Overexpression of HER2 and HER3 reverses miR-199a- and miR-125b-inhibited tumor angiogenesis.

<p>(A) Immunoblotting to confirm establishment of an A2780 cell line stably overexpressing HRE2 and OVCAR-3 cell line stably overexpressing HER3 using pBABEpuro and pReceiver-Lv105 vector, respectively. (B) Tube formation assay using HUVEC cells was described as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056647#pone-0056647-g002" target="_blank">Figure 2</a>. (C) Total tube lengths for each treatment were analyzed and presented as mean ± SE (millimeter) from six replicates for each treatment. *Significantly different vs vector+miR-control (P<0.05). (D, E) A2780 and OVCAR cells were transfected pre-miR-control, pre-miR-199a or pre-miR-125b, respectively; and implanted onto the CAMs to perform angiogenesis assay as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056647#pone-0056647-g002" target="_blank">Figure 2</a>. The representative images from each group were shown here. The total number of blood vessels in each group was quantified. *,**Significantly different compared with that of the same cell line transfected with pre-miR-control with *P<0.05 and **P<0.01.</p

MiR-199a and miR-125b suppress tumor angiogenesis associated with reduction of HIF-1α and VEGF expression.

<p>(A) HUVEC cells were cultured in serum free medium overnight and re-suspended in basic EBM-2 medium. To perform the tube formation assay, HUVEC cells were incubated in basic EBM-2 medium; conditioned medium prepared from OVCAR-3 or A2780 transfected with pre-miR-control, pre-miR-125b and pre-miR-199a, respectively. Tube formation was determined under light microscope in 12 h. Total tube length (mm) was presented as mean ± SE from six replicates for each treatment. **Significantly different compared with OVCAR-3 control (P<0.01). ^##Significantly different compared with A2780 control (P<0.01). (B) A2780 cells were transfected with 30 nM pre-miR-125b, pre-miR-199a and pre-miR-control precursor, respectively. After transfection (24 h), 2×10⁶ cells were trypsinized, suspended, and mixed with equal volume of Matrigel, and implanted onto the chicken CAMs of 10-day-old chicken embryos. The branches of blood vessels were counted as the index of angiogenesis was obtained from the CAMs of 8–10 embryos per treatment 96 h after implantation. The data represent as mean ± SE of blood vessel numbers, which were normalized to that of the control. **Significantly different compared with that of the control (P<0.05). (C) A2780 cells and OVCAR-3 cells were cultured under normoxic condition, and transiently transfected as above to analyze HIF-1α and β-actin protein expression by immunoblotting and VEGF mRNA levels (VEGF-165 and VEGF-121 isoforms) by RT-PCR. Quantification was performed by scanning densitometry. The results are obtained from triplicate experiments and presented as mean ± SE. *Significantly difference compared with the same cell lines transfected with miR-cont. (D) VEGF mRNA levels in human ovarian cancer tissues (n = 33) were analyzed by SYBR Green qRT-PCR. The Pearson correlations were analyzed between miR-125b or miR-199a expression and its corresponding VEGF expression in cancer tissues.</p

Overexpression of HER2 or HER3 in ovarian cancer cells reversed miR-199a and miR-125b inhibitory effects on Akt/p70S6K1/HIF-1α/VEGF pathway.

<p>(A, B) A2780 vector, A2780 HER2, OVCAR-3 vector and OVCAR-3 HER3 cells were cultured under normoxic condition, and were transfected with pre-miR control, pre-miR-199a and pre-miR-125b precursor at the final concentration of 30 nM. The cells were harvested and analyzed 70 h after transfection for protein levels of p-Akt, total Akt, p-p70S6K1, p70S6K1, HIF-1α and HIF-1β. (C) RT-PCR was used to assess the VEGF mRNA levels in stable cells transfected with miR-control, miR-199a or miR-125b. (D) Cells were seeded in 12-well plates and transiently co-transfected with human VEGF luciferase reporter, pre-miRNAs and β-galactosidase plasmids. The cells were cultured for 48 h after transfection and relative luciferase activities were measured as described in Method section. (E) A2780 cells stably overexpressing HIF-1α was established by infecting A2780 cells with retrovirus carrying HIF-1α. (F) A2780 cells and A2780/HIF-1α cells were transiently transfected with the indicated miRNAs, and harvested 70 h later to analyze VEGF mRNA levels by RT-PCR as indicated in the figure. (G) A2780 vector control cells and A2780 HIF-1α cells were transfected with pre-miR-control, pre-miR-199a or pre-miR-125b, respectively; then 2×10⁶ cells were used to perform angiogenesis assay as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056647#pone-0056647-g002" target="_blank">Figure 2</a>. *Significantly different compared with that of the vector cell line transfected with pre-miR-control (P<0.05).</p

MiR-199a and miR-125b are down-regulated in ovarian cancer tissues and cells.

<p>(A) The expression levels of miR-199a and miR-125b in human epithelial ovarian carcinoma tissues and normal ovarian tissues determined by RT-PCR. Total of 33 ovarian epithelial cancer tissues and 7 normal ovary tissues were analyzed. Representative samples (n = 6 for each group) are shown. The expression level of U6 was used as loading control. (B) Taqman RT-PCR was performed to assess miR-125b and miR-199a expression levels using 33 ovarian cancer tissues and 7 normal tissues. The bar refers to the mean for each group. The values were normalized to the mean of cancer tissues. *Significantly different compared with that of control (P<0.05). (C) The miR-199a and miR-125b expression levels were analyzed using Taqman qRT-PCR in ovarian cancer cell lines. Relative expression levels were represented as RQ using of 2^−ΔΔCT methods. Each data sample was normalized to the U6 expression level and that in OVCAR-3 cells. Mean ± SE values were from three separate experiments. ^#Significantly different compared with OVCAR-3 (P<0.05). *Significantly different compared with A2780 (P<0.05).</p

HER2 and HER3 protein expression levels in ovarian cancer tissues.

<p>(A) The protein levels of HER2 and HER3 were evaluated in human ovarian cancer tissues and normal ovarian tissues by immunoblotting (6 representatives HER2 or HER3 positive samples are shown for both groups). The level of β-actin was used as loading control. (B) Scanning densitometry was used to quantify the relative expression levels of HER2 or HER3. The scattered graphs showed HER2 or HER3 expression levels in HER2 or HER3 positive samples in normal tissues and cancer tissues. The values were normalized to the average value of normal tissues. (C) Expression levels of HER2 and HER3 in ovarian cancer cell lines SKOV3, A2780, OVCAR-3, and immortalized ovarian epithelial cell lines IOSE397, IOSE396 were analyzed by immunoblotting.</p