productive interaction.

SummaryA cell line with characteristics of the periodontal ligament fibroblasts is negatively regulated for mineralization and Runx2/Cbfa1/Osf2 activity, part of which can be overcome by bone morphogenetic protein-2

Cathepsin K is a recently identified cysteine protease which is abundantly and selectively expressed in osteoclasts. To evaluate the contribution of cathepsin K to bone resorption processes, we investigated the effect of cathepsin K antisense phosphothiorate oligodeoxynucleotide (S-ODN) on the bone-resorbing activity of osteoclasts. Rabbit osteoclasts were cultured on dentine slices for 24 h in the presence or absence of antisense S-ODN in a medium containing 100 nM Tfx TM -50, polycationic liposome, as a carrier of the S-ODN. Uptake of the S-ODN by osteoclasts was confirmed microscopically using fluorescein-labeled S-ODN. The treatment with antisense significantly decreased the amount of cathepsin K protein in osteoclasts. The antisense inhibited the osteoclastic pit formation in a concentration-dependent fashion. At 10 M the antisense reduced the total pit number and area and average pit depth by 46, 52, and 30%, respectively. The sense and mismatch SODNs, which were used as negative controls, had no effect on either the cathepsin K protein level or the pit formation. A nonspecific cysteine protease inhibitor, E-64, also reduced pit formation in a concentration-dependent manner with maximum reductions at 1 M of 46, 48, and 35% in the above pit parameters. The inhibitory effect of the antisense almost equal to that of E-64 demonstrates that cathepsin K is a cysteine protease playing a crucial role in osteoclastic bone resorption.Bone tissue is a composite matrix comprising of hydroxyapatite and fibrous proteins (mainly Type I collagen) and is constantly subjected to a cycle of bone resorption and bone formation (1). Bone resorption is mainly carried out by osteoclasts which are multinucleate giant cells. In osteoclastic bone resorption, demineralization, in which osteoclasts release protons to solubilize the inorganic salt (2), is followed by the degradation of the protein fibers with cysteine proteases (1, 3). The involvement of the cysteine proteases has been verified in both in vitro and in vivo studies showing that various types of cysteine protease inhibitors reduce bone resorption (1,(3)(4)(5)(6)(7)(8)(9)(10). From studies based on substrate preference, inhibitor preference, and immunoreactivity, the cathepsins L and B were suggested to be responsible for osteoclastic bone resorption processes (8 -14).Recently, several research laboratories (including our own) have successfully cloned cDNAs for a novel cysteine protease, namely cathepsin K, from rabbit and human cDNA libraries (15)(16)(17)(18)(19), and its role in bone resorption has been the focus of recent attention. Human cathepsin K is highly and selectively expressed in osteoclasts (16 -21); in fact its expression level is much greater than those of cathepsins B, L, and S (20, 21). Brömme et al. (22) and Bossard et al. (23) showed that cathepsin K expresses a potent proteolytic activity against Type I collagen. Saneshige et al. (24) demonstrated that retinoic acid, a vitamin A metabolite, both up-regulates the gene expression of cathepsin K in ost...

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