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目的 :建立吉非替尼获得性耐药非小细胞肺癌细胞株,并筛选和分析耐药前后差异表达的环状RNA(circular RNA,circ RNA)。方法:利用吉非替尼敏感性非小细胞肺癌细胞株H292(简称为H292_S),采用逐步加药法建立吉非替尼获得性耐药株(命名为H292_R)。CCK-8法检测吉非替尼对2种细胞增殖的抑制作用;RNA测序(RNA sequencing,RNA-seq)法筛选2种细胞中差异表达的circRNA;实时荧光定量PCR法验证RNA-seq结果,并结合统计学和生物信息学方法对差异circRNA来源基因的生物学功能进行分析。结果:吉非替尼对H292_S和H292_R细胞增殖的半数抑制浓度分别为(108.63±0.32)nmol/L和(982.37±2.62)nmol/L,2者间差异有统计学意义(P值均<0.05),耐药细胞H292_R对吉非替尼的耐药指数为9.04。2种细胞中存在36个差异表达circRNA,其中耐药细胞中24个circRNA表达上调(P值均<0.05),12个circRNA表达下调(P值均<0.05);GO富集分析发现,异常表达的circRNA主要涉及调节细胞生长增殖、蛋白转运和基因转录等生物学过程;KEGG通路分析发现,异常circRNA的信号通路主要涉及细胞质DNA感受通路、核因子κB(nuclear factor-kappa B,NF-κB)、胞吞和凋亡等通路。实时荧光定量PCR法验证2种细胞中差异表达circRNA hsa_circ_0000567和hsa_circ_0000620的表达水平差异,结果显示耐药细胞中这2个circRNA水平均明显上调(P值均<0.05),与RNA-seq结果相符。结论:筛选出吉非替尼获得性耐药相关的差异表达circRNA,这有助于更深入地阐明非小细胞肺癌耐药的机制,并可能为其早期诊断提供生物学标志。
OBJECTIVE: To establish gefitinib-resistant non-small cell lung cancer cell lines and to screen and analyze differentially expressed circular RNA (circ RNA) before and after drug resistance. Methods: The gefitinib-resistant non-small cell lung cancer cell line H292 (referred to as H292_S) was established by stepwise dosing method. CCK-8 assay was used to detect the inhibitory effect of gefitinib on the proliferation of the two cell lines; RNA sequencing (RNA-seq) was used to screen the circRNAs differentially expressed in the two cell types; RNA-seq was detected by real- The biological functions of the genes of different circRNAs were analyzed with statistical methods and bioinformatics methods. Results: The half inhibitory concentrations of gefitinib on H292_S and H292_R cells were (108.63 ± 0.32) nmol / L and (982.37 ± 2.62) nmol / L respectively, with significant difference between the two groups (P <0.05) ). The drug resistance index of resistant cell H292_R to gefitinib was 9.04. There were 36 differentially expressed circulating circRNAs in 24 kinds of cells, of which 24 circRNAs were up-regulated (P <0.05), and 12 circRNAs The results of GO enrichment analysis showed that abnormally expressed circRNA mainly involved in the regulation of cell proliferation and proliferation, protein transport and gene transcription and other biological processes; KEGG pathway analysis found that aberrant circRNA signaling pathway mainly involved the cytoplasm DNA sensing pathway, nuclear factor-kappa B (NF-κB), endocytosis and apoptosis pathways. Real-time fluorescent quantitative PCR method was used to verify the difference of expression levels of circRNA hsa_circ_0000567 and hsa_circ_0000620 between the two cell lines. The results showed that the levels of these two circRNAs were all significantly up-regulated (P <0.05), which was consistent with the results of RNA-seq. CONCLUSIONS: CircRNA, which is differentially expressed related to acquired resistance to gefitinib, was screened out, which may help elucidate the mechanism of resistance to non-small cell lung cancer more deeply and may provide biological markers for its early diagnosis.