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
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/G...
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| Published in | 中华肿瘤杂志 Vol. 45; no. 9; pp. 779 - 786 |
|---|---|
| Main Authors | , , , , , , |
| Format | Journal Article |
| Language | Chinese |
| Published |
河北医科大学第四医院肿瘤免疫治疗科，石家庄 050011%河北医科大学第四医院肿瘤内科，石家庄 050011%河北医科大学第四医院放疗科，石家庄 050011%河北医科大学第四医院病理科，石家庄 050011
01.09.2023
|
| Subjects | |
| Online Access | Get full text |
| ISSN | 0253-3766 |
| DOI | 10.3760/cma.j.cn112152-20220715-00493 |
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| Abstract | 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 |
|---|---|
| AbstractList | 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 |
| Abstract_FL | 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. |
| Author | 郑博阳 刘月平 刘丽华 程子硕 贾云泷 甄书漫 赵妍 |
| AuthorAffiliation | 河北医科大学第四医院肿瘤免疫治疗科，石家庄 050011%河北医科大学第四医院肿瘤内科，石家庄 050011%河北医科大学第四医院放疗科，石家庄 050011%河北医科大学第四医院病理科，石家庄 050011 |
| AuthorAffiliation_xml | – name: 河北医科大学第四医院肿瘤免疫治疗科，石家庄 050011%河北医科大学第四医院肿瘤内科，石家庄 050011%河北医科大学第四医院放疗科，石家庄 050011%河北医科大学第四医院病理科，石家庄 050011 |
| Author_FL | Jia Yunlong Zhen Shuman Liu Yueping Zhao Yan Zheng Boyang Cheng Zishuo Liu Lihua |
| Author_FL_xml | – sequence: 1 fullname: Jia Yunlong – sequence: 2 fullname: Zhao Yan – sequence: 3 fullname: Zhen Shuman – sequence: 4 fullname: Cheng Zishuo – sequence: 5 fullname: Zheng Boyang – sequence: 6 fullname: Liu Yueping – sequence: 7 fullname: Liu Lihua |
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| Keywords | Adenocarcinoma 腺癌 溶质运载体家族7成员11 Solute carrier family 7 member 11 Ferroptosis 吉非替尼 肺肿瘤 铁死亡 Gefitinib 耐药 Lung neoplasms Treatment-resistance |
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| Title | 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 |
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