缺氧诱导因子2α对肝细胞癌化疗耐药性的影响

doi:10.12092/j.issn.1009-2501.2019.06.005

中国临床药理学与治疗学 ›› 2019, Vol. 24 ›› Issue (6): 630-636.doi: 10.12092/j.issn.1009-2501.2019.06.005

缺氧诱导因子2α对肝细胞癌化疗耐药性的影响

杨书才^1,6，张莉^1,6，刘利平³，邓唯杰^4,6，周杰^1,6，刘慧^5,6，张鲍虎^1,6，金涛²

¹广东省深圳市坪山区人民医院检验科，深圳 518118，广东； ²华中科技大学同济医学院附属梨园医院肿瘤科，武汉 430030，湖北；³广东省深圳市人民医院肝胆外科，深圳 518020，广东；⁴广东省深圳市坪山区人民医院麻醉科，⁵药剂科，深圳 518118，广东；⁶湖北医药学院第八临床学院，深圳 518118，广东

收稿日期:2019-02-13 修回日期:2019-05-12 出版日期:2019-06-26 发布日期:2019-06-25
通讯作者: 金涛，男，本科，主治医师，研究方向：肿瘤。 Tel：13995696582 E-mail：13995696582@qq.com
作者简介:杨书才，男，博士，主管技师，研究方向：肿瘤与微生物。 Tel：0755-89263726 E-mail: 3506074@qq.com
基金资助:
广东省自然科学基金项目（2017A030313846）；深圳市科技创新委员会基础研究项目（JCYJ20160429173544879）；广东省深圳市坪山区卫生系统科研项目（201725）

Effect of hypoxia-inducible factor 2α on chemoresistance of hepatocellular carcinoma

YANG Shucai ^1,6，ZHANG Li ^1,6，LIU Liping ³，DENG Weijie ^4,6，ZHOU Jie ^1,6，LIU Hui ^5,6，ZHANG Baohu ^1,6，JIN Tao ²

¹Department of Laboratory Medicine, Pingshan District People's Hospital, Shenzhen 518118, Guangdong, China; ²Department of Oncology, Liyuan Hospital, Tongji Medical College of HUST, Wuhan 430030, Hubei, China; ³Department of Hepatobiliary Surgery, Shenzhen People's Hospital, Shenzhen 518020, Guangdong, China; ⁴Department of Anesthesiology, ⁵Department of Pharmacy, Pingshan District People's Hospital, Shenzhen 518118, Guangdong, China; ⁶The 8th Clinical School of Hubei Medical College, Shenzhen 518118, Guangdong, China

Received:2019-02-13 Revised:2019-05-12 Online:2019-06-26 Published:2019-06-25

摘要/Abstract

摘要：

目的:研究缺氧诱导因子2α（HIF-2α）对肝细胞癌化疗耐药性的影响及其机制。方法:选取6种肝细胞癌细胞株，Western blot方法检测HIF-2α的表达；制备过表达HIF-2α慢病毒颗粒并感染人肝细胞癌细胞株HepG2和PLC/PRF/5细胞，制备稳定表达HIF-2α的细胞株HepG2-HIF-2α和PLC/PRF/5-HIF-2α，Western blot法验证其表达；配置6种常规化疗药物氟尿嘧啶、环磷酰胺、盐酸表柔比星、丝裂霉素、甲氨蝶呤、索拉菲尼的5个浓度，并加入HepG2-HIF-2α及对照细胞中，MTT法检测细胞抑制率；Western blot检测两种过表达HIF-2α的肝细胞癌细胞株及相应对照细胞中耐药基因MDR1、LRP及MRP1的水平。结果:Western blot结果显示HIF-2α在6种肝细胞癌细胞株中均有表达；HIF-2α慢病毒颗粒感染肝细胞癌细胞株后能够在肝细胞癌细胞中稳定过表达HIF-2α；MTT结果显示，与对照组相比，环磷酰胺、甲氨蝶呤和索拉菲尼对HepG2-HIF-2α抑制率明显较低（P<0.05），提示HIF-2α的表达引起耐药性的增加；而氟尿嘧啶、盐酸表柔比星和丝裂霉素对HepG2-HIF-2α细胞的抑制率与对照组无统计学差异（P>0.05）；Western blot法进一步分析了HIF-2α在肝细胞癌细胞中的表达导致耐药性增加的原因，结果显示，在HepG2-HIF-2α细胞及PLC/PRF/5-HIF-2α细胞中，耐药相关基因MDR1、LRP、MRP1的表达水平均高于对照组（P<0.05）。结论:在肝细胞癌中，HIF-2α能够通过上调MDR1、LRP、MRP1耐药基因的表达引起肝细胞癌细胞的耐药性的增加。

关键词: 肝细胞癌, HIF-2α, MDR1, LRP, MRP1

Abstract:

AIM: To study the effect of hypoxia-inducible factor 2α (HIF-2α) on chemoresistance of hepatocellular carcinoma and its mechanism. METHODS: Six hepatocellular carcinoma cell lines were selected and the expression of HIF-2α were detected by Western blot. The HIF-2α lentiviral particles were produced and infected with HepG2 and PLC/PRF/5 cells to prepare a cell line stably expressing HIF-2α, and Western blot was used to verify its expression. Six conventional chemotherapy drugs (fluorouracil, cyclophosphamide, epirubicin hydrochloride, mitomycin, methotrexate, and sorafenib) in five concentrations were added to HepG2-HIF2α and Control cells. MTT assay was used to detect cell inhibition rate. To study the mechanism, Western blot was used to detect the expression of drug resistance genes MDR1, LRP and MRP1 in HIF-2α overexpressing HCC cells and the corresponding control cells. RESULTS:Western blot result showed that HIF-2α was expressed in 6 hepatocellular carcinoma cell lines. The ectopic expression of HIF-2α in lentivirus-infected HepG2 and PLC/PRF/5 cells were validated by Western blot. MTT results showed that the inhibitory rate of cyclophosphamide, methotrexate and sorafenib on HepG2-HIF-2α was significantly lower than control cells (P<0.05), suggesting that the expression of HIF-2α caused chemotherapy resistance. However, the inhibition rate of fluorouracil, epirubicin hydrochloride and mitomycin on HepG2-HIF-2α cells was not different from that of the control group (P>0.05). Western blot analysis further analyzed the cause of HIF-2α on the increase of chemotherapy resistance in hepatocellular carcinoma cells. The results showed that the expression levels of drug resistance-related genes MDR1, LRP and MRP1 were higher in HepG2-HIF-2α cells and PLC/PRF/5-HIF-2α cells than those in the control group (P<0.05). CONCLUSION: In hepatocellular carcinoma, HIF-2α can increase the chemotherapy resistance of hepatocellular carcinoma cells by up-regulating the expression of MDR1, LRP and MRP1 resistance genes.

Key words: hepatocellular carcinoma, HIF-2α, MDR1, LRP, MRP1

中图分类号:

R965.2

杨书才，张莉，刘利平，邓唯杰，周杰，刘慧，张鲍虎，金涛. 缺氧诱导因子2α对肝细胞癌化疗耐药性的影响[J]. 中国临床药理学与治疗学, 2019, 24(6): 630-636.

YANG Shucai，ZHANG Li，LIU Liping，DENG Weijie，ZHOU Jie，LIU Hui，ZHANG Baohu，JIN Tao. Effect of hypoxia-inducible factor 2α on chemoresistance of hepatocellular carcinoma[J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2019, 24(6): 630-636.

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缺氧诱导因子2α对肝细胞癌化疗耐药性的影响

Effect of hypoxia-inducible factor 2α on chemoresistance of hepatocellular carcinoma

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