Knockdown of long non-coding RNA HOTAIR suppresses tumor invasion and reverses epithelial-mesenchymal transition in gastric cancer

Abstract

Background: Over-expression of long non-coding RNA HOTAIR has been reported in several types of cancer. Yet its involvement in gastric cancer (GC) has not been well understood. The aim of present study was to examine the expression pattern of HOTAIR in GC patients, then, explore its role in promoting cancer invasion and underlying molecular mechanism.

Methods: The expression level of HOTAIR in the tumor specimens of GC patients was quantified by Realtime RT-PCR. The correlation between HOTAIR level and clinicopathological factors as well as prognosis was then examined. Down-regulation of HOTAIR by RNA interference was applied to investigate its roles in tumor invasiveness via the view of Epithelial-to-mesenchymal transition (EMT).

Results: The expression level of HOTAIR in cancer tissues was higher than that in adjacent noncancerous tissues. Expression level of HOTAIR was significantly correlated with lymph node metastasis and TNM stage. Furthermore, high expression level of HOTAIR was a predictor of poor over-all survival in GC patients. In vitro, inhibition of HOTAIR in GC cells could reduce invasiveness, as well as the expression of MMP1 and MMP3. In addition, suppression of HOTAIR could reverse EMT process.

Conclusions: HOTAIR could act as a potential predictor for over-all survival in patients with GC. Inhibition of HOTAIR could reduce invasiveness and reverse EMT process in GC cells, indicating the potential role of HOTAIR in GC diagnostics and therapeutics.

Keywords: Epithelial-mesenchymal Transition; Gastric cancer; HOTAIR; Long non-coding RNA; Lymphatic node metastasis..

Fig 1

HOTAIR expression and its clinical significance. (A) The relative expression of HOTAIR was measured by real-time RT-PCR in tumorous and adjacent non-tumorous tissues. (B) The relative expression of HOTAIR was measured in various cell lines, including one immortalized gastric cell lines (GES-1) and 5 GC cell lines (AGS, SGC-7901, MKN-45, HGC-27, MGC-803). (C) The relative expression of HOTAIR mRNA in LNs with or without metastasis. (D) ROC analysis of HOTAIR expression in predicting the existence of LNs metastasis.

Fig 2

Expression level of EMT- and metastasis-related genes in two groups of patients. (A) The Heat-map representation of the EMT-related genes. (B) The Heat-map representation of the metastasis-related genes. Genes are plotted from the minimal level of expression (green) to the maximal level (red).

Fig 3

Effect of HOTAIR knockdown on the invasiveness. (A) Knockdown of HOTAIR expression in AGS cells by siRNAs was confirmed by real-time RT-PCR. (B) Transwell matrigel invasion assays of AGS cells after transfected with siRNA-2 and siRNA-3. (C) Western-blot tested the expression level of some MMPs in the AGS cells after HOTAIR knockdown.

Fig 4

Effect of HOTAIR knockdown on the EMT progress. (A) Morphological change of AGS after transfected with HOTAIR-siRNA-3. (B) Western-blot analysis of phenotypic markers after HOTAIR knockdown in GC cells. (C) Western-blot analysis of EMT-related TFs after HOTAIR knockdown in GC cells. (D) Confocal microscopy analysis of phenotypic markers in AGS cells. The green signal represented the staining of corresponding proteins, and the red signal represented the nuclear DNA staining by PI. (Magnification:×1200).

Fig 5

Effect of exogenous snail or twist on invasiveness and EMT of GC cells. (A) Transwell matrigel invasion assays of AGS cells after transfected with negative control, HOTAIR-siRNA-3 alone or HOTAIR-siRNA-3 combined with exogenous snail or twist. (B) The efficiency of transfections was confirmed by real-time RT-PCR. (C) Realtime RT-PCR analysis of phenotypic markers of AGS cells after transfected with negative control, HOTAIR-siRNA-3 alone or. HOTAIR-siRNA-3 combined with exogenous snail (※: the difference, compared with control group, was significant).

Fig 6

Overall survival curves of GC patients according to the HOTAIR expression levels. (A) Kaplan-Meier survival curves of the whole cohort of 83 patients (B) Kaplan-Meier survival curves of the patients in N2 stage. (C) Kaplan-Meier survival curves of the patients in N3a stage.