The cellular and molecular mechanisms responsible for pregnancy-related disorders remain unclear. tolerance. Problems with the development and maintenance of the placenta can therefore result in pregnancy-related disorders, such as preeclampsia or foetal growth restriction. However, the mechanisms responsible for such pregnancy-related disorders remain poorly comprehended. MicroRNAs (miRNAs) regulate various important physiological and pathological processes, including embryonic development1. The pregnancy-associated chromosome 14 miRNA cluster (C14MC) and chromosome 19 miRNA cluster (C19MC) miRNAs are predominantly expressed in human placental tissue during pregnancy and play a crucial role in placental development2,3. C19MC miRNAs have exhibited buy Fexofenadine HCl an association with buy Fexofenadine HCl preeclampsia caused by abnormal development of placental vessels in early pregnancy4. Furthermore, trophoblast cells may release exosomes made up of C19MC miRNAs, which enable foetoplacentalCmaternal communication by affecting both local and distant target tissues3,5. Pregnancy-associated miRNAs have been identified in the maternal blood circulation6, and we have previously reported that C19MC miRNAs in maternal plasma may serve as a useful biomarker for pregnancy-related disorders7,8,9,10,11. Pregnancy-related disorders are thought to be associated with biological abnormalities of trophoblast cells. However, the use of primary trophoblast cells to study such disorders has been limited by their short life span and poor proliferation expansion As a potential tool for investigating the mechanisms responsible for pregnancy-related disorders, it is usually essential to understand the stability of pregnancy-associated miRNA expression levels in these placenta-derived MSCs. We therefore decided if the expression of pregnancy-associated miRNAs changed during the expansion process. The expression of miR-323-3p in CP-MSCs and CV-MSCs was significantly higher in later (p8) compared with earlier passage (p2) cells (p?0.001 expansion (Fig. 5b,c). Physique 5 Changes in expression levels of pregnancy-associated miRNAs in mesenchymal stem cells (MSCs) during expansion. We also primarily expanded and compared the properties of first- and third-trimester CV-MSCs, and showed that they had comparable morphological features and cellular properties (Fig. 6a,w). However, first-trimester CV-MSCs showed relatively higher expression of miR-323-3p compared with third-trimester CV-MSCs, though the difference was not significant (Fig. 6c), while miR-518b and -517a levels were comparable in cells from both trimesters (Fig. 6c). Physique 6 Mesenchymal stem cells (MSCs) primarily expanded from chorionic villi (CV-MSCs) from first-trimester placental tissues. We decided the effects of siRNA transfection in twice-passaged CP-MSCs and CV-MSCs derived from third-trimester placenta. siRNA transfection had little toxic effect (Fig. 7a). Furthermore, expression levels of miR-518b and miR-323-3p were effectively facilitated and suppressed by transfection with miRNA mimic and miRNA inhibitor, respectively (Fig. 7b). Physique 7 Efficiency of siRNA transfection of twice-passaged mesenchymal stem cells (MSCs) from chorionic plate Rabbit Polyclonal to Fos (CP-MSCs) and chorionic villi (CV-MSCs) from term placentas. Screening for miR-518b target genes associated with pregnancy-related disorders The main function of miRNAs is usually to regulate gene expression via antisense complimentarily to one or more messenger RNAs (mRNAs)17,18,19. We initially screened for miR-518b target genes by transfection of twice-passaged CV-MSCs with an miR-518b mimic. Microarray analysis indicated a number of genes that were up- or down-regulated by the miR-518b buy Fexofenadine HCl mimic. Among the 124 target genes down-regulated more than 2-fold (Supplementary Table S1), two genes (tyrosine hydroxylase: and hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1: and stanniocalcin 1: expansion process (within approximately 60 days). We also compared the expression of C19MC miRNAs between primarily expanded MSCs and their original tissues, and showed lower expression levels in CP-MSCs and CV-MSCs compared with the equivalent original tissues. Given that C19MC miRNAs are highly expressed in trophoblast cells53, it is usually not surprising that they were less enriched in placenta-derived MSCs compared with their original tissues. Various miRNAs, especially pregnancy-associated miRNAs, have been implicated in pregnancy-related disorders, such as preeclampsia and foetal growth restriction8,10,52. MiRNAs have also frequently been used in overexpression or knockdown experiments of targeted genes to elucidate miRNA functions in the placenta. We confirmed the efficiency of siRNA transfection in these primarily expanded placental MSCs, with no buy Fexofenadine HCl obvious toxic effects. By transfection of CV-MSCs with the miR-518b mimic, we screened for potential miR-518b target genes by microarray analysis. miR-518b seems to control multiple target genes located on various chromosomes. Interestingly, some miR-518b target genes were previously exhibited to associate with preeclampsia (e.g., and for down-regulated genes, and and for up-regulated genes)20,21,22,35,36,37,38 and with preeclampsia.