miRNAs associated with endoplasmic reticulum stress and unfolded protein response during decidualization.

RESEARCH QUESTION: Do microRNAs (miRNAs) play a role in regulating endoplasmic reticulum stress (ERS) and unfolded protein response (UPR) in decidualized cells and endometrium associated with reproductive failures? DESIGN: Endometrial stromal cell line St-T1b was decidualized in vitro with 8-Br-cAMP over 5 days, or treated with the ERS inducer thapsigargin. Expression of ERS sensors, UPR markers and potential miRNA regulators was analysed by quantitative PCR. Endometrial biopsies from patients with recurrent pregnancy loss (RPL) and recurrent implantation failure (RIF) were investigated for the location of miRNA expression. RESULTS: Decidualization of St-T1b cells resulted in increased expression of ERS sensors including ATF6α, PERK and IRE1α, and the UPR marker, CHOP. TXNIP, which serves as a link between the ERS pathway and inflammation, as well as inflammasome NLRP3 and interleukin 1ß expression increased in decidualized cells. An in-silico analysis identified miR-17-5p, miR-21-5p and miR-193b-3p as miRNAs potentially involved in regulation of the ERS/UPR pathways and inflammation associated with embryo implantation. Their expression decreased significantly (P ≤ 0.0391) in non-decidualized cells in the presence of thapsigargin. Finally, expression of the selected miRNAs was localized by in-situ hybridization in stromal and glandular epithelial cells in endometrial samples from patients with RPL and RIF. Expression in stroma cells from patients with RPL was lower in comparison with stroma cells from patients with RIF. CONCLUSIONS: Decidualization in St-T1b cells is accompanied by ERS/UPR processes, associated with an inflammatory response that is potentially influenced by miR-17-5p, miR-21-5p and miR-193b-3p. These miRNAs are expressed differentially in stromal cells from patients with RPL and RIF, indicating an alteration in regulation of the ERS/UPR pathways.

Doxorubicin induces cytotoxicity and miR-132 expression in granulosa cells.

Doxorubicin (DOX) is one of the most commonly used drugs for the treatment of childhood cancers, including leukemia and lymphomas. Despite the high survival rate, female leukemia survivors are at higher risk of ovarian failure and infertility later in life. Treatment with chemotherapeutic drugs like DOX is associated with damage in ovarian follicles, but the affectation grade of granulosa cells remains unclear. To assess and avoid the possible side-effects of DOX, early biomarkers of ovarian injury and chemotherapy-induced ovarian toxicity should be identified. MicroRNAs (miRNAs) have emerged in recent years as a promising new class of biomarkers for drug-induced tissue toxicity. In this study, the effects of DOX on cell viability, steroidogenesis, and miRNA expression were studied in primary granulosa cells (GCs) and in two cellular models (COV434 and KGN cells). We report that compared to other chemotherapeutic drugs, DOX treatment is more detrimental to granulosa cells as observed by decrease of cell viability. Treatment with DOX changes the expression of the aromatase gene (CYP19A1) and the secretion of 17ß-estradiol (E2) in a cell-specific manner. miR-132-3p is dose-dependently increased by DOX in all cellular models. In absence of DOX, miR-132-3p overexpression in COV434 cells has no effect on E2 secretion or CYP19A1 expression. Altogether, these findings contribute to understanding the hormonal disbalance caused by DOX in human ovarian cells and suggest miR-132 as a putative sensor to predict DOX-induced ovarian toxicity.

Identification of miRNAs and associated pathways regulated by Leukemia Inhibitory Factor in trophoblastic cell lines.

INTRODUCTION: Leukemia Inhibitory Factor (LIF) regulates behavior of trophoblast cells and their interaction with immune and endothelial cells. In vitro, trophoblast cell response to LIF may vary depending on the cell model. Reported differences in the miRNA profile of trophoblastic cells may be responsible for these observations. Therefore, miRNA expression was investigated in four trophoblastic cell lines under LIF stimulation followed by in silico analysis of altered miRNAs and their associated pathways. METHODS: Low density TaqMan miRNA assays were used to quantify levels of 762 mature miRNAs under LIF stimulation in three choriocarcinoma-derived (JEG-3, ACH-3P and AC1-M59) and a trophoblast immortalized (HTR-8/SVneo) cell lines. Expression of selected miRNAs was confirmed in primary trophoblast cells and cell lines by qPCR. Targets and associated pathways of the differentially expressed miRNAs were inferred from the miRTarBase followed by a KEGG Pathway Enrichment Analysis. HTR-8/SVneo and JEG-3 cells were transfected with miR-21-mimics and expression of miR-21 targets was assessed by qPCR. RESULTS: A similar number of miRNAs changed in each tested cell line upon LIF stimulation, however, low coincidence of individual miRNA species was observed and occurred more often among choriocarcinoma-derived cells (complete data set at http://www.ncbi.nlm.nih.gov/geo/ under GEO accession number GSE130489). Altered miRNAs were categorized into pathways involved in human diseases, cellular processes and signal transduction. Six cascades were identified as significantly enriched, including JAK/STAT and TGFB-SMAD. Upregulation of miR-21-3p was validated in all cell lines and primary cells and STAT3 was confirmed as its target. DISCUSSION: Dissimilar miRNA responses may be involved in differences of LIF effects on trophoblastic cell lines.