肺腺癌转移相关转录本1诱导肺癌HCC827细胞对奥希替尼耐药的作用机制

Objective: To test the effect of metastasis associated in lung adenocarcinoma transcript 1 (MALAT1) and/or osimertinib on the proliferation and apoptosis of HCC827 cells, and explore the potential mechanism of MALAT1 induced resistance to osimertinib. Methods: We transfected HCC827 cells with LV-vector or LV-over/MALAT1. Stable transfected cells (HCC827/Vector, HCC827/MALAT1) were selected by adding puromycin. HCC827/MALAT1 cells were further transfected with the shRNA-negative control (NC) or shRNA-human epidermal growth factor receptor 3 (ERBB3) plasmid. The effects of overexpression of MALAT1, knockdown of ERBB3 and/or osimertinib on the proliferation of HCC827 cells were evaluated by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H tetrazolium bromide (MTT) assay. Cell apoptosis induced by MALAT1 overexpression, knockdown of ERBB3 and/or osimertinib treatment were analyzed by flow cytometry analysis. The expressions of EGFR and ERBB3 signal pathway related proteins in HCC827 cells treated with overexpression of MALAT1, knockdown of ERBB3 and/or osimertinib treatment were detected by western blot. Results: The MTT assay showed that sensitivity to osimertinib of HCC827/MALAT1 cells were significantly repressed. The 50% inhibitive concentration (IC(50)) of osimertinib >4 000 nmol/L in HCC827/MALAT1 cells. However, knockdown of ERBB3 facilitated the anti-proliferation effect of osimertinib, and the IC(50) of osimertinib in shRNA-ERBB3 cells was (17.27±3.21) nmol/L. The results of flow cytometry analysis showed that the apoptotic rate of HCC827/MALAT1 cells induced by 10 nmol/L osimertinib was (8.38±0.92)%, significantly lower than (27.17±5.83)% of knockdown of ERBB3 (P<0.01). Western blotting showed that the expression of p-ERBB3, p-AKT and p-extracellular regulated protein kinases (ERK) in HCC827/MALAT1 cells was markedly up-regulated, while the expression of p-epithelial growth factor receptor (EGFR) was inhibited. The expressions of p-ERBB3, p-AKT and p-ERK were marginally affected by osimertinb. However, osimertinib downregulated the expressions of p-EGFR, p-ERBB3, p-AKT and p-ERK in ERBB3 deleted cells. Conclusions: MALAT1 confers resistance to osimertinb in HCC827 cells by activating of the ERBB3/PI3K/AKT and ERBB3/MAPK/ERK signaling pathways.目的： 探讨肺腺癌转移相关转录本1(MALAT1)和奥希替尼对肺癌细胞HCC827增殖和凋亡的影响以及MALAT1介导奥希替尼耐药的机制。 方法： 应用LV－vector和LV－over/MALAT1转染HCC827细胞，建立稳定细胞株HCC827/Vector和HCC827/MALAT1。应用短发夹RNA阴性对照(shRNA－NC)、shRNA人表皮生长因子受体3(shRNA－ERBB3)转染HCC827/MALAT1细胞，应用四甲基偶氮唑蓝(MTT)法检测MALAT1、奥希替尼和shRNA－ERBB3对细胞增殖的影响，应用流式细胞术检测MALAT1、奥希替尼和shRNA－ERBB3对细胞凋亡的影响。应用Western blot检测MALAT1、奥希替尼和shRNA－ERBB3作用后，肺癌细胞中表皮生长因子受体(EGFR)和ERBB3信号通路相关蛋白的表达。 结果： MTT结果显示，奥希替尼对HCC827/MALAT1细胞增殖的抑制作用显著降低，半数抑制浓度(IC(50))>4 000 nmol/L；但干扰ERBB3后，HCC827/MALAT1细胞对奥希替尼再次敏感，IC(50)为(17.27±3.21)nmol/L。细胞凋亡实验显示，10 nmol/L奥希替尼干预HCC827/MALAT1细胞后，凋亡率为(8.38±0.92)%；但干扰ERBB3后，奥希替尼能促进HCC827/MALAT1细胞凋亡，凋亡率为(27.17±5.83)%，差异有统计学意义(P<0.01)；Western blot检测结果显示，HCC827/MALAT1细胞中磷酸化ERBB3(p－ERBB3)、p－AKT和p－ERK蛋白明显上调，奥希替尼能抑制磷酸化EGFR(p－EGFR)蛋白的表达，但对p－ERBB3、p－AKT和p－ERK蛋白的表达无影响。敲减ERBB3后，奥希替尼能下调p－EGFR、p－ERBB3、p－AKT和p－ERK蛋白的表达水平。 结论： MALAT1可能是通过激活ERBB3/PI3K/AKT和ERBB3/MAPK/ERK信号通路介导了奥希替尼耐药。.