Over-expressed long noncoding RNA HOXA11-AS promotes cell cycle progression and metastasis in gastric cancer

Abstract

Background: Long noncoding RNAs (lncRNAs) have emerged as critical regulators in a variety of human cancers, including gastric cancer (GC). However, the function and mechanisms responsible for these molecules in GC are not fully understood. In our previous study, we found that GC associated lncRNA HOXA11-AS is significantly upregulated in GC tissues. Over-expressed HOXA11-AS promotes GC cells proliferation and invasion through scaffolding the chromatin modification factors PRC2, LSD1 and DNMT1.

Methods: HOXA11-AS expression levels in GC cells was detected by quantitative real-time PCR (qPCR). HOXA11-AS siRNAs and overexpression vector were transfected into GC cells to down-regulate or up-regulate HOXA11-AS expression. In vitro and in vivo assays were performed to investigate the functional role of HOXA11-AS in GC cells cell cycle progression, invasion and metastasis. RIP and ChIP assays were used to determine the mechanism of HOXA11-AS's regulation of underlying targets.

Results: We found that knockdown of HOXA11-AS induced GC cells G0/G1 phase arrest and suppressed GC cells migration, invasion and metastasis in vivo. Moreover, mechanistic investigation showed that HOXA11-AS could interact with WDR5 and promote β-catenin transcription, bind with EZH2 and repress P21 transcription, and induce KLF2 mRNA degradation via interacting with STAU1.

Conclusions: Taken together, these findings show that HOXA11-AS not only could promote GC cells migration and invasion in vitro, but also promotes GC cells metastasis in vivo, at least in part, by regulating β-catenin and KLF2.

Keywords: Cell cycle progression; Gastric cancer; HOXA11-AS; Metastasis; lncRNA.

Fig. 1

The effect of HOXA11-AS on gastric cancer cells proliferation and cell cycle progression. a qRT–PCR analysis of HOXA11-AS expression in BGC823, SGC7901 cells transfected with HOXA11-AS or NC siRNAs, and in AGS cells transfected with HOXA11-AS over-expression vector. b Growth curves of BGC823, SGC7901 and AGS cells after transfection with HOXA11-AS siRNAs or vector were determined by CCK8 assays. Values represented the mean ± s.e. from three independent experiments. c, d The cell cycle progression of BGC823 and SGC7901 cells was evaluated 48 h after transfection with HOXA11-AS siRNAs or NC using Flow cytometry assays. e The cell cycle progression of AGS cells was evaluated 48 h after transfection with HOXA11-AS vector or empty vector using Flow cytometry assays. The bar chart represented the percentage of cells in G0/G1, S, or G2/M phase, as indicated. f, g The cyclinD1, CDK2, CDK4 and CDK6 protein levels were detected in BGC823 and SGC7901 cells after transfection with HOXA11-AS siRNAs or NC using western blot. *P < 0.05, **P < 0.01

Fig. 2

Knockdown of HOXA11-AS inhibits cell migration and invasion in GC. a, b The effect of HOXA11-AS knockdown on BGC823 cells migration and invasion was determined by transwell assays. c, d The effect of HOXA11-AS knockdown on SGC7901 cells migration and invasion was determined by transwell assays. e, f The effect of HOXA11-AS over-expression on AGS cells migration and invasion was determined by transwell assays. *P < 0.05, **P < 0.01

Fig. 3

HOXA11-AS downregulation inhibits GC cells metastasis in vivo. a Analysis of an experimental metastasis animal model was performed by injecting HOXA11-AS stably downregulated BGC823 cells into nude mice. lungs from mice in each experimental group. b The numbers of metastais tumor nodules on lung surfaces were shown. c Visualization of the HE-stained lung sections. d, e The E-cadherin, N-cadherin, Vimentin and β-catenin protein levels were detected in BGC823 and SGC7901 cells after transfection with HOXA11-AS siRNAs or NC using western blot. f The β-catenin protein levels were detected in BGC823 cells after transfection with HOXA11-AS siRNAs or NC using Immunofluorescence.*P < 0.05, **P < 0.01

Fig. 4

β-catenin, KLF2 and P21 are downstream targets of HOXA11-AS. a HOXA11-AS RNA levels in immunoprecipitates were determined by qRT-PCR. Expression levels of HOXA11-AS RNA were presented as fold enrichment relative to IgG immunoprecipitates. b qRT-PCR analysis of β-catenin, KLF2 and P21 expression in BGC823 and SGC7901 cells after transfection with HOXA11-AS or NC siRNA. c, d The β-catenin, KLF2 and P21 protein levels were detected in BGC823 and SGC7901 after transfection with HOXA11-AS siRNAs or NC using western blot. *P < 0.05, **P < 0.01

Fig. 5

HOXA11-AS promotes β-catenin transcription by binding with WDR5. a, b Western blot analysis of WDR5 protein levels in BGC823 and SGC7901 cells after transfection with WDR5 or NC siRNA. c qRT-PCR analysis of β-catenin expression in BGC823 and SGC7901 cells after transfection with WDR5 or NC siRNA. d ChIP-qPCR analysis of WDR5, and H3K4me3 occupancy in the β-catenin promoter in BGC823 and SGC7901 cells. e ChIP-qPCR analysis of WDR5, and H3K4me3 occupancy in the β-catenin promoter in BGC823 and SGC7901 cells after transfection with HOXA11-AS or NC siRNA. *P < 0.05, **P < 0.01

Fig. 6

HOXA11-AS represses P21 transcription and KLF2 expression by binding with EZH2 and STAU1. a qRT-PCR analysis of STAU1 and KLF2 expression in BGC823 and SGC7901 cells after transfection with STAU1 or NC siRNA. b ChIP-qPCR analysis of EZH2, and H3K27me3 occupancy in the P21 promoter in BGC823 and SGC7901 cells. c ChIP-qPCR analysis of EZH2, and H3K27me3 occupancy in the P21 promoter in BGC823 and SGC7901 cells after transfection with HOXA11-AS or NC siRNA. d qRT-PCR analysis of EZH2 and P21 expression in BGC823 and SGC7901 cells after transfection with EZH2 or NC siRNA. e KLF2 mRNA levels in immunoprecipitates were determined by qRT-PCR. Expression levels of KLF2 mRNA were presented as fold enrichment relative to IgG immunoprecipitates. f KLF2 mRNA levels in immunoprecipitates were determined by qRT-PCR after transfection with HOXA11-AS or NC siRNA. *P < 0.05, **P < 0.01