Objectives: 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) for 2 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, and 1.6 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 mRNA expressions of LSINCT5, phosphatidylinositol 3-kinase (PI3K) and protein kinase B (Akt) were measured by real-time PCR. The protein levels of PI3K, 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, PI3K, 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).
Conclusions: The expression of LSINCT5 is high in the erlotinib-resistant cells. Interference with LSINCT5 may inhibit the expression and activity of Akt and promote the cell sensitivity to erlotinib.
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 stress-induced 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1.6 μmol/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1.6 μmol/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。.
Keywords: drug resistance; erlotinib; long non-coding RNA; lung cancer.