赖氨酸羟化酶2诱导肺癌HCC827细胞对奥希替尼耐药的机制研究

Mechanism of PLOD2 induced osimertinib resistance in non-small cell lung cancer HCC827 cells

目的探讨奥希替尼对过表达赖氨酸羟化酶2(PLOD2)的HCC827细胞增殖、迁移和侵袭的影响及其PLOD2诱导奥希替尼耐药的作用机制。方法应用LV-vector和LV-over/PLOD2慢病毒载体转染肺癌HCC827细胞,应用实时荧光定量聚合酶链反应(qRT-PCR)和Western blot法检测PLOD2的表达情况,应用四甲基偶氮唑蓝(MTT)法检测奥希替尼的抗细胞增殖能力,应用细胞划痕实验和Transwell实验检测肿瘤细胞的迁移和侵袭能力,应用免疫荧光法检测E-cadherin和vimentin的表达,应用Western blot法检测上皮间质转化(EMT)、FAK-PI3K/AKT和MAPK信号通路相关蛋白的表达。结果MTT检测结果显示,过表达PLOD2的HCC827-PLOD2细胞对奥希替尼的敏感性降低,半数抑制浓度>1000 nmol/L,耐药指数>100。细胞划痕实验结果显示,HCC827-PLOD2细胞迁移的距离为HCC827-vector细胞的(2.13±0.21)倍。Transwell结果显示,HCC827-PLOD2细胞的穿膜细胞数为(212.78±10.43)个,与对照组HCC827-vector细胞[(101.32±12.52)个]比较,差异有统计学意义(P<0.01)。免疫荧光检测结果显示,与HCC827-vector细胞比较,HCC827-PLOD2细胞中E-cadherin的表达下调,vimentin的表达上调;奥希替尼可下调HCC827-vector细胞中vimentin的表达,上调E-cadherin蛋白的表达,但对HCC827-PLOD2细胞中E-cadherin和vimentin的表达无明显调控作用。Western blot结果显示,过表达PLOD2可下调E-cadherin蛋白的表达,上调vimentin蛋白的表达,奥希替尼可抑制HCC827-PLOD2细胞中磷酸化表皮生长因子受体的表达,但对PLOD2、p-FAK、p-AKT、p-ERK和vimentin等蛋白的表达无明显抑制作用,对E-cadherin蛋白的表达亦无上调作用。结论PLOD2可能通过促进EMT的表达,激活FAK-PI3K/AKT及MAPK信号通路诱导奥希替尼耐药。

Objective To investigate the effects of osimertinib on proliferation,migration and invasion of procollagen-lysine 2-oxoglutarate 5-dioxygenase 2(PLOD2)overexpressing HCC827 cells and explore the potential mechanism of PLOD2 induced osimertinib resistance.Methods We transfected HCC827 cells with LV-vector and LV-over/PLOD2.The expression of PLOD2 was detected by quantitative real time polymerase chain reaction(qRT-PCR)and western blotting.The effects of osimertinib on the proliferation of HCC827-vector and HCC827-PLOD2 cells were evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide(MTT)assay.The effects of osimertinib on the migration and invasion of HCC827-vector and HCC827-PLOD2 cells were determined by Transwell assays.The expressions of E-cadherin and vimentin in cells were detected by immunofluorescence(IF).The expressions of epithelial-mesenchymal transition(EMT),FAK-PI3K/AKT and MAPK signal pathway related proteins were detected by western blotting.Results The MTT assay showed that HCC827-PLOD2 cells were hyposensitive to osimertinib.The 50%inhibitory concentration(IC50)and resistance index of osimertinib for HCC827-PLOD2 cells was over 1000 nmol/L and over 100,respectively.The result of wound healing assay showed that the migration distance of HCC827-PLOD2 was about(2.13±0.21)fold changes as that of HCC827-vector cells.The result of Transwell assay showed that the numbers of HCC827-PLOD2 passing through the matrix membrane were(212.78±10.43),significantly higher than(101.32±12.52)of HCC827-vector cells(P<0.01).The result of IF showed that compared with HCC827-vector cells,the expression of E-cadherin was down-regulated while vimentin was up-regulated in HCC827-PLOD2 cells.Osimertinb downregulated E-cadherin and upregulated vimentin expression in HCC827-vector cells but had limited effect in HCC827-PLOD2 cells.The result of western blotting showed that PLOD2 significantly increased vimentin expression level while decreased E-cadherin expression level.Osimertinib inhibited the expression of p-EGFR,but did not affect the expressions of PLOD2,p-FAK,p-AKT,p-ERK,vimentin and E-cadherin in HCC827-PLOD2 cells.Conclusion PLOD2 confers resistance to osimertinib in HCC827 cells by regulating EMT,FAK-PI3K/AKT and MAPK signal pathways.