KHSRP has oncogenic functions and regulates the expression and alternative splicing of DNA repair genes in breast cancer MDA-MB-231 cells

Breast cancer has become the most common type of cancers worldwide. Its high prevalence and malignant features are associated with various environmental factors and molecules. The KH-type splicing regulatory protein (KHSRP) participates in the development of breast cancer, while the underlying mechanisms are largely unknown. In this study, we silenced KHSRP expression in MDA-MB-231 cells by small interfering RNA (siKHSRP), and then assessed its effects on cellular features. Finally, we performed whole transcriptome sequencing (RNA-seq) experiments to explore the downstream targets of KHSRP, and validated their changed pattern using quantitative polymerase chain reaction. We found KHSRP showed higher expression level and was associated with worse prognosis in breast cancer patients. In siKHSRP samples, the proliferation, invasion, and migration abilities were significantly repressed compared with negative control (NC) samples, while the apoptosis level was increased. By investigating the RNA-seq data, we found KHSRP globally regulates the expression and alternative splicing profiles of MDA-MB-231 cells by identifying 1632 differentially expressed genes (DEGs) and 1630 HKSRP-regulated AS events (RASEs). Functional enriched analysis of DEGs demonstrated that cilium assembly and movement and extracellular matrix organization pathways were specifically enriched in up DEGs, consistent with the repressed migration and invasion abilities in siKHSRP cells. Interestingly, the cell cycle and DNA damage and repair associated pathways were enriched in both down DEGs and RASE genes, suggesting that KHSRP may modulate cell proliferation by regulating genes in these pathways. Finally, we validated the changed expression and AS patterns of genes in cell cycle and DNA damage/repair pathways. Expression levels of BIRC5, CCNA2, CDK1, FEN1, FOXM1, PTTG1, and UHRF1 were downregulated in siKHSRP samples. The AS patterns of PARK7, UBE2A, UBE2D3, NPM1, ERCC1, CSNK1E, CENPX, and SWI5 were also dysregulated in siKHSRP samples. In summary, our study comprehensively explored the downstream targets and their functions of KHSRP in breast cancer cells, highlighting the molecular mechanisms of KHSRP on the oncogenic features of breast cancer. The identified molecular targets could be served as potential therapeutic targets for breast cancer in future. Overall design: In this study, we silenced KHSRP expression in MDA-MB-231 cells by small interfering RNA (siKHSRP), and then we performed whole transcriptome sequencing (RNA-seq) experiments to explore the downstream targets of KHSRP.

乳腺癌现已成为全球范围内最常见的恶性肿瘤类型。其高患病率与恶性表型与多种环境因素及分子事件密切相关。KH型剪接调控蛋白（KH-type splicing regulatory protein, KHSRP）参与乳腺癌的发生发展，但其具体分子机制目前尚未完全阐明。
本研究通过靶向KHSRP的小干扰RNA（small interfering RNA, siKHSRP）沉默MDA-MB-231细胞中KHSRP的表达，进而评估其对细胞表型的影响；随后开展全转录组测序（whole transcriptome sequencing, RNA-seq）实验以探究KHSRP的下游调控靶点，并采用定量聚合酶链反应验证靶基因的表达变化。
研究发现，KHSRP在乳腺癌患者中呈高表达，且与不良预后显著相关。与阴性对照（negative control, NC）组相比，siKHSRP组细胞的增殖、侵袭及迁移能力均显著受抑，而细胞凋亡水平则明显升高。
通过分析RNA-seq数据，我们鉴定得到1632个差异表达基因（differentially expressed genes, DEGs）以及1630个KHSRP调控的可变剪接事件（RASEs），证实KHSRP可全局性调控MDA-MB-231细胞的基因表达与可变剪接谱。对差异表达基因的功能富集分析显示，上调差异基因显著富集于纤毛组装与运动、细胞外基质组织等通路，这与siKHSRP组细胞迁移、侵袭能力受抑的表型一致。有趣的是，细胞周期、DNA损伤修复相关通路同时富集于下调差异基因及KHSRP调控可变剪接事件的靶基因中，提示KHSRP可能通过调控上述通路中的基因表达来影响细胞增殖。
最后，我们验证了细胞周期及DNA损伤/修复通路中基因的表达与可变剪接模式变化：siKHSRP组中BIRC5、CCNA2、CDK1、FEN1、FOXM1、PTTG1及UHRF1的表达水平均显著下调；PARK7、UBE2A、UBE2D3、NPM1、ERCC1、CSNK1E、CENPX及SWI5的可变剪接模式同样出现异常。
综上，本研究全面探究了KHSRP在乳腺癌细胞中的下游调控靶点及其功能，阐明了KHSRP影响乳腺癌恶性表型的分子机制，所鉴定得到的分子靶点有望成为未来乳腺癌治疗的潜在靶标。
实验设计：本研究通过小干扰RNA沉默MDA-MB-231细胞中KHSRP的表达，随后开展全转录组测序以探究KHSRP的下游调控靶点。