缺氧诱导因子2α对肝细胞癌化疗耐药性的影响
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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。
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.
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.
引文
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[22] 王寅声,李晓智,沈杰,等.姜黄素对乳腺癌MCF-7与MCF-7/DOX细胞中P-糖蛋白表达的影响及其机制研究[J].中国临床药理学与治疗学,2015,20(7):769-774.
[23] Stefan SM,Wiese M.Small-molecule inhibitors of multidrug resistance-associated protein 1 and related processes:A historic approach and recent advances[J].Med Res Rev,2019,39(1):176-264.
[24] 张菁华,崔国忠,李永生,等.MMP-9、BSP与LRP在乳腺癌组织中表达的研究[J].现代肿瘤医学,2014,22(6):1324-1327.
[2] Shindoh J,Kaseb A,Vauthey JN.Surgical strategy for liver cancers in the era of effective chemotherapy [J].Liver Cancer,2013,2(1):47-54.
[3] 方长英,朱传卫,张青松,等.原发性肝癌组织中长链非编码RNA外泌体复合物7的表达及临床意义[J].临床肝胆病杂志,2019,35(1):123-126.
[4] 廖维荣,顾超,张大伟.索拉非尼治疗原发性肝癌的效果及对患者血清血管内皮生长因子受体2、胎盘生长因子水平的影响[J].临床肝胆病杂志,2018,34(6):1220-1224.
[5] Kamimura K,Suda T,Tamura Y,et al.Phase I study of miriplatin combined with transarterial chemotherapy using CDDP powder in patients with hepatocellular carcinoma[J].BMC Gastroenterol,2012,12:127.
[6] Murugesan T,Rajajeyabalachandran G,Kumar S,et al.Targeting HIF-2α as therapy for advanced cancers[J].Drug Discovery Today,2018,23(7):1444-1451.
[7] Wang D,Zhang X,Lu Y,et al.Hypoxia inducible factor 1alpha in hepatocellular carcinoma with cirrhosis:Association with prognosis[J].Pathol Res Pract,2018,214(12):1987-1992.
[8] Feng W,Xue T,Huang S,et al.HIF-1alpha promotes the migration and invasion of hepatocellular carcinoma cells via the IL-8-NF-kappaB axis[J].Cell Mol Biol Lett,2018,23:26.
[9] 刘林森,何前进,张悦,等.ZBP-89在肝癌组织中的表达及同缺氧诱导因子-2α的相关性分析[J].现代肿瘤医学,2017,25(8):1264-1267.
[10] 潘婷婷,马杰,徐治军,等.缺氧诱导因子2在肝细胞癌中的表达及临床意义[J].肝胆外科杂志,2016,24(5):392-395.
[11] Zheng X,Zhang Y,Liu Y,et al.HIF-2alpha activated lncRNA NEAT1 promotes hepatocellular carcinoma cell invasion and metastasis by affecting the epithelial-mesenchymal transition[J].J Cell Biochem,2018,119(4):3247-3256.
[12] Wang X,Dong J,Jia L,et al.HIF-2-dependent expression of stem cell factor promotes metastasis in hepatocellular carcinoma[J].Cancer Lett,2017,393:113-124.
[13] Zhu YJ,Zheng B,Wang HY,et al.New knowledge of the mechanisms of sorafenib resistance in liver cancer[J].Acta Pharmacol Sin,2017,38(5):614-622.
[14] Bray F,Ferlay J,Soerjomataram I,et al.Global cancer statistics 2018:GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[J].CA Cancer J Clin,2018,68(6):394-424.
[15] Donadon M,Solbiati L,Dawson L,et al.Hepatocellular carcinoma:the role of interventional oncology [J].Liver Cancer,2016,6(1):34-43.
[16] Dong X,Mumper RJ.Nanomedicinal strategies to treat multidrug-resistant tumors:current progress[J].Nanomedicine (Lond),2010,5(4):597-615.
[17] Uchida T,Rossignol F,Matthay MA,et al.Prolonged hypoxia differentially regulates hypoxia-inducible factor (HIF)-1alpha and HIF-2alpha expression in lung epithelial cells:implication of natural antisense HIF-1alpha [J].J Biol Chem,2004,279(15):14871-14878.
[18] Zhao J,Du F,Luo Y,et al.The emerging role of hypoxia-inducible factor-2 involved in chemo/radioresistance in solid tumors[J].Cancer Treat Rev,2015,41(7):623-633.
[19] He C,Sun X,Qiao H,et al.Downregulating hypoxia-inducible factor-2α improves the efficacy of doxorubicin in the treatment of hepatocellular carcinoma[J].Cancer Sci,2012,103(3):528-534.
[20] Cannito S,Turato C,Paternostro C,et al.Hypoxia up-regulates SERPINB3 through HIF-2alpha in human liver cancer cells[J].Oncotarget,2015,6(4):2206-2221.
[21] Zhao D,Zhai B,He C,et al.Upregulation of HIF-2α induced by sorafenib contributes to the resistance by activating the TGF-α/EGFR pathway in hepatocellular carcinoma cells[J].Cell Signal,2014,26(5):1030-1039.
[22] 王寅声,李晓智,沈杰,等.姜黄素对乳腺癌MCF-7与MCF-7/DOX细胞中P-糖蛋白表达的影响及其机制研究[J].中国临床药理学与治疗学,2015,20(7):769-774.
[23] Stefan SM,Wiese M.Small-molecule inhibitors of multidrug resistance-associated protein 1 and related processes:A historic approach and recent advances[J].Med Res Rev,2019,39(1):176-264.
[24] 张菁华,崔国忠,李永生,等.MMP-9、BSP与LRP在乳腺癌组织中表达的研究[J].现代肿瘤医学,2014,22(6):1324-1327.