文章：

精子相关抗原5敲除降低三阴性乳腺癌MDA-MB-231与BT549细胞对阿霉素及多西他赛的耐药性

Sperm-Associated Antigen 5 Knockout Reduces Doxorubicin and Docetaxel Resistance in Triple-Negative Breast Cancer MDA-MB-231 and BT549 Cells

原文发布日期：24 March 2024

DOI: 10.3390/cancers16071269

类型: Article

开放获取: 是

英文摘要：

Sperm-associated antigen 5 (SPAG5), also known as Astrin, was previously demonstrated as a biomarker for cellular resistance to major breast cancer therapies, including chemo-, endocrine- and targeted therapy. However, the contribution of SPAG5 to anthracycline- and taxane-based chemotherapy in triple-negative breast cancer (TNBC) remains controversial. In the present study, theSPAG5knockout cell model was established by using clustered regularly interspaced palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system in MDA-MB-231 and BT549 TNBC cell lines. The knockout of SPAG5 was confirmed on both gene and protein levels using genomic PCR, DNA sequencing and western blotting. The functional loss of SPAG5 was determined by colony-formation assay. SPAG5-regulated doxorubicin- and docetaxel-resistance was assessed by MTT and apoptosis assays. The results indicated that all theSPAG5knockout MDA-MB-231 and BT549 clones were biallelic, where one allele was replaced by the donor template, and the other allele had the same “T” insertion (indel) adjacent to the cutting sites of gRNAs at the exon 1 boundary, irrespective of the gRNAs and cell lines. The locus of indel interrupted theSPAG5transcription by damaging the GT-AG mRNA processing rule. Deletion of SPAG5 decreased clonogenicity in both MDA-MB-231 and BT549 cells. SPAG5 was able to regulate the resistance and the drug-induced apoptosis of both doxorubicin and docetaxel. In conclusion, recombinant plasmid-based CRISPR-Cas9 technology can be used to delete theSPAG5gene in the TNBC cell lines. SPAG5 has an important role in regulating cell proliferation and doxorubicin- and docetaxel-resistance in MDA-MB-231 and BT549 cells.

摘要翻译： 

精子相关抗原5（SPAG5），亦称Astrin，既往研究已证实其可作为细胞对乳腺癌主要治疗方式（包括化疗、内分泌治疗及靶向治疗）产生耐药的生物标志物。然而，SPAG5在三阴性乳腺癌（TNBC）蒽环类和紫杉类药物化疗中的作用仍存在争议。本研究利用成簇规律间隔短回文重复序列（CRISPR）-CRISPR相关蛋白9（Cas9）系统，在MDA-MB-231和BT549三阴性乳腺癌细胞系中建立了SPAG5基因敲除细胞模型。通过基因组PCR、DNA测序及蛋白质印迹法在基因和蛋白水平验证了SPAG5的敲除效果。采用克隆形成实验确认SPAG5功能缺失，并通过MTT法和细胞凋亡实验评估SPAG5调控多柔比星及多西他赛耐药的作用。结果显示：所有SPAG5敲除的MDA-MB-231和BT549克隆均为双等位基因修饰，其中一个等位基因被供体模板替换，另一个等位基因在外显子1边界gRNA切割位点附近均存在相同的“T”插入（indel），该现象与所用gRNA及细胞系无关。该indel位点通过破坏GT-AG mRNA加工规则而中断SPAG5转录。SPAG5缺失降低了MDA-MB-231和BT549细胞的克隆形成能力。SPAG5能够调控多柔比星和多西他赛的耐药性及药物诱导的细胞凋亡。综上所述，基于重组质粒的CRISPR-Cas9技术可用于敲除三阴性乳腺癌细胞系中的SPAG5基因。SPAG5在调控MDA-MB-231和BT549细胞增殖及多柔比星/多西他赛耐药中具有重要作用。

原文链接：

Sperm-Associated Antigen 5 Knockout Reduces Doxorubicin and Docetaxel Resistance in Triple-Negative Breast Cancer MDA-MB-231 and BT549 Cells