Cancer Res Treat.  2017 Jul;49(3):656-668. 10.4143/crt.2016.263.

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

• Yim GW
• Kim HJ
• Kim LK
• Kim SW
• Kim S
• Nam EJ
• Kim YT

• KMID: 2388311
• DOI: http://doi.org/10.4143/crt.2016.263

Abstract

PURPOSE
The biological function of long non-coding RNAs (lncRNAs) is only partially understood; therefore, in this study, we investigated the expression of the novel HOXA11 antisense (HOXA11as) lncRNA and its oncogenic role in serous ovarian cancer (SOC).
MATERIALS AND METHODS
HOXA11as expression was examined in 129 SOC tissue samples by real time reverse transcription polymerase chain reaction. Clinicopathological factors and patient survival were compared between the high (n=27) and low HOXA11as expression group (n=102). To investigate the role of HOXA11as in cell proliferation, invasion, and migration, HOXA11as expression in ovarian cancer cells was knocked down using RNA interference.
RESULTS
HOXA11as expression in cancer tissue was 77-fold higher than that of noncancerous tissue (p < 0.05). Higher HOXA11as expression was significantly correlated with histological grade (p=0.017) and preoperative cancer antigen 125 (p=0.048). HOXA11as overexpression in SOC cells led to increased cell proliferation, invasion, and migration. Moreover, HOXA11as was associated with the expression of genes involved in cell invasion, migration, and epithelial-mesenchymal transition (EMT), including vascular endothelial growth factor, matrix metalloproteinase 9 (MMP-9), B-catenin, E-cadherin, Snail, Twist, and vimentin. Multivariate analysis revealed that HOXA11as was a prognostic factor of progressive disease and mortality (hazard ratio [HR], 1.730; p=0.043 and HR, 2.170; p=0.033, respectively). Progression-free and overall survival were significantly shorter in patients with high HOXA11as expression.
CONCLUSION
These findings highlight the clinical significance of HOXA11as to predicting the prognosis of SOC patients and suggest its potential in promoting tumor aggressiveness via regulation of vascular endothelial growth factor (VEGF), MMP-9, and EMT-related mechanisms.

Keyword

Cell proliferation; Long noncoding RNA; Ovarian neoplasms; Prognosis

MeSH Terms

Cadherins
Cell Proliferation*
Epithelial-Mesenchymal Transition
Humans
Matrix Metalloproteinase 9
Mortality
Multivariate Analysis
Ovarian Neoplasms*
Polymerase Chain Reaction
Prognosis*
Reverse Transcription
RNA Interference
RNA, Long Noncoding*
Snails
Vascular Endothelial Growth Factor A
Vimentin
Cadherins
Matrix Metalloproteinase 9
RNA, Long Noncoding
Vascular Endothelial Growth Factor A
Vimentin

Figure

• 

  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.

Reference

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