Comprehensive analysis of hub genes associated with cisplatin-resistance in ovarian cancer and screening of therapeutic drugs through bioinformatics and experimental validation

Abstract

Background: To identify key genes associated with cisplatin resistance in ovarian cancer, a comprehensive analysis was conducted on three datasets from the GEO database and through experimental validation.

Methods: Gene expression profiles were retrieved from the GEO database. DEGs were identified by comparing gene expression profiles between cisplatin-sensitive and resistant ovarian cancer cell lines. The identified genes were further subjected to GO, KEGG, and PPI network analysis. Potential inhibitors of key genes were identified through methods such as LibDock nuclear molecular docking. In vitro assays and RT-qPCR were performed to assess the expression levels of key genes in ovarian cancer cell lines. The sensitivity of cells to chemotherapy and proliferation of key gene knockout cells were evaluated through CCK8 and Clonogenic assays.

Results: Results showed that 12 genes influenced the chemosensitivity of the ovarian cancer cell line SKOV3, and 9 genes were associated with the prognosis and survival outcomes of ovarian cancer patients. RT-qPCR results revealed NDRG1, CYBRD1, MT2A, CNIH3, DPYSL3, and CARMIL1 were upregulated, whereas ERBB4, ANK3, B2M, LRRTM4, EYA4, and SLIT2 were downregulated in cisplatin-resistant cell lines. NDRG1, CYBRD1, and DPYSL3 knock-down significantly inhibited the proliferation of cisplatin-resistant cell line SKOV3. Finally, photofrin, a small-molecule compound targeting CYBRD1, was identified.

Conclusion: This study reveals changes in the expression level of some genes associated with cisplatin-resistant ovarian cancer. In addition, a new small molecule compound was identified for the treatment of cisplatin-resistant ovarian cancer.

Keywords: Bioinformatics analysis; Cisplatin resistance; Molecular docking; Ovarian cancer; miRNA-mRNA pairs.

Fig. 1

DEGs analysis. A DEGs between the cisplatin-sensitive and cisplatin-resistant groups are shown on a volcano plot. B Venn diagram shows common DEGs

Fig. 2

Functional analysis. A GO terms (Biological Process, BP). B GO terms (Cellular Component, CC). C GO terms (Molecular Function, MF). D KEGG Enrichment. E DEGs were filtered into the PPI network through STRING database

Fig. 3

Prognostic survival analysis. A OS of NDRG1. B PFS, OS and PPS of CYBRD1. C PFS of DYBRD1. D PFS of ERBB4. E PFS, OS and PPS of ANK3. F PFS and PPS of MT2A. G OS and PPS of LRRTM4. H PFS, OS and PPS of EYA4. I PFS, OS and PPS of SLIT2

Fig. 4

Information on the genetic alterations of the hub genes. A The genetic alterations related to the hub genes are shown through a visual summary across a set of ovarian serous cystadenocarcinoma samples. B An overview of the alterations of hub genes in the genomics datasets of ovarian serous cystadenocarcinoma in the TCGA database

Fig. 5

Construction of miRNA-hub gene regulatory network. The collected common miRNA and hub genes were connected to construct a miRNA-gene regulatory network

Fig. 6

In vitro validation of hub genes. A 14 genes in SKOV3 cells and SKOV3/DDP cells were measured by qRT‐PCR. B The expression of NDRG1, CYBRD1 and MT2A genes detected in SKOV3/DDP cells by qRT‐PCR. C Left panel: The cell viability of SKOV3 and SKOV3/DDP cells was assessed using CCK-8 assay at 48 hours.The IC50s of DDP for SKOV3 and SKOV3/DDP cells were 3.431 and 15.76 μM/L, respectively. Right panel: The cell viability of SKOV3/DDP cells and 3 gene knockdown SKOV3/DDP cell lines were assessed using CCK-8 assay at 48 hours D Colony formation of untreated SKOV3/DDP cells and SKOV3/DDP cells+5 μmol DDP and statistical analysis from 3 independent experiments .(****P < 0.0001; ***P < 0.001; **P < 0.01;*P < 0.05, Student’s t-test, Error bars are±SEM)

Fig. 7

Virtual screening of small molecules inhibitors. A The crystal structure of CYBRD1. B the predicted binding modes of photofrin with CYBRD1. C The docking detail between photofrin with CYBRD1. D Analyze thebond-forming relationships between photofrin and CYBRD1. E Calculate the binding free energy for the docked pose