hnRNPA2B1通过抑制转铁蛋白受体增强胰腺癌细胞对铁死亡的抵抗

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摘要目的：探讨异质核核糖核蛋白A2B1(heterogeneous nuclear ribonucleoprotein A2B1，hnRNPA2B1)对胰腺癌细胞铁死亡的影响及其潜在机制。方法：将3种胰腺癌PaTu8988、MIA-paca2、PANC1细胞用不同浓度Erastin处理3 d，CCK8法检测细胞活性，计算3种细胞Erastin半数抑制浓度(IC50)并比较其对Erastin的抵抗力；荧光实时定量PCR(qRT-PCR)和蛋白免疫印迹法分别检测3种胰腺癌细胞中hnRNPA2B1 mRNA和蛋白的相对表达。通过GEPIA平台分析hnRNPA2B1在胰腺癌组织和正常组织中的差异性表达，通过GEO数据库下载临床数据集分析差异性表达hnRNPA2B1患者的预后。在PaTu8988细胞中干扰hnRNPA2B1表达，分别转染sh-Control、sh-hnRNPA2B1质粒；在PANC1细胞中过表达hnRNPA2B1，分别转染Vector、Flag-hnRNPA2B1质粒；分别采用qRT-PCR和蛋白免疫印迹检测转染效率，通过Transwell实验检测hnRNPA2B1对胰腺癌细胞迁移和侵袭的影响，通过CCK8法检测hnRNPA2B1对胰腺癌细胞增殖的影响；对sh-Control组和sh-hnRNPA2B1组、Vector组和Flag-hnRNPA2B1组，分别予以对照(0 μmol/L Erastin)、Erastin(IC50浓度)处理，通过流式细胞术检测脂质过氧化物含量，比色法检测丙二醛和组织铁含量，此外shhnRNPA2B1组和Flag-hnRNPA2B1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ZVAD-FMK，台酚蓝染色法检测细胞活性；qRT-PCR和蛋白免疫印迹法分别检测胰腺癌细胞转铁蛋白受体(transferrin receptor，TFRC)、铁蛋白重链、谷胱甘肽过氧化物酶4、溶质载体家族7成员11等mRNA和蛋白相对表达水平。结果： 3种胰腺癌细胞对Erastin抗性由高到低依次是PaTu8988、MIA-paca2、PANC1细胞，其Erastin IC50依次为18.020、15.760、2.947 μmol/L；hnRNPA2B1表达量由高到低的趋势与Erastin IC50趋势相同，于PaTu8988细胞中最高，MIA-paca2细胞中次之，PANC1细胞中最低(P均<0.05)；胰腺癌组织中hnRNPA2B1表达水平明显高于正常组织，高表达hnRNPA2B1患者预后较差(P均<0.05)。下调hnRNPA2B1表达后，PaTu8988细胞迁移、侵袭和增殖能力明显降低，上调则与之相反(P均<0.05)。经Erastin处理后shhnRNPA2B1组和Flag-hnRNPA2B1组降低的细胞活性仅可被Ferrostatin-1挽救(P<0.05)；与sh-Control组相比，sh-hnRNPA2B1组脂质过氧化物、丙二醛和组织铁含量明显增高(P均<0.05)；与Vector组相比，Flag-hnRNPA2B1组脂质过氧化物、丙二醛和组织铁含量明显降低(P均<0.05)。hnRNPA2B1可抑制TFRC表达，干扰hnRNPA2B1则与之相反(P均<0.05)。结论： hnRNPA2B1通过抑制TFRC表达阻止铁蓄积，从而促进胰腺癌细胞铁死亡抵抗。

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	李政1
	刘雅雯1
	陈家希2
	王慧之1
	于正悦1
	王舜宇1
	龚爱华1
	徐岷3

关键词 ： hnRNPA2B1font-family: ????, font-weight: 700, text-align: -webkit-right, ')" href="#">background-color: rgb(212, 226, 255);">, 胰腺癌font-family: ????, font-weight: 700, text-align: -webkit-right, ')" href="#">background-color: rgb(212, 226, 255);">, 铁死亡font-family: ????, font-weight: 700, text-align: -webkit-right, ')" href="#">background-color: rgb(212, 226, 255);">, 铁代谢font-family: ????, font-weight: 700, text-align: -webkit-right, ')" href="#">background-color: rgb(212, 226, 255);">, 转铁蛋白受体

Abstract： Objective： To investigate the role of heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) in ferroptosis of pancreatic cancer cells and its potential mechanisms. Methods： Three pancreatic cancer cell lines PaTu8988, MIA-paca2 and PANC1 were treated with different concentrations of Erastin for 3 days. The cell viability was detected by CCK8 method. The half inhibitory concentrations (IC50) of Erastin in three kinds of pancreatic cancer cell lines were calculated and their resistance capabilities to Erastin were compared; qRT-PCR and Western blotting were used to detect the relative expression levels of mRNA and protein of hnRNPA2B1 in pancreatic cancer cells. The differential expression of the hnRNPA2B1 in pancreatic cancer tissues and normal tissues was analyzed by GEPIA, and the survival prognosis of patients with differential expression of hnRNPA2B1 was analyzed from clinical data sets from GEO database. PaTu8988 cells was transfected with sh-Control and sh-hnRNPA2B1 plasmid, respectively, to knock down hnRNPA2B1; PANC1 cells was transfected with Vector and Flag-hnRNPA2B1 plasmid, respectively, to overexpress hnRNPA2B1; and the transfection efficiency was verfied by qRT-PCR and Western blotting, respectively; the effects of knockdown or overexpression of hnRNPA2B1 on the migration and invasion were detected by Transwell assay, and the effects on the proliferation of pancreatic cancer cells were detected by CCK8 assay. The cells in sh-Control, sh-hnRNPA2B1, Vector and Flag-hnRNPA2B1 group were treated with control (0 μmol/L Erastin) and Erastin (IC50 concentration), respectively, then flow cytometry was used to determine the lipid peroxidation level, and colorimetry was used to determine the malindialdehyde (MDA) and tissue iron concentration; in addition to Erastin, the sh-hnRNPA2B1 and Flag-hnRNPA2B1 groups were added with Ferrostatin-1, Necrostatin-1, ZVAD-FMK, respectively, the cells activity was detected by trypan blue dye exclusion. The relative expression levels of RNA and protein of transferrin receptor (TFRC), ferritin heavy chain, glutathione peroxidase 4 and solute carrier family 7 member 11 weredetected by qRT-PCR and Western blotting, respectively. Results： The resistance of three types of pancreatic cancer cells to Erastin from high to low were PaTu8988, MIA-paca2 and PANC1, corresponding Erastin IC50 was 18.020, 15.760 and 2.947 μmol/L, respectively (P<0.05). The relative expression level of hnRNPA2B1 was the highest in PaTu8988 cells, followed by MIA-paca2 cells and the lowest in PANC1 cells (P<0.05), which was the same with Erastin IC50. The expression level of hnRNPA2B1 in pancreatic cancer tissues was significantly higher than that in normol tissues, and the prognosis of patients with higher expression of hnRNPA2B1 was poor (P<0.05). Down-regulating the expression of hnRNPA2B1, the migration, invasion and proliferation ability of PaTu8988 cells were greatly reduced, while up-regulating was the opposite (P<0-05). Erastin-induced cell death was significantly restored by Ferrostatin-1 not by ZVAD-FMK or Necrostatin-1 (P<0.05); compared with sh-Control group, the level of lipid peroxidation, MDA and iron concentration in sh-hnRNPA2B1 group were significantly increased (P<0.05); compared with Vector group, Flag-hnRNPA2B1 group showed lower level of lipid peroxidation, MDA and ironconcentration (P<0.05). Upregulation of hnRNPA2B1 inhibited the expression of TFRC, while interference with hnRNPA2B1 produced the opposite effect (P<0.05). Conclusion： hnRNPA2B1 could inhibit t he intracelluar accumulation of iron by inhibiting the expression of TFRC, thus promoting the resistance of pancreatic cancer cells to ferroptosis.

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收稿日期: 2022-12-30

基金资助:

国家自然科学基金面上项目(82072754)；江苏省卫生健康委员会面上项目(M2020011)；江苏省重点研发计划社会发展项目(BE2018689)；镇江市重点研发计划社会发展项目(SH2018033)

通讯作者: 徐岷(通讯作者)，主任医师，教授，博士生导师，E-mail:peterxu1974@163.com

作者简介: 李政(1997—)，女，硕士研究生；

引用本文:

李政1，刘雅雯1，陈家希2，王慧之1，于正悦1，王舜宇1，龚爱华1，徐岷3. hnRNPA2B1通过抑制转铁蛋白受体增强胰腺癌细胞对铁死亡的抵抗[J]. 江苏大学学报:医学版, 2024, 24(01): 1-10. LI Zheng1, LIU Yawen1, CHEN Jiaxi2, WANG Huizhi1, YU Zhengyue1, WANG Shunyu1, GONG Aihua1, XU Min3. hnRNPA2B1 enhances the resistance of pancreatic cancer cells to ferroptosis by inhibiting transferrin receptor. Journal of Jiangsu University(Medicine Edition), 2024, 24(01): 1-10.

链接本文:

http://zzs.ujs.edu.cn/xbyxb/CN/ 或 http://zzs.ujs.edu.cn/xbyxb/CN/Y2024/V24/I01/1

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