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. 2022 Aug 16;14(16):3959.
doi: 10.3390/cancers14163959.

Transcriptomic Profiling Analysis of Castration-Resistant Prostate Cancer Cell Lines Treated with Chronic Intermittent Hypoxia

Chung Lyul Lee  1 , Minji Lee  2 , Ji Yong Lee  1 , Sin-Hyoung Hong  2   3 , Seung Woo Yang  1 , Ji-Hyeon Min  3   4 , Dong-Eon Lee  2   3 , Joonyoung Baek  3   4 , Chanseul Kim  3   4 , Jae Sung Lim  1 , Ki Hak Song  1 , Ju Hyun Shin  1 , Gun-Hwa Kim  2   3   4
Affiliations

Transcriptomic Profiling Analysis of Castration-Resistant Prostate Cancer Cell Lines Treated with Chronic Intermittent Hypoxia

Chung Lyul Lee et al. Cancers (Basel). .

Abstract

Castration-resistant prostate cancer (CRPC) is still a major concern in men's health, with 375,000 cancer deaths annually. Hypoxia, which is a marked characteristic of advanced solid tumors, has been suggested to induce prostate cancer towards CRPC, metastasis and treatment resistance. To evaluate the effect of hypoxia on prostate cancer, two and five cycles of hypoxia and reoxygenation were administered using 22Rv1 cell lines and denominated as 22Rv1-CI and 22Rv1-PCI, respectively. Cancer cell migration was promoted in 22Rv1-CI compared to controls, and the expression of COL13A1 was significantly up-regulated in 22Rv1-CI according to differentially expressed gene analysis of RNA sequencing among groups. Cancer cell migration was impeded in a wound healing assay after transfecting si-COL13A1. Moreover, the expression of COL13A1 was also higher in the cell line originating from bone metastatic prostate cancer compared to other cell lines. Using the open database GEO, we also confirmed that the expression of COL13A1 was higher in bone metastatic prostate cancer tissue than in localized prostate cancer tissue in patients. Therefore, COL13A1 may be closely related to the bony metastasis of prostate cancer, and our findings may provide valuable information on the pathophysiology of the metastatic niche induced by hypoxia in patients with CRPC.

Keywords: RNA-seq; castration-resistant prostate cancer; hypoxia; hypoxia-resistant cell lines.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Characterization of hypoxia-resistant cell lines. (A) Identification of cell viability in hypoxic environment using MTT assay in 22Rv1 cells or hypoxia-resistant cells (22Rv1-CI and 22Rv1-PCI cell lines). (B) mRNA expression and Western blot for HIF-1α expression in hypoxia-resistant cells (22Rv1-CI and 22Rv1-PCI cell lines) compared with 22Rv1 cells. Data are mean ± SEM. * p < 0.05; ns, not significant.
Figure 2
Figure 2
Overview of transcriptome profiles between 22Rv1 cells and hypoxia-resistant cells. (A) Schematic outline of RNA sequencing in 22Rv1 cells or hypoxia-resistant cells. (B,C) Volcano plot in 22rv1-CI and 22rv1-PCI displaying fold change versus adjusted p-values. Red dots represent the significant DEGs (|FC| > 1.5, p-value < 0.05). (D) GSEA enrichment score curve of hypoxia- or migration-related genes in 22rv1-CI. (E) Top significantly affected canonical pathway analysis in 22rv1-CI. (F) Gene network analysis associated with hypoxia and wound healing signaling in 22rv1-CI. Red, upregulated; green, downregulated. (G) Representative gene networks of Col13A1 in 22rv1-CI. (H) Top significantly affected canonical pathway analysis in 22rv1-PCI. (I) PTEN canonical pathway in 22rv1-PCI. (J) Wnt pathway in 22rv1-PCI.
Figure 2
Figure 2
Overview of transcriptome profiles between 22Rv1 cells and hypoxia-resistant cells. (A) Schematic outline of RNA sequencing in 22Rv1 cells or hypoxia-resistant cells. (B,C) Volcano plot in 22rv1-CI and 22rv1-PCI displaying fold change versus adjusted p-values. Red dots represent the significant DEGs (|FC| > 1.5, p-value < 0.05). (D) GSEA enrichment score curve of hypoxia- or migration-related genes in 22rv1-CI. (E) Top significantly affected canonical pathway analysis in 22rv1-CI. (F) Gene network analysis associated with hypoxia and wound healing signaling in 22rv1-CI. Red, upregulated; green, downregulated. (G) Representative gene networks of Col13A1 in 22rv1-CI. (H) Top significantly affected canonical pathway analysis in 22rv1-PCI. (I) PTEN canonical pathway in 22rv1-PCI. (J) Wnt pathway in 22rv1-PCI.
Figure 2
Figure 2
Overview of transcriptome profiles between 22Rv1 cells and hypoxia-resistant cells. (A) Schematic outline of RNA sequencing in 22Rv1 cells or hypoxia-resistant cells. (B,C) Volcano plot in 22rv1-CI and 22rv1-PCI displaying fold change versus adjusted p-values. Red dots represent the significant DEGs (|FC| > 1.5, p-value < 0.05). (D) GSEA enrichment score curve of hypoxia- or migration-related genes in 22rv1-CI. (E) Top significantly affected canonical pathway analysis in 22rv1-CI. (F) Gene network analysis associated with hypoxia and wound healing signaling in 22rv1-CI. Red, upregulated; green, downregulated. (G) Representative gene networks of Col13A1 in 22rv1-CI. (H) Top significantly affected canonical pathway analysis in 22rv1-PCI. (I) PTEN canonical pathway in 22rv1-PCI. (J) Wnt pathway in 22rv1-PCI.
Figure 2
Figure 2
Overview of transcriptome profiles between 22Rv1 cells and hypoxia-resistant cells. (A) Schematic outline of RNA sequencing in 22Rv1 cells or hypoxia-resistant cells. (B,C) Volcano plot in 22rv1-CI and 22rv1-PCI displaying fold change versus adjusted p-values. Red dots represent the significant DEGs (|FC| > 1.5, p-value < 0.05). (D) GSEA enrichment score curve of hypoxia- or migration-related genes in 22rv1-CI. (E) Top significantly affected canonical pathway analysis in 22rv1-CI. (F) Gene network analysis associated with hypoxia and wound healing signaling in 22rv1-CI. Red, upregulated; green, downregulated. (G) Representative gene networks of Col13A1 in 22rv1-CI. (H) Top significantly affected canonical pathway analysis in 22rv1-PCI. (I) PTEN canonical pathway in 22rv1-PCI. (J) Wnt pathway in 22rv1-PCI.
Figure 3
Figure 3
(A) Cell migration of 22Rv1-CI or 22Rv1-PCI cells compared with 22Rv1 cells. (B) Bar graph represents the percentage of scratch closure. Data are mean ± SEM. * p < 0.05; ns, not significant.
Figure 4
Figure 4
mRNA expression of COL13A1 22Rv1-CI, and 22Rv1-PCI cells compared with 22Rv1 cells. (A) mRNA expression of COL13A1 in 22Rv1 cells or cells with repeated H–R cycles. (B) Bar graph represents the expression of COL13A1. Data are mean ± SEM. * p < 0.05.
Figure 5
Figure 5
Wound healing assay of 22Rv1, 22Rv1-CI, and 22Rv1-PCI cells transfected with si-NC or si-COL13A1. (A) Representative image of wound healing assay in control and hypoxia-resistant cells. (B) Bar graph represents the percentage of scratch closure. (C) Cell viability assay of 22Rv1, 22Rv1-CI, and 22Rv1-PCI cells transfected with si-NC and si-COL13A1. Data are mean ± SEM. * p < 0.05; ns, not significant.
Figure 6
Figure 6
Expression of COL13A1 in LNCaP, DU145, and PC3 prostate cancer cell lines. (A) mRNA expression of COL13A1 in three differently originated prostate cancer cell lines. (B) Bar graph of the relative expression of COL13A1 in several prostate cancer cell lines. (C) Representative image of wound healing assay in control and hypoxia-resistant cells. (D) Bar graph represents the percentage of scratch closure. Data are mean ± SEM. * p < 0.05.
Figure 7
Figure 7
Expression of COL13A1 and COL6A1 in patients with primary localized prostate cancer and bone metastatic prostate cancer (GSE32269). The expression of COL13A1 and COL6A1 was high in DEGs of bone metastatic prostate cancer (cut-off value: log2FC > 2, p-value < 0.05). Data are mean ± SEM. ** p < 0.01, *** p < 0.001.
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