Long non-coding RNA HOTAIR promotes carcinogenesis and invasion of gastric adenocarcinoma

https://doi.org/10.1016/j.bbrc.2014.07.067Get rights and content

Highlights

  • HOTAIR expression was tested in fifty patients with gastric cancer.

  • Cell proliferation was measured after HOTAIR silencing in gastric cancer cell line.

  • siRNA–HOTAIR suppresses cell invasiveness and capacity of migration.

  • Knock down of HOTAR leads to decreased expression of EMT markers.

  • Inhibition of HOTAIR induces apoptosis and cell cycle arrest.

Abstract

Gastric cancer is one of the major causes of cancer death worldwide; however, the mechanism of carcinogenesis is complex and poorly understood. Long non-coding RNA HOTAIR (HOX transcript antisense RNA) recently emerged as a promoter of metastasis in various cancers including gastric cancer. Here we investigated the impact of HOTAIR on apoptosis, cell proliferation and cell cycle to dissect the carcinogenesis of gastric cancer. We examined the mechanism of invasion and metastasis and analyzed the clinical significance of HOTAIR. Downregulation of HOTAIR was confirmed by two different siRNAs. The expression of HOTAIR was significantly elevated in various gastric cancer cell lines and tissues compared to normal control. si-HOTAIR significantly reduced viability in MKN 28, MKN 74, and KATO III cells but not in AGS cells. si-HOTAIR induced apoptosis in KATO III cells. Lymphovascular invasion and lymph node metastasis were more common in the high level of HOTAIR group. si-HOTAIR significantly decreased invasiveness and migration. si-HOTAIR led to differential expression of epithelial to mesenchymal transition markers. We found that HOTAIR was involved in inhibition of apoptosis and promoted invasiveness, supporting a role for HOTAIR in carcinogenesis and progression of gastric cancer.

Introduction

Gastric cancer is one of the major causes of cancer death worldwide, with almost 990,000 cases detected annually [1]. The incidence of gastric cancer varies with geographic location, and is highest in Eastern Asia including Korea, Japan and China. Despite its prevalence, there is still no curative modality for late-diagnosed gastric cancer. The mechanism of gastric carcinogenesis is complex and poorly understood, and is influenced by both infection with Helicobacter pylori and genetic factors. Gastric cancer appears to be caused by the accumulation of both genetic and epigenetic changes [2]. Non-coding RNAs (ncRNAs) like microRNA are major components of epigenetic regulatory networks, and have been shown to be deregulated in gastric cancer [3], [4], [5], [6].

A new class of ncRNA that have been receiving increased attention are long non-coding RNA (lncRNA), with lengths ranging from 200 bp to 100 kbp [7]. HOX transcript antisense intergenic RNA (HOTAIR) is one of the well-studied lncRNAs that regulate gene expression by mediating the modulation of chromatin structure [7], [8]. HOTAIR acts as a scaffold of histone modification complexes to coordinately interact with PRC2 complex and LSD1 histone modifiers [9]. HOTAIR promotes metastasis of breast cancer through the repression of multiple metastasis suppressor genes by interaction with PRC2 complex [10]. HOTAIR levels are also elevated in both primary and metastasized tumors of multiple other cancer types such as colorectal cancer, hepatocellular carcinoma, nasopharyngeal carcinoma and gastric carcinoma [11], [12], [13], [14].

Altered apoptosis, cell proliferation and cell cycle regulation are key features of carcinogenesis of gastric cancer [2], [15]. Although elevated HOTAIR is known to be related to invasiveness [11], metastasis, and poor prognosis, little is known about its relationship with carcinogenesis in gastric cancer.

In this study, we investigated the impact of HOTAIR on apoptosis, cell proliferation and cell cycle as indicators of the carcinogenesis of gastric cancer. We examined the mechanism of invasion and metastasis and analyzed the clinical significance of HOTAIR in patients with gastric cancer.

Section snippets

Patients and tissue samples

Fifty fresh gastric cancer tissue and paired adjacent gastric tissue samples were obtained from 50 patients who underwent surgical resection for gastric cancer at Severance Hospital, Yonsei University College of Medicine. All samples were frozen in liquid nitrogen immediately after resection and stored at −80 °C until use. The mean age of patients was 60.7 (39–79) years and the male/female ratio was 2.2/1. This study was approved by the Ethics Committee of Yonsei University.

Cell lines and cell culture

A total of 22 gastric

Altered expression of HOTAIR in gastric cancer cell lines and gastric cancer tissues

HOTAIR was expressed at various levels in 22 gastric cancer cell lines compared to normal gastric RNA (Fig. 1A). Nineteen cell lines showed elevated HOTAIR expression compared to normal gastric RNA. Gastric cancer cell lines had an average of 43-fold increase in HOTAIR expression compared to normal gastric RNA. HOTAIR was detected in 48 of 50 gastric cancer tissues and the expression of HOTAIR was significantly higher in cancer tissues compared to adjacent normal tissues (P = 0.007) (Fig. 1B).

The expression of HOTAIR and clinicopathologic characteristics of gastric cancer

Discussion

In this study, HOTAIR was elevated in gastric cancer compared to normal tissue and this finding was supported by our results showing that HOTAIR could inhibit apoptosis in gastric cancer cell lines. We also clearly showed that higher expression of HOTAIR was associated with lymphovascular invasion, lymph node metastasis, advanced stage and poor clinical outcomes. Lastly, HOTAIR promoted the EMT through regulation of E-cadherin, N-cadherin, ZEB1 and Snail.

A considerable number of lncRNAs have

Acknowledgments

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2012R1A1A2001479).

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    This work was supported by the Brain Korea 21 PLUS Project for Medical Science, Yonsei University.

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