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Link to original content: http://pubmed.ncbi.nlm.nih.gov/38883382/
RACGAP1 knockdown synergizes and enhances the effects of chemotherapeutics on ovarian cancer - PubMed Skip to main page content
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. 2024 May 15;16(5):2132-2146.
doi: 10.62347/QNZU1402. eCollection 2024.

RACGAP1 knockdown synergizes and enhances the effects of chemotherapeutics on ovarian cancer

Jun Ye  1 , Xiang Zhang  2 , Jia-Xuan Xie  2 , Yue Hou  1 , Wei-Min Fan  1 , Xiao-Qin Wang  1 , Li-Wen Zhang  1 , Xiao-Mei Yang  2 , Jun Li  2 , He Fei  1
Affiliations

RACGAP1 knockdown synergizes and enhances the effects of chemotherapeutics on ovarian cancer

Jun Ye et al. Am J Transl Res. .

Abstract

Among the three most prevalent cancers affecting the female reproductive system, ovarian cancer (OV) ranks as the second most frequently diagnosed. It is important to investigate the genomic complexity of OV to develop diagnostic and therapeutic strategies. Through the utilization of bioinformatics analysis, it was determined that RacGTPase Activating Protein 1 (RACGAP1) holds significant significance in the field of OV chemotherapeutics, an aspect that has not been thoroughly explored in prior investigations. In our study, a notable increase in RACGAP1 expression was detected in ovarian cancer, demonstrating a robust association with clinicopathological features and patient prognosis. In vivo and in vitro testing revealed that RACGAP1 acts synergistically with chemotherapeutics to enhance their effects on ovarian cancer. Furthermore, an interaction between RACGAP1 and the subunit G2 of the condensin II complex, known as non-SMC condensin II complex subunit G2 (NCAPG2), has been identified. Our findings may provide new insight for improving therapeutic strategies for OV.

Keywords: NCAPG2; Ovarian cancer; RACGAP1; chemotherapeutics.

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Conflict of interest statement

None.

Figures

Figure 1
Figure 1
RACGAP1 expression is significantly upregulated in OV. (A) The comparison of RACGAP1 expression in tumor and normal tissues using TCGA data through GEPIA for a comprehensive analysis across multiple cancer types. (B-E) RACGAP1 was significantly upregulated in tumor tissues compared with normal tissues from TCGA and GEO database. (F, G) The expression of RACGAP1 was detected using a tissue microarray. (H) The expression of RACGAP1 was found to be upregulated in 71% of OV patients (high), no change in 18% of OV patients (medium) and downregulated in 11% of OV patients (low). (I, J) An increased level of RACGAP1 mRNA was observed to be significantly associated with reduced OS and PFS. Scale bar indicates 1.5 mm in (F), and 400 µm in (G). RACGAP1, RacGTPase activating protein 1; OV, ovarian cancer; OS, overall survival; PFS, progression-free survival; N, normal tissues; T, tumor tissues. The Student’s t-test was employed to compare the groups, and P value is labeled in the graph.
Figure 2
Figure 2
RACGAP1 knockdown could enhance the cytotoxic effects of cisplatin or Taxol on OV cells in vitro. (A, B) RACGAP1 expression was closely related to mismatch repair, nucleotide excision repair, and drug metabolism cytochrome P450 by GO analysis. (C-E) The GSEA analysis revealed a significant positive association between the expression of RACGAP1 and the signals generated by chemotherapeutic agents used in ovarian cancer treatment. (F) The initial assessment of RACGAP1 expression was conducted in SKOV3, HEY, COV318, HO-8910PM, and OVCAR3 cell lines. The y-axis in (F) illustrates the comparative mRNA expression level of RACGAP1 across different OV cell lines. (G) The interference efficiency of RACGAP1 in SKOV3 and HO-8910PM was detected by real-time PCR. The y-axis in (G) represents the mRNA expression level of RACGAP1 after transfection by siRNA. All the values were normalized to GAPDH. (H-K) Cells were divided into 6 groups: vehicle, vehicle+cisplatin, vehicle+Taxol, vehicle+si-RACGAP1, vehicle+si-RACGAP1 plus cisplatin, and vehicle+si-RACGAP1 plus Taxol. Cell viability of each group was measured at 0, 1, 2, 3, and 4 days. It was indicated that Knockdown of RACGAP1 further enhanced the effects of cisplatin or Taxol on OV cells analyzed by CCK8 assay. RACGAP1, RacGTPase activating protein 1; OV, ovarian cancer; GO, Gene Ontology; GSEA, Gene Set Enrichment Analysis. We employed the Student’s t test to compare groups, with a significance threshold of P < 0.01. Significance levels were indicated as **P < 0.01.
Figure 3
Figure 3
Knockdown of RACGAP1 enhances the effects of cisplatin or Taxol on OV in vivo. A. SKOV3 and HO-8910PM cells transfected with sh-NC or sh-RACGAP1 were subcutaneously inoculated into nude mice. The nude mice were divided into 6 groups (n=6 in each group). The drugs were injected twice a week while the subcutaneous tumor grew to 5 mm3. The dosage of cisplatin given was 25 mg per kilogram, while the dosage of Taxol administered was 10 mg per kilogram. Tumors were harvested and measured in the fifth week. B, C. Tumor volume was measured at 0, 1, 3 and 5 week, respectively. Comparing the tumor volume of these groups, it was known that RACGAP1 knockdown further synergized and enhanced the inhibitory effects of cisplatin or Taxol on OV. D, E. RACGAP1 knockdown could enhance the inhibitory effects of cisplatin or Taxol on the proliferation of OV cells by Ki67 staining. F, G. Reducing the expression of RACGAP1 could enhance the apoptotic effects of cisplatin or Taxol on OV cells, as evidenced by TUNEL staining. Scale bar indicates 100 µm. RACGAP1, RacGTPase activating protein 1; OV, ovarian cancer. We employed the Student’s t test to compare groups, with a significance threshold of P < 0.01. Significance levels were indicated as **P < 0.01.
Figure 4
Figure 4
RACGAP1 interacts with NCAPG2 and regulates PI3K/AKT pathway in cisplatin treated ovarian cancer cells. A. OV patient cohort data obtained from TCGA in the LinkedOmics online database were used to explore RACGAP1-related genes. B. The GEO datasets were used for Pearson correlation analysis to obtain the related genes of RACGAP1 (cor > 0.5). C. NCAPG2 was co-expressed by the Venn tool. D. NCAPG2 may be closely related to RACGAP1 in OV progression. E, F. NCAPG2 is closely related to RACGAP1 in OV tissue detected by qPCR and CO-IP. G, H. The expression of NCAPG2 mRNA was found to be significantly correlated with decreased OS and PFS, as demonstrated by the Kaplan-Meier plotter analysis. I. Double knockout of RACGAP1 and NCAPG2 caused pPI3K and pAKT expression decreased significantly with cisplatin treatment. RACGAP1, RacGTPase activating protein 1; OV, ovarian cancer; NCAPG2, non-SMC condensin II complex subunit G2; OS, overall survival; PFS, progression-free survival. The Student’s t-test was employed to compare the groups, and P value is labeled in the graph.
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