IGFBP3基因启动子高甲基化所致表达下降促进胃癌化疗耐药
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摘要
目的:研究胰岛素样生长因子结合蛋白3(IGFBP3)在胃癌化疗敏感及化疗耐药细胞和组织中的表达情况,初步探讨其在胃癌化疗耐药中的作用及表达异常机制。方法:采用荧光实时定量PCR(qRT-PCR)及蛋白质免疫印迹实验(WB)的方法检测IGFBP3在化疗药物敏感的胃癌细胞AZ521、SC-M1,对应顺铂耐药细胞AZ521/cisplatin、SC-M1/cisplatin和胃癌组织中的表达水平;在降低和增加IGFBP3表达量后,采用细胞计数试剂盒-8(CCK-8)试剂及流式细胞术(FCM)检测胃癌细胞对化疗药物的敏感性的变化;甲基化特异性PCR(MSP)检测IGFBP3基因启动子区甲基化水平。结果:IGFBP3在化疗耐受的胃癌细胞及组织中表达低于化疗敏感的胃癌细胞及组织(P<0.05);在敏感细胞中干扰IGFBP3表达可促进胃癌细胞的耐药性,而在耐药细胞中恢复IGFBP3表达可显著逆转耐药性;MSP结果显示,IGFBP3的表达受DNA甲基化调控,耐药细胞中IGFBP3启动子高甲基化导致其表达下降(P<0.05);甲基转移酶抑制剂地西他滨(DAC)处理耐药的胃癌细胞,可恢复IGFBP3表达,并提高其对化疗药物的敏感性(P<0.05)。结论:IGFBP3启动子区发生DNA高甲基化导致其表达下降,使得化疗药物诱导胃癌细胞发生凋亡的能力下降,最终导致胃癌细胞的化疗耐药。
Objective: To study the expression of insulin-like growth factor binding protein 3(IGFBP3) in chemosensitivity and chemosensitivity cells and tissues of gastric cancer, and to explore its function and mechanism of abnormal expression in the chemoresistance of gastric cancer. Methods: The expression of IGFBP3 in chemotherapeutic drug-sensitive gastric cancer cells AZ521 and SC-M1, expressions of cisplatin-resistant cells AZ521/cisplatin, SC-M1/cisplatin and gastric cancer were detected by real-time quantitative fluorescence polymerase chain reaction(qRT-PCR) and Western blot assay(WB). After decreasing and increasing the expression of IGFBP3, the sensitivity of gastric cancer cells to chemotherapeutic drugs was detected by cell counting kit-8(CCK-8)reagent and flow cytometry(FCM). The promoter methylation level of IGFBP3 was determined by the methylation specific PCR(MSP)assay. Results: The expression of IGFBP3 in chemo-tolerant gastric cancer cells and tissues was lower than that in chemo-sensitive gastric cancer cells and tissues(P <0.05). Interference of IGFBP3 expression in sensitive cells can promote drug resistance of gastric cancer cells. While recovery of IGFBP3 expression in drug-resistant cells can significantly reversed drug resistance. MSP results showed that the expression of IGFBP3 was regulated by DNA methylation. Higher methylation of IGFBP3 promoter in drug-resistant cells resulted in decreased expression of IGFBP3(P <0.05). The drug-resistant gastric cancer cells were treatment with methyltransferase inhibitor dicetabine(DAC), the expression of IGFBP3 was restored, its sensitivity to chemotherapeutic drugs were increased(P<0.05).Conclusion: DNA hypermethylation in the promoter region of IGFBP3 leads to a decrease in its expression, which leads to a decrease in the ability of chemotherapeutic drugs to induce apoptosis of gastric cancer cells, and ultimately leads to chemotherapeutic resistance of gastric cancer cells.
Objective: To study the expression of insulin-like growth factor binding protein 3(IGFBP3) in chemosensitivity and chemosensitivity cells and tissues of gastric cancer, and to explore its function and mechanism of abnormal expression in the chemoresistance of gastric cancer. Methods: The expression of IGFBP3 in chemotherapeutic drug-sensitive gastric cancer cells AZ521 and SC-M1, expressions of cisplatin-resistant cells AZ521/cisplatin, SC-M1/cisplatin and gastric cancer were detected by real-time quantitative fluorescence polymerase chain reaction(qRT-PCR) and Western blot assay(WB). After decreasing and increasing the expression of IGFBP3, the sensitivity of gastric cancer cells to chemotherapeutic drugs was detected by cell counting kit-8(CCK-8)reagent and flow cytometry(FCM). The promoter methylation level of IGFBP3 was determined by the methylation specific PCR(MSP)assay. Results: The expression of IGFBP3 in chemo-tolerant gastric cancer cells and tissues was lower than that in chemo-sensitive gastric cancer cells and tissues(P <0.05). Interference of IGFBP3 expression in sensitive cells can promote drug resistance of gastric cancer cells. While recovery of IGFBP3 expression in drug-resistant cells can significantly reversed drug resistance. MSP results showed that the expression of IGFBP3 was regulated by DNA methylation. Higher methylation of IGFBP3 promoter in drug-resistant cells resulted in decreased expression of IGFBP3(P <0.05). The drug-resistant gastric cancer cells were treatment with methyltransferase inhibitor dicetabine(DAC), the expression of IGFBP3 was restored, its sensitivity to chemotherapeutic drugs were increased(P<0.05).Conclusion: DNA hypermethylation in the promoter region of IGFBP3 leads to a decrease in its expression, which leads to a decrease in the ability of chemotherapeutic drugs to induce apoptosis of gastric cancer cells, and ultimately leads to chemotherapeutic resistance of gastric cancer cells.
引文
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[11]Han JJ,Xue DW,Han QR,et al.Induction of apoptosis by IGFBP3overexpression in hepatocellular carcinoma cells[J].Asian Pac JCancer Prev,2014,15(23):10085-10089
[12]Ipsaro JJ,Shen C,Arai E,et al.Rapid generation of drug-resistance alleles at endogenous loci using CRISPR-Cas9 indel mutagenesis[J].PLo S One,2017,12(2):e0172177
[13]董媛,师贞宗,魏刚,等.PRKAA1和UNC5CL基因位点多态性与胃癌的相关性研究[J].现代生物医学进展,2018,18(6):1112-1115,1175
[14]Datta S,Choudhury D,Das A,et al.Paclitaxel resistance development is associated with biphasic changes in reactive oxygen species,mitochondrial membrane potential and autophagy with elevated energy production capacity in lung cancer cells:Achronological study[J].Tumour Biol,2017,39(2):1010428317694314
[15]Zhang K,Yuan Q.Current mechanism of acquired resistance to epidermal growth factor receptor-tyrosine kinase inhibitors and updated therapy strategies in human nonsmall cell lung cancer[J].JCancer Res Ther,2016,12(Supplement):C131-C137
[16]殷鹏飞,杨小翠,贺永锋,等.WWOX基因过表达对人胃癌细胞增殖、凋亡能力的影响及机制研究[J].胃肠病学和肝病学杂志,2017,26(12):1350-1354
[17]李君,刘恩令,杨丽,等.抑制IGFBP2、IGFBP3在卵巢肿瘤中的表达对卵巢癌的迁移、侵袭的影响[J].中国比较医学杂志,2017,27(1):32-36,48
[18]Yunusova NV,Villert AB,Spirina LV,et al.Insulin-Like Growth Factors and Their Binding Proteins in Tumors and Ascites of Ovarian Cancer Patients:Association With Response To Neoadjuvant Chemotherapy[J].Asian Pac J Cancer Prev,2016,17(12):6215-6220
[19]Feng X,Lin J,Xing S,et al.Higher IGFBP-1 to IGF-1 serum ratio predicts unfavourable survival in patients with nasopharyngeal carcinoma[J].BMC Cancer,2017,17(1):90
[20]Canel M,Byron A,Sims AH,et al.Nuclear FAK and Runx1Cooperate to Regulate IGFBP3,Cell-Cycle Progression,and Tumor Growth[J].Cancer Res,2017,77(19):5301-5312
[21]Baxter RC.IGF binding proteins in cancer:mechanistic and clinical insights[J].Nat Rev Cancer,2014,14(5):329-341
[22]张瑜,冯颖.IGFBP-2、-3、-5表达变化与非小细胞肺癌患者化疗敏感性的关系[J].临床与实验病理学杂志,2016,32(6):656-659
[23]张静,邓存良.胰岛素样生长因子结合蛋白与肝纤维化的研究进展[J].海南医学,2017,28(3):459-461,462
[24]Lee KH,Shin TJ,Kim WH,et al.Methylation of LINE-1 in cell-free?DNA serves as a liquid biopsy biomarker for human breast cancers and dog mammary tumors[J].Sci Rep,2019,9(1):175
[25]Feinberg AP,Koldobskiy MA,G觟nd觟r A.Epigenetic modulators,modifiers and mediators in cancer aetiology and progression[J].Nat Rev Genet,2016,17(5):284-299
[26]García-Martínez A,Sottile J,Sánchez-Tejada L,et al.DNAMethylation of Tumor Suppressor Genes in Pituitary Neuroendocrine Tumors[J].J Clin Endocrinol Metab,2019,104(4):1272-1282
[27]李利玲,王少帅.宫颈癌组织中DNA甲基转移酶表达及其与HPV感染、肿瘤恶性程度的关系[J].海南医学院学报,2017,23(3):408-410,414
[28]Gnyszka A,Jastrzebski Z,Flis S.DNA methyltransferase inhibitors and their emerging role in epigenetic therapy of cancer[J].Anticancer Res,2013,33(8):2989-2996
[29]Choi SJ,Jung SW,Huh S,et al.Alteration of DNA Methylation in Gastric Cancer with Chemotherapy[J].J Microbiol Biotechnol,2017,27(8):1367-1378
[30]Maeda M,Moro H,Ushijima T.Mechanisms for the induction of gastric cancer by Helicobacter pylori infection:aberrant DNAmethylation pathway[J].Gastric Cancer,2017,20(Suppl 1):8-15
[2]婷婷,郑荣寿,曾红梅,等.中国胃癌流行病学现状[J].中国肿瘤临床,2017,44(1):52-58
[3]春雷,张忠泰,高红,等.胃癌组织中生存素和基质金属蛋白酶-9的表达及其与胃癌临床病理特点的关系[J].中华实验外科杂志,2017,34(2):214-216
[4]Bach LA.Insulin-Like Growth Factor Binding Proteins--an Update[J].Pediatr Endocrinol Rev,2015,13(2):521-530
[5]Ranke MB.Insulin-like growth factor binding-protein-3(IGFBP-3)[J].Best Pract Res Clin Endocrinol Metab,2015,29(5):701-711
[6]Bao L,Liu H,You B,et al.Overexpression of IGFBP3 is associated with poor prognosis and tumor metastasis in nasopharyngeal carcinoma[J].Tumour Biol,2016,37(11):15043-15052
[7]Milju觢G,Malenkovi V,ukanovi B,et al.IGFBP-3/transferrin/transferrin receptor 1 complexes as principal mediators of IGFBP-3 delivery to colon cells in non-cancer and cancer tissues[J].Exp Mol Pathol,2015,98(3):431-438
[8]Yang CH,Yue J,Pfeffer SR,et al.MicroRNA-21 promotes glioblastoma tumorigenesis by down-regulating insulin-like growth factor-binding protein-3(IGFBP3)[J].J Biol Chem,2014,289(36):25079-25087
[9]Du Y,Long Q,Shi Y,et al.Insulin-like growth factor binding protein-3 mediates interleukin-24-induced apoptosis through inhibition of the mTOR pathway in prostate cancer[J].Oncol Rep,2015,34(5):2273-2281
[10]Croce CM,Reed JC.Finally,An Apoptosis-Targeting Therapeutic for Cancer[J].Cancer Res,2016,76(20):5914-5920
[11]Han JJ,Xue DW,Han QR,et al.Induction of apoptosis by IGFBP3overexpression in hepatocellular carcinoma cells[J].Asian Pac JCancer Prev,2014,15(23):10085-10089
[12]Ipsaro JJ,Shen C,Arai E,et al.Rapid generation of drug-resistance alleles at endogenous loci using CRISPR-Cas9 indel mutagenesis[J].PLo S One,2017,12(2):e0172177
[13]董媛,师贞宗,魏刚,等.PRKAA1和UNC5CL基因位点多态性与胃癌的相关性研究[J].现代生物医学进展,2018,18(6):1112-1115,1175
[14]Datta S,Choudhury D,Das A,et al.Paclitaxel resistance development is associated with biphasic changes in reactive oxygen species,mitochondrial membrane potential and autophagy with elevated energy production capacity in lung cancer cells:Achronological study[J].Tumour Biol,2017,39(2):1010428317694314
[15]Zhang K,Yuan Q.Current mechanism of acquired resistance to epidermal growth factor receptor-tyrosine kinase inhibitors and updated therapy strategies in human nonsmall cell lung cancer[J].JCancer Res Ther,2016,12(Supplement):C131-C137
[16]殷鹏飞,杨小翠,贺永锋,等.WWOX基因过表达对人胃癌细胞增殖、凋亡能力的影响及机制研究[J].胃肠病学和肝病学杂志,2017,26(12):1350-1354
[17]李君,刘恩令,杨丽,等.抑制IGFBP2、IGFBP3在卵巢肿瘤中的表达对卵巢癌的迁移、侵袭的影响[J].中国比较医学杂志,2017,27(1):32-36,48
[18]Yunusova NV,Villert AB,Spirina LV,et al.Insulin-Like Growth Factors and Their Binding Proteins in Tumors and Ascites of Ovarian Cancer Patients:Association With Response To Neoadjuvant Chemotherapy[J].Asian Pac J Cancer Prev,2016,17(12):6215-6220
[19]Feng X,Lin J,Xing S,et al.Higher IGFBP-1 to IGF-1 serum ratio predicts unfavourable survival in patients with nasopharyngeal carcinoma[J].BMC Cancer,2017,17(1):90
[20]Canel M,Byron A,Sims AH,et al.Nuclear FAK and Runx1Cooperate to Regulate IGFBP3,Cell-Cycle Progression,and Tumor Growth[J].Cancer Res,2017,77(19):5301-5312
[21]Baxter RC.IGF binding proteins in cancer:mechanistic and clinical insights[J].Nat Rev Cancer,2014,14(5):329-341
[22]张瑜,冯颖.IGFBP-2、-3、-5表达变化与非小细胞肺癌患者化疗敏感性的关系[J].临床与实验病理学杂志,2016,32(6):656-659
[23]张静,邓存良.胰岛素样生长因子结合蛋白与肝纤维化的研究进展[J].海南医学,2017,28(3):459-461,462
[24]Lee KH,Shin TJ,Kim WH,et al.Methylation of LINE-1 in cell-free?DNA serves as a liquid biopsy biomarker for human breast cancers and dog mammary tumors[J].Sci Rep,2019,9(1):175
[25]Feinberg AP,Koldobskiy MA,G觟nd觟r A.Epigenetic modulators,modifiers and mediators in cancer aetiology and progression[J].Nat Rev Genet,2016,17(5):284-299
[26]García-Martínez A,Sottile J,Sánchez-Tejada L,et al.DNAMethylation of Tumor Suppressor Genes in Pituitary Neuroendocrine Tumors[J].J Clin Endocrinol Metab,2019,104(4):1272-1282
[27]李利玲,王少帅.宫颈癌组织中DNA甲基转移酶表达及其与HPV感染、肿瘤恶性程度的关系[J].海南医学院学报,2017,23(3):408-410,414
[28]Gnyszka A,Jastrzebski Z,Flis S.DNA methyltransferase inhibitors and their emerging role in epigenetic therapy of cancer[J].Anticancer Res,2013,33(8):2989-2996
[29]Choi SJ,Jung SW,Huh S,et al.Alteration of DNA Methylation in Gastric Cancer with Chemotherapy[J].J Microbiol Biotechnol,2017,27(8):1367-1378
[30]Maeda M,Moro H,Ushijima T.Mechanisms for the induction of gastric cancer by Helicobacter pylori infection:aberrant DNAmethylation pathway[J].Gastric Cancer,2017,20(Suppl 1):8-15