Human exosomal placenta-associated miR-517a-3p modulates the expression of PRKG1 mRNA in Jurkat cells.

During pregnancy, human placenta-associated microRNAs (miRNAs) derived from the miRNA cluster in human chromosome 19 are expressed in villous trophoblasts and secreted into maternal circulation via exosomes; however, little is known about whether circulating placenta-associated miRNAs are transferred into maternal immune cells via exosomes, and modulate expression of target genes in the recipient cells. We employed an in vitro model of trophoblast-immune cell communication using BeWo cells (a human trophoblast cell line) and Jurkat cells (a human leukemic T-cell line) and investigated whether BeWo exosomal placenta-associated miRNAs can suppress expression of target genes in the recipient Jurkat cells. Using this system, we identified PRKG1 as a target gene of placenta-associated miRNA miR-517a-3p. Moreover, we demonstrated that BeWo exosomal miR-517a-3p was internalized into Jurkat cells and subsequently suppressed the expression of PRKG1 in recipient Jurkat cells. Furthermore, using peripheral blood natural killer (NK) cells in vivo, we confirmed that circulating miR-517a-3p was delivered into maternal NK cells as it was into Jurkat cells in vitro. Placenta-associated miR-517a-3p was incorporated into maternal NK cells in the third trimester, and it was rapidly cleared after delivery. Expression levels of miR-517a-3p and its target mRNA PRKG1 were inversely correlated in NK cells before and after delivery. These in vitro and in vivo results suggest that exosome-mediated transfer of placenta-associated miRNAs and subsequent modulation of their target genes occur in maternal NK cells. The present study provides novel insight into our understanding of placenta-maternal communication.

Chondromodulin-1 directly suppresses growth of human cancer cells.

BACKGROUND: Chondromodulin-1 (ChM1), an endogenous anti-angiogenic factor expressed in cartilage, has been suggested to inhibit invasion of endothelial cells into cartilage. In addition, the ectopic administration of ChM1 has been reported to suppress tumorigenesis in vivo. However, it is unclear whether the anti-tumor effect is due to not only the anti-vascularization effect of ChM1, but also its direct action against oncocytes. In the present study, we sought to determine whether ChM1 has a direct action on tumor cells. METHODS: BrdU incorporation assay was performed on human umbilical vein endothelial cells (HUVECs), normal human dermal fibroblasts (NHDFs), HepG2 cells and HeLa cells in the presence or absence of recombinant human ChM1 (rhChM1). An adenovirus that expresses ChM1, Ad-ChM1, was established and applied to the tumor xenografted in vivo, and to in vitro tumor cells cultured on plates or in soft agar. Cell cycle-related proteins and the phosphorylation of Erk, Akt, and GSK3beta, the downstream molecules of the extracellular matrix-integrin signaling pathways, in HepG2 cells treated with or without Ad-ChM1 were detected by western blot analysis. Luciferase reporter assays of STAT, GAS, and ISRE, which participate in another cytokine signaling pathway, ware performed in HepG2, HeLa, and HUVEC cells. RESULTS: ChM1 suppressed BrdU incorporation in HUVECs and in HepG2 cells dose-dependently, but did not suppress BrdU incorporation in NHDFs and HeLa cells cultured on plates. In soft agar, however, ChM1 suppressed the growth of HeLa cells, as well as HepG2 cells. Western blot analyses demonstrated that ChM1 decreased the levels of cyclin D1, cyclin D3, and cdk6 and increased those of p21cip1 without affecting the phosphorylation levels of Erk, Akt, and GSK3beta in HepG2 cells. The luciferase reporter assay demonstrated that ChM1 suppressed the transcriptional activities of STAT and GAS but not of ISRE. CONCLUSION: ChM1 directly suppressed the proliferation of tumor cells in an anchorage-independent manner. However, ChM1 did not alter the phosphorylation of downstream molecules, at which the signaling pathways through growth factor and cytokine receptors converge with the anchorage-dependent pathway. Our results show that ChM1 has a direct anti-tumor effect; moreover, this effect occurs by inhibiting the STAT signaling pathway.

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.

PIASxbeta is a key regulator of osterix transcriptional activity and matrix mineralization in osteoblasts.

We recently reported that tensile stress induces osteoblast differentiation and osteogenesis in the mouse calvarial suture in vitro. Using this experimental system, we identified PIASxbeta, a splice isoform of Pias2, as one of the genes most highly upregulated by tensile stress. Further study using cell culture revealed that this upregulation was transient and was accompanied by upregulation of other differentiation markers, including osterix, whereas expression of Runx2 was unaffected. Runx2 and osterix are the two master proteins controlling osteoblast differentiation, with Runx2 being upstream of osterix. Targeted knockdown of PIASxbeta by small interfering RNA (siRNA) markedly suppressed osteoblastic differentiation and matrix mineralization, whereas transient overexpression of PIASxbeta caused the exact opposite effects. Regardless of PIASxbeta expression level, Runx2 expression remained constant. Reporter assays demonstrated that osterix enhanced its own promoter activity, which was further stimulated by PIASxbeta but not by its sumoylation-defective mutant. NFATc1 and NFATc3 additionally increased osterix transcriptional activity when co-transfected with PIASxbeta. Because osterix has no consensus motif for sumoylation, other proteins are probably involved in the PIASxbeta-mediated activation and NFAT proteins may be among such targets. This study provides the first line of evidence that PIASxbeta is indispensable for osteoblast differentiation and matrix mineralization, and that this signaling molecule is located between Runx2 and osterix.