下调溶质运载体家族7成员11对表皮生长因子受体基因19号外显子突变肺腺癌细胞吉非替尼耐药性的影响及其机制

Effect of SLC7A11 gene downregulation on the gefitinib resistance of lung adenocarcinoma PC9/GR cells and its mechanism

目的筛选促进表皮生长因子受体(EGFR)基因19号外显子突变肺腺癌吉非替尼耐药细胞PC9/GR的关键基因,探讨下调溶质运载体家族7成员11(SLC7A11)对PC9/GR细胞吉非替尼耐药性的影响及其机制。方法采用RNA微阵列技术检测PC9细胞和PC9/GR细胞的基因表达,使用R语言的limma包筛选差异基因并使用clusterProfiler包进行京都基因与基因组百科全书(KEGG)基因富集分析。使用Western blot法检测SLC7A11蛋白在PC9和PC9/GR细胞中的表达,采用慢病毒包装体系构建下调SLC7A11的短发卡RNA(shRNA)和阴性对照shRNA慢病毒并对PC9/GR细胞进行转染,实时荧光定量聚合酶链反应检测shRNA对SLC7A11 mRNA的下调效率,细胞计数试剂盒8法检测吉非替尼对PC9/GR细胞的杀伤能力,线粒体红色荧光探针和丙二醛(MDA)检测试剂盒检测PC9/GR细胞中吉非替尼介导的铁死亡,免疫组化检测SLC7A11在携带EGFR基因19号外显子突变的晚期肺腺癌患者(选取2016—2020年于河北医科大学第四医院接受EGFR酪氨酸激酶抑制剂作为一线治疗方案的晚期肺癌患者36例)肿瘤组织中的表达,绘制Kaplan-Meier生存曲线分析SLC7A11与患者无进展生存时间(PFS)的相关性。结果RNA微阵列结果显示,PC9和PC9/GR细胞的差异表达基因共2888个,KEGG富集分析结果显示,铁死亡相关基因是PC9和PC9/GR细胞差异表达基因的主要富集区域之一,共包含13个基因,其中SLC7A11表达差异最大。Western blot结果显示,PC9/GR细胞中SLC7A11蛋白的相对表达水平为0.76±0.03,高于PC9细胞(0.19±0.02,P<0.001)。吉非替尼干预sh-SLC7A11组和sh-NC组PC9/GR细胞的半数抑制浓度值分别为35.08和64.01μmol/L。吉非替尼干预后,与sh-NC组比较,sh-SLC7A11组PC9/GR细胞的线粒体荧光强度降低(分别为213.77±26.50和47.88±4.55,P<0.001),MDA含量增加[分别为(15.43±1.60)μmol/mg和(82.18±7.77)μmol/mg,P<0.001]。SLC7A11低表达组(n=18)患者的中位PFS为16.77个月,优于SLC7A11高表达组(n=18)患者(9.14个月,P<0.001)。结论下调SLC7A11能够通过促进铁死亡提高PC9/GR细胞对吉非替尼的敏感性。

Objective To screen the key genes involved in gefitinib resistance of lung adenocarcinoma PC9/GR cells which harbored 19 exon mutation of epidermal growth factor receptor(EGFR)gene,and discuss the effect and mechanism of downregulation of solute carrier family 7 member 11(SLC7A11)on the gefitinib resistance of PC9/GR cells.Methods RNA microarray was conducted to detect the gene expressions in PC9 and PC9/GR cells.The differently expressed genes were screened by using limma package of R language and analyzed by Kyoto encyclopedia of genes and genomes(KEGG)pathway enrichment analysis.Western blotting was performed to determine the expression of SLC7A11 protein in PC9 and PC9/GR cells.PC9/GR cells were infected with lentivirus plasmid containing short hairpin RNA(shRNA)targeting SLC7A11 or negative control shRNA(sh-NC),respectively.Real-time quantitative polymerase chain reaction(RT-qPCR)was performed to evaluate the efficacy of shRNA on the expression of SLC7A11 mRNA.Cell counting kit-8(CCK-8)assay was conducted to determine the suppressing effect of gefitinib on PC9/GR cells.Mito-Tracker Red CMXRos probe and malondialdehyde(MDA)assay kit were used to evaluate gefitinib-induced ferroptosis in PC9/GR cells.Immunohistochemistry(IHC)was conducted to detect the expression of SLC7A11 protein in the tumor tissues of advanced stage lung adenocarcinoma patients harboring 19 exon mutation of EGFR gene.Thirty-six advanced stage lung adenocarcinoma patients who received EGFR-tyrosihe kinase inhibitor(TKI)as first-line treatment in Fourth Hospital of Hebei Medical Unviersity were enrolled.Kaplan-Meier survival curve was drawn to analyze the correlation between SLC7A11 expression and progression-free survival(PFS)of the patients.Results RNA array demonstrated that 2888 genes were differently expressed between PC9 and PC9/GR cells.KEGG analysis showed that ferroptosis-related gene was one of the most enriched region of the differently expressed genes between PC9 and PC9/GR cells.These ferroptosis-related gene cohort contained 13 genes,among which SLC7A11 exhibited the most significant difference.Western blotting showed that the expression of SLC7A11 protein in PC9/GR cells was significantly higher than that in PC9 cells(0.76±0.03 vs.0.19±0.02,P<0.001).The 50%inhibiting concentration(IC50)of gefitinib was 35.08μmol/L and 64.01μmol/L for sh-SLC7A11 and sh-NC group PC9/GR cells,respectively.PC9/GR cells in sh-SLC7A11 group exhibited significantly lower density of mitochondria fluorescence after gefitinib treatment,compared to the sh-NC group(213.77±26.50 vs.47.88±4.55,P<0.001).In addition,PC9/GR cells in sh-SLC7A11 group exhibited significantly higher MDA after gefitinib treatment,compared to the sh-NC group[(15.43±1.60)μmol/mg vs.(82.18±7.77)μmol/mg,P<0.001].The PFS of the patients with low expression of SLC7A11(n=18)was significantly longer than the patients with high expression of SLC7A11(n=18,16.77 months vs.9.14 months,P<0.001).Conclusion Downregulation of SLC7A11 could increase the sensitivity of PC9/GR cells to gefitinib by promoting ferroptosis.