【摘要】：目的 :建立吉非替尼獲得性耐藥非小細(xì)胞肺癌細(xì)胞株,并篩選和分析耐藥前后差異表達(dá)的環(huán)狀RNA(circular RNA,circ RNA)。方法:利用吉非替尼敏感性非小細(xì)胞肺癌細(xì)胞株H292(簡(jiǎn)稱(chēng)為H292_S),采用逐步加藥法建立吉非替尼獲得性耐藥株(命名為H292_R)。CCK-8法檢測(cè)吉非替尼對(duì)2種細(xì)胞增殖的抑制作用;RNA測(cè)序(RNA sequencing,RNA-seq)法篩選2種細(xì)胞中差異表達(dá)的circRNA;實(shí)時(shí)熒光定量PCR法驗(yàn)證RNA-seq結(jié)果,并結(jié)合統(tǒng)計(jì)學(xué)和生物信息學(xué)方法對(duì)差異circRNA來(lái)源基因的生物學(xué)功能進(jìn)行分析。結(jié)果:吉非替尼對(duì)H292_S和H292_R細(xì)胞增殖的半數(shù)抑制濃度分別為(108.63±0.32)nmol/L和(982.37±2.62)nmol/L,2者間差異有統(tǒng)計(jì)學(xué)意義(P值均0.05),耐藥細(xì)胞H292_R對(duì)吉非替尼的耐藥指數(shù)為9.04。2種細(xì)胞中存在36個(gè)差異表達(dá)circRNA,其中耐藥細(xì)胞中24個(gè)circRNA表達(dá)上調(diào)(P值均0.05),12個(gè)circRNA表達(dá)下調(diào)(P值均0.05);GO富集分析發(fā)現(xiàn),異常表達(dá)的circRNA主要涉及調(diào)節(jié)細(xì)胞生長(zhǎng)增殖、蛋白轉(zhuǎn)運(yùn)和基因轉(zhuǎn)錄等生物學(xué)過(guò)程;KEGG通路分析發(fā)現(xiàn),異常circRNA的信號(hào)通路主要涉及細(xì)胞質(zhì)DNA感受通路、核因子κB(nuclear factor-kappa B,NF-κB)、胞吞和凋亡等通路。實(shí)時(shí)熒光定量PCR法驗(yàn)證2種細(xì)胞中差異表達(dá)circRNA hsa_circ_0000567和hsa_circ_0000620的表達(dá)水平差異,結(jié)果顯示耐藥細(xì)胞中這2個(gè)circRNA水平均明顯上調(diào)(P值均0.05),與RNA-seq結(jié)果相符。結(jié)論:篩選出吉非替尼獲得性耐藥相關(guān)的差異表達(dá)circRNA,這有助于更深入地闡明非小細(xì)胞肺癌耐藥的機(jī)制,并可能為其早期診斷提供生物學(xué)標(biāo)志。
[Abstract]:Objective: to establish a gefitinib acquired drug-resistant non-small cell lung cancer cell line and screen and analyze the differentially expressed cyclic RNA (circular RNA,circ RNA). Before and after drug resistance. Methods: Gifetinib sensitive non-small cell lung cancer cell line H292 (abbreviated as H292 鈮，

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