Non­viral transfection methods optimized for miRNA delivery to human dermal fibroblasts.

Fibroblasts are beneficial model cells for in vitro studies and are frequently used in tissue engineering. A number of transfection reagents have been employed to deliver microRNAs (miRNAs/miRs) into cells for genetic manipulation. The present study aimed to establish an effective method of transient miRNA mimic transfection into human dermal fibroblasts. The experimental conditions included three different methods: Physical/mechanical nucleofection, and two lipid­based methods, Viromer® Blue and INTERFERin®. To evaluate the impact of these methods, cell viability and cytotoxicity assays were performed. The silencing effect of miR­302b­3p was revealed to alter the expression levels of its target gene carnitine O­octanoyltransferase (CROT) by reverse transcription­quantitative PCR. The present study showed that all selected non­viral transient transfection systems exhibited good efficiency. It was also confirmed that nucleofection, for which a 21.4­fold decrease in the expression of the CROT gene was observed 4 h after 50 nM hsa­miR­302b­3p transfection, was the most effective method. However, these results indicated that lipid­based reagents can maintain the silencing effect of miRNAs up to 72 h after transfection. In summary, these results indicated that nucleofection may be the optimal method for the transport of small miRNA mimics. However, lipid­based methods allow for the use of lower concentrations of miRNA and maintain longer­lasting effects.

The effect of triclosan on hormone secretion and viability of human choriocarcinoma JEG-3 cells.

Triclosan is an antimicrobial agent frequently used in pharmaceuticals and personal care products. We analyzed triclosan for its action on placental secretion of progesterone, estradiol and human chorionic gonadotropin in vitro in the JEG-3 cells. We also investigated its action on cell viability, proliferation and apoptosis. The JEG-3 cells were cultured with increasing doses of triclosan (1×10(-9)-1×10(-4) M) for 24, 48 and 72 h. Triclosan was found to increase estradiol and progesterone secretion after short- and long-term exposure. The stimulatory effect was observed up to 10 µM after short- and long-term exposure to triclosan. In addition, triclosan caused an adverse effect on ß-hCG secretion. The highest doses of triclosan (50 and 100 µM) showed a strong cytotoxic effect. Anti proliferative and pro-apoptotic effects were also observed. Overall, this study demonstrates that triclosan may indirectly disrupt steroidogenesis which may, in turn, affect placental development and consequently fetal growth.

Effects of two isomers of DDT and their metabolite DDE on CYP1A1 and AhR function in human placental cells.

The aim of this study was to investigate the actions of two isomers of DDT (p,p'-DDT, o,p'-DDT) and DDE (p,p'-DDE, o,p'-DDE) on the human placenta. We studied the effects of DDT and its metabolite DDE on CYP1A1 activity and on CYP1A1 and aryl hydrocarbon receptor (AhR) protein expression in placental cells. We used explants from third-trimester human placental tissue and JEG-3 cells, which are first-trimester human placenta cells. The main finding of this study was that the activity of CYP1A1 in the human placenta, measured in terms of ethoxyresorufin-O-deethylase (EROD) activity, was suppressed by treatment of 1, 10, and 100 ng/ml p,p'-DDT, o,p'-DDT, p,p'-DDE and o,p'-DDE. Immunoblot analyses indicated that both isomers of DDT and DDE inhibited the expression of CYP1A1 most effectively at 48 h and/or 72 h after the treatment. Because CYP1A1 activity is mediated by AhR, we evaluated the expression of AhR in placental tissue exposed to DDT and DDE for 1 h to 72 h. Our data showed that DDT and DDE gradually decreased the level of AhR protein, starting at 3 h or 24 h after the start of the experiment. Our results strongly support the involvement of the AhR/CYP1A1 signaling pathway in the mechanism of action of DDT and DDE in the human placenta.