STAT3-mediated upregulation of LINC00520 contributed to temozolomide chemoresistance in glioblastoma by interacting with RNA-binding protein LIN28B

Abstract

A considerable number of glioblastoma (GBM) patients developed drug resistance to Temozolomide (TMZ) during chemotherapy, resulting in therapeutic failure and tumor recurrence. However, the exact mechanism of TMZ chemoresistance in GBM is still poorly clarified. As a novel identified lncRNA, LINC00520 was located on chromosome 14 and overexpressed in multiple human cancers. This study was designed and conducted to investigate the role and underlying mechanism of LINC00520 in GBM chemoresistance to TMZ. The qRT-PCR assay demonstrated that LINC00520 was significantly overexpressed in TMZ-sensitive and/or TMZ-resistant GBM cells (P < 0.001). The silencing of LINC00520 markedly reduced the cell viability, suppressed colony formation, induced cell apoptosis and G1/S phase arrest in TMZ-resistant cells (P < 0.001). In contrast, overexpression of LINC00520 conferred TMZ-resistant phenotype of GBM cells in vitro (P < 0.001). The orthotopic xenograft model was established and the results indicated that the volume of tumor xenografts in vivo was markedly inhibited by TMZ treatment after the silencing of LINC00520 (P < 0.001). Luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay revealed a strong affinity of transcription factor STAT3 to the promoter regions of LINC00520, suggesting that STAT3 mediated the aberrant expression of LINC00520 in GBM. Further experiments demonstrated that LINC00520 could interact with RNA-binding protein LIN28B to inhibit autophagy and reduce DNA damage, thereby contributing to TMZ chemoresistance in GBM. These findings suggested that STAT3/LINC00520/LIN28B axis might be a promising target to improve TMZ chemoresistance of GBM.

Keywords: Chemoresistance; Glioblastoma (GBM); LIN28B; LINC00520; STAT3; Temozolomide (TMZ).

Fig. 1

The expression of LINC00520 was increased in TMZ-resistant cells and its silencing improved TMZ chemoresistance in GBM. A The expression of LINC00520 in primary and recurrent GBM tissues was detected by FISH. B The expression levels of LINC00520 in TMZ-sensitive and TMZ-resistant cells were detected by qRT-PCR assay. C The efficiency of LINC00520 knockdown in U251/TMZ and SKMG-1/TMZ cells was detected after transfecting with siRNA for LINC00520. D LINC00520 knockdown decreased the cell viability of U251/TMZ and SKMG-1/TMZ under TMZ treatment (200 µM). E LINC00520 knockdown suppressed colony formation of U251/TMZ and SKMG-1/TMZ under TMZ treatment (200 µM). The effect of LINC00520 knockdown on cell apoptosis of U251/TMZ and SKMG-1/TMZ was investigated by flow cytometry (F) and TUNEL assay (G), respectively. H The distribution of cell cycle in U251/TMZ and SKMG-1/TMZ cells transfected with si-LINC00520 or si-NC were analyzed using flow cytometer

Fig. 2

Overexpression of LINC00520 contributed to TMZ chemoresistance of GBM cells. A The efficiency of LINC00520 overexpression in U251 and SKMG-1 cells was detected by qRT-PCR analysis. B The viability of GBM cells treated with TMZ was significantly increased by LINC00520 overexpression. C LINC00520 overexpression promoted colony formation of U251 and SKMG-1 cells treated with 200 µM TMZ. The effect of LINC00520 overexpression on cell apoptosis of GBM was analyzed by flow cytometry (D) and TUNEL assay (E), respectively. F LINC00520 overexpression elevated the proportion of G1 phase in U251 and SKMG-1 cells treated with 200 µM TMZ

Fig. 3

STAT3 promoted the transcription of LINC00520 via binding to its promoter. A Luciferase reporter assays revealed that LINC00520 expression in TMZ-sensitive and TMZ-resistant cells could be regulated by transcription factor STAT3. B ChIP assay further supported that STAT3 was a key regulator of LINC00520 transcription (PCR 1 was conducted using the specific primer for LINC00520, and PCR 2 was conducted using the control primer)

Fig. 4

LINC00520 contributed to TMZ chemoresistance by interacting with LIN28B. A LIN28B was overexpressed in recurrent GBM samples compared to primary GBM samples. B RNA immunoprecipitation assay revealed that LINC00520 was markedly enriched in both TMZ-sensitive and TMZ-resistant cells using anti-LIN28B antibody, suggesting an interaction between LINC00520 and LIN28B. qRT-PCR (C) and Western blot assay (D) consistently demonstrated that the expression of LIN28B was positively related to LINC00520 expression in GBM cells

Fig. 5

LIN28B contributed to TMZ chemoresistance by inhibiting cell autophagy and reducing DNA-damage response. A The effects of LINC00520/LIN28B axis on cell autophagy of GBM were measured by immunofluorescence labeled with GFP-LC3. B The expression of autophagy-related proteins, including LC3II, LC3I and Beclin-1, were detected by Western blot. C The alkaline comet assay indicated that LINC00520/LIN28B axis contributed to TMZ chemoresistance in GBM via reducing DNA damage response

Fig. 6

Silencing of LINC00520 improved the response to TMZ chemotherapy in vivo. A Representative images of intracranial xenografts originated from U251/TMZ cells transfected with si-LINC00520 vector or si-NC vector under TMZ treatment. B The volume of tumor xenografts were evidently reduced by transfecting with si-LINC00520, suggesting that silencing of LINC00520 improve TMZ chemoresistance of GBM cells in vivo