TRHDE-AS1对肺癌细胞迁移侵袭和JAK/STAT通路的影响

Effects of TRHDE-AS1 on migration,invasion and JAK/STAT pathway of lung cancer cells

目的探讨长非编码RNA促甲状腺激素释放激素降解酶反义RNA 1(TRHDE-AS1)在非小细胞肺癌(NSCLC)侵袭和迁移过程中的分子功能及对Janus激酶/信号转导和转录活化因子(JAK/STAT)通路的影响。方法利用GEPIA在线分析TCGA数据库中NSCLC组织的TRHDE-AS1水平,采用实时定量PCR(qPCR)检测4种NSCLC细胞株(ANIP-973、NCI-H157、NCI-H1975和A549)和人正常肺上皮细胞BEAS-2B的TRHDE-AS1水平。将A549细胞作为后续功能验证实验对象并分为3组:对照组(未转染作空白对照)、pcDNA3.1组(转染pcDNA3.1空质粒作阴性对照)和pcDNA3.1-TRHDE-AS1组(转染pcDNA3.1-TRHDE-AS1质粒作过表达处理)。MTT法、划痕实验和Transwell小室实验分别检测TRHDE-AS1在A549细胞增殖、侵袭和迁移中的作用,qPCR和Western blotting检测JAK1、STAT3、p-JAK1和p-STAT3水平。结果NSCLC组织的TRHDE-AS1水平低于正常组织(P<0.05),且与BEAS-2B细胞(1.000±0.057)相比,TRHDE-AS1在NSCLC细胞ANIP-973(0.643±0.042)、NCI-H157(0.552±0.048)、NCI-H1975(0.446±0.038)和A549(0.391±0.027)的水平均呈现低表达(P<0.05)。转染处理后,pcDNA3.1-TRHDE-AS1组的TRHDE-AS1水平较对照组(1.000±0.058)和pcDNA3.1组(1.062±0.064)升高至1034.430±70.276(P<0.05)。pcDNA3.1-TRHDE-AS1组在转染48和72 h后的细胞增殖活性均低于对照组和pcDNA3.1组(P<0.05)。pcDNA3.1-TRHDE-AS1组的划痕愈合率和穿膜细胞数分别为(24.116±3.378)%和(101.243±9.092)个,均低于对照组的(76.826±7.909)%和(165.482±14.582)个及pcDNA3.1组的(78.724±8.578)%和(168.232±18.083)个(P<0.05)。与其余两组相比,pcDNA3.1-TRHDE-AS1组A549细胞JAK1和STAT3水平的差异无统计学意义(P>0.05),而p-JAK1和p-STAT3水平降低(P<0.05)。结论NSCLC组织和细胞的TRHDE-AS1水平降低,且过表达TRHDE-AS1可在体外抑制NSCLC的侵袭和迁移并失活JAK/STAT通路,为NSCLC的治疗提供可能的干预手段。

Objective To uncover the molecular functions of long non-coding RNA thyrotropin releasing hormone degrading enzyme antisense RNA 1(TRHDE-AS1)in invasion and migration progression and its effects on Janus kinase/signal transducer and activator of transcription(JAK/STAT)pathway of non-small cell lung cancer(NSCLC)cells.Methods TRHDE-AS1 level in NSCLC tissues of TCGA database was analyzed by GEPIA online database.Real-time quantitative PCR(qPCR)was performed to detect TRHDE-AS1 levels in four NSCLC cell lines(ANIP-973,NCI-H157,NCI-H1975 and A549)and normal lung epithelial cell line BEAS-2B.A549 cell was allocated into three groups:control(cells without transfection as blank)group,pcDNA3.1(cells transfected with pcDNA3.1 as negative control)group and pcDNA3.1-TRHDE-AS1(cells transfected with pcDNA3.1-TRHDE-AS1)group.Besides,the function of TRHDE-AS1 in proliferation,invasion and migration were detected through MTT assay,Transwell assay and scratch assay in A549 cells.Levels of JAK1,STAT3,p-JAK1 and p-STAT3 were evaluated by qPCR and Western blotting.Results The TRHDE-AS1 level in NSCLC tissues was lower than that in normal tissues(P<0.05).Compared with 1.000±0.057 in BEAS-2B cells,TRHDE-AS1 levels were remarkably downregulated in NSCLC cells including ANIP-973(0.643±0.042),NCI-H157(0.552±0.048),NCI-H1975(0.446±0.038)and A549(0.391±0.027)cells.Compared with control group(1.000±0.058)and pcDNA3.1 group(1.062±0.064),the TRHDE-AS1 level in pcDNA3.1-TRHDE-AS1 group was up-regulated to 1034.430±70.276(P<0.05).The proliferation activity after 48 and 72 h-transfection of the pcDNA3.1-TRHDE-AS1 group were lower than those of control group and pcDNA3.1 group(P<0.05).The scratch healing rate and number of transmembrane cells of pcDNA3.1-TRHDE-AS1 group were(24.116±3.378)%and 101.243±9.092,lower than(76.826±7.909)%and 165.482±14.582 of control group and(78.724±8.578)%and 168.232±18.083 of pcDNA3.1 group(P<0.05).No differences were observed among three groups in levels of JAK1 and STAT3,while levels of p-JAK1 and p-STAT3 were significantly decreased in pcDNA3.1-TRHDE-AS1 group compared with other two group(P<0.05).Conclusion Levels of TRHDE-AS1 in NSCLC tissues and cells were decreased.Overexpression of TRHDE-AS1 can inhibit the invasion and migration of NSCLC in vitro and inactivate JAK/STAT pathway,providing a possible intervention for the treatment of NSCLC.