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. 2019 Oct 30;11(1):153.
doi: 10.1186/s13148-019-0748-4.

Hypermethylation of mismatch repair gene hMSH2 associates with platinum-resistant disease in epithelial ovarian cancer

Hua Tian  1   2   3 , Li Yan  1 , Li Xiao-Fei  3 , Sun Hai-Yan  3 , Chen Juan  3 , Kang Shan  4
Affiliations

Hypermethylation of mismatch repair gene hMSH2 associates with platinum-resistant disease in epithelial ovarian cancer

Hua Tian et al. Clin Epigenetics. .

Abstract

Purpose: One major reason of the high mortality of epithelial ovarian cancer (EOC) is due to platinum-based chemotherapy resistance. Aberrant DNA methylation may be a potential mechanism underlying the development of platinum resistance in EOC. The purpose of this study is to discover potential aberrant DNA methylation that contributes to drug resistance.

Methods: By initially screening of 16 platinum-sensitive/resistant samples from EOC patients with reduced representation bisulfite sequencing (RRBS), the upstream region of the hMSH2 gene was discovered hypermethylated in the platinum-resistant group. The effect of hMSH2 methylation on the cellular response to cisplatin was explored by demethylation and knockdown assays in ovarian cancer cell line A2780. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry was employed to examine the methylation levels of hMSH2 upstream region in additional 40 EOC patient samples. RT-qPCR and IHC assay was used to detect the hMSH2 mRNA and protein expression in extended 150 patients.

Results: RRBS assay discovered an upstream region from - 1193 to - 1125 of hMSH2 was significant hypermethylated in resistant EOC patients (P = 1.06 × 10-14). In vitro analysis demonstrated that global demethylation increased cisplatin sensitivity along with a higher expression of the hMSH2 mRNA and protein. Knockdown hMSH2 reduced the cell sensitivity to cisplatin. MALDI-TOF mass spectrometry assay validated the strong association of hypermethylation of hMSH2 upstream region with platinum resistance. Spearman's correlation analysis revealed a significantly negative connection between methylation level of hMSH2 upstream region and its expression. The Kaplan-Meier analyses showed the high methylation of hMSH2 promoter region, and its low expressions are associated with worse survival. In multivariable models, hMSH2 low expression was an independent factor predicting poor outcome (P = 0.03, HR = 1.91, 95%CI = 1.85-2.31).

Conclusion: The hypermethylation of hMSH2 upstream region is associated with platinum resistant in EOC, and low expression of hMSH2 may be an index for the poor prognosis.

Keywords: DNA methylation; Mismatch repair; Prognosis; RRBS; hMSH2.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Effects of 5-aza-dC treatment on the ovarian cancer cellular sensitivity to cisplatin. a The alterations of the methylation level of hMSH2 promoter in A2780 cells after 5-aza-dC treatment (15 μM) for 72 h. b The change of hMSH2 mRNA expression after treated with the increasing concentrations of 5-aza-dC for 72 h. c, d Western bolt assay showed the increase trend of hMSH2 protein in A2780 cells after treated with the increasing concentrations of 5-aza-dC for 72 h. e CCK-8 assays suggested A2780 cells pre-treated with 5-aza-dC (15 μM) for 72 h showed the obvious decreased proliferation rates to cisplatin compared to control cells (DMSO treatment). f The cell apoptosis rates were significantly increased in A2780 pre-treated with 15 μM 5-aza-dC for 72 h than that in control cells (DMSO treatment) at several cisplatin concentrations by flow cytometry. *P < 0.05; **P < 0.01; ***P < 0.001. Each assay was performed in triplicate
Fig. 2
Fig. 2
The alteration of the sensitivity to cisplatin after a knockdown of hMSH2 expression in A2780 cells. a, b RT-qPCR and western blot assay showed the reduced expression of hMSH2 in sh-hMSH2 cells compared to shNC cells. c CCK-8 assays showed a significant increase in the proliferation rates in the shRNA-hMSH2 cells compared with the shNC cells after cisplatin treatment at several concentrations for 24 h. d Flow cytometry showed that the apoptosis rate in the shRNA-hMSH2 cells was significantly lower than that in the shNC cells after exposure to cisplatin at the 20 μM concentration for 24 h. The A2780 cell apoptosis rates in each group. *P < 0.05; **P < 0.01. The experiments were repeated three times
Fig. 3
Fig. 3
The methylation level of hMSH2 promoter was associated with platinum-resistant in EOC patients. a Reduced representation bisulfite sequencing (RRBS) assay showed the region (− 1193 to − 1125 upstream) within the promoter of hMSH2 was hypermethylated in platinum-resistant patients compared with platinum-sensitive patients (P = 1.06 × 10−14). b The methylation of − 1164 CpG site was significantly hypermethylated in platinum-resistant patients compared with platinum-sensitive patients by MALDI-TOF mass. c The mRNA expression of hMSH2 in platinum-resistant patients and platinum-sensitive patients. All experiments were repeated three times. d Images shown the expression of hMSH2 protein in EOC tumor tissues. *P < 0.05; **P < 0.01
Fig. 4
Fig. 4
The high methylation of hMSH2 and its low mRNA expression are associated with poor survival in EOC patients. a Kaplan-Meier analysis of 150 EOC patients’ survival according to the hMSH2 expression. b Kaplan-Meier analysis of PFS and OS according to the hMSH2 methylation level in 40 EOC patients
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