LncRNA XIST promotes human lung adenocarcinoma cells to cisplatin resistance via let-7i/BAG-1 axis

Abstract

Long noncoding RNAs (lncRNAs) have been identified as oncogenes or tumor suppressors that are involved in tumorigenesis and chemoresistance. LncRNA XIST expression is upregulated in several cancers, however, its biologic role in the development of the chemotherapy of human lung adenocarcinoma (LAD) has not been elucidated. This study aimed to observe the expression of LncRNA XIST in LAD and to evaluate its biologic role and clinical significance in the resistance of LAD cells to cisplatin. LncRNA XIST expression was markedly increased in cisplatin-resistant A549/DDP cells compared with parental A549 cells as shown by qRT-PCR. LncRNA XIST overexpression in A549 cells increased their chemosensitivity to cisplatin both in vitro and in vivo by protecting cells from apoptosis and promoting cell proliferation. By contrast, LncRNA XIST knockdown in A549/DDP cells decreased the chemoresistance. We revealed that XIST functioned as competing endogenous RNA to repress let-7i, which controlled its down-stream target BAG-1. We proposed that XIST was responsible for cisplatin resistance of LAD cells and XIST exerted its function through the let-7i/BAG-1 axis. Our findings suggested that lncRNA XIST may be a new marker of poor response to cisplatin and could be a potential therapeutic target for LAD chemotherapy.

Keywords: BAG-1; LncRNA XIST; chemoresistance; human lung adenocarcinoma; let-7i.

Figure 1

. The level of lncRNA XIST expression in LAD cells. (A) qRT-PCR analysis of lncRNA XIST expression levels in LAD patients' tumor tissues; (B) qRT-PCR analysis of lncRNA XIST expression levels in A549 and A549/DDP cells; (C) MTT assay of the IC₅₀ values of A549 and A549/DDP cells to cisplatin; (D) qRT-PCR analysis of lncRNA XIST expression levels in XIST overexpression A549 cells; (E) MTT assay of the IC₅₀ values of XIST overexpression A549 cells to cisplatin; (F) qRT-PCR analysis of lncRNA XIST expression levels in XIST knockdown A549/DDP cells; (G) MTT assay of the IC₅₀ values of XIST knockdown A549/DDP cells to cisplatin. ** P < 0.01, ***P < 0.001.

Figure 2.

The LncRNA XIST promotes human lung adenocarcinoma cells to cisplatin resistance. (A) Flow cytometry analysis of apoptosis of XIST overexpression A549 cells in combination with increasing concentrations of cisplatin (0.0, 4.0, and 8.0 µg/ml); (B) TUNEL assay for cell apoptosis of XIST overexpression A549 cells in combination with increasing concentrations of cisplatin (0.0, 2.0, and 4.0 µg/ml); (C) MTT assay of XIST overexpression A549 cells proliferation with or without 2 µg /ml cisplatin; (D) Colony formation analysis of cell proliferation in combination with increasing concentrations of cisplatin (0.0, 2.0, and 4.0 µg/ml); (E) Flow cytometry analysis of apoptosis of XIST knockdown A549 cells in combination with increasing concentrations of cisplatin (0.0, 4.0, and 8.0 µg/ml); (F) MTT assay of XIST knockdown A549 cells proliferation with or without 2 µg /ml cisplatin; (G) Tumor volumes and (H) tumor weights from xenografts with XIST overexpression A549 cells and negative control A549 cells. *P < 0.05, **P < 0.01.

Figure 3.

Reciprocal repression between XIST and let-7i. (A) Schematic representation of the predicted binding sites between let-7i and XIST, and the mutagenesis design for the reporter assays; (B) qRT-PCR analysis of let-7i expression levels in A549 and A549/DDP cells; (C) Luciferase reporter assay in human embryonic kidney (HEK) 293T cells, co-transfected with the reporter plasmid (or the corresponding mutant reporter) and the indicated miRNAs; (D) Effects of let-7i mimics or inhibitors on XIST expression in A549 cells; (E) Relative let-7i and XIST expression, presented as fold enrichment in Ago2 relative to normal IgG immunoprecipitates. * P < 0.05, **P < 0.01.

Figure 4.

Let-7i/BAG-1 axis mediated the cisplatin resistance of lncRNA XIST on LAD. (A) MTT assays and (B) colony formation assay revealed that knockdown of XIST decreased cell proliferation and increase apoptosis, while anti-let-7i treatment rescued this effect; (C) Schematic representation of the predicted binding sites between let-7i and BAG-1, and the mutagenesis design for the reporter assays; (D) The luciferase assay showed that cells transfected with let-7i had less luciferase activity than those transfected with miR-ctrl. (E) The WB assay showed that cells transfected with let-7i repressed BAG-1 expression; (F) BAG-1 expression in A549 and A549/DDP cells; (G) MTT assays and (H) colony formation assay revealed that knockdown of BAG-1 decreased cell proliferation and increase apoptosis in XIST overexpression A549 cells. *P < 0.05, **P < 0.01.