Long Non-coding RNA HOXA11 Antisense Promotes Cell Proliferation and Invasion and Predicts Patient Prognosis in Serous Ovarian Cancer (original) (raw)

Fig. 1. HOXA11 antisense (HOXA11as) expression in human ovarian tumor tissue. (A) Relative expression of HOXA11as was significantly higher in serous ovarian cancer (SOC) tissues (n=129) than in noncancerous tissues (n=38). HOXA11as expression was determined using quantitative real time polymerase chain reaction with U6 as an internal control. (B) HOXA11as expression was classified into two groups according to the expression level of SOC tissues. Data are expressed as mean±standard deviation. *p < 0.05 vs. non-tumor control.

Fig. 2. Knockdown of HOXA11 antisense (HOXA11as) inhibits serous ovarian cancer cell proliferation. (A) Expression of HOXA11as in human ovarian surface epithelial cell line (HOSE) and six ovarian cancer cell lines determined by quantitative real time polymerase chain reaction (qRT-PCR). (B) Knockdown efficiency was determined by qRT-PCR analysis in OVCA429 and SKOV3 cells. Cells were transfected with HOXA11as-specific siRNA (siHOXA11as) and negative control siRNA (siNC). (C, D) Knockdown of HOXA11as significantly reduced cell proliferation in OVCA429 and SKOV3 cells as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Bars indicate mean±standard deviation of three independent experiments performed in triplicate. *p < 0.05 vs. siNC. siHOXA11as, HOXA11as-specific siRNA.

Fig. 3. HOXA11 antisense (HOXA11as) promotes migration and invasion of ovarian cancer cells. (A, B) Wound healing assay was used to determine migration in HOXA11as-specific siRNA (siHOXA11as)-transfected OVCA429 and SKOV3 cells (×200). (C) Matrigel invasion assay was used to determine invasion after 24 hours in OVCA429 cells. (D) Overexpression of HOXA11as in OVCA429 cells analyzed by quantitative real time polymerase chain reaction. (E, F) Migration and invasion assay after overexpressing HOXA11as expression in OVCA429 cells. Overexpression of HOXA11as in OVCA429 cells increased the invasive capacity after 48 hours. Each assay was performed in triplicate. Data are mean±standard deviation. *p < 0.05 vs. siNC, vector.

Fig. 4. Knockdown of HOXA11 antisense (HOXA11as) inhibits matrix metalloproteinase (MMP) 2, MMP-9, and vascular endothelial growth factor (VEGF) expression in ovarian cancer cells. Protein lysates were obtained from HOXA11as-specific siRNA (siHOXA11as) and negative control siRNA (siNC)-transfected OVCA429 cells 48-hour post-transfection. MMP-2, MMP-9, and VEGF expression were analyzed by quantitative real time polymerase chain reaction (A) and western blotting (B). Each assay was performed in triplicate. Band intensities were quantitated, and MMP-2, MMP-9, and VEGF protein levels were normalized to that of β-actin. Each assay was performed in triplicate. Data are mean±standard deviation. *p < 0.05 vs. siNC.

Fig. 5. Expression of HOXA11 antisense (HOXA11as) knockdown on the epithelial-mesenchymal transition–related genes in OVCA429 cells. OVCA429 cells were transfected with HOXA11as-specific siRNA (siHOXA11as) and negative control siRNA (siNC) for 48 hours. E-cadherin, β-catenin, N-cadherin, vimentin, Snail, and Twist expression were analyzed by quantitative real time polymerase chain reaction (A) and western blotting (B). Each assay was performed in triplicate. Data are mean±standard deviation. *p < 0.05 vs. siNC.

Fig. 6. Correlation of HOXA11 antisense (HOXA11as) expression with patient survival. Kaplan-Meier curves for progression- free survival (A) and overall survival (B) in serous ovarian cancer patients with different expression levels of HOXA11as. (C) Receiver operating characteristic (ROC) curve for prognosis prediction of patients using HOXA11as level. The area under curve (AUC) is shown in plots.