Long non-coding RNA HOTAIR mediates the switching of histone H3 lysine 27 acetylation to methylation to promote epithelial-to-mesenchymal transition in gastric cancer

Abstract

HOX transcript antisense intergenic RNA (HOTAIR), a well‑known long non‑coding RNA, plays an important role in the regulation of epithelial‑to‑mesenchymal transition (EMT). In this study, we propose a novel mechanism through which HOTAIR promotes EMT by switching histone H3 lysine 27 acetylation to methylation at the E‑cadherin promoter, which induces the transcriptional inhibition of E‑cadherin. HOTAIR recruits polycomb repressive complex 2 (PRC2) to catalyze H3K27me3; however, whether HOTAIR is associated with the acetylation of histone H3 lysine 27, a marker of transcriptional activation, and the mechanisms through which HOTAIR triggers the metastasis of gastric cancer (GC) by epigenetic regulation remain largely unknown. In this study, HOTAIR knockdown significantly reversed EMT by increasing the expression of E‑cadherin in GC cells. Additionally, the loss of PRC2 activity induced by HOTAIR knockdown resulted in a global decrease in H3K27 methylation and an increase in H3K27 acetylation. Furthermore, HOTAIR recruits PRC2 (which consists of H3K27 methyltransferase EZH2, SUZ12 and EED), which may inhibit the reaction between the acetyltransferase CBP and H3K27 acetylation. On the whole, the findings of this study suggested that the HOTAIR‑mediated acetylation to methylation switch was associated with the transcriptional inhibition of E‑cadherin. HOTAIR can promote the development of GC through the epigenetic regulation of E‑cadherin, switching the state of the E‑cadherin promoter from the transcriptionally active to the transcriptionally repressive state.

Keywords: HOTAIR; gastric cancer; histone modification; epithelial-to-mesenchymal transition; molecular biomarker.

Figure 1

HOTAIR knockdown inhibits the malignant properties of gastric cancer cells. (A) RT-qPCR was used to detect HOTAIR expression in SGC-7901 and MGC-803 cells infected with Lenti-HOTAIR si or Lenti-HOTAIR (^***P<0.001). (B) Transwell assays indicated that the knockdown of HOTAIR by infection with Lenti-HOTAIR si inhibited the invasion of SGC-7901 and MGC-803 cells (magnification, ×200, ^***P<0.001). (C) Silencing of HOTAIR suppressed the proliferation of SGC-7901 and MGC-803 cells as determined by CCK-8 assay (^*P<0.05 and ^**P<0.01 vs. control).

Figure 2

HOTAIR negatively regulates E-cadherin expression. (A) RT-qPCR was used to detect CDH1 expression in SGC-7901 and MGC-803 cells infected with Lenti-HOTAIR si or Lenti-HOTAIR (^***P<0.001). (B) Western blot analysis was performed to examine the expression of E-cadherin, N-cadherin, snail, slug, twist and β-catenin in the SGC-7901 and MGC-803 cells. All experiments were performed in triplicate with 3 technical replicates. GAPDH was used as a control. (C) Immunofluorescence staining revealed the levels of cytoplasmic E-cadherin following infection with Lenti-HOTAIR si or Lenti-HOTAIR in MGC-803 gastric cancer cells (magnification, ×1,000).

Figure 3

HOTAIR regulates the histone methylation and acetylation of H3K4 and H3K27. Western blot analysis was used to examine the expression levels of H3K4me1/me2/me3/ac and H3K27me1/me2/me3/ac. Histone H3 was used as the nuclear extraction loading control. (A) Infection with Lenti-HOTAIR si or Lenti-HOTAIR regulated the expression of histone H3K4/K27 modification. (B) The gray value of H3K27me3/ac following infection of the gastric cancer cells with Lenti-HOTAIR si or Lenti-HOTAIR.(^**P<0.01 and ^***P<0.001).

Figure 4

HOTAIR is associated with an antagonistic switch between histone H3K27 methylation and acetylation. (A and B) Western blot analysis was used to examine the expression levels of EZH2, SUZ12, CBP, H3K27me3 and H3K27ac in the SGC-7901 and MGC-803 cells infected with Lenti-HOTAIR si, Lenti-HOTAIR, si-EZH2 and si-SUZ12. GAPDH was used as a loading control. (C and D) Immunofluorescence staining revealed the expression of H3K27me3 and H3K27ac located in the nuclei of gastric cancer cells infected with Lenti-HOTAIR si or Lenti- (magnification, ×1,000).

Figure 5

Epigenetic regulation of E-cadherin expression by the methylation and acetylation of H3K27. (A) Genome browser analysis of H3K27me3 and H3K27ac enrichment peaks in the E-cadherin promoter region. (B) ChIP analysis of SGC-7901 and MGC-803 cells infected with Lenti-HOTAIR si was conducted on the E-cadherin promoter. (C) ChIP assay demonstrated that the level of H3K27me3 in the E-cadherin promoter was decreased following infection with Lenti-HOTAIR si (^***P<0.001). (D) ChIP demonstrated that the level of H3K27ac in the E-cadherin promoter was increased following infection with Lenti-HOTAIR si (^***P<0.001). The H3K27me3 or H3K27ac binding to the E-cadherin promoters was assessed by ChIP assays followed by qPCR as described in the Materials and methods.

Figure 6

HOTAIR knockdown inhibits gastric cancer growth in vivo. The 2 mouse groups: the MGC-803/Lenti-NC and MGC-803 Lenti-HOTAIR si groups. (A) Images of and tumor tissue specimens in vivo. (B) The mouse body weights, and tumor volume and weight were monitored in the MGC-803 Lenti-NC and Lenti-HOTAIR si orthotopic gastric cancer models. (C) Representative images of immunohistochemical staining of E-cadherin, H3K27me3 and H3K27ac in tissues from mice with orthotopic tumors derived from MGC-803 cells infected with Lenti-NC or Lenti-HOTAIR si (magnification, ×200).

Figure 7

HOTAIR promotes epithelial-mesenchymal transition (EMT) of gastric cancer by inhibiting the expression of E-cadherin through an antagonistic switch of histone H3K27 acetylation to methylation at the E-cadherin promoter.