长链非编码RNA LINC00628通过竞争性结合miR-145促进肺腺癌细胞吉非替尼抵抗

目的探究长链非编码RNA(long non-coding RNA,lncRNA)LINC00628及其潜在靶基因microRNA-145(miR-145)对肺腺癌细胞吉非替尼抵抗的影响。方法收集54例肺腺癌患者的肿瘤及癌旁组织。以人肺腺癌细胞系PC9为体外模型构建吉非替尼抵抗细胞系(Gefitinib resistance,GR组)及相应的对照细胞系(Control组),慢病毒转染随机对照干扰载体及LINC00628小干扰RNA(small interference LINC00628,siLINC00628)载体于GR组细胞中作为干扰对照细胞系(siControl组)及干扰细胞系(siLINC组),转染空白载体、LINC00628过表达载体及miR-145过表达载体于GR细胞中作为空白细胞系(Blank组)、过表达细胞系(miR-145组)及回复组细胞系(Rescue组);实时荧光定量PCR(qRT-PCR)技术检测LINC00628、miR-145、c-Myc及AKT1 mRNA表达水平,Western Blot实验检测c-Myc及AKT1蛋白表达水平,CCK-8实验检测细胞吉非替尼的半抑制浓度(50%inhibitory concentration,IC₅₀),荧光素酶报告基因实验验证LINC00628与miR-145的结合作用。结果相比癌旁组织,LINC00628在肺腺癌中表达显著升高,且与患者预后不良相关;体外实验中,相比Control组,GR组细胞吉非替尼IC₅₀显著增高,LINC00628、AKT1及AKT1表达显著上调,miR-145表达显著降低;相比siControl组,siLINC组细胞吉非替尼IC₅₀显著降低,miR-145表达显著增高,LINC00628、c-Myc及AKT1表达显著下调;相比Blank组,miR-145组吉非替尼IC₅₀显著降低,miR-145表达显著增高,c-Myc及AKT1表达显著下调;相比miR-145组,Rescue组吉非替尼IC₅₀显著升高,miR-145表达显著降低,LINC00628、c-Myc及AKT1表达显著上调。相比NC mimics,miR-145 mimics可使携带LINC00628预测位点的psiCHECK2载体荧光强度显著降低。结论LINC00628可与miR-145相互结合,促进c-Myc及AKT1基因表达及肺腺癌细胞吉非替尼抵抗。

Long non-coding RNA LINC00628 promotes gefitinib resistance in lung adenocarcinoma cells through competitive binding to miR-145

Objective To investigate the effect of long non-coding RNA(lncRNA)LINC00628 and its potential target gene microRNA-145(miR-145)on gefitinib resistance in lung adenocarcinoma cells.Methods Tumor and paracancerous tissues from 54 patients with lung adenocarcinoma were collected.Gefitinib resistance(the GR group)and corresponding control cell line(the control group)were constructed using human lung adenocarcinoma cell line PC9 as in vitro model.Random control interference vector and LINC00628 small interference RNA(small interference RNA)were transferred to GR cells via lentivirus as the si-control group and the si-LINC group.Blank vector,LINC00628 overexpression vector and miR-145 over-expression vector were transferred to GR cells as the blank group,the miR-145 group and the rescue group.The expressions of LINC00628,miR-145,AKT1 and c-Myc were detected by real-time fluorescent quantitative PCR(qRT-PCR).The protein of c-Myc and AKT1 was detected by Western blot,and inhibitory concentration 50%(IC₅₀)of gefitinib was detected by CCK-8 assay.The binding of LINC00628 to miR-145 was verified by luciferase reporter gene assay.Results The expression of LINC00628 in lung adenocarcinoma was significantly higher than that in adjacent tissues and was associated with poor prognosis.In vitro,compared with the control group,IC₅₀ of gefitinib in the GR group was significantly increased,LINC00628,AKT1 and c-Myc were significantly increased,while miR-145 was significantly decreased in the GR group.Compared with the si-control group,IC₅₀ of gefitinib in the GR group was significantly decreased,miR-145 was significantly was up-regulated and LINC00628,AKT1 and c-Myc were significantly down-regulated.Compared with the blank group,IC₅₀ of gefitinib in the miR-145 group was significantly decreased,miR-145 expression was significantly increased and c-Myc and AKT1 expression were significantly down-regulated.Compared with the miR-145 group,IC₅₀ of gefitinib in the rescue group was significantly increased,miR-145 expression was significantly decreased,and c-Myc and AKT1 expression were significantly up-regulated.Compared with the NC mimics,miR-145 mimics significantly reduced the fluorescence intensity of psi-Check vector carrying the site of LINC00628 binding to miR-145.Conclusion Linc00628 can interact with miR-145 to promote c-Myc and AKT1 gene and gefitinib resistance in lung adenocarcinoma cells.