乳腺癌已成为全球最常见的癌症类型。其高患病率和恶性特征与各种环境因素和分子有关。KH型剪接调节蛋白(KHSRP)参与乳腺癌的发生发展,而潜在的机制在很大程度上是未知的。在这项研究中,我们通过小干扰RNA(siKHSRP)沉默MDA-MB-231细胞中KHSRP的表达,然后评估其对细胞特征的影响。最后,我们进行了全转录组测序(RNA-seq)实验来探索KHSRP的下游靶标,并使用定量聚合酶链反应验证了它们的变化模式。我们发现KHSRP在乳腺癌患者中表达水平较高,并且与预后较差有关。在siKHSRP样本中,扩散,入侵,与阴性对照(NC)样品相比,迁移能力显着抑制,而细胞凋亡水平升高。通过研究RNA-seq数据,我们发现KHSRP通过鉴定1632个差异表达基因(DEGs)和1630个HKSRP调节的AS事件(RASEs),在全球范围内调节MDA-MB-231细胞的表达和可变剪接谱.DEGs的功能富集分析表明,纤毛组装和运动以及细胞外基质组织途径在上DEGs中特异性富集,与siKHSRP细胞中抑制的迁移和侵袭能力一致。有趣的是,细胞周期和DNA损伤和修复相关的途径在DEGs和RASE基因中都富集,提示KHSRP可能通过调节这些途径中的基因来调节细胞增殖。最后,我们验证了细胞周期和DNA损伤/修复途径中基因表达和AS模式的变化。SiKHSRP样品中BIRC5、CCNA2、CDK1、FEN1、FOXM1、PTTG1和UHRF1的表达水平下调。PARK7、ERCC1、CENPX、和UBE2A在siKHSRP样品中也失调,并证实了PCR实验。总之,我们的研究全面探讨了KHSRP在乳腺癌细胞中的下游靶标及其功能,强调KHSRP对乳腺癌致癌特征的分子机制。确定的分子靶标可以作为未来乳腺癌的潜在治疗靶标。
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, ERCC1, CENPX, and UBE2A were also dysregulated in siKHSRP samples and confirmed PCR experiments. 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.