The targeting of DAPK1 with miR-190a-3p promotes autophagy in trophoblast cells.

Pre-eclampsia (PE) is a dangerous pathological status that occurs during pregnancy and is a leading reason for both maternal and fetal death. Autophagy is necessary for cellular survival in the face of environmental stress as well as cellular homeostasis and energy management. Aberrant microRNA (miRNA) expression is crucial in the pathophysiology of PE. Although studies have shown that miRNA (miR)-190a-3p function is tissue-specific, the precise involvement of miR-190a-3p in PE has yet to be determined. We discovered that miR-190a-3p was significantly lower and death-associated protein kinase 1 (DAPK1) was significantly higher in PE placental tissues compared to normal tissues, which is consistent with the results in cells. The luciferase analyses demonstrated the target-regulatory relationship between miR-190a-3p and DAPK1. The inhibitory effect of miR-190a-3p on autophagy was reversed by co-transfection of si-DAPK1 and miR-190a-3p inhibitors. Thus, our data indicate that the hypoxia-dependent miR-190a-3p/DAPK1 regulatory pathway is implicated in the development and progression of PE by promoting autophagy in trophoblast cells.

Functional mechanism and clinical implications of miR-141 in human cancers.

Cancer is caused by the abnormal proliferation of local tissue cells under the control of many oncogenic factors. MicroRNAs (miRNAs) are a class of evolutionarily conserved, approximately 22-nucleotide noncoding small RNAs that influence transcriptional regulationby binding to the 3'-untranslated region of target messenger RNA. As a member of the miRNA family, miR-141 acts as a suppressor or an oncomiR in various cancers and regulates cancer cell proliferation, apoptosis, invasion, and metastasis through a variety of signaling pathways, such as phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) and constitutive activation of nuclear factor-κB (NF-κB). Target gene validation and pathway analysis have provided mechanistic insight into the role of this miRNA in different tissues. This review also outlines novel findings that suggest miR-141 may be useful as a noninvasive biomarker and as a therapeutic target in several cancers.

Placenta-specific lncRNA 1600012P17Rik is expressed in spongiotrophoblast and glycogen trophoblast cells of mouse placenta.

A few long noncoding RNAs (long ncRNAs, lncRNAs) exhibit trophoblast cell type-specific expression patterns and functional roles in mouse placenta. However, the cell- and stage-specific expression patterns and functions of most placenta-derived lncRNAs remain unclear. In this study, we explored mouse placenta-associated lncRNAs using a combined bioinformatic and experimental approach. We used the FANTOM5 database to survey lncRNA expression in mouse placenta and found that 1600012P17Rik (MGI: 1919275, designated P17Rik), a long intergenic ncRNA, was the most highly expressed lncRNA at gestational day 17. Polymerase chain reaction analysis confirmed that P17Rik was exclusively expressed in placenta and not in any of the adult organs examined in this study. In situ hybridization analysis revealed that it was highly expressed in spongiotrophoblast cells and to a lesser extent in glycogen trophoblast cells, including migratory glycogen trophoblast cells invading the decidua. Moreover, we found that it is a polyadenylated lncRNA localized mainly to the cytoplasm of these trophoblast cells. As these trophoblast cells also expressed the neighboring protein-coding gene, pappalysin 2 (Pappa2), we investigated the effects of P17Rik on Pappa2 expression using Pappa2-expressing MC3T3-E1 cells and found that P17Rik transfection increased the messenger RNA (mRNA) and protein levels of Pappa2. These results indicate that mouse placenta-specific lncRNA P17Rik modulates the expression of the neighboring protein-coding gene Pappa2 in spongiotrophoblast and glycogen trophoblast cells of mouse placenta during late gestation.

Comprehensive circular RNA profiling reveals that circular RNA100783 is involved in chronic CD28-associated CD8(+)T cell ageing.

BACKGROUND: Ageing brings about the gradual deterioration of the immune system, also known as immunosenescence. The role of non-coding circular RNA in immunosenescence is under studied. Using circular RNA microarray data, we assembled Comparison groups (C1, C2, C3 and C4) that allowed us to compare the circular RNA expression profiles between CD28(+)CD8(+) T cells and CD28(-)CD8(+) T cells isolated from healthy elderly or adult control subjects. Using a step-wise biomathematical strategy, the differentially-expressed circRNAs were identified in C1 (CD28(+)CD8(+) vs CD28(-)CD8(+)T cells in the elderly) and C4 (CD28(-)CD8(+)T cells in the elderly vs in the adult), and the commonly-expressed circRNA species from these profiles were optimized as immunosenescence biomarkers. RESULTS: Four overlapping upregulated circular RNAs (100550, 100783, 101328 and 102592) expressed in cross-comparison between C1 and C4 were validated using quantitative polymerase chain reaction. Of these, only circular RNA100783 exhibited significant validation. None of the down-regulated circular RNAs were expressed in the C1 and the C4 cross-comparisons. Therefore, we further predicted circular RNA100783-targeted miRNA-gene interactions using online DAVID annotation. The analysis revealed that a circular RNA100783-targeted miRNA-mRNA network may be involved in alternative splicing, the production of splice variants, and in the regulation of phosphoprotein expression. Considering the hypothesis of splicing-related biogenesis of circRNAs, we propose that circular RNA100783 may play a role in phosphoprotein-associated functions duringCD28-related CD8(+) T cell ageing. CONCLUSIONS: This study is the first to employ circular RNA profiling to investigate circular RNA-micro RNA interactions in ageing human CD8(+)T cell populations and the accompanying loss of CD28 expression. The overlapping expression of circular RNA100783 may represent a novel biomarker for the longitudinal tracking ofCD28-related CD8(+) T cell ageing and global immunosenescence.

Differentiated miRNA expression and validation of signaling pathways in apoE gene knockout mice by cross-verification microarray platform.

The microRNA (miRNA) regulation mechanisms associated with atherosclerosis are largely undocumented. Specific selection and efficient validation of miRNA regulation pathways involved in atherosclerosis development may be better assessed by contemporary microarray platforms applying cross-verification methodology. A screening platform was established using both miRNA and genomic microarrays. Microarray analysis was then simultaneously performed on pooled atherosclerotic aortic tissues from 10 Apolipoprotein E (apoE) knockout mice (apoE-/-) and 10 healthy C57BL/6 (B6) mice. Differentiated miRNAs were screened and cross-verified against an mRNA screen database to explore integrative mRNA-miRNA regulation. Gene set enrichment analysis was conducted to describe the potential pathways regulated by these mRNA-miRNA interactions. High-throughput data analysis of miRNA and genomic microarrays of knockout and healthy control mice revealed 75 differentially expressed miRNAs in apoE-/- mice at a threshold value of 2. The six miRNAs with the greatest differentiation expression were confirmed by real-time quantitative reverse-transcription PCR (qRT-PCR) in atherosclerotic tissues. Significantly enriched pathways, such as the type 2 diabetes mellitus pathway, were observed by a gene-set enrichment analysis. The enriched molecular pathways were confirmed through qRT-PCR evaluation by observing the presence of suppressor of cytokine signaling 3 (SOCS3) and SOCS3-related miRNAs, miR-30a, miR-30e and miR-19b. Cross-verified high-throughput microarrays are optimally accurate and effective screening methods for miRNA regulation profiles associated with atherosclerosis. The identified SOCS3 pathway is a potentially valuable target for future development of targeted miRNA therapies to control atherosclerosis development and progression.

Multilevel model estimation of age-dependent individual-specific trajectories for left ventricular echocardiographic indexes in an asymptomatic elderly cohort.

Few studies have been performed on the individual-specific trajectory of left ventricular aging as assessed by echocardiography in an asymptomatic elderly cohort. In the present study, a representative cohort of elderly men, who were long-term asymptomatic for cardiovascular issues, were selected from an ongoing observational aging study. Annual echocardiographic data were used to establish an age-dependent hierarchical model. Based on two-level linear regression results, four echocardiographic indexes [left ventricular mass (LVmass; -1.872 g/yr), posterior ventricular wall thickness (-0.048 mm/yr), fraction shortening (0.097/yr), and transmitral peak A velocity (-0.006 m·s(-1)·yr(-1))] changed significantly with increasing age and were age- and subject-dependent. The most characterized results of the study were the significant, age-related, within-individual variances in echocardiographic results, which were observed using the likelihood ratio test at an occasion-dependent level. Of these, fluctuated amplitudes of two systolic variables [i.e., LVmass (con/age = -0.012 ± 0.004; P = 0.0007) and fraction shortening (con/age = -0.001 ± 0.004; P = 0.05)] were significantly attenuated with increasing age within individuals. On the other hand, the age-related variability of four diastolic Doppler variables [i.e., peak A velocity (con/age = 0.003 ± 0.002; P = 0.0009), peak E velocity (con/age = 0.004 ± 0.003; P = 0.01), E/A ratio (con/age = 0.007 ± 0.003; P = 0.0002), and deceleration time of E wave (con/age = 0.025 ± 0.007; P < 0.0001)] significantly increased with increasing age within individuals. The age-related individual variability of left ventricular indexes observed in this continuous asymptomatic cohort may reflect the mechanism of preclinical, individualized heart aging. In conclusion, successfully fitted multilevel models were applied as a valuable tool to determine the mechanism of individual cardiac aging in the elderly.

Hydroxysteroid (17-ß) dehydrogenase 1 is dysregulated by miR-210 and miR-518c that are aberrantly expressed in preeclamptic placentas: a novel marker for predicting preeclampsia.

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-ß) 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 value=21.9 ng/mL) and 0.88/0.51 (cutoff value=30.5 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.

Short RNA duplexes elicit RIG-I-mediated apoptosis in a cell type- and length-dependent manner.

Short double-stranded RNAs (dsRNAs) induce type I interferon (IFN)-mediated innate immune responses. In functional studies with short interfering RNAs or synthetic mimics of microRNA precursors in vitro, we found that short dsRNAs readily induced apoptosis in cells derived from human granulosa cell tumors, but not in other cell types. Apoptosis was independent of the sequence of the dsRNA, but depended on its length, and was induced by 23- and 24-nucleotide (nt) dsRNAs, but not by shorter dsRNAs (< 22 nt) or by the long dsRNA polyinosinic-polycytidylic acid. Microarray analysis revealed that apoptosis was accompanied by the increased expression of IFN-stimulated genes; however, several lines of evidence showed that IFNs did not directly induce apoptosis. Subsequent analyses revealed that the short dsRNAs increased the expression of retinoic acid-inducible gene I (RIG-I) through dsRNA-activated protein kinase (PKR). Although these dsRNAs bore 3' overhangs and nontriphosphate 5' termini, which are not thought to be RIG-I-activating structures, the dsRNAs bound to RIG-I and triggered proapoptotic signaling mostly by activating RIG-I, which was followed by activation of the mitogen-activated protein kinase p38. Thus, we suggest that ligand recognition and subsequent signaling by RNA sensors are more complicated than previously believed. In addition, short dsRNAs may serve as pharmacological agents to target specific tumors, such as granulosa cell tumors.

Human villous trophoblasts express and secrete placenta-specific microRNAs into maternal circulation via exosomes.

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.

MicroRNA (miRNA) cloning analysis reveals sex differences in miRNA expression profiles between adult mouse testis and ovary.

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.

Expression of heat shock proteins and stress fiber formation within the arterial vascular tree of adult rats under normal physiological conditions.

In this study, we investigated heat shock protein (HSP) expression and stress fiber (SF) formation in endothelial cells (ECs) within the arterial vascular tree of adult rats under normal physiological conditions. Using quantitative immunofluorescence microscopy, we found no significant differences in expression of HSPs 25, 60, 70, and 90 among ECs in the straight portions of rat arteries. In these regions, ECs appeared spindle-shaped and contained short bundles of central SFs. In contrast, ECs in the curved portions or the branch sites of the arteries, exhibited striking differences in HSP expression. ECs with higher HSP expression were localized at the lesser curvature in the curved portions or the distal site of the branch ostia. Moreover, the ECs became polygonal and contained irregular central SFs at the lesser curvature. At the branch sites, downstream ECs became spear-shaped and contained long, thick bundles of central SFs. Curved portions or branch sites are the regions of disturbed flow at which early atherosclerotic lesions are often found. Our results demonstrate these positional differences in HSP expression associated with changes in SF formation within the arterial vascular tree under non-pathological conditions. Our study provides basic information for understanding stress responses via HSP expression and SF formation in vascular ECs and the pathogenesis of atherosclerotic disease.

Role of heat shock protein 70 in induction of stress fiber formation in rat arterial endothelial cells in response to stretch stress.

We investigated the mechanism by which endothelial cells (ECs) resist various forms of physical stress using an experimental system consisting of rat arterial EC sheets. Formation of actin stress fibers (SFs) and expression of endothelial heat-shock stress proteins (HSPs) in response to mechanical stretch stress were assessed by immunofluorescence microscopy. Stretch stimulation increased expression of HSPs 25 and 70, but not that of HSP 90. Treatment with SB203580, a p38 MAP kinase inhibitor that acts upstream of the HSP 25 activation cascade, or with geldanamycin, an inhibitor of HSP 90, had no effect on the SF formation response to mechanical stretch stress. In contrast, treatment with quercetin, an HSP 70 inhibitor, inhibited both upregulation of endothelial HSP 70 and formation of SFs in response to tensile stress. In addition, treatment of stretched ECs with cytochalasin D, which disrupts SF formation, did not adversely affect stretch-induced upregulation of endothelial HSP 70. Our data suggest that endothelial HSP 70 plays an important role in inducing SF formation in response to tensile stress.

The cytotrophoblast layer of human chorionic villi becomes thinner but maintains its structural integrity during gestation.

Chorionic villi in the human placenta serve as essential structures in fetomaternal exchanges. According to the embryology and placentology literature, during the first trimester, the cytotrophoblast (CTB) layer that is subjacent to the syncytiotrophoblast (STB) and supported by a basal lamina is nearly complete, but later, it becomes discontinuous. In the present study, we investigated the structural integrity of the CTB layer in the normal villous tree by advanced microscopy techniques using an antibody to hepatocyte growth factor (HGF) activator inhibitor type 1 (SPINT1), a potent inhibitor of HGF activators expressed exclusively on villous CTB. In full-term placenta, the cell surface of the CTB layer was spread over the basal lamina but was not interrupted. Morphometric analysis showed that throughout the villous tree, 80% of the continuity of the CTB layer of full-term placenta and 90% of that of first-trimester placenta were preserved. Gestation was accompanied by unique structural change in the basal domain of the trophoblast layer. The initially cuboidal-shaped CTB cells were transformed to flat cells with many cellular processes that, together with those of the adjacent STB, eventually covered the trophoblast basal lamina in a complex network of interdigitations. In addition, the expression levels of SPINT1, ST14, HGF, and MET mRNAs in the villous tree increased over the course of gestation. These results suggest that the structural integrity of the SPINT1-positive CTB layer may play an important role in villous differentiation and in maintenance of the villous tree via the HGF signaling system during gestation.