mRNA and methylation profiling of radioresistant esophageal cancer cells: the involvement of Sall2 in acquired aggressive phenotypes

Abstract

Esophageal squamous cell carcinoma (ESCC) is one of the deadliest malignancies worldwide. Radiotherapy plays a critical role in the curative management of inoperable ESCC patients. However, radioresistance restricts the efficacy of radiotherapy for ESCC patients. The molecules involved in radioresistance remain largely unknown, and new approaches to sensitize cells to irradiation are in demand. Technical advances in analysis of mRNA and methylation have enabled the exploration of the etiology of diseases and have the potential to broaden our understanding of the molecular pathways of ESCC radioresistance. In this study, we constructed radioresistant TE-1 and Eca-109 cell lines (TE-1/R and Eca-109/R, respectively). The radioresistant cells showed an increased migration ability but reduced apoptosis and cisplatin sensitivity compared with their parent cells. mRNA and methylation profiling by microarray revealed 1192 preferentially expressed mRNAs and 8841 aberrantly methylated regions between TE-1/R and TE-1 cells. By integrating the mRNA and methylation profiles, we related the decreased expression of transcription factor Sall2 with a corresponding increase in its methylation in TE-1/R cells, indicating its involvement in radioresistance. Upregulation of Sall2 decreased the growth and migration advantage of radioresistant ESCC cells. Taken together, our present findings illustrate the mRNA and DNA methylation changes during the radioresistance of ESCC and the important role of Sall2 in esophageal cancer malignancy.

Keywords: Esophageal squamous cell carcinoma (ESCC); promoter methylation, Sall2; radioresistance.

Fig 1

Clonogenic cell survival curves of the parent and radioresistant esophageal cancer cells. (A) Clonogenic cell survival curves from TE-1 and radioresistant TE-1 (TE-1/R) cells were generated, and D0, Dq and SER values were calculated according to the multi-target single-hit model. (B) Representative clonogenic plates of TE-1 and TE-1/R cells after 0 or 4 Gy irradiation. (C) Clonogenic cell survival curves from Eca-109 and radioresistant Eca-109 (Eca-109/R) cells. (D) Representative clonogenic plates of Eca-109 and Eca-109/R cells after 0 or 4 Gy irradiation. (E) MTT assay of TE-1 cells after 0, 3 and 6 Gy irradiation. (F) MTT assay of Eca-109 cells after 0, 3 and 6 Gy irradiation. Data are normalized to the control cells and presented as the mean ± SEM of three independent experiments, * P < 0.05; ** P < 0.01.

Fig 2

Radioresistant TE-1 and Eca-109 cells showed more aggressive malignancies. (A) Wound-healing assay of TE-1 and TE-1/R cells. (B) Wound-healing assay of Eca-109 and Eca-109/R cells. Wound healing was observed 24 h after the treatment. (C) Induction of apoptosis by radiation in TE-1 and TE-1/R cells. (D) Induction of apoptosis by radiation in Eca-109 and Eca-109/R cells. Cells were treated with 6 Gy irradiation, and the apoptosis was measured using propidium iodide (PI)/Annexin-V double staining. Data are normalized to the control cells and presented as the mean ± SEM of three independent experiments, * P < 0.05; ** P < 0.01.

Fig 3

Radioresistant TE-1 and Eca-109 cells were resistant to cisplatin. Cells were treated with (A) cisplatin, (B) oxaliplatin, (C) 5-FU and (D) paclitaxel for 48 h. MTT assay of cell viability. The data are shown as the mean ± SEM for three independent experiments. Statistical analysis between the groups was determined by ANOVA; *P < 0.05.

Fig 4

Significant pathways affected in the mRNA and methylation profiling. (A) Heat map of gene expression between TE-1 and TE-1/R cells. (B) Predicted significant pathways for dysregulated genes. (C) Heat map of methylation profiling between TE-1 and TE-1/R cells. (D) Predicted significant pathways for the genes with dysregulated promoter methylation.

Fig 5

Methylation status of Sall2 in parent and radioresistant esophageal cancer cells. (A) Western blot analysis of Sall2 in parent and radioresistant esophageal cancer cells. (B) Sequences for methylation analysis of Sall2 gene. CpG dinucleotides are shaded in yellow. Translational start site for Sall2 is shown in red. The arrows indicate primers used for amplification of the CpG-rich region. (C) Schematic map of Sall2 gene. Exon 1 of Sall2, which contains 13 CpG sites, was predicted as a potential CpG island. After bisulfite treatment of DNA, ten clones of each PCR product were sequenced. (D) The methylation percentage of Sall2 CpG sites in the parent, 2 Gy-treated and radioresistant TE-1 cells. Blue and yellow represent the percentage of methylation for each CpG site. (E) The methylation percentage of Sall2 CpG sites in the parent, 2 Gy-treated and radioresistant Eca-109 cells.

Fig 6

The effect of Sall2 overexpression on the apoptosis, migration and cisplatin sensitivity of esophageal cancer cells. (A) Cells were transiently transfected with control vector or Sall2-overexpression vector (pcDNA3.1-Sall2). The expression of Sall2 was validated by Western blot. Apoptosis of (B) TE-1/R and (C) Eca-109/R cells was measured using propidium iodide (PI)/Annexin-V double staining. (D) Wound-healing assay of cells after the indicated transfection in TE-1/R cells. (E) Wound-healing assay of Eca-109/R cells after the indicated transfection. Wound-healing was observed 24 h after the treatment. (F) TE-1 and (G) Eca-109 cells were treated with cisplatin for 24 h. MTT assay of cell viability. The data are shown as the mean ± SEM for three independent experiments. Statistical analysis between the groups was determined by ANOVA; *P < 0.05; ** P < 0.01.