铁死亡抑制蛋白1调控增殖细胞核抗原表达促进乳腺癌细胞增殖

Ferroptosis suppressor protein 1 prompts the proliferation of breast cancer cells by regulating the expression of proliferating cell nuclear antigen

摘要:

目的 探究铁死亡抑制蛋白1(ferroptosis suppressor protein 1, FSP1)在乳腺癌中的表达及其对乳腺癌细胞增殖能力的影响，并初步探索其分子机制。 方法 借助CCLE数据库分析FSP1基因在乳腺癌细胞系中表达水平，构建FSP1沉默或过表达乳腺癌细胞模型；利用Western印迹法、qRT-PCR和细胞免疫荧光染色实验检测FSP1沉默或过表达后FSP1和增殖细胞核抗原(proliferating cell nuclear antigen, PCNA)表达情况；通过集落形成实验和EdU细胞增殖实验检测FSP1沉默或过表达乳腺癌细胞增殖能力，并进一步探索FSP1是否通过调控PCNA表达影响乳腺癌细胞增殖能力。比较FSP1在乳腺癌组织及癌旁正常组织中的表达差异，并利用免疫组织化学染色方法检测了92例三阴性乳腺癌癌组织与癌旁正常组织中FSP1蛋白的表达水平，同时对这92例三阴性乳腺癌癌组织中FSP1和细胞增殖标志物PCNA的表达关系进行了分析。使用K-M plotter在线数据库分析FSP1表达和PCNA表达与乳腺癌患者无进展生存期的关系。 结果 CCLE数据库分析结果显示，MDA-MB-231细胞中FSP1表达较高，而在T47D细胞中表达较低。在T47D细胞中过表达FSP1后，细胞增殖能力明显增强(P<0.001)；而在MDA-MB-231细胞中，沉默FSP1则可以抑制细胞增殖能力(P<0.001)。FSP1可以促进乳腺癌细胞中PCNA表达，过表达FSP1的乳腺癌细胞中，当PCNA表达量被沉默后，细胞增殖能力受到抑制(P<0.001)。在92例三阴性乳腺癌患者的癌组织中，FSP1和PCNA蛋白的表达均高于癌旁正常组织(P<0.001)，而且癌组织中FSP1和PCNA的表达呈正相关(r=0.887,P<0001)。K-M plotter在线数据库的生存分析结果显示，FSP1高表达及PCNA高表达的乳腺癌患者无进展生存期均较短(FSP1: P=0.003 9;PCNA: P<0.001)。

Abstract:

Objective To investigate the expression of ferroptosis suppressor protein 1(FSP1) in breast cancer tissues and its effects on breast cancer proliferation.Methods Using the CCLE database, the gene expression level of FSP1was analyzed in various breast cancer cell lines. The FSP1 silencing or overexpressing breast cancer cellular models were constructed. Then the expression of FSP1 and cell proliferation marker proliferating cell nuclear antigen(PCNA) was detected in the breast cancer cellular models by using Western blot assay, quantitative real-time polymerase chain reaction(qRT-PCR) assay, and cell immunofluorescence staining assay. The proliferation ability of FSP1 silenced or overexpressed breast cancer cells was examined via conducting colony-forming and EdU cell proliferation assays. Further, it was explored if FSP1 mediates the proliferation ability of breast cancer cells through the regulation of PCNA. The difference in FSP1 expression between breast cancer tissues and normal adjacent tissues was checked. The expression of FSP1 protein in cancer tissues and adjacent normal tissues of 92 cases of triple negative breast cancer was detected by immunohistochemical staining, and the relationship between FSP1 and PCNA, a marker of cell proliferation, was analyzed. The progression-free survival in breast cancer patients was analyzed with the K-M plotter online database. Results The CCLE database revealed a high level of FSP1 expression in MDA-MB-231 cells as compared to T47D cells.The proliferation ability of T47D cells was enhanced when the FSP1 was over-expressed(P<0.001), while the FSP1 silencing inhibited the proliferation ability of MDA-MB-231 cells(P<0.001). FSP1 promoted the expression of PCNA in breast cancer cells. In breast cancer cells with overexpressed FSP1, when the expression level of PCNA was silenced, cell proliferation was inhibited(P<0.001). The protein expressions of FSP1 and PCNA were elevated in 92 triple-negative breast cancer patients’ tumor tissues when compared with those in normal adjacent tissues(P<0.001), and the expression of FSP1 and PCNA in the tumor tissues showed a positive correlation(r=0.887, P<0.001). Breast cancer patients with elevated FSP1 and PCNA expressions had a shorter progression-free survival time according to the K-M plotter website(FSP1: P=0.003 9; PCNA: P<0.001). Conclusion FSP1 enhances the proliferation capacity of breast cancer cells by positively regulating PCNA expression. In triple-negative breast cancer, high expression of FSP1 is positively correlated with PCNA expression, and the progression-free survival of breast cancer patients with high expression of FSP1 is shorter.