Antiphospholipid antibody-induced miR-146a-3p drives trophoblast interleukin-8 secretion through activation of Toll-like receptor 8.
What is the role of microRNAs (miRs) in antiphospholipid antibody (aPL)-induced trophoblast inflammation?
aPL-induced up-regulation of trophoblast miR-146a-3p is mediated by Toll-like receptor 4 (TLR4), and miR-146a-3p in turn drives the cells to secrete interleukin (IL)-8 by activating the RNA sensor, TLR8.
Obstetric antiphospholipid syndrome (APS) is an autoimmune disorder characterized by circulating aPL and an increased risk of pregnancy complications. We previously showed that aPL recognizing beta2 glycoprotein I (β2GPI) elicit human first trimester trophoblast secretion of IL-8 by activating TLR4. Since some miRs control TLR responses, their regulation in trophoblast cells by aPL and functional role in the aPL-mediated inflammatory response was investigated. miRs can be released from cells via exosomes, and therefore, miR exosome expression was also examined. A panel of miRs was selected based on their involvement with TLR signaling: miR-9; miR-146a-5p and its isomiR, miR-146a-3p; miR-155, miR-210; and Let-7c. Since certain miRs can activate the RNA sensor, TLR8, this was also investigated.
For in vitro studies, the human first trimester extravillous trophoblast cell line, HTR8 was studied. HTR8 cells transfected to express a TLR8 dominant negative (DN) were also used. Plasma was evaluated from pregnant women who have aPL, either with or without systemic lupus erythematous (SLE) (n = 39); SLE patients without aPL (n = 30); and healthy pregnant controls (n = 20).
Trophoblast HTR8 wildtype and TLR8-DN cells were incubated with or without aPL (mouse anti-human β2GPI mAb) for 48-72 h. HTR8 cells were also treated with or without aPL in the presence and the absence of a TLR4 antagonist (lipopolysaccharide from Rhodobacter sphaeroides; LPS-RS), specific miR inhibitors or specific miR mimics. miR expression levels in trophoblast cells, trophoblast-derived exosomes and exosomes isolated from patient plasma were measured by qPCR. Trophoblast IL-8 secretion was measured by ELISA.
aPL significantly increased trophoblast cellular and exosome expression of miR-146a-5p, miR-146a-3p, miR-155 and miR-210. aPL-induced up-regulation of trophoblast miR-146a-5p, miR-146a-3p and miR-210, but not miR-155, was inhibited by the TLR4 antagonist, LPS-RS. While inhibition or overexpression of miR-146a-5p had no effect on aPL-induced trophoblast IL-8 secretion, miR-146a-3p inhibition significantly reduced this response. aPL-induced trophoblast IL-8 secretion was inhibited by the presence of the TLR8-DN. In the absence of aPL, transfection of trophoblast cells with a miR-146a-3p mimic significantly increased IL-8 secretion and this was inhibited by the presence of the TLR8-DN. Patients with aPL and adverse pregnancy outcomes (APOs) expressed significantly higher levels of circulating miR-146a-3p compared with healthy pregnant controls with no pregnancy complications (P < 0.05).
While the enrichment of miR-146a-3p in trophoblast-derived exosomes support the role of this miR acting in a paracrine or endocrine manner through exosome delivery, this has not been demonstrated. However, miR-146a-3p may also exert its pro-inflammatory effect intracellularly within the same trophoblast cell targeted by aPL.
These findings provide a novel mechanism of trophoblast inflammation through miRs activating RNA-sensing receptors. Furthermore, circulating exosomal-associated miR-146a-3p in APS patients may serve clinically as a biomarker for related APOs.
This study was supported in part by grants from the American Heart Association (#10GRNT3640032 to V.M.A.), the March of Dimes Foundation (Gene Discovery and Translational Research Grant #6-FY12-255 to V.M.A.), NICHD, NIH (R01HD049446 to V.M.A.), the Gina M. Finzi Memorial Student Summer Fellowship from the Lupus Foundation of America (to S.M.G.), and the Yale University School of Medicine Medical Student Fellowship (to S.M.G.). The authors declare no competing financial interests.
N/A.
Gysler SM
，Mulla MJ
，Guerra M
，Brosens JJ
，Salmon JE
，Chamley LW
，Abrahams VM
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MicroRNA-141-3p and miR-200a-3p regulate insulin-like growth factor 2 during mouse placental development.
Insulin-like growth factor 2 (IGF2) plays a vital role in fetal and placental development throughout gestation. Placental expression of IGF2 decreases substantially in intra-uterine growth restriction (IUGR) and Igf2 null mice develop small placentas. In this report, we examined the role of microRNAs in regulating Igf2 gene expression during mouse placental development. Using bioinformatic analysis, we have identified microRNAs that have conserved binding sites in the 3'-UTR of Igf2. Using luciferase reporter assay, we demonstrated that miR141-3p and miR-200a-3p mimics substantially down regulated relative luciferase activity by binding to 3'-UTR of Igf2, which was reversed by using miR141-3p and miR-200a-3p inhibitors. Furthermore, in a similar assay, use of Igf2 3'-UTR that lacked the binding site for the microRNAs did not have any effect on luceiferase activity. Interestingly, the expression of miR141-3p and miR-200a-3p were inversely and temporally correlated to the expression of IGF2 during mouse placental development. Overexpression of miR141-3p and miR-200a-3p in mouse trophoblast stem cells suppressed endogenous expression of IGF2. Consequently, IGF2 silencing by miR141-3p and miR-200a-3p diminished Akt activation in mouse trophoblast stem cells. Our study provides evidence for regulation of Igf2 by microRNAs and further elucidates the role of miR141-3p and miR-200a-3p in the mouse placental development.
Saha S
，Choudhury J
，Ain R
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Elevated microRNA-141-3p in placenta of non-diabetic macrosomia regulate trophoblast proliferation.
Several studies have reported microRNAs (miRNAs) could regulate the placental development, though the role and mechanism of miRNAs in the development of non-diabetic macrosomia (NDFMS) remains unclear.
To identify the aberrantly expressed key miRNAs in placenta of NDFMS, we employed a strategy consisting of initial screening with miRNA microarray and further validation with quantitative RT-PCR assay (qRT-PCR). In vitro cellular model and a mouse pregnancy model were used to delineate the functional effects of key miRNA on proliferation, invasion, and migration.
miR-141-3p was identified as the key miRNA with expression level significantly higher in placentas of NDFMS compared with those from normal controls. Overexpressed miR-141-3p in HTR-8/SVneo cells contributed to increased cell proliferation, invasion, and migration. miR-141-3p inhibition in HTR-8/SVneo cells resulted in decreased cell proliferation and invasion. Significantly increased infant birth weight was observed in late pregnancy of C57BL/6J mice treated with miR-141-3p agomir. However, no significant difference was found in early pregnancy of C57BL/6J mice treated with miR-141-3p agomir.
miR-141-3p could stimulate placental cell proliferation to participate in the occurrence and development of NDFMS.
Guo D
，Jiang H
，Chen Y
，Yang J
，Fu Z
，Li J
，Han X
，Wu X
，Xia Y
，Wang X
，Chen L
，Tang Q
，Wu W
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