Background: Drug resistance is the main cause of high mortality of lung cancer. This study was conducted to investigate the effect of folic acid (FA) on the resistance of non-small cell lung cancer (NSCLC) cells to Osimertinib (OSM) by regulating the methylation of dual specificity phosphatase 1 (DUSP1).
Methods: The OSM resistant NSCLC cell line PC9R was establishd by gradually escalation of OSM concentration in PC9 cells. PC9R cells were randomly grouped into Control group, OSM group (5 μmol/L OSM), FA group (600 nmol/L FA), methylation inhibitor decitabine (DAC) group (10 μmol/L DAC), FA+OSM group (600 nmol/L FA+5 μmol/L OSM), and FA+OSM+DAC group (600 nmol/L FA+5 μmol/L OSM+10 μmol/L DAC). CCK-8 method was applied to detect cell proliferation ability. Scratch test was applied to test the ability of cell migration. Transwell assay was applied to detect cell invasion ability. Flow cytometry was applied to measure and analyze the apoptosis rate of cells in each group. Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) method was applied to detect the expression level of DUSP1 mRNA in cells. Methylation specific PCR (MSP) was applied to detect the methylation status of the DUSP1 promoter region in each group. Western blot was applied to analyze the expression levels of DUSP1 protein and key proteins in the DUSP1 downstream mitogen-activated protein kinase (MAPK) signaling pathway in each group.
Results: Compared with the Control group, the cell OD450 values (48 h, 72 h), scratch healing rate, number of cell invasions, and expression of DUSP1 in the OSM group were obviously decreased (P<0.05); the apoptosis rate, the methylation level of DUSP1, the expression of p38 MAPK protein, and the phosphorylation level of extracellular regulated protein kinases (ERK) were obviously increased (P<0.05); the cell OD450 values (48, 72 h), scratch healing rate, number of cell invasions, and expression of DUSP1 in the DAC group were obviously increased (P<0.05); the apoptosis rate, the expression of p38 MAPK protein, the phosphorylation level of ERK, and the methylation level of DUSP1 were obviously reduced (P<0.05). Compared with the OSM group, the cell OD450 values (48, 72 h), scratch healing rate, number of cell invasions, and expression of DUSP1 in the FA+OSM group were obviously decreased (P<0.05); the apoptosis rate, the methylation level of DUSP1, the expression of p38 MAPK protein, and the phosphorylation level of ERK were obviously increased (P<0.05). Compared with the FA+OSM group, the cell OD450 values (48, 72 h), scratch healing rate, number of cell invasions, and expression of DUSP1 in the FA+OSM+DAC group were obviously increased; the apoptosis rate, the methylation level of DUSP1, the expression of p38 MAPK protein, and the phosphorylation level of ERK were obviously reduced (P<0.05).
Conclusions: FA may inhibit DUSP1 expression by enhancing DUSP1 methylation, regulate downstream MAPK signal pathway, then promote apoptosis, inhibit cell invasion and metastasis, and ultimately reduce OSM resistance in NSCLC cells.
【中文题目：叶酸调控DUSP1甲基化对非小细胞肺癌细胞奥西替尼耐药性的影响】 【中文摘要：背景与目的 耐药的产生是肺癌死亡率居高不下的主要原因，本研究旨在探究叶酸（folic acid, FA）调控双特异性磷酸酶1（dual specificity phosphatase 1, DUSP1）甲基化对非小细胞肺癌（non-small cell lung cancer, NSCLC）细胞奥西替尼（Osimertinib, OSM）耐药性的影响。方法 浓度梯度递增法建立OSM耐药NSCLC细胞株PC9R。将PC9R细胞随机分为对照组、OSM组（5 μmol/L OSM）、FA组（600 nmol/L FA）、甲基化抑制剂地西他滨组（Decitabine, DAC）（10 μmol/L DAC）、FA+OSM组（600 nmol/L FA+5 μmol/L OSM）和FA+OSM+DAC组（600 nmol/L FA+5 μmol/L OSM+10 μmol/L DAC）。CCK-8法检测细胞增殖能力。划痕实验检测细胞迁移能力。Transwell实验检测细胞侵袭能力。流式细胞术测定分析各组细胞凋亡率。实时荧光定量聚合酶链式反应（real-time fluorescence quantitative polymerase chain reaction, RT-qPCR）法检测细胞DUSP1 mRNA的表达水平。甲基化特异性PCR（methylation specific PCR, MSP）检测各组细胞中DUSP1启动子区甲基化状态。Western blot分析各组细胞DUSP1蛋白、DUSP1下游丝裂原活化蛋白激酶（mitogen-activated protein kinase, MAPK）信号通路关键蛋白的表达水平。结果 与对照组相比，OSM组细胞OD450值（48、72 h）、划痕愈合率、细胞侵袭数目、DUSP1表达显著下降（P<0.05），细胞凋亡率、DUSP1甲基化水平、p38 MAPK蛋白表达、细胞外调节蛋白激酶（extracellular regulated protein kinases, ERK）磷酸化水平显著上升（P<0.05）；DAC组细胞OD450值（48、72 h）、划痕愈合率、细胞侵袭数目、DUSP1表达显著上升（P<0.05），细胞凋亡率、p38 MAPK蛋白表达、ERK磷酸化水平、DUSP1甲基化水平显著下降（P<0.05）。与OSM组相比，FA+OSM组细胞OD450值（48、72 h）、划痕愈合率、细胞侵袭数目、DUSP1表达显著下降（P<0.05），细胞凋亡率、DUSP1甲基化水平、p38 MAPK蛋白表达、ERK磷酸化水平显著上升（P<0.05）。与FA+OSM组相比，FA+OSM+DAC组细胞OD450值（48、72 h）、划痕愈合率、细胞侵袭数目、DUSP1水平显著升高，细胞凋亡率、DUSP1甲基化水平、p38 MAPK蛋白表达、ERK磷酸化水平显著降低（P<0.05）。结论 FA可能通过增强DUSP1甲基化抑制DUSP1表达，调控下游MAPK信号通路，进而促进细胞凋亡，抑制细胞侵袭转移，最终减弱NSCLC细胞OSM耐药性。 】 【中文关键词：叶酸；双特异性磷酸酶1；甲基化；奥西替尼；肺肿瘤；耐药性】.
Keywords: Dual specificity phosphatase 1; Folic acid; Lung neoplasms; Methylation; Osimertinib; Resistance.