Dampening HOTAIR sensitizes the gastric cancer cells to oxaliplatin through miR-195-5p and ABCG2 pathway

Abstract

Long non-coding RNAs (lncRNA) have an extensive role in the progression and chemoresistance of gastric cancer (GC). Deeply study the regulatory role of lncRNAs could provide potential therapeutic targets. The aim of this study is to explore the regulatory role of HOTAIR in the progression and oxaliplatin resistance of GC. The expression of HOTAIR in GC and cell lines were detected by using qRT-PCR. Cell proliferation and apoptosis were analysed by CCK-8, EdU incorporation and flow cytometry. Luciferase reporter assay was used to identify the interaction between HOTAIR and ABCG2 (ATP-binding cassette (ABC) superfamily G member 2, ABCG2) via miR-195-5p. The regulatory functions were verified by using molecular biology experiments. HOTAIR was significantly overexpressed in GC and associated with poor prognosis. Knock-down of HOTAIR inhibited the GC cells proliferation and oxaliplatin resistance, while overexpression of HOTAIR showed opposite functions. Further studies found that HOTAIR acted as a competing endogenous RNA (ceRNA) to absorb miR-195-5p and elevated the expression of ABCG2, which leads to resistance of GC cells to oxaliplatin. Taken together, our findings demonstrated that HOTAIR regulates ABCG2 induced resistance of GC to oxaliplatin through miR-195-5p signalling and illustrate the great potential of developing new therapeutic targets for GC patients.

Keywords: ABCG2; HOTAIR; chemoresistance; gastric cancer.

FIGURE 1

HOTAIR was highly expressed in GC and correlated with poor prognosis. (A–C) Heat map, volcano and scatter map of the significantly differentially expressed lncRNAs between 20 pairs of normal tissues and GC tissues (GSE70880, fold change≥2; p ≤ 0.05). HOTAIR was overexpressed with fold change of 2.06 in gastric cancer. (D) Relative expression of HOTAIR in GC and normal tissues (n = 50). (E–G) Prognosis analysis of overall survival (E), progression‐free survival (F) and post progression survival (G) in HOTAIR high expression and low expression group from Kaplan–Meier database.

FIGURE 2

HOTAIR promotes gastric cancer cells proliferation. (A, B) The efficacy of knockdown and overexpression of HOTAIR. (C, D) CCK‐8 assay of AGS and HGC‐27 cells after knockdown (C) and overexpression (D) of HOTAIR. Data were expressed as the mean ± SD (*p < 0.05; ** p < 0.01; *** p < 0.001).

FIGURE 3

EdU assay of AGS and HGC‐27 cells with knockdown (A, B) and overexpression(C, D) of HOTAIR. Data were expressed as mean ± SD (*p < 0.05).

FIGURE 4

Apoptosis assay of AGS and HGC‐27 with HOTAIR knockdown (A, B) and overexpression (C, D). Data were expressed as mean ± SD (**p < 0.01; ***p < 0.001).

FIGURE 5

HOTAIR elevated the resistance of gastric cancer cells to oxaliplatin. (A–C) The survival of gastric cancer cells treated with increasing concentration of oxaliplatin. EdU incorporation (A, B), CCK‐8 assay (C). (D–G) CCK‐8 assay of AGS (D) and HGC‐27 (F) with HOTAIR knockdown and overexpression (E, G), treated with increasing concentration of oxaliplatin (*p < 0.05; **p < 0.01; ***p < 0.001).

FIGURE 6

Apoptosis assay of HGC‐27 and AGS with HOTAIR knockdown (A, B) and overexpression (C, D), treated with increasing concentration of oxaliplatin. Data were expressed as mean ± SD (***p < 0.001).

FIGURE 7

HOTAIR acts as a ceRNA and competitively sponge miR‐195‐5p. (A) The predicted miR‐195‐5p binding sites in HOTAIR. (B) The luciferase activities in gastric cancer cells. (C–F) The relative expression of miR‐195‐5p in AGS and HGC‐27 with HOTAIR knockdown (C, D) and overexpression (E, F).

FIGURE 8

ABCG2 competitively sponges miR‐195‐5p. (A) The predicted miR‐195‐5p binding sites in ABCG2. (B) The luciferase activities in gastric cancer cells. (C–F) The relative expression of ABCG2 in gastric cancer cells transfected with miR‐195‐5p mimics (C, D) and inhibitors (E, F). (G–J) The relative expression of ABCG2 in gastric cancer cells with HOTAIR knockdown (G, H) and overexpression (I, J).

FIGURE 9

Silencing of ABCG2 partially reduced the proliferation and promoted apoptosis of AGS transfected with homo‐HOTAIR. (A, B) CCK‐8 assay showed that knockdown of ABCG2 partially reduced the proliferation of AGS transfected with homo‐HOTAIR. (C, D) Apoptosis assay showed that the elevated apoptosis rates of AGS in the same above groups.