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

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 2 888 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 (IC(50)) of gefitinib was 35.08 μmol/L and 64.01 μmol/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.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2 888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64.01 μmol/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。.