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Abstract：
BACKGROUND: Gemcitabine is a cornerstone drug for the treatment of all stages of pancreatic cancer and can prolong the survival of patients with pancreatic cancer, but resistance to gemcitabine in pancreatic cancer patients hinders its efficacy. The overexpression of Early growth response 1(EGR1) in pancreatic ductal adenocarcinoma as a mechanism of gemcitabine chemoresistance in pancreatic cancer has not been explored. The major mechanisms of gemcitabine chemoresistance are related to drug uptake, metabolism, and action. One of the common causes of tumor multidrug resistance (MDR) to chemotherapy in cancer cells is that transporter proteins increase intracellular drug efflux and decrease drug concentrations by inducing anti-apoptotic mechanisms. It has been reported that gemcitabine binds to MDR1 with high affinity. The purpose of this research was to investigate the potential mechanisms by which EGR1 associates with MDR1 to regulate gemcitabine resistance in pancreatic cancer cells.
METHODS: The following in vitro and in vivo techniques were used in this research to explore the potential mechanisms by which EGR1 binds to MDR1 to regulate gemcitabine resistance in pancreatic cancer cells. Cell culture; in vitro and in vivo study of EGR1 function by loss of function analysis. Binding of EGR1 to the MDR1 promoter was detected using the ChIP assay. qRT-PCR, Western blot assays to detect protein and mRNA expression; use of Annexin V apoptosis detection assay to test apoptosis; CCK8, Edu assay to test cell proliferation viability. The animal model of pancreatic cancer subcutaneous allograft was constructed and the tumours were stained with hematoxylin eosin and Ki-67 expression was detected using immunohistochemistry.
RESULTS: We revealed that EGR1 expression was increased in different pancreatic cancer cell lines compared to normal pancreatic ductal epithelial cells. Moreover, gemcitabine treatment induced upregulation of EGR1 expression in a dose- and time-dependent manner. EGR1 is significantly enriched in the MDR1 promoter sequence.Upon knockdown of EGR1, cell proliferation was impaired in CFPAC-1 and PANC-1 cell lines, apoptosis was enhanced and MDR1 expression was decreased, thereby partially reversing gemcitabine chemoresistance. In animal experiments, knockdown of EGR1 enhanced the inhibitory effect of gemcitabine on tumor growth compared with the sh-NC group.
CONCLUSIONS: Our study suggests that EGR1 may be involved in the regulation of MDR1 to enhance gemcitabine resistance in pancreatic cancer cells. EGR1 could be a novel therapeutic target to overcome gemcitabine resistance in pancreatic cancer.
METHODS: The following in vitro and in vivo techniques were used in this research to explore the potential mechanisms by which EGR1 binds to MDR1 to regulate gemcitabine resistance in pancreatic cancer cells. Cell culture; in vitro and in vivo study of EGR1 function by loss of function analysis. Binding of EGR1 to the MDR1 promoter was detected using the ChIP assay. qRT-PCR, Western blot assays to detect protein and mRNA expression; use of Annexin V apoptosis detection assay to test apoptosis; CCK8, Edu assay to test cell proliferation viability. The animal model of pancreatic cancer subcutaneous allograft was constructed and the tumours were stained with hematoxylin eosin and Ki-67 expression was detected using immunohistochemistry.
RESULTS: We revealed that EGR1 expression was increased in different pancreatic cancer cell lines compared to normal pancreatic ductal epithelial cells. Moreover, gemcitabine treatment induced upregulation of EGR1 expression in a dose- and time-dependent manner. EGR1 is significantly enriched in the MDR1 promoter sequence.Upon knockdown of EGR1, cell proliferation was impaired in CFPAC-1 and PANC-1 cell lines, apoptosis was enhanced and MDR1 expression was decreased, thereby partially reversing gemcitabine chemoresistance. In animal experiments, knockdown of EGR1 enhanced the inhibitory effect of gemcitabine on tumor growth compared with the sh-NC group.
CONCLUSIONS: Our study suggests that EGR1 may be involved in the regulation of MDR1 to enhance gemcitabine resistance in pancreatic cancer cells. EGR1 could be a novel therapeutic target to overcome gemcitabine resistance in pancreatic cancer.
摘要：
背景：吉西他滨是治疗胰腺癌所有阶段的基石药物，可以延长胰腺癌患者的生存期，但是胰腺癌患者对吉西他滨的耐药性阻碍了其疗效。尚未探索胰腺导管腺癌中早期生长反应1（EGR1）的过表达作为胰腺癌吉西他滨化学耐药的机制。吉西他滨化疗耐药的主要机制与药物摄取有关。新陈代谢，和行动。肿瘤多药耐药（MDR）在癌细胞中化疗的常见原因之一是转运蛋白通过诱导抗凋亡机制增加细胞内药物流出并降低药物浓度。据报道,吉西他滨以高亲和力结合MDR1。这项研究的目的是研究EGR1与MDR1联合调节胰腺癌细胞吉西他滨耐药性的潜在机制。
方法：本研究使用以下体外和体内技术来探索EGR1与MDR1结合以调节胰腺癌细胞吉西他滨耐药性的潜在机制。细胞培养;通过功能分析的EGR1功能的体外和体内研究。使用ChIP测定法检测EGR1与MDR1启动子的结合。qRT-PCR,Westernblot法检测蛋白和mRNA表达；运用AnnexinV凋亡检测法检测细胞凋亡；CCK8、Edu法检测细胞增殖活力。建立胰腺癌皮下移植瘤动物模型,用苏木素伊红染色,免疫组化检测Ki-67表达。
结果：我们发现，与正常胰腺导管上皮细胞相比，EGR1在不同胰腺癌细胞系中的表达增加。此外，吉西他滨治疗以剂量和时间依赖性方式诱导EGR1表达上调。EGR1在MDR1启动子序列中显著富集。敲低EGR1后，CFPAC-1和PANC-1细胞系中的细胞增殖受损，细胞凋亡增强，MDR1表达降低，从而部分逆转吉西他滨化学耐药。在动物实验中,与sh-NC组相比，EGR1敲除增强了吉西他滨对肿瘤生长的抑制作用。
结论：我们的研究表明，EGR1可能参与MDR1的调节，以增强胰腺癌细胞的吉西他滨耐药性。EGR1可能是克服胰腺癌吉西他滨耐药的新治疗靶点。
方法：本研究使用以下体外和体内技术来探索EGR1与MDR1结合以调节胰腺癌细胞吉西他滨耐药性的潜在机制。细胞培养;通过功能分析的EGR1功能的体外和体内研究。使用ChIP测定法检测EGR1与MDR1启动子的结合。qRT-PCR,Westernblot法检测蛋白和mRNA表达；运用AnnexinV凋亡检测法检测细胞凋亡；CCK8、Edu法检测细胞增殖活力。建立胰腺癌皮下移植瘤动物模型,用苏木素伊红染色,免疫组化检测Ki-67表达。
结果：我们发现，与正常胰腺导管上皮细胞相比，EGR1在不同胰腺癌细胞系中的表达增加。此外，吉西他滨治疗以剂量和时间依赖性方式诱导EGR1表达上调。EGR1在MDR1启动子序列中显著富集。敲低EGR1后，CFPAC-1和PANC-1细胞系中的细胞增殖受损，细胞凋亡增强，MDR1表达降低，从而部分逆转吉西他滨化学耐药。在动物实验中,与sh-NC组相比，EGR1敲除增强了吉西他滨对肿瘤生长的抑制作用。
结论：我们的研究表明，EGR1可能参与MDR1的调节，以增强胰腺癌细胞的吉西他滨耐药性。EGR1可能是克服胰腺癌吉西他滨耐药的新治疗靶点。
