长链非编码RNA应激诱导长链非编码转录本5对肺癌细胞厄洛替尼敏感性的影响及其机制  

Effect of long non-coding RNA long stress-induced noncoding transcript 5 on erlotinib resistance to lung cancer cells and the underlying mechanisms

作　　者：陈一川[1] 刘嘉苗 卢婷[1] 唐敬群[3] 李乐之[4] 刘芳[4] CHEN Yichuan;LIU Jiamiao;LU Ting;TANG Jingqun;LI Lezhi;LIU Fang(Department of Cardiovascular Surgery,Second Xiangya Hospital,Central South University,Changsha 410011;Five Year Program of Clinical Medicine,Xiangya School of Medicine,Central South University,Changsha 410078;Department of Thoracic Surgery,Second Xiangya Hospital,Central South University,Changsha 410011;Clinic Nursing Teaching and Research Section,Second Xiangya Hospital,Central South University,Changsha 410011,China)

机构地区：[1]中南大学湘雅二医院心血管外科,长沙410011 [2]中南大学湘雅医学院,长沙410078 [3]中南大学湘雅二医院胸外科,长沙410011 [4]中南大学湘雅二医院临床护理教研室,长沙410011

出　　处：《中南大学学报（医学版）》2020年第8期886-891,共6页Journal of Central South University ：Medical Science

摘　　要：目的:探究长链非编码RNA(long non-coding RNA,lncRNA)应激诱导长链非编码转录本5(long stressinduced noncoding transcript 5,LSINCT5)与肺癌细胞厄洛替尼抵抗的关系及潜在机制。方法:收集并培养人肺癌细胞系A549,H520,H358,H1299,SPCA1和PC9。将表皮生长因子受体(epidermal growth factor receptor,EGFR)突变型肺癌细胞系PC9分为对照组、抵抗组、干扰Ⅰ组和Ⅱ组。对照组采用二甲基亚砜(dimethylsulfoxide,DMSO)处理10周后,再用慢病毒转染无意义对照靶向序列表达载体;抵抗组采用梯度浓度的厄洛替尼(浓度分别为0.1,0.2,0.4,0.8和1.6μmol/L)分别处理2周后,再用慢病毒转染无意义对照靶向序列表达载体;干扰I组和II组采用梯度浓度的厄洛替尼(浓度分别为0.1,0.2,0.4,0.8和1.6μmol/L)分别处理2周后,再用慢病毒转染shRNA靶向LSINCT5表达载体1号和2号。细胞计数试剂盒8(cell counting kit-8,CCK-8)检测细胞厄洛替尼半抑制浓度(50%inhibitory concentration,IC50);real-time PCR检测细胞中LSINCT5,磷脂酰肌醇-3激酶(phosphatidylinositol 3-kinase,PI3K)和蛋白激酶B(protein kinase B,Akt) mRNA表达;蛋白质印迹法检测细胞PI3K,Akt和磷酸化Akt(phospho-Akt,p-Akt)蛋白表达;RNA结合蛋白免疫沉淀(RNA immunoprecipitation,RNA-IP)实验检测LSINCT5与Akt和IgG结合作用的差异。结果:PC9细胞中LSINCT5表达显著高于其他人肺癌细胞系(均P<0.05);与对照组相比,抵抗组厄洛替尼IC50,LSINCT5,PI3K和Akt mRNA和蛋白表达均显著升高(均P<0.05),干扰Ⅰ组和Ⅱ组厄洛替尼IC50,LSINCT5,Akt和p-Akt蛋白表达均显著降低(均P<0.05)。与IgG结合相比,LSINCT5和Akt的结合显著增多(P<0.05)。结论:LSINCT5在厄洛替尼抵抗细胞中呈高表达,干扰其表达可抑制Akt的表达及活性,并能促进细胞对厄洛替尼的敏感性。Objective: To explore the relationship between long non-coding RNA(lncRNA) long stress-induced noncoding transcript 5(LSINCT5) and erotinib resistance to lung cancer cells and the potential mechanisms.Methods: Human lung cancer cell line A549, H520, H358, H1299, SPCA1, and PC9 were collected and cultured. Epidermal growth factor receptor(EGFR) mutant lung cancer cell line PC9 was divided into a control group, a resistance group, a interference group Ⅰ and Ⅱ.The control group was treated with dimethylsulfoxide(DMSO) for 10 weeks and then was transfected with control target sequence expression vector. The resistant group was treated with erlotinib at gradient concentration(0.1, 0.2, 0.4, 0.8, and 1.6 μmol/L, respectively) for2 weeks and then transfected with control target sequence expression vector. Interference group Ⅰ and Ⅱ were treated with erlotinib at gradient concentration(0.1, 0.2, 0.4, 0.8, and1.6 μmol/L, respectively) for 2 weeks and then transfected with the shRNA targeting expression vectors 1 and 2. 50% inhibitory concentration(IC50) of erlotinib was detected by cell counting kit-8(CCK-8) assay. The m RNA expressions of LSINCT5,phosphatidylinositol 3-kinase(PI3 K) and protein kinase B(Akt) were measured by realtime PCR. The protein levels of PI3 K, Akt, and phospho-Akt(p-Akt) were detected by Western blotting. The divergences of Akt and IgG binding to LSINCT5 were detected by RNA immunoprecipitation(RNA-IP) experiment.Results: The expression of LSINCT5 in PC9 cells was significantly higher than that in other lung cancer cell lines(all P<0.05). Compared with the control group, the IC50 of erotinib and the expression of LSINCT5, PI3 K, and Akt mRNA and protein in the resistance group were significantly higher(all P<0.05), and the IC50 of erotinib and the expression of LSINCT5, Akt, and p-Akt in the interference group I and II were significantly lower(all P<0.05). Compared with IgG, LSINCT5 binding to Akt was increased significantly(P<0.05).Conclusion: The expression of LSINCT5 is high in the er

关 键 词：肺癌 长链非编码RNA 厄洛替尼 药物抵抗 

分 类 号：R734.2[医药卫生—肿瘤]