PTEN is a target gene of mmu-miR-141.

<p>Mmu-miR-141 expression (A) and PTEN mRNA level (B) were detected by real-time PCR in endometrial stromal cells transfected with mmu-miR-141 mimic or inhibitor. (C) PTEN protein level was detected by Western blot analysis. N: untreated cells. MC: control of mimic. IC: control of inhibitor. I: inhibitor. M: mimic. (D) EGFP fluorescence reporter vector analysis. The constructs MU-pcDNA3/EGFP/PTEN, pcDNA3/EGFP/PTEN, pcDNA3.1(+), and pcDNA3.1/pri-miR-141 were transfected into HEC-1B cells, and fluorescence was measured after 48 h. *P<0.05 compared with untreated cells.</p

In-situ hybridization to determine mmu-miR-141 localization in the uterus during embryo implantation.

<p>mmu-miR-141 was primarily located in stromal cells. mmu-miR-141 expression was much higher in second decidual zone (SDZ) than that in primary decidual zone (PDZ) on D6 implantation sites. Bluish-violet staining was determined as positive. (×200).</p

Primers used for Real-time RT-PCR.

<p>Primers used for Real-time RT-PCR.</p

Real-time PCR analysis of mmu-miR-141 expression.

<p>The mmu-miR-141 expression level was significantly lower in D6 than in D4 (A). mmu-miR-141 expression in progesterone treated group was the most higher than the other groups (B). *P<0.05.</p

mmu-miR-141 inhibitor inhibited stromal cell growth.

<p>MTT assay revealed reduced cell viability in the mmu-miR-141 inhibitor-transfected stromal cells as compared to the control. This decrease in cell viability was observed at all concentrations of mmu-miR-141 (50, 100, and 150 nM) and at all incubation times (24, 48, and 72 h). *P<0.05, **P<0.01.</p

Expression of mmu-miR-141 and PTEN after injection.

<p>(A) mRNA levels of mmu-miR-141 and PTEN by Real-time PCR. (B) Protein levels of PTEN by western blot. *P<0.05 compared with untreated uteri horns.</p

Effect of mmu-miR-141 inhibitor on cell cycle.

<p>Endometrial stromal cells transfected with mmu-miR-141 inhibitor exhibited S phase arrest, while G1 and G2 phases in these cells remained unaltered. (A) normal endometrial stromal cells. (B) negative control. (C) miR-141inhibitor.</p

Effect of mmu-miR-141 inhibitor on apoptosis of endometrial stromal cells.

<p>Compared with normal endometrial stromal cells and negative control, the apoptotic rate of endometrium stromal cells transfected by mmu-miR-141 inhibitor was significantly increased. (A) normal endometrial stromal cells. (B) negative control. (C) miR-141 inhibitor. (D) quantification of the apoptotic rate. **p<0.01 as compared to the control.</p

Sequences of primers.

<p>Sequences of primers.</p