Skip to main content
. 2015 Mar 15;6(13):11342–11356. doi: 10.18632/oncotarget.3604

Figure 1. Hypoxia downregulates miR-199a-3p expression, and the restoration of miR-199a-3p inhibits ovarian cancer invasion induced by hypoxia.

Figure 1

(A) In vitro adhesion assay. Ovarian cancer cells (SKOV3ip1, CaOV3, and RMUG-S; 5 × 104 cells) were plated onto 50 μg/mL fibronectin- or collagen type 1–coated 96-well plates under 20% O2 or 1% O2. After 75 minutes incubation, plates were washed, fixed, and stained with Giemsa solution. The number of adherent cells was counted under a light microscope. Data represent mean ± SEM; n = 5. (B) Matrigel invasion assay. Ovarian cancer cells (5 × 104 cells) were plated in serum-free medium into a modified Boyden chamber system coated with 25 μg Matrigel and the allowed to invade the lower chamber, which contained Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, for 24 hours under 20% O2 or 1% O2. Noninvading cells were removed with a cotton swab, and invading cells on the underside of the filter were counted. Representative images are shown. Bar represents 200 μm. Data represent mean ± SEM; n = 10. (C) Venn diagrams showing microRNAs (miRNAs) commonly differentially expressed between CaOV3 and RMUG-S cells. CaOV3 and RMUG-S cells were incubated under 20% O2 or 1% O2 for 48 hours. Total RNA was collected using TRI Reagent and subjected to TaqMan miRNA RT-PCR arrays. Green circles represent miRNAs which expression levels were decreased by <0.5-fold under hypoxia. Red circles show miRNAs which expression levels were increased by >2.0-fold under hypoxia. (D) Heat Map representing color-coded expression levels of 23 miRNAs commonly differentially expressed under hypoxia. (E) Results of the miRNA real-time RT-quantitative PCR. The expression of miR-199a-3p was downregulated under hypoxia in 5 ovarian cancer cells. The 2−ΔΔCT method was used to determine the relative abundance of miR-199a-3p with respect to RNU6B expression. (F) Matrigel invasion assay. SKOV3ip1 and CaOV3 cells were transfected with either pre-miR-199a-3p or the negative control miRNA, and allowed to invade for 24 hours. The results show that miR-199a-3p inhibits cell invasion induced by hypoxia. *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant.