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. 2024 Dec 1;25(23):12929.
doi: 10.3390/ijms252312929.

Integrated Analysis of Single-Cell and Bulk RNA Sequencing Reveals HSD3B7 as a Prognostic Biomarker and Potential Therapeutic Target in ccRCC

Guicen Liu  1 , Qichen Liu  2 , Jiawei Zhao  1 , Ruyue Luo  1 , Yuan Wan  1 , Zhongli Luo  1
Affiliations

Integrated Analysis of Single-Cell and Bulk RNA Sequencing Reveals HSD3B7 as a Prognostic Biomarker and Potential Therapeutic Target in ccRCC

Guicen Liu et al. Int J Mol Sci. .

Abstract

Clear cell renal cell carcinoma (ccRCC) is the most common kidney malignancy, with a poor prognosis for advanced-stage patients. Identifying key biomarkers involved in tumor progression is crucial for improving treatment outcomes. In this study, we employed an integrated approach combining single-cell RNA sequencing (scRNA-seq) and bulk RNA sequencing (bulk RNA-seq) to identify biomarkers associated with ccRCC progression and prognosis. Single-cell transcriptomic data were obtained from publicly available datasets, and genes related to tumor progression were screened using Monocle2. Bulk RNA-seq data for ccRCC were retrieved from The Cancer Genome Atlas (TCGA) and integrated with scRNA-seq data to explore tumor heterogeneity. We identified 3 beta-hydroxy steroid dehydrogenase type 7 (HSD3B7) as a candidate biomarker for ccRCC, associated with poor overall survival, disease-specific survival, and progression-free interval. Elevated HSD3B7 expression correlated with aggressive clinical features such as advanced TNM stages, histologic grades, and metastasis. Functional studies demonstrated that HSD3B7 promotes cell proliferation, migration, and invasion in vitro, while its silencing significantly inhibits tumor growth in vivo. Our findings reveal that HSD3B7 is a novel biomarker for ccRCC, providing insights into its role in tumor progression and potential as a target for therapy. This study highlights the value of integrating scRNA-seq and bulk RNA-seq data to uncover key regulators of tumor biology and lays the foundation for developing personalized therapeutic strategies for ccRCC patients.

Keywords: HSD3B7; bulk RNA-seq; ccRCC; scRNA-seq.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Single-cell transcriptomic atlas following the integration of multiple datasets. (A,C) Kernel density plots illustrating cell type clustering across the integrated datasets, where each point represents a single cell, and each color corresponds to a specific cell type. Each contour line represents areas of different cell densities, with closer lines indicating higher cell densities in that region. (B,D) Violin plots displaying the expression levels of specific marker genes for each cell type, where violins of the same color represent marker genes for the same cell type.
Figure 2
Figure 2
Identification of HSD3B7. (A) Developmental trajectory of epithelial cells based on Monocle2 analysis. Each dot represents a cell, and the lines represent differentiation trajectories. (B) The upper panel displays the distribution of epithelial cells along pseudotime. Heatmap of differentially expressed genes, ordered according to their common expression variation through pseudotime (gene sets 1–5). (C) Expression pattern of the HSD3B7 gene along the pseudotime.
Figure 3
Figure 3
Upregulation of HSD3B7 in ccRCC. Differential expression of HSD3B7 in paired (A) and unpaired (B) normal adjacent and tumor samples from TCGA-KIRC dataset. Each dot represents a sample. Relative expression of HSD3B7 in ccRCC cells compared to normal kidney cells was determined by quantitative real-time PCR (qRT-PCR) (C) and Western blot (WB) (D). *** p < 0.001, **** p < 0.0001.
Figure 4
Figure 4
Association of HSD3B7 expression with survival outcomes and clinical features. (A) Kaplan–Meier curves comparing OS, DSS, and PFI between patients with high and low HSD3B7 expression. (B–E) Expression levels of HSD3B7 across various pathological conditions and primary therapy outcomes. * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 5
Figure 5
Effects of HSD3B7 knockdown on cell proliferation. (A) qRT-PCR indicated a knockdown efficiency exceeding 50% in 769-P cells. (B) WB revealed the knockdown efficiency of HSD3B7 in 769-P cells. (C) The proliferative activity of cells was measured using the CCK-8 assay following knockdown of HSD3B7 expression. (D) The impact of HSD3B7 on the proliferative capacity of 769-P cells was evaluated using a colony formation assay. ** p < 0.01, **** p < 0.0001.
Figure 6
Figure 6
Impact of HSD3B7 knockdown on apoptosis, cell cycle, migration, and invasion. (A,B) Apoptosis and cell cycle alterations in ccRCC cells following HSD3B7 knockdown were assessed using flow cytometry. (C) The migratory and invasive capabilities of 769-P cells under varying transfection conditions were analyzed via Transwell assay. (D) The cell scratch assay revealed a significant decrease in migration ability in the HSD3B7 knockdown group compared to the control group. * p < 0.05, ** p < 0.01, **** p < 0.0001.
Figure 7
Figure 7
Effects of HSD3B7 overexpression on renal carcinoma cell proliferation, colony formation, and migration. (A,B) The relative expression of HSD3B7 in A498 cells transfected with the indicated vectors was assessed using qRT-PCR and WB. (C) The proliferative activity of the cells following HSD3B7 overexpression was measured using the CCK-8 assay. (D) The impact of HSD3B7 on the proliferative capacity of A498 cells was evaluated using a colony formation assay. (E) The effect of HSD3B7 on the migratory capacity of A498 cells was assessed using a wound healing assay. * p < 0.05, ** p < 0.01, **** p < 0.0001.
Figure 8
Figure 8
Effects of HSD3B7 silencing on tumor growth in a xenograft model using nude mice. (A) Representative images of nude mice in each group (n = 6). (B) Representative bioluminescence images of the subcutaneous tumor model in each group of nude mice. (C) Comparison of tumor weights across groups (n = 6). (D) Weekly measurements of tumor xenograft growth curves for nude mice in each group (n = 6). (E) WB analysis of HSD3B7 protein levels in tumor tissues from the nude mouse xenograft model following silencing. ** p < 0.01, **** p < 0.0001.
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