吉非替尼获得性耐药非小细胞肺癌细胞中差异表达环状RNA分析

Differential expression of circular RNAs in gefitinib-acquired resistant non-small cell lung cancer cells

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-acquired resistant non-small cell lung cancer cell lines, then to screen and analyze the differentially expressed circular RNAs （circRNAs） before and after drug resistance. Methods： The gefitinib-sensitive non-small cell lung cancer cell line H292 （named as H292_S） was used to establish gefitinib-acquired resistant cell line （named as H292_R） by stepwise dose escalation of gefitinib. The inhibitory effect of gefitinib on proliferation of the two cell lines was detected by CCK-8 assay. The differentially expressed circRNA in the two cell lines was screened by RNA sequencing （RNA-Seq） method and confirmed by real-time fluorescent quantitative PCR. The biological functions of host genes of differentially expressed circRNAs were analyzed by statistics and bioinformatics methods. Results： The half inhibition concentrations （ICs0） of gefitinib on the proliferation of H292_S and H292_R cells were （108.63＋_0.32） nmol/L and （982.37_＋2.62） nmol/L, respectively; the difference was statistically significant （P 〈 0.05）. The drug-resistant index of H292_P, cells to gefitinib was 9.04. There were 36 differentially expressed circRNAs between the two cell lines （all P 〈 0.05） , including 24 up-regulated circRNAs and 12 clown-regulated circRNAs in H292_R cells. Gene Ontology （GO） enrichment analysis showed that these abnormal expressed circRNAs mainly involved in the regulations of cell growth and proliferation, protein transport, gene transcription and other biological processes. Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway analysis showed that these abnormal circRNAs mainly involved in cytoplasmic DNA-sensing pathway, nuclear factor-kappa B （NF-~B） signaling pathway, endocytosis, apoptosis and so on. Real-time fluorescent quantitative PCR verified that the expressions of hsa_circ_0000567 and hsa_circ_O000620 were significantly up- regulated in drug-resistant H292R cells （both P 〈 0.05）, which was consistent with RNA-seq results. Conclusion： The differentially expressed circRNAs related to gefitinib-acquired resistance are screened out, which maybe helpful to elucidate the mechanism of drug resistance in non- small cell lung cancer, and provide a biological marker for early diagnosis of drug-resistant non-small cell lung cancer.