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.
Zhong Nan Da Xue Xue Bao Yi Xue Ban. 2020 Aug 28;45(8):886-891. doi: 10.11817/j.issn.1672-7347.2020.190705.
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.
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 (IC) 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.
The expression of LSINCT5 in PC9 cells was significantly higher than that in other lung cancer cell lines (all <0.05). Compared with the control group, the IC of erotinib and the expression of LSINCT5, PI3K, and Akt mRNA and protein in the resistance group were significantly higher (all <0.05), and the IC of erotinib and the expression of LSINCT5, Akt, and p-Akt in the interference group I and II were significantly lower (all <0.05). Compared with IgG, LSINCT5 binding to Akt was increased significantly (<0.05).
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.
探讨长链非编码RNA（lncRNA）长应激诱导非编码转录本5（LSINCT5）与肺癌细胞对厄洛替尼耐药性之间的关系及潜在机制。
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（EGFR）突变的肺癌细胞系PC9分为对照组、耐药组、干扰组I和干扰组II。对照组用二甲基亚砜（DMSO）处理10周，然后转染对照靶序列表达载体。耐药组用梯度浓度（分别为0.1、0.2、0.4、0.8和1.6 μmol/L）的厄洛替尼处理2周，然后转染对照靶序列表达载体。干扰组I和干扰组II用梯度浓度（分别为0.1、0.2、0.4、0.8和1.6 μmol/L）的厄洛替尼处理2周，然后转染靶向shRNA表达载体1和2。采用细胞计数试剂盒-8（CCK-8）法检测厄洛替尼的半数抑制浓度（IC）。通过实时PCR检测LSINCT5、磷脂酰肌醇3激酶（PI3K）和蛋白激酶B（Akt）的mRNA表达。通过蛋白质印迹法检测PI3K、Akt和磷酸化Akt（p-Akt）的蛋白水平。通过RNA免疫沉淀（RNA-IP）实验检测Akt与LSINCT5结合的差异及IgG情况。
PC9细胞中LSINCT5的表达明显高于其他肺癌细胞系（均P<0.05）。与对照组相比，耐药组中厄洛替尼的IC以及LSINCT5、PI3K和Akt的mRNA及蛋白表达均显著升高（均P<0.05），干扰组I和干扰组II中厄洛替尼的IC以及LSINCT5、Akt和p-Akt的表达均显著降低（均P<0.05）。与IgG相比，LSINCT5与Akt的结合显著增加（P<0.05）。
LSINCT5在厄洛替尼耐药细胞中表达较高。干扰LSINCT5可能抑制Akt的表达和活性，提高细胞对厄洛替尼的敏感性。
