DNA methylome profiling identifies novel methylated genes in epithelial ovarian cancer patients with platinum resistance.

AIM: Platinum-based chemotherapy is widely used for epithelial ovarian cancer (EOC). As high as 20-25% of EOC patients will not respond to the initial chemotherapy. Accumulated evidences have implied that DNA methylation may serve as a potential bio-marker for chemotherapy-resistant phenotypic screening; however, the pattern underlying primary platinum resistance remains unclear. METHODS: Reduced representation bisulfite sequencing (RRBS) analysis was performed to identify differences in methylation status between primary platinum-resistant patients Progression free survival (PFS) (PFS < 6 months, n = 8) and extreme sensitive patients (PFS ≥ 24 months, n = 8). The Qubit 3.0 Fluorometer was used for the quantification of RRBS library. The RRBS library was sequenced on Illumina HiSeq2500 sequencer as 50 bp paired-end reads. RESULTS: After screening, 94 valid hyper-/hypo-methylated regions were identified to be located within 94 gene promoter and exon regions (adjusted q ≤ 0.5), which were primarily associated with cell-cell adhesion, B cell activation and lymphocyte activation according to GO analysis. The 19 differentially methylated regions (DMR) located in the promoter region including TRC-GCA11-1, LOC105370912, ANO7P1, DHX4,MSH2, CDCP2, CCNL1, ARHGAP42P2, PRDM13, LOC101928344, USP29, ZIC5,IL1RAPL1, EVX2, ABR, MGRN1, UBALD1, LINC00261, and ISL2 were identified according to the order of P-values from low to high, of which MSH2, LINC00261, MGRN1, ZIC5, EVX2, CCNL1, and DHX40 were presented to play a variety of roles in cancers process based on the previous studies. CONCLUSION: DNA methylome profiling based on RRBS assay is an effective method for screening aberrantly methylated genes in primary platinum-resistant patients, which may serve as a potential epigenetic bio-marker for the prediction of primary platinum resistance.

Deciphering the expression patterns of homologous recombination-related lncRNAs identifies new molecular subtypes and emerging therapeutic opportunities in epithelial ovarian cancer.

Epithelial ovarian cancer (EOC) is the leading killer among women with gynecologic malignancies. Homologous recombination deficiency (HRD) has attracted increasing attention due to its significant implication in the prediction of prognosis and response to treatments. In addition to the germline and somatic mutations of homologous recombination (HR) repair genes, to widely and deeply understand the molecular characteristics of HRD, we sought to screen the long non-coding RNAs (lncRNAs) with regard to HR repair genes and to establish a prognostic risk model for EOC. Herein, we retrieved the transcriptome data from the Genotype-Tissue Expression Project (GTEx) and The Cancer Genome Atlas (TCGA) databases. HR-related lncRNAs (HRRlncRNAs) associated with prognosis were identified by co-expression and univariate Cox regression analyses. The least absolute shrinkage and selection operator (LASSO) and multivariate stepwise Cox regression were performed to construct an HRRlncRNA risk model containing AC138904.1, AP001001.1, AL603832.1, AC138932.1, and AC040169.1. Next, Kaplan-Meier analysis, time-dependent receiver operating characteristics (ROC), nomogram, calibration, and DCA curves were made to verify and evaluate the model. Gene set enrichment analysis (GSEA), immune analysis, and prediction of the half-maximal inhibitory concentration (IC50) in the risk groups were also analyzed. The calibration plots showed a good concordance with the prognosis prediction. ROCs of 1-, 3-, and 5-year survival confirmed the well-predictive efficacy of this model in EOC. The risk score was used to divide the patients into high-risk and low-risk subgroups. The low-risk group patients tended to exhibit a lower immune infiltration status and a higher HRD score. Furthermore, consensus clustering analysis was employed to divide patients with EOC into three clusters based on the expression of the five HRRlncRNAs, which exhibited a significant difference in checkpoints' expression levels and the tumor microenvironment (TME) status. Taken together, the results of this project supported that the five HRRlncRNA models might function as a biomarker and prognostic indicator with respect to predicting the PARP inhibitor and immune treatment in EOC.

The HSP70 gene predicts prognosis and response to chemotherapy in epithelial ovarian cancer.

BACKGROUND: Chemotherapy resistance is an intractable problem in treating patients with epithelial ovarian cancer (EOC). Heat shock proteins (HSPs) act as apoptosis inhibitors and are highly conserved genetically. Most HSPs have strong cytoprotective effects, and their overexpression inhibits apoptosis. This has been demonstrated for HSP70. Heat shock protein 70 (HSP70) expression is abnormally upregulated in malignant cells. Furthermore, HSP70 can inhibit cell death and promote chemotherapeutic resistance. In our study, the relationship between the HSP70 gene and primary chemotherapy resistance and clinical outcome in patients with EOC was explored. METHODS: Quantitative real-time polymerase chain (qRT-PCR) was applied to determine HSP70 messenger RNA (mRNA) levels, and immunohistochemistry assay was conducted to determine HSP70 protein level. HSP70 overexpression was assessed to clarify its role on chemotherapy resistance to cisplatin in SKOV3 cell lines. RESULTS: RT-qPCR assay indicated a strong relationship between HSP70 expression and chemotherapy resistance in patients with EOC. In cultured SKOV3 cells, overexpression of HSP70 inhibited cell sensitivity to cisplatin. Kaplan-Meier analysis demonstrated high HSP70 expression was associated with poor outcome of EOC patients. In multivariate models, high HSP70 expression independently predicted this poor outcome. CONCLUSIONS: HSP70 predicts the prognosis and response to chemotherapy in EOC patients.

Genetic Variation of Glutathione S-Transferase M1 Is Associated with Patients with Ovarian Endometriosis and Endometriosis-Related Primary Infertility.

BACKGROUND: The aim of the study was to investigate the role of the genetic variation of glutathione S-transferase M1 (GSTM1) in the development of ovarian endometriosis and endometriosis-related primary infertility risk. METHODS: This case-control study included 564 women with ovarian endometriosis and 576 normal women in the control group in northern China. The polymorphism of GSTM1 was genotyped by polymerase chain reaction (PCR)/ligase detection reaction method. To assess the biological significance of polymorphisms, the level of GSTM1 mRNA expression in patients' endometrial tissues with different genotypes was detected by quantitative real-time PCR (qRT-PCR). RESULTS: Compared with the positive genotype, the null genotype of GSTM1 was associated with the risk of developing ovarian endometriosis (OR = 1.29, 95% CI = 1.02-1.62). Further analysis showed that patients with a null genotype also had a significantly higher risk of primary infertility than patients with positive genotypes (OR = 1.59, 95% CI = 1.01-2.49). In addition, we found that GSTM1 mRNA expression was present in the endometrial tissue of all patients, but the expression level of patients with a positive genotype was nearly 10 times higher than that of patients with a negative genotype. CONCLUSION: Our results suggest that the GSTM1 polymorphism is not only related to the genetic susceptibility to ovarian endometriosis but also a potential molecular marker of primary infertility in patients with ovarian endometriosis.

Promoter Methylation of the MGRN1 Gene Predicts Prognosis and Response to Chemotherapy of High-Grade Serous Ovarian Cancer Patients.

Aberrant DNA methylation is considered to play a critical role in the chemoresistance of epithelial ovarian cancer (EOC). In this study, we explored the relationship between hypermethylation of the Mahogunin Ring Finger 1 (MGRN1) gene promoter and primary chemoresistance and clinical outcomes in high-grade serous ovarian cancer (HGSOC) patients. The MALDI-TOF mass spectrometry assays revealed a strong association between hypermethylation of the MGRN1 upstream region and platinum resistance in HGSOC patients. Spearman's correlation analysis revealed a significantly negative connection between the methylation level of MGRN1 and its expression in HGSOC. In vitro analysis demonstrated that knockdown of MGRN1 reduced the sensitivity of cells to cisplatin and that expression of EGR1 was significantly decreased in SKOV3 cells with low levels of MGRN1 expression. Similarly, EGR1 mRNA expression was lower in platinum-resistant HGSOC patients and was positively correlated with MGRN1 mRNA expression. Kaplan-Meier analyses showed that high methylation of the MGRN1 promoter region and low expression of MGRN1 were associated with worse survival of HGSOC patients. In multivariable models, low MGRN1 expression was an independent factor predicting poor outcome. Furthermore, low expression of EGR1 was also been confirmed to be significantly related to the poor prognosis of HGSOC patients by Kaplan-Meier. The hypermethylation of the MGRN1 promoter region and low expression of MGRN1 were associated with platinum resistance and poor outcomes in HGSOC patients, probably by altering EGR1 expression.

FOXP4-AS1 is a favorable prognostic-related enhancer RNA in ovarian cancer.

Ovarian cancer (OV) is the main cause of deaths worldwide in female reproductive system malignancies. Enhancer RNAs (eRNAs) are derived from the transcription of enhancers and has attracted increasing attention in cancers recently. However, the biological functions and clinical significance of eRNAs in OV have not been well described presently. We used an integrated data analysis to identify prognostic-related eRNAs in OV. Tissue-specific enhancer-derived RNAs and their regulating genes were considered as putative eRNA-target pairs using the computational pipeline PreSTIGE. Gene expression profiles and clinical data of OV and 32 other cancer types were obtained from the UCSC Xena platform. Altogether, 71 eRNAs candidates showed significant correlation with overall survival (OS) of OV samples (Kaplan-Meier log-rank test, P<0.05). Among which, 23 were determined to be correlated with their potential target genes (Spearman's r > 0.3, P<0.001). It was found that among the 23 prognostic-related eRNAs, the expression of forkhead box P4 antisense RNA 1 (FOXP4-AS1) had the highest positive correlation with its predicted target gene FOXP4 (Spearman's r = 0.61). Moreover, the results were further validated by RT-qPCR analysis in an independent OV cohort. Our results suggested the eRNA FOXP4-AS1 expression index may be a favorable independent prognostic biomarker candidate in OV.