CBX8抑制前列腺癌细胞侵袭的机制研究

doi:10.3969/j.issn.1674-8115.2023.12.005

摘要/Abstract

摘要：

目的·探究染色质组织调节框同源蛋白8（chromobox protein homolog 8，CBX8）在前列腺癌中的生物学功能，并通过转录组及表观修饰分析揭示CBX8在前列腺癌转移中的作用机制。方法·利用cBioPortal数据库对癌症基因组图谱（The Cancer Genome Atlas，TCGA）中前列腺腺癌（prostate adenocarcinoma，PRAD）患者样本数据集进行CBX家族蛋白mRNA表达分析。采用短发夹RNA技术敲低DU145前列腺癌细胞系中的CBX8，通过CCK-8和Transwell实验检测细胞增殖和侵袭水平的变化。使用RNA转录组测序（RNA-seq）分析敲低CBX8后影响的差异表达基因。对这些差异表达基因进行基因集富集分析（Gene Set Enrichment Analysis，GSEA）、基因本体论（Gene Ontology，GO）功能分析以及京都基因和基因组百科全书（Kyoto Encyclopedia of Genes and Genomes，KEGG）信号通路富集分析。通过染色质免疫沉淀测序（ChIP-seq）观察和测定敲低CBX8后基因组H3K27me3甲基化水平的变化。结果·根据对TCGA-PRAD患者样本数据的分析，发现CBX8 mRNA在前列腺癌中高表达。在前列腺癌细胞系DU145中敲低CBX8后，细胞的增殖能力没有显著变化（P>0.05），但其侵袭能力却显著提高（P<0.05）。RNA-seq分析显示CBX8敲低导致750个基因表达上调，951个基因表达下调；其中，与多种肿瘤转移有关的支链氨基酸转氨酶1（branched-chain-amino-acid aminotransferase 1，BCAT1）在敲除CBX8后表达明显上升。GSEA显示表达水平受影响的基因与多梳蛋白复合体1（polycomb repressive complex 1，PRC1）的功能有关。同时，通过GO和KEGG信号通路富集分析发现受影响的生物过程包括转运RNA（transfer RNA，tRNA）氨酰化、DNA复制、氨酰基-tRNA连接酶活性变化以及钙黏蛋白的结合等；特别是在GO功能分析的细胞组分方面富集了与肿瘤转移有关的细胞-基底连接相关基因。利用ChIP-seq对表观修饰的研究显示，在敲低CBX8后全基因组的H3K27me3水平有所下降；并鉴定了97个位于CBX8敲低后转录上调基因附近的位点，其中包括BCAT1转录起始位点。结论·CBX8在人前列腺癌中高表达。CBX8具有抑制肿瘤细胞侵袭的功能。其机制可能是CBX8/PRC1复合体结合于BCAT1转录起始位点并抑制BCAT1转录。

关键词: 染色质组织调节框同源蛋白8, 前列腺癌, 细胞增殖, 肿瘤侵袭

Abstract:

Objective ·To elucidate the regulatory mechanisms of the chromobox protein homolog 8 (CBX8) in prostate cancer metastasis from transcriptome and epigenetic modification perspectives. Methods ·The correlation between the expression of CBX proteins and prostate adenocarcinoma (PRAD) in The Cancer Genome Atlas (TCGA) was examined through an analysis based on cBioPortal database. A stable CBX8 knockdown DU145 prostate cancer cell line was established via short hairpin RNA (shRNA) transfection. Subsequently, the proliferation and invasion of the CBX8 knockdown cells were analyzed by CCK-8 assay and Transwell assay, respectively. Transcriptome changes of the CBX8 knockdown cells were investigated through RNA sequencing (RNA-seq) coupled with Gene Set Enrichment Analysis (GSEA). To further evaluate the functional implications of these transcriptomic alterations, Gene Ontology (GO) for functional analysis was deployed. Moreover, to identify potentially affected signalling pathways, the Kyoto Encyclopedia of Genes and Genomes (KEGG) was utilized for pathway enrichment analysis. Lastly, the levels of H3K27me3, a key histone modification associated with CBX8, in the knockdown cells were determined by chromatin immunoprecipitation sequencing (ChIP-seq). Results ·Bioinformatic analysis with cBioPortal database, based on TCGA-PRAD cohorts, unveiled a high CBX8 mRNA expression in PRAD. Knockdown of CBX8 did not significantly affect the proliferation of DU145 cells (P>0.05), but caused a a significant increase in their invasiveness (P<0.05). The RNA-seq analysis revealed that CBX8 knockdown led to the upregulation of 750 genes and the downregulation of 951 genes. Notably, branched-chain-amino-acid aminotransferase 1 (BCAT1), a gene implicated in the metastasis of various types of cancers, showed a significant increase in expression following CBX8 knockdown. GSEA showed that the expression levels were of the affected genes were related to the functions of the polycomb repressive complex 1 (PRC1). A further investigation using GO and KEGG analyses identified several enriched pathways in the CBX8 knockdown cells, including transfer RNA (tRNA) aminoacylation, DNA replication, changes in aminoacyl-tRNA ligase activity, and cadherin binding. Interestingly, in terms of cell component of GO functional analysis, cell-substrate junction-related genes associated with tumor metastasis appeared to be enriched. ChIP-seq results showed a global decrease in H3K27me3 levels. Significantly, 97 reduced H3K27me3 peaks were found located nearby genes that were upregulated upon CBX8 knockdown, including the transcriptional start site of BCAT1. Conclusion ·CBX8 is highly expressed in prostate cancer. CBX8 suppresses prostate cancer cell invasion, possibly by recruiting the transcriptional repressive PRC1 complex to the transcription site of BCAT1, thereby inhibiting BCAT1 transcription and tumor metastasis.

Key words: chromobox protein homolog 8 (CBX8), prostate cancer, cell proliferation, tumor invasion

中图分类号:

杨万里, 宋娟, 李兵, 劳一敏. CBX8抑制前列腺癌细胞侵袭的机制研究[J]. 上海交通大学学报（医学版）, 2023, 43(12): 1507-1519.

YANG Wanli, SONG Juan, LI Bing, LAO Yimin. Deciphering the suppressive effects of CBX8 on prostate cancer cell invasion[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2023, 43(12): 1507-1519.

图/表 5

图1 CBX家族在前列腺癌中mRNA的表达水平与突变情况

Fig 1 Expression levels of mRNA and mutations of CBX family in prostate cancer

图2 敲低 CBX8 对前列腺癌细胞增殖、侵袭能力的影响Note：A. Western blotting analysis demonstrating the expression levels of CBX8 after its stable knockdown in DU145 cell line. Selected cell lines for subsequent experiments are marked with red box. B. Effects of CBX8 knockdown on the proliferation of DU145 cell line were evaluated via CCK8 assay. C. The impact of CBX8 knockdown on the invasive potential of DU145 cell line was depicted by the Transwell invasion assay. Scale bar=100 μm. D. Statistical analysis derived from the aforementioned Transwell invasion assay results. ns—no statistical significance.

Fig 2 Effects of knocking down of CBX8 on the proliferation and invasion ability of prostate cancer cells

图3 敲低 CBX8 的前列腺癌细胞的基因表达谱变化和GSEA富集分析Note： A. The heatmap of the differentially expressed genes after CBX8 knocking down in DU145 cell line. Color bar means log fold change. B. Volcano plot of differentially expressed genes after CBX8 knocking down in DU145 cell line. C. GSEA showed that genes differentially expressed after CBX8 knocking down were enriched in gene sets of upregulated genes when knocking down PCGF2 (MEL18). D. GSEA showed that genes differentially expressed after CBX8 knocking down were enriched in gene sets of downregulated genes when knocking down PCGF4 (BMI1). Gene sets in Fig 3C and D were obtained from MSigDB (The Molecular Signatures Database). NES—normalized enrichment score.

Fig 3 Gene expression profiling and GSEA of CBX8 knocking down prostate cancer cells

图4 敲低 CBX8 前列腺癌细胞的基因表达谱变化的功能注释与富集分析Note：A. GO biological process (BP) analysis of differentially expressed genes in CBX8 knocking down DU145 cell line. B. GO molecular function (MF) analysis of differentially expressed genes in CBX8 knocking down DU145 cell line. C. GO cell component (CC) analysis of differentially expressed genes in CBX8 knocking down DU145 cell line. D. KEGG analysis of differentially expressed genes in CBX8 knocking down DU145 cell line. P adj—adjusted P value. E. GSEA showed that genes differentially expressed after CBX8 knocking down were enriched in gene sets related to cell cycle. NES—normalized enrichment score. F. Heat map of genes differentially expressed in cell-substrate junction gene set after CBX8 knocking down.

Fig 4 Functional annotation and enrichment analysis of differentially expressed genes in CBX8 knocking down prostate cancer cells

图5 敲低 CBX8 对前列腺癌细胞H3K27me3修饰水平的影响Note：A. The Venn diagram of unique and shared genes between upregulated genes and genes with decreased H3K27me3 peaks after CBX8 knocking down. B. Peak intensity analysis of H3K27me3 in shCT and shCBX8-treated DU145 cells. C. Heatmap analysis of H3K27me3 enrichment in shCT and shCBX8-treated DU145 cells. D. Overview of H3K27me3 occupancy at the BCAT1 promoter region in shCT and shCBX8-treated DU145 cells.

Fig 5 Effect of CBX8 knocking down on H3K27me3 modification in prostate cancer cells

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