非小细胞肺癌PC-9细胞厄洛替尼耐药潜在机制的RNA测序分析

Potential mechanism for the resistance of non-small cell lung cancer PC-9 cells to erlotinib analyzed through RNA sequencing

目的:通过RNA测序分析比较亲本PC-9细胞和厄洛替尼获得性耐药PC-9细胞(PC-9/ER)表达谱的差异,揭示非小细胞肺癌(NSCLC)表皮生长因子受体酪氨酸激酶抑制剂(EGFR-TKI)耐药的潜在机制。方法:采用间歇诱导方法,建立PC-9/ER耐药细胞株,通过MTT实验绘制厄洛替尼药物浓度-细胞存活率曲线。通过RNA测序分析PC-9/ER细胞的差异表达基因并进行GO和KEGG功能富集分析;通过qRT-PCR进一步筛选可能参与EGFR-TKI耐药的潜在基因或可能的靶点。结果:厄洛替尼对PC-9/ER细胞增殖的抑制率显著低于PC-9细胞的抑制率,PC-9/ER细胞的耐药指数为41.92。与PC-9细胞相比,RNA测序PC-9/ER细胞筛选出1028个差异表达基因,其中720个基因表达上调,308个基因表达下调,而且差异表达基因显著富集在PI3K-AKT通路和癌症通路。qRT-PCR验证差异表达基因的转录水平与测序结果基本一致。结论:ST6GALNAC3、CYP1A1、PAPPA2、INHBE和ACSS3等基因可能参与EGFR-TKI的耐药过程,针对PC-9/ER细胞差异表达基因的后续实验可能为将来改善NSCLC的靶向治疗进一步提供实验和理论依据。

Objective:To analyze and compare the gene expression profiles of parental PC-9 cells and acquired erlotinib-resistant PC-9(PC-9/ER)cells through RNA sequencing,and to reveal the potential mechanism of epidermal growth factor receptor tyrosine kinase inhibitor(EGFR-TKI)resistance for non-small cell lung cancer(NSCLC).Methods:Intermittent exposure to erlotinib was used to establish erlotinib-resistant cell line PC-9/ER,which was validated by the curve of erlotinib concentration-cell survival rate.The differentially expressed genes of PC-9/ER cells were analyzed with GO and KEGG functional enrichment analysis.Furthermore,qRT-PCR assay was subsequently utilized to screen potential genes or possible targets participated in EGFR-TKI resistance.Results:The inhibitory effect of erlobinib on PC-9/ER cells was much less than on PC-9 cells,with the resistance index of PC-9/ER cells to erlotinib being 41.92 based on the drug concentration-cell survival rate curve.A total of 1028 differentially expressed genes were determined in PC-9/ER cells compared with PC-9 cells,with 720 genes up-regulated and 308 genes down-regulated,which were significantly enriched in PI3K-AKT signaling pathway and pathways in cancer.The qRT-PCR verification demonstrated that the transcriptional levels of differentially expressed genes were basically consistent with the sequencing results.Conclusion:Genes including ST6GALNAC3,CYP1A1,PAPPA2,INHBE and ACSS3 might be involved in the resistance to EGFR-TKI.Follow-up experiments for the differentially express genes may provide further experimental and theoretical basis to improve the target therapy for NSCLC.