BOLA family genes are the drivers and potential biomarkers of survival in kidney renal clear cell carcinoma patients

Abstract

Objectives: To analyze the diagnostic and prognostic potential of the BOLA gene family in kidney renal clear cell carcinoma (KIRC).

Methods: This study is an observational study. The whole study was carried out at Prince Sattam bin Abdulaziz University, Al-Kharj, Saudi Arabia from January to November 2023. As it involves in silico and in vitro methods, ethical consent was not required. Various in silico and molecular experimental techniques were employed in this study.

Results: Significant up-regulation and hypomethylation of BOLA1, BOLA2, and BOLA3 were observed across 12 KIRC cell lines. Retrospective analysis of the cancer genome atlas program (TCGA)-KIRC cohorts confirmed hypomethylation of these genes in KIRC tissues. The cBioPortal analysis showed minimal genetic alterations, with amplification being the most common. Kaplan-Meier plotter data revealed that high BOLA1, BOLA2, and BOLA3 expression correlated with shorter overall survival and relapse-free survival in KIRC. Tumor-immune system interactions database analysis linked BOLA1 expression to immune and molecular subtypes. Gene silencing of BOLA1, BOLA2, and BOLA3 in 786-0 cells reduced cell growth and proliferation, enhancing wound healing capacity.

Conclusion: BOLA genes may serve as diagnostic and prognostic markers in KIRC, offering insights into therapeutic targets and disease progression.

Keywords: BOLA genes; carcinoma; gene expression regulation; molecular targeted therapy; prognostic biomarkers.

Figure 1

- Profiling the expression, promoter methylation, and receiver operating characteristic (ROC) analysis of BOLA1, BOLA2, and BOLA3 genes in kidney renal clear cell carcinoma (KIRC) and control cell lines. A) Real-time quantitative polymerase chain reaction-based expression profiling of BOLA1, BOLA2, and BOLA3 genes in KIRC and control cell lines. B) ROC curves based on gene expressions of BOLA1, BOLA2, and BOLA3. C) Bisulfite sequencing-based promoter methylation profiling of BOLA1, BOLA2, and BOLA3 genes in KIRC and control cell lines. D) ROC curves based on promoter methylation level of BOLA1, BOLA2, and BOLA3. ***p<0.001. KIRC: kidney renal clear cell carcinoma, AUC: area under the curve

Figure 2

- Validation of BOLA1, BOLA2, and BOLA3 promoter methylation levels and mutational analysis in extended kidney renal clear cell carcinoma (KIRC) cohorts via the OncoDB, UALCAN, GSCA, and cBioPortal databases. A) Methylation analysis via the OncoDB database. B) Methylation analysis via the UALCAN database. C) Methylation analysis via the GSCA database. D-E) Frequency of genetic mutations in BOLA1, BOLA2, and BOLA3 across KIRC patients. F) Effect of the genetic mutations on the overall survival of the KIRC patients. *p<0.05.

Figure 3

- Survival analysis and correlation of BOLA1, BOLA2, and BOLA3 expression with diverse immune and molecular subtypes of kidney renal clear cell carcinoma (KIRC). A) Effect of the up-regulated BOLA1, BOLA2, and BOLA3 on the overall survival (OS) of KIRC patients. B) Effect of the up-regulated BOLA1, BOLA2, and BOLA3 on the relapse-free (RFS) survival of KIRC patients. C) Correlation of BOLA1, BOLA2, and BOLA3 expression with diverse immune subtypes of KIRC. D) Correlation of BOLA1, BOLA2, and BOLA3 expression with diverse molecular subtypes of KIRC. P<0.05.

Figure 4

- Gene enrichment analysis of BOLA1, BOLA2, and BOLA3 using the DAVID tool. A) Cellular component (CC) terms. B) Biological process (BP) terms. C) Molecular Function (MF) terms. D) Kyoto encyclopedia of genes and genomes (KEGG) terms. P<0.05.

Figure 5

- Knockdown of BOLA1, BOLA2, and BOLA3 impairs the growth and metastatic potential and enhances wound healing ability of the 786-O cells. A-B) The transfection efficiency of BOLA1, BOLA2, and BOLA3 was checked using real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot analyses. C) 786-O control and transfected cells were analyzed for colony formation. D-F) Proliferation and wound healing assays. ***P-value of <0.001.