Three independent cultures have been performed for each time point. Differences in the quantified proliferation rates of JEG-3 cells were statistically assessed by Student’s t-test and considered significant find protocol when P < 0.05. JEG-3 cells were stimulated up to 24 hr with 10 ng/mL LIF, and the expression of miRNAs was assessed at five different time points by real-time PCR. LIF stimulation significantly reduces the expression of miR-141 after 4 and 6 hr compared with the respective basal expression levels. MiR-93 increases at all time points (significantly after 2 and 24 hr of LIF stimulation up to 9.2-fold), and miR-21 increases significantly after 1, 6, and 24 hr with a maximum

Decitabine cost of 19.8-fold. After 4 hr of LIF stimulation, miR-21 expression is significantly reduced compared with that at the aforementioned time points. This

strong reduction has been obvious in each individual experiment. All other changes, including the 2.3-fold increase in let-7g expression at 2 hr LIF stimulation, were not significant (Fig. 1). Because we have observed the most stable LIF-induced changes in miR-141, we decided to analyze its impact on proliferation by silencing and over-expression in JEG-3 cells. Transfection of JEG-3 cells with control substances reduces proliferation at all analyzed time points. Only silencing of miR-141 leads to a block of proliferation, when compared with its respective control, and is, after 48 hr, approximately 50% lower than in cells transfected with a non-genomic control sequence. In all other settings, proliferation is time-dependent. Over-expression of miR-141 does not lead to a further increase in proliferation (Fig. 2). We have observed a significant influence of LIF on the expression of the miRNAs miR-21, miR-93 (upregulation), and miR-141 (downregulation). The strongest effects were observable 4 and 6 hr after stimulation with LIF

when miR-141 was downregulated by far more than 50%. A surprising result was the downregulation of miR-21 after 4 hr of LIF stimulation compared with the earlier and later analyses. Silencing of miR-141 inhibits proliferation of JEG-3 cells, while over-expression does not further induce proliferation. To the best of our knowledge, P-type ATPase thus far, no studies have been published on LIF-induced miRNA in any cell type, but several STAT3-induced miRNAs have been described. LIF is well known to phosphorylate and activate STAT3 in a variety of cells including trophoblastic cells, where it induces invasiveness.3 In our experiments, LIF stimulation of JEG-3 cells significantly increased miR-21 expression. This is compatible with previous reports that in head and neck carcinoma, osteosarcoma, ovarian carcinomas, and others, miR-21 promotes proliferation, migration, and invasion.