吡咯啉-5-羧酸合成酶在人肝癌组织中表达及其对人肝癌细胞系铁死亡的调控作用观察

Expression of pyrroline-5-carboxylate synthase in hepatocellular carcinoma tissues and its regulatory effect on ferroptosis in human hepatoma cell lines

目的 观察吡咯啉-5-羧酸合成酶(Pyrroline-5-carboxylate synthase,P5CS)在肝细胞癌(Hepatocellular car-cinoma,HCC)组织中的表达变化及其对人肝癌细胞系铁死亡的调控作用。方法 (1)HCC及癌旁组织P5CS检测:选择10例HCC患者的癌组织及癌旁组织,采用免疫组化法检测HCC及癌旁组织P5CS蛋白;(2)P5CS对人肝癌细胞系Li-7、Huh-7铁死亡的调控作用观察:取对数生长期Li-7、Huh-7,用si ALDH18A1[乙醛脱氢酶家族18成员A1(AL-DH18A1)可编码P5CS蛋白]转染细胞,采用q RT-PCR法检测转染前后Li-7及Huh-7细胞脂质过氧化物合成相关基因ACSL4、LPCAT3、LOX-15,采用WESTERN Blotting法检测转染前后Li-7及Huh-7细胞铁死亡相关蛋白溶质载体家族7成员11(SLC7A11)、谷胱甘肽过氧化物酶(GPX4)。取转染前后Li-7及Huh-7细胞各分为2组,分别加入5μmol/L的铁死亡诱导剂Erastin、同体积DMSO,采用C11-BODIPY荧光探针检测各组细胞脂质过氧化物含量。取转染前、后Li-7及Huh-7细胞各分为2组,分别加入5μmol/L的GPX4靶向抑制剂RSL-3、5μmol/L的Erastin、同体积DMSO,采用C11-BODIPY荧光探针检测各组细胞脂质过氧化物含量。结果 HCC及癌旁组织P5CS蛋白相对表达量分别为6.80±2.10、0.40±0.20,二者比较,P<0.05。与转染前比较,转染后Li-7及Huh-7细胞SLC7A11、GPX4蛋白相对表达量低(P均<0.05)。与转染前比较,转染后加入Erastin、RSL-3的Li-7、Huh-7细胞脂质过氧化物含量均升高(P均<0.05)。与加入Erastin者比较,转染后加入RSL-3的Li-7、Huh-7细胞脂质过氧化物含量高、细胞活性低(P均<0.05)。结论 HCC组织中P5CS表达升高。P5CS可抑制Li-7及Huh-7细胞的铁死亡,P5CS可能通过抑制细胞GPX4表达,调控Li-7及Huh-7细胞的铁死亡。

Objective To observe the expression changes of pyrroline-5-carboxylate synthase(P5CS) in hepatocel-lular carcinoma(HCC) tissues and its regulatory effect on ferroptosis in human hepatoma cell lines.Methods(1)Detec-tion of P5CS in HCC and adjacent tissues:The cancer tissues and adjacent tissues of 10 patients with HCC were collected,and P5CS was detected by immunohistochemistry.(2)The regulatory effect of P5CS on ferroptosis of human hepatoma cell lines Li-7 and Huh-7:Human hepatoma cell lines Li-7 and Huh-7 in the logarithmic growth phase were transfected with si-ALDH18A1(targeting P5CS coding gene ALDH18A1).Before and after the transfection of si ALDH18A1 in Li-7 and Huh-7 cells,q RT-PCR was used to detect the expression levels of ACSL4,LPCAT3 and LOX-15 genes related to lipid peroxide synthesis,and Western blotting was used to detect the content of ferroptosis-related protein solute carrier family 7 member 11(SLC7A11) and glutathione peroxidase(GPX4).Li-7 and Huh-7 cells with or without si ALDH18A1 transfection were divided into two groups,and 5 μmol/L Erastin or the same volume of DMSO were added,respectively.The content of lip-id peroxide in each group was detected by C11-BODIPY fluorescent probe.Another group of cells with or without si-ALDH18A1 transfection were divided into two groups,and 5 μmol/L GPX4 targeted inhibitor RSL-3,5 μmol/L Erastin or the same volume of DMSO were added,respectively.The content of lipid peroxide in each group was detected by C11-BODIPY fluorescent probe.Results The expression of P5CS protein of HCC and adjacent tissues were 6.80 ± 2.10 and 0.40 ± 0.20,respectively(P<0.05).The relative expression levels of SLC7A11 and GPX4 protein were lower in compari-son with those before inhibition(both P<0.05).Compared with those before inhibition,the lipid peroxide content in Li-7 and Huh-7 cells added with Erastin or RSL-3 after transfection increased(all P<0.05).The lipid peroxide content of Li-7 and Huh-7 cells added with RSL-3 after transfection was higher and the cell activity was lower in comparison with those of cells added with Erastin(all P<0.05).Conclusions P5CS was highly expressed in HCC tissues.P5CS inhibits ferropto-sis of Li-7 and Huh-7 cells possibly by inhibiting GPX4 expression.