环状RNA FGGY在肝癌中的表达及其对HepG2细胞铁死亡的影响

Expression of FGGY circular RNA in liver cancer and its effect on ferroptosis in HepG2 cells

目的 探讨环状RNA FGGY (circFGGY)在肝癌中的表达以及对肝癌HepG2细胞铁死亡的影响及其相关机制。方法 应用实时荧光定量PCR检测circFGGY在正常肝细胞和肝癌HepG2细胞中的表达;应用RNA免疫沉淀(IP)实验验证musahi RNA结合蛋白2 (MSI2)与circFGGY,以及circFGGY与铁死亡抑制蛋白1 (FSP1)的结合作用;应用甲基化RNA免疫沉淀(MeRIP)实验验证甲基转移酶3 (METTL3)与circFGGY的结合作用;应用CCK-8法、脂质过氧化物试剂盒、C11-BODIPY染色、流式细胞仪、谷胱甘肽试剂盒以及铁测定试剂盒方法,检测沉默circFGGY对肝癌HepG2细胞铁死亡的影响;应用Western blotting检测沉默circFGGY对FSP1蛋白表达水平的影响。结果 circFGGY在肝癌细胞中高表达,外源性敲减其表达能促进HepG2细胞的铁死亡。MSI2能够结合并促进circFGGY的形成。高表达的METTL3促进circFGGY m6A甲基化修饰,进而促进其细胞核质重分布。结论 MSI2可促进circFGGY形成,m6A甲基化修饰增多的circFGGY出核调控FSP1蛋白的表达,进而调控HepG2细胞铁死亡。

Objective To study the expression of FGGY circular RNA(circRNA) in liver cancer,its effect on ferroptosis in HepG2 cells,and its related mechanism. Methods Real-time PCR detected the expression of circFGGY in normal liver cells and HepG2 liver cancer cells. RNA immunoprecipitation(IP) was used to detect the binding of MSI2 and circFGGY,and binding of circFGGY and FSP1.Methylated RNA IP(MeRIP) was used to detect binding of METTL3 and circFGGY. CCK8,lipid mass peroxide,glutathione,and iron assays,C11-BODIPY staining,and flow cytometry were used to detect the effect of silencing circFGGY on ferroptosis in HepG2 cells.The effect of silencing circFGGY on FSP1 protein expression was detected by Western blotting method. Results circFGGY was highly expressed in hepatoma cells. Exogenous knockdown of circFGGY expression promoted ferroptosis in HepG2 cells. MSI2 bound to and promoted the formation of circFGGY. High expression of METTL3 promoted circFGGY m6A methylation modification and its nucleoplasmic redistribution. Conclusion MSI2 promotes the formation of circFGGY. The nuclear export of circFGGY with increased m6A methylation regulates the expression of FSP1 protein and ferroptosis in HepG2 cells.