血管抑制蛋白2在三阴性乳腺癌中的功能及其调控可变剪接机制

doi:10.3969/j.issn.1674-8115.2024.12.005

摘要/Abstract

摘要：

目的·探索血管抑制蛋白2（vasohibin-2，VASH2）在三阴性乳腺癌（triple-negative breast cancer，TNBC）中的表达及VASH2通过调控基因表达和可变剪接在TNBC发生发展中的作用机制。方法·通过基因型-组织表达数据库（Genotype-Tissue Expression，GTEx）联合癌症基因组图谱数据库（The Cancer Genome Atlas，TCGA）比较VASH2在TNBC中与正常乳腺组织中的表达差异，并分析VASH2在TNBC中的甲基化水平。在TNBC细胞系MDA-MB-231中对VASH2过表达，并进行转录组测序，分析受VASH2所调控的差异表达基因及可变剪接基因。结果·VASH2在TNBC组织中与正常组织及其他乳腺癌分型比较表达水平显著提高。VASH2基因的低甲基化可能是VASH2在TNBC 中表达上调的原因之一。VASH2过表达后引起81个基因显著差异表达，其中上调的基因23个，下调的基因58个。VASH2过表达后可变剪接水平发生显著变化的基因主要富集在细胞周期、p53信号通路上。结论·VASH2可能通过调控TNBC中基因可变剪接促进其发生和发展。

关键词: 血管抑制蛋白2, 三阴性乳腺癌, 可变剪接, p53信号通路, 细胞周期

Abstract:

Objective ·To explore the role of vasohibin-2 (VASH2) in the regulation of proliferation and metastasis of triple-negative breast cancer (TNBC) cells, and explore the mechanism of VASH2 in the occurrence and development of TNBC through regulation of gene expression and alternative splicing. Methods ·TCGA-GTEx was used to analyze the expression of VASH2 in TNBC. VASH2 methylation levels in TNBC were also analyzed. VASH2 was overexpressed in the MDA-MB-231 human TNBC cell line and transcriptome sequencing was performed. Differentially expressed genes and alternatively spliced genes regulated by VASH2 were analyzed to explore the mechanism of action of VASH2 in TNBC. Results ·VASH2 was significantly overexpressed in TNBC compared to the normal tissues. Hypomethylation of the VASH2 gene was implicated in the upregulation of VASH2 expression in TNBC. Overexpression of VASH2 caused significant differential expression of 81 genes, of which 23 genes were up-regulated and 58 genes were down-regulated. Genes with significantly altered alternative splicing levels due to VASH2 overexpressed were enriched in cell cycle and p53 signaling pathways. Conclusion ·VASH2 regulates the alternative splicing of TNBC oncogenes and promotes TNBC occurrence and development.

Key words: vasohibin-2 (VASH2), triple-negative breast cancer (TNBC), alternative splicing, p53 signaling pathway, cell cycle

中图分类号:

R737.9

王卫, 王红丽, 阿力比亚提·艾尼, 衣力亚尔·肉苏, 阿依努尔, 杨亮. 血管抑制蛋白2在三阴性乳腺癌中的功能及其调控可变剪接机制[J]. 上海交通大学学报（医学版）, 2024, 44(12): 1526-1535.

WANG Wei, WANG Hongli, ALIBIYATI·i Ain, YILIYAER· Rousu, AYI NUER, YANG Liang. Function of vasohibin-2 and the mechanism of alternative splicing in triple-negative breast cancer[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2024, 44(12): 1526-1535.

图/表 6

图1 VASH2 的差异表达Note： A. Differential expression between TNBC and normal breast tissues. B. Differential expression between TNBC and paired cancer-adjacent tissues. C. Comparative analysis between TNBC and nonTNBC. ①P<0.001, ②P=0.035.

Fig 1 Differential expression of VASH2

图2 VASH2 在TNBC中的甲基化分析Note： A. Methylation site correlation analysis in the VASH2 promoter. B. Methylation levels corresponded to VASH2 expression. C. Correlation analysis between the degree of methylation sites and the expression of VASH2. ①P<0.001, ②P=0.011, ③P=0.029, ④P=0.004, ⑤P=0.002.

Fig 2 Methylated analysis of VASH2 in TNBC

图3 验证VASH2在MDA-MB-231细胞中高表达Note： A. RT-qPCR results of OE-VASH2 and the control groups. B. Western blotting results. ①P<0.001, compared with the control groups.

Fig 3 Verification of high expression of VASH2 in MDA-MB-231 cells

图4 VASH2 调控转录组的RNA-seq分析Note： A. Volcano map based on differentially expressed genes (DEGs). B. Heat map of expression levels of DEGs. C. GO function enrichment bar chart. D/E. Bar charts showing KEGG pathway enrichment of downregulated DEGs (D) and upregulated DEGs (E).

Fig 4 RNA-seq analysis of VASH2-regulated transcriptome

图5 MDA-MB-231细胞中 VASH2 调控的可变剪接事件及功能分析Note： A. Classification of RASEs. B?D. Regulation of top 10 enriched GO biological processes for VASH2. E. Top 10 enriched KEGG pathways for VASH2.

Fig 5 RASEs in MDA-MB-231 cells and function analysis

图6 富含细胞凋亡事件的可变剪接基因Note： A. Wayne diagram of the gene set with significantly altered AS expression levels in the cell cycle and p53 signaling pathway. B. ASE of the VASH2-regulated gene TSC2 and IGV-sashimi plot showing A3SS & ES events. C.ASE of the VASH2-regulated gene SMAD2 and IGV-sashimi plot showing ES events. D. ASE of the VASH2-regulated gene CASP8. ①P=0.006, ②P=0.017, ③P=0.027.

Fig 6 Apoptosis-enriched alternative splicing genes

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