LncRNA ABHD11-AS1在Erastin诱导的胰腺癌细胞铁死亡中的作用及其机制

［摘要］目的： 探讨长链非编码RNA(long non coding RNA,lncRNA)α/β 水解酶结构域蛋白11的反义链1(α/β hydrolase domain containing protein 11 antisense strand 1，ABHD11 AS1)在Erastin诱导的胰腺癌细胞铁死亡中的作用及其潜在机制。方法: 通过GEPIA平台分析ABHD11 AS1在胰腺癌中的表达和患者预后。采用荧光实时定量PCR(qRT PCR)检测人胰腺癌PANC1、MIApaca 2和PaTu8988细胞中ABHD11 AS1表达，CCK8法计算Erastin半数抑制浓度(IC50)。在PANC1细胞中过表达ABHD11 AS1，分别转染pcDNA3.1、pcDNA3.1 ABHD11 AS1质粒；在PaTu8988细胞中干扰ABHD11 AS1表达，分别转染siControl、siRNA1、siRNA2，qRT PCR检测转染效率，分别予以对照(0 μmol/L Erastin)、Erastin(IC50浓度)处理，CCK8法检测细胞活性，ELISA法检测丙二醛和还原型谷胱甘肽(glutathione，GSH)含量；另在pcDNA3-1-ABHD11-AS1和siRNA1组加入Erastin的同时加入铁死亡挽救剂Ferrostatin 1，坏死挽救剂Necrostatin-1或凋亡挽救剂Z-VAD-FMK，CCK8法检测细胞活性；免疫印迹法检测5组胰腺癌细胞中谷胱甘肽过氧化物酶4(glutathione peroxidase 4，GPX4)、雷帕霉素靶蛋白(mammalian target of rapamycin，mTOR)以及磷酸化mTOR(p-mTOR)表达水平。结果: 胰腺癌组织中ABHD11-AS1表达水平明显高于癌旁组织，高表达ABHD11-AS1组患者预后较差(P<0.05)。ABHD11-AS1相对表达量由低到高依次是人胰腺癌PANC1、MIApaca 2和PaTu8988细胞，Erasin IC50趋势与ABHD11 AS1表达量相同，依次是2.245、11.760、17.120 μmol/L。与pcDNA3.1组相比，pcDNA3.1 ABHD11 AS1组细胞活性及GSH含量明显增高，而丙二醛含量明显降低(P<0.05)；与siControl组相比，siRNA1组和siRNA2组细胞活性及GSH含量明显降低，丙二醛含量明显增高(P均<0.05)。仅Ferrostatin-1可以挽救pcDNA3.1-ABHD11 AS1和siRNA1组细胞活性(P<0.05)。过表达ABHD11 AS1可促进mTOR活化，增强GPX4表达(P<0.05)；而干扰ABHD11 AS1则相反。结论: LncRNA ABHD11 AS1可通过活化mTOR促进GPX4表达，从而促进胰腺癌细胞铁死亡抵抗。

Role of LncRNA ABHD11 AS1 in Erastin induced ferroptosis of pancreatic cancer cells and its mechanism

 ［Abstract］Objective To investigate the role of long non-coding RNA (lncRNA) α/β-hydrolase domain containing protein 11-antisense strand 1 (ABHD11-AS1) in Erastin-induced ferroptosis of pancreatic cancer cells and its potential mechanism. Methods The expression of ABHD11-AS1 and prognosis of patients in pancreatic cancer were analyzed by GEPIA platform. qRT-PCR was used to detect ABHD11-AS1 expression in human pancreatic cancer cells PANC1, MIApaca 2 and PaTu8988, and CCK8 assay was used to calculate the IC50 of Erastin. PANC1 cells was transfected with pcDNA3.1 or PCDNA3.1-ABHD11-AS1 plasmid to overexpress ABHD11-AS1. PaTu8988 cells was transfected with siControl, siRNA1 and siRNA2 to interfere ABHD11-AS1; and the transfection efficiency was detected by qRT-PCR. The cells were treated with control (0 μmol/L Erastin), Erastin (IC50 concentration), respectively; and cell viability was detected by CCK8 assay. The content of malondialdehyde (MDA) and glutathione (GSH) were determined by ELISA. In addition, in pcDNA3.1 ABHD11-AS1 and siRNA1 groups, Erastin was added and ferroptosis inhibiting agent Ferrostatin-1, necrosis salvage agent Necrosstatin-1 or apoptosis inhibitor Z VAD FMK were added at the same time, and cell viability was detected by CCK8 assay. Western blotting was used to detect the protein expression of glutathione peroxidase 4 (GPX4), mammalian target of rapamycin (mTOR), and phosphorylated mTOR (p-mTOR) in pancreatic cancer cells of 5 groups. Results The expression level of ABHD11-AS1 in pancreatic cancer tissues was significantly higher than that in adjacent tissues, and the prognosis of patients with higher expression of ABHD11-AS1 was poorer (P<0.05). The expression level of ABHD11-AS1 from low to high in human pancreatic cancer cells were PANC1, MIApaca-2, and PaTu8988. The trend of Erasin IC50 was the same as that of ABHD11-AS1, which was 2.245, 11.760, 17.120 μmol/L, respectively. Compared with the pcDNA3.1 group, PCDNA3.1 ABHD11 AS1 group showed higher levels of cell viability and GSH content, while lower content of MDA (all P<0.05). Compared with the siControl group, the cell viability and GSH content in siRNA1 group and siRNA2 group decreased significantly, and the MDA content increased significantly (P<0.05). Only Ferrostatin-1 could rescue the cell viability of pcDNA3.1 ABHD11-AS1 and siRNA1 groups (P<0.05). ABHD11-AS1 increased cell viability under Erastin treatment by enhancing ferroptosis resistance. Overexpression of ABHD11-AS1 could promote the activation of mTOR and enhance the expression of GPX4 (P<0.05); knockdown of ABHD11-AS1 produced the opposite effect. Conclusion LncRNA ABHD11-AS1 could promote the expression of GPX4 by activating mTOR1, thus promoting ferroptosis resistance of pancreatic cancer cells.