WBSCR22基因调控人恶性肿瘤细胞铁死亡的研究

Study on the Regulation of Ferroptosis in Cancer Cells by WBSCR22 Gene

关键词:  WBSCR22  铁死亡  肿瘤细胞  脂质过氧化  Fe²⁺

Key words:  WBSCR22  ferroptosis  cancer cell  lipid peroxidation  Fe²⁺

摘要:

目的 研究WBSCR22是否参与调控细胞铁死亡。方法 设计合成靶向人WBSCR22基因的siRNA (siWBSCR22)，瞬时转染敲低WBSCR22 mRNA；RT-qPCR、Western blotting分别检测WBSCR22 mRNA、蛋白表达水平；铁死亡诱导剂Erastin、RSL3单独或联合抗氧化剂NAC、坏死抑制剂NSA、凋亡抑制剂ZVAD-FMK、自噬抑制剂3-MA作用于人结直肠癌细胞系HCT116和RKO、人肺癌细胞系H460、人肝癌细胞系SMMC7721，CCK8法检测细胞死亡率；丙二醛法、菲罗嗪法分别检测细胞脂质过氧化水平、Fe²⁺浓度。结果 siWBSCR22瞬时转染显著降低WBSCR22 mRNA及蛋白水平；WBSCR22敲低显著促进Erastin、RSL3诱导的HCT116、RKO、H460、SMMC7721细胞铁死亡；加入NAC后显著降低Erastin、RSL3诱导的WBSCR22敲低细胞铁死亡，而加入NSA、ZVAD-FMK、3-MA对Erastin、RSL3诱导的WBSCR22敲低细胞铁死亡无影响；Erastin、RSL3诱导后细胞内的Fe²⁺浓度无明显变化，脂质过氧化水平显著增加。结论 WBSCR22基因参与调控肿瘤细胞铁死亡。

Abstract:

OBJECTIVE To investigate whether WBSCR22 was involved in ferroptosis. METHODS The siRNA targeting human WBSCR22 gene(siWBSCR22) was designed and synthesized, which was transient transfected to knockdown WBSCR22 mRNA. The level of WBSCR22 mRNA and protein were measured by RT-qPCR, Western blotting, respectively. The human colorectal cancer cell lines HCT116, RKO, lung cancer cell lines H460, and hepatoma cell line SMMC7721 were treated with the ferroptosis inducer Erastin, RSL3 alone or in combination with antioxidant NAC, necrosis inhibitor NSA, apoptosis inhibitor ZVAD-FMK, autophagy inhibitor 3-MA, and cell death rate was measured by CCK8. In addition, the cellular lipid peroxidation was measured by MDA assay and the cellular concentration of Fe²⁺ was measured by Fe assay. RESULTS Transient transfection of siWBSCR22 significantly reduced the mRNA and protein levels of WBSCR22. WBSCR22 knockdown significantly promoted the ferroptosis induced by Erastin and RSL3 in HCT116, RKO, H460, and SMMC7721 cells; the addition of NAC significantly reduced ferroptosis, while NSA, ZVAD-FMK, and 3-MA had no effect on ferroptosis. Erastin or RSL3 treatment significantly increased lipid peroxidation in cancer cells, which led to ferroptosis. However, Erastin or RSL3 did not alter the Fe²⁺ concentration in cells. CONCLUSION WBSCR22 gene regulates ferroptosis in cancer cells.