微小RNA-301a对肺癌细胞上皮间质转化和Smad4表达的影响  

Effect of microRNA-301a on the epithelial mesenchymal transition and Smad4 expression of lung cancer cells

作　　者：郝攀 张春丽[2] 闫平 彭如臣[1] HAO Pan;ZHANG Chunli;YAN Ping;PENG Ruchen(Department of Radiology,Beijing Luhe Hospital Affiliated to Capital Medical University,Beijing 101149,China)

机构地区：[1]首都医科大学附属北京潞河医院放射科,北京101149 [2]北京大学第一医院核医学科,100034

出　　处：《临床肿瘤学杂志》2020年第2期103-108,共6页Chinese Clinical Oncology

摘　　要：目的探讨微小RNA-301a(miR-301a)对非小细胞肺癌(NSCLC)细胞上皮间质转化(EMT)和SMAD家族成员4(Smad4)表达的影响。方法采用实时定量PCR(QPCR)检测30对NSCLC组织和癌旁组织的miR-301a和Smad4水平;脂质体法向A549细胞转染miR-301a抑制剂(抑制组)或阴性对照(NC组),另以未行转染的细胞为对照组,采用QPCR、活细胞计数(CCK-8)、划痕实验、Transwell小室实验和Western blotting检测miR-301a水平、增殖活力、划痕愈合率、穿膜细胞数和Smad4水平,QPCR和Western blotting检测EMT相关标志物[上皮型钙黏蛋白(E-cad)、神经型钙黏蛋白(N-cad)和波形蛋白(Vim)]的水平,生物信息学预测和双荧光素酶报告验证miR-301a和Smad4间的靶向关系。结果NSCLC组织的miR-301a水平为2.157±0.597,高于癌旁组织的1.023±0.229,癌组织的Smad4水平为0.648±0.162,低于癌旁组织的1.159±0.164,差异均有统计学意义(P<0.05)。抑制组的miR-301a水平、划痕愈合率和穿膜细胞数分别为0.166±0.075、(31.518±4.306)%和(89.744±15.060)个,均低于对照组的1.032±0.097、(52.461±6.373)%和(139.339±17.871)个及NC组的0.981±0.089、(54.791±4.528)%和(145.243±19.782)个,而抑制组的Smad4水平为0.712±0.121,高于对照组的0.428±0.163和NC组的0.390±0.096,上述差异均有统计学意义(P<0.05)。与其余两组相比,抑制组转染24、48 h的增殖活力及Vim和N-cad水平降低,而E-cad水平升高(P<0.05)。Smad4野生型质粒与miR-301a模拟物共转染A549细胞后,荧光素酶活性下降(P<0.05),而Smad4突变型质粒和miR-301a模拟物共转染后,荧光素酶活性无明显变化(P>0.05)。结论NSCLC中miR-301a高表达且发挥类似癌基因的作用,下调该miRNA水平可抑制增殖和迁移侵袭能力并逆转EMT,可能通过靶向Smad4来发挥作用,有望成为NSCLC诊治的潜在新靶标。Objective To investigate the effect of microRNA-301a(miR-301a)on epithelial mesenchymal transition(EMT)and expression of SMAD family member 4(Smad4)in non-small cell lung cancer(NSCLC)cells.Methods Levels of miR-301a and Smad4 in 30 pairs of NSCLC tissues and adjacent tissues were detected by real-time quantitative PCR(QPCR).A549 cells were transfected with miR-301a inhibitor(Inhibition group)or negative control(NC group)by liposome method,and the untransfected cells were used as Control group.The miR-301a level,proliferation viability,scratch healing rate,number of transmembrane cells and Smad4 level were detected by QPCR,Cell Count Kit(CCK-8)assay,scratch test,Transwell chamber test and Western blotting.Levels of EMT-related markers including vimentin(Vim),E-cadherin(E-cad)and N-cadherin(N-cad)were measured via QPCR and Western blotting.The targeting relationship between miR-301a and Smad4 was verified by bioinformatics and double Luciferase Report.Results The miR-301a level in NSCLC tissues was 2.157±0.597,higher than 1.023±0.229 in the adjacent tissues,and the Smad4 level in NSCLC tissues was 0.648±0.162,lower than 1.159±0.164 in the adjacent tissues(P<0.05).The miR-301a level,scratch healing rate and number of membrane penetrating cells in the Inhibition group were 0.166±0.075,(31.518±4.306)%and 89.744±15.060,lower than 1.032±0.097,(52.461±6.373)%and 139.339±17.871 in Control group and 0.981±0.089,(54.791±4.528)%and 145.243±19.782 in NC group(P<0.05).The Smad4 level in Inhibition group was 0.712±0.121,higher than 0.428±0.163 in Control group and 0.390±0.096 in NC group(P<0.05).Compared with other two groups,the proliferation activity and levels of Vim and N-cad in the Inhibition group were decreased,while level of E-cad was increased(P<0.05).The luciferase activity of A549 cells co-transfected with smad4 wild type and miR-301a mimics decreased significantly(P<0.05),while the luciferase activity of A549 cells co-transfected with smad4 mutant type and miR-301a mimics did not change significantl

关 键 词：非小细胞肺癌 微小RNA-301a SMAD家族成员4 上皮间质转化 

分 类 号：R734.2[医药卫生—肿瘤]