Long non-coding RNA HOTAIR is associated with human cervical cancer progression

Abstract

The functions of many long non-coding RNAs (lncRNAs) in human cancers remain to be clarified. The lncRNA Hox transcript antisense intergenic RNA (HOTAIR) has been reported to reprogram chromatin organization and promote breast and colorectal cancer metastasis, the involvement of lncRNAs in cervical cancer is just beginning to be studied. In the present study, we examined the expression and the functional role of HOTAIR in cervical cancer. HOTAIR expression was determined in cervical cancer tissues (n=111) and corresponding normal tissues (n=40) by using real-time polymerase chain reaction, and its correlation with clinical parameters and prognosis were analyzed. To determine the effect of HOTAIR knockdown and overexpression in cervical cancer cell lines, we used the CCK-8 assay, wound healing migration and matrigel invasion assay. The expression level of HOTAIR in cervical cancer tissues was higher than that in corresponding non-cancerous tissues. High HOTAIR expression correlated with lymph node metastasis, and reduced overall survival. A multivariate analysis showed that HOTAIR was a prognostic factor for predicting cervical cancer recurrence. Knockdown of HOTAIR reduced cell proliferation, migration, and invasion in cervical cancer cell lines. Moreover, HOTAIR regulated the expression of vascular endothelial growth factor, matrix metalloproteinase-9 and epithelial-to-mesenchymal transition (EMT)-related genes, which are important for cell motility and metastasis. Therefore, HOTAIR may promote tumor aggressiveness through the upregulation of VEGF and MMP-9 and EMT-related genes. These findings indicate that HOTAIR may represent a novel biomarker for predicting recurrence and prognosis and serve as a promising therapeutic target in cervical cancer.

Figure 1

Relative HOTAIR expression and its clinical significance. (A) HOTAIR expression was significantly higher in cervical cancer tissues (n=111) than in non-cancerous tissues (n=40). Relative HOTAIR expression was determined using qRT-PCR with U6 as an internal control. Data are expressed as mean ± SD. ^*P<0.05 vs. non-tumor control. (B) Kaplan-Meier overall survival curves of the patients with cervical cancer and different levels of HOTAIR (log-rank test; P=0.035).

Figure 2

Knockdown of HOTAIR inhibits the cell proliferation of cervical cancer cells. (A) Expression of HOTAIR in cervical cancer cells. HOTAIR expression was evaluated using qRT-PCR with U6 as an internal control. (B) Cells were transfected with HOTAIR-specific siRNA and negative control siRNA (siNC), and knockdown efficiency was determined by qRT-PCR analysis. (C) Knockdown of HOTAIR decreases cell proliferation in HeLa, SiHa and CasKi cells. The proliferation of cervical cancer cells transfected with siHOTAIR and negative control siRNA (siNC) was determined using the CCK-8 assay. Bars indicate mean ± SD of three independent experiments performed in triplicate. ^*P<0.05 vs. siNC.

Figure 3

HOTAIR knockdown inhibits the migration and invasion of cervical cancer cells. (A) Wound healing assay was used to determine migration in siHOTAIR-transfected HeLa SiHa and CasKi cells (magnification, ×200). (B) Matrigel invasion assay was used to determine invasion after 48 h in HeLa cells. (C) Overexpression of HOTAIR in SiHa cells analyzed by qRT-PCR. (D) Cell invasion was evaluated using Matrigel invasion chamber. Overexpression of HOTAIR in SiHa cells increased the invasive capacity after 48 h. Each assay was performed in triplicate. Data are mean ± SD. ^*P<0.05 vs. siNC.

Figure 3

HOTAIR knockdown inhibits the migration and invasion of cervical cancer cells. (A) Wound healing assay was used to determine migration in siHOTAIR-transfected HeLa SiHa and CasKi cells (magnification, ×200). (B) Matrigel invasion assay was used to determine invasion after 48 h in HeLa cells. (C) Overexpression of HOTAIR in SiHa cells analyzed by qRT-PCR. (D) Cell invasion was evaluated using Matrigel invasion chamber. Overexpression of HOTAIR in SiHa cells increased the invasive capacity after 48 h. Each assay was performed in triplicate. Data are mean ± SD. ^*P<0.05 vs. siNC.

Figure 4

HOTAIR increases VEGF and MMP-9 expression in cervical cancer cells. Protein lysates were obtained from siHOTAIR and siNC-transfected HeLa cells 48 h post-transfection. (A) VEGF and (B) MMP-9 expression were analyzed by western blotting. (C) VEGF and MMP-9 levels were analyzed by western blotting in HOTAIR overexpression SiHa cells. Band intensities were quantitated, and VEGF and MMP-9 expression were normalized to that of β-actin. (D) VEGF and MMP-9 levels were determined by qRT-PCR in low groups and high HOTAIR expression groups of cervical cancer tissues. Each assay was performed in triplicate. Data are mean ± SD. ^*P<0.05 vs. siNC.

Figure 5

Expression of HOTAIR knockdown on the EMT-related genes in HeLa cells. (A) HeLa cells were transfected with HOTAIR-specific siRNA and siNC for 48 h. E-cadherin, β-catenin, Vimentin, Snail and Twist expression were analyzed by (A) qRT-PCR and (B) western blotting. Each assay was performed in triplicate. Data are mean ± SD. ^*P<0.05 vs. siNC, ^**P<0.001 vs. siNC.