lncRNA LUCAT1影响自噬促进肺腺癌PC-9/GR细胞对吉非替尼耐药的研究  

lncRNA LUCAT1 promotes gefitinib resistance in lung adenocarcinoma PC-9/GR cells by affecting autophagy

作　　者：姜梅 王鑫 李雷 李乐瑶 姚雨君 李亚军 JIANG Mei;WANG Xin;LI Lei;LI Leyao;YAO Yujun;LI Yajun(Oncology Department,the Third Affiliated Hospital of Zunyi Medical University/the First People's Hospital of Zunyi,Guizhou Zunyi 563000,China;Oncology Department,the Third Affiliated Hospital of Zunyi Medical University/Scientific Research Center,the First People's Hospital of Zunyi,Guizhou Zunyi 563000,China)

机构地区：[1]遵义医科大学第三附属医院肿瘤科/遵义市第一人民医院,贵州遵义563000 [2]遵义医科大学第三附属医院肿瘤科/遵义市第一人民医院中心实验室,贵州遵义563000

出　　处：《现代肿瘤医学》2024年第2期255-261,共7页Journal of Modern Oncology

基　　金：国家自然科学基金(编号:82060544);贵州省科技计划项目(编号:黔科合基础-ZK[2023]一般486);遵义市第一人民医院研究与试验发展课题(编号:院科字(2020)10号)。

摘　　要：目的:探索lncRNA LUCAT1对肺腺癌PC-9/GR细胞耐药性的影响,并研究其潜在的耐药机制。方法:CCK-8、克隆形成及Transwell迁移实验检测细胞的药物敏感性、增殖能力、克隆形成能力及迁移能力,qRT-PCR检测细胞中lncRNA LUCAT1的mRNA表达,Western Blot检测自噬关键蛋白p62、Beclin1、LC3-Ⅱ/LC3-Ⅰ的表达,透射电子显微镜观察细胞中自噬小体数量变化。慢病毒转染构建敲低lncRNA LUCAT1和对照组细胞(PC-9/GR-shLUCAT1、PC-9/GR-NC),48 h后荧光显微镜观察慢病毒转染情况,进一步采用qRT-PCR检测敲低LUCAT1转染效果,检测调控LUCAT1前后各组细胞耐药性、克隆及迁移能力的改变,Western Blot实验检测各组细胞自噬关键蛋白表达情况。结果:PC-9/GR细胞较亲本细胞PC-9具有较强耐药性和增殖、迁移能力(P<0.05);PC-9/GR细胞中LUCAT1的mRNA相对表达量高于亲本细胞(P<0.05),并且自噬关键基因LC3-II/LC3-I的蛋白表达量较高,p62蛋白表达较其亲本细胞偏低(P<0.05),而Beclin1蛋白在PC-9/GR和PC-9细胞中表达无明显差异性(P>0.05);PC-9/GR细胞中自噬小体数量明显多于亲本细胞(P<0.05)。敲低LUCAT1后PC-9/GR细胞的耐药性、克隆形成能力及迁移能力均较对照组细胞明显减弱(P<0.05),同时自噬关键基因LC3-II/LC3-I下降、p62蛋白升高(P<0.05)。另外我们发现在PC-9/GR细胞中敲低LUCAT1不影响自噬关键基因Beclin1的蛋白表达(P>0.05),可能在继发性耐药细胞中LUCAT1不是通过影响Beclin1蛋白来调控自噬。结论:肺腺癌PC-9/GR细胞高表达lncRNA LUCAT1及具有高水平自噬,LUCAT1可能通过影响LC3B/p62介导的自噬通路激活自噬进而促进肺腺癌细胞对吉非替尼发生耐药。Objective:To explore the effect of lncRNA LUCAT1 on drug resistance of lung adenocarcinoma PC-9/GR cells and to study its potential mechanism of drug resistance.Methods:CCK-8,colony formation and Transwell migration assay were used to detect the drug sensitivity,proliferation,clone formation and migration of cells.The mRNA expression of lncRNA LUCAT1 in cells was detected by qRT-PCR.The expression of key autophagy proteins p62,Beclin1,LC3-Ⅱ/LC3-Ⅰwas detected by Western Blot,and the number of autophagosomes in the cells was observed by transmission electron microscope.Lentiviral transfection was used to knock down lncRNA LUCAT1 and control cells(PC-9/GR-shLUCAT1,PC-9/GR-NC).After 48 h,the transfection of lentivirus was observed by fluorescence microscope.The transfection effect of knockdown LUCAT1 was further detected by qRT-PCR.The changes of drug resistance,cloning and migration ability of cells in each group before and after regulation of LUCAT1 were detected and Western Blot assay was used to detect the expression of autophagy key proteins in each group.Results:Compared with parent cells PC-9,PC-9/GR cells had stronger drug resistance,proliferation and migration ability(P<0.05).The relative mRNA expression of lncRNA LUCAT1 in PC-9/GR cells was higher than that in parent cells(P<0.05),and the protein expression of autophagy key gene LC3-II/LC3-I was higher,and the protein expression of p62 was lower than that in parent cells(P<0.05),but there was no significant difference in the expression of Beclin1 protein between PC-9/GR and PC-9 cells(P>0.05).The number of autophagosomes in PC-9/GR cells was significantly higher than that in parent cells(P<0.05).After knocking down LUCAT1,the drug resistance,colony formation ability and migration ability of PC-9/GR cells were significantly weaker than those of the control group(P<0.05).At the same time,the autophagy key gene LC3-II/LC3-I decreased and p62 protein increased(P<0.05).In addition,we found that knocking down LUCAT1 in PC-9/GR cells did not affect the protein

关 键 词：肺腺癌 lncRNA LUCAT1 自噬 吉非替尼 获得性耐药 

分 类 号：R734.2[医药卫生—肿瘤]