【摘要】 目的探讨长基因间非编码RNA 472(LINC00472)靶向调控微小RNA-942-5p(miR-942-5p)表达及对非小细胞肺癌(NSCLC)细胞上皮间质转化(EMT)的影响。方法采用GEPIA分析来自TCGA数据库的969例NSCLC组织(483例腺癌+486例鳞癌)的LINC00472水平,实时荧光定量PCR(q PCR)检测正常肺上皮细胞BEAS-2B及NSCLC细胞系(NCIH157、NCI-H1975和A549)的LINC00472水平。构建LINC00472过表达质粒pc DNA3.1-LINC00472并采用脂质体法转染A549细胞,根据转染质粒分为3组:未转染任何质粒的空白对照组、转染pc DNA3.1质粒的阴性对照组和转染pc DNA3.1-LINC00472质粒的过表达组。MTT比色法、划痕实验和Transwell小室实验检测A549细胞的增殖活力、划痕愈合率和穿膜细胞数,q PCR和Western blotting检测EMT相关因子E-钙黏蛋白(E-cad)、N-钙黏蛋白(N-cad)和波形蛋白(Vim)的水平,Lnc Base预测LINC00472与miR-942-5p的结合序列并经荧光素酶报告基因实验验证两者的靶向关系。结果 GEPIA在线分析显示,NSCLC组织的LINC00472水平低于正常组织(P<0.05); NSCLC细胞的LINC00472水平均低于BEAS-2B细胞(P<0.05),选取水平最低的A549细胞进行后续功能实验。过表达组的LINC00472和E-cad水平升高,而miR-942-5p、N-cad和Vim水平降低(P<0.05)。过表达组转染48、72 h后的增殖活力低于其余两组(P<0.05)。过表达组的划痕愈合率为(29.148±3.034)%,低于空白对照组的(84.517±8.722)%和阴性对照组的(87.479±5.672)%,穿膜细胞数为(144.437±12.178)个,亦低于空白对照组的(240.365±14.156)个和阴性对照组的(224.713±17.805)个,差异有统计学意义(P<0.05)。荧光素酶活性检测结果显示,miR-942-5p模拟物能抑制LINC00472野生型质粒的荧光素酶活性(P<0.05),但对突变型无影响(P>0.05)。空白对照组和阴性对照组上述指标的差异无统计学意义(P>0.05)。结论 LINC00472在NSCLC组织和细胞中表达均下调,增强LINC00472表达可抑制NSCLC细胞的EMT和侵袭迁移能力,可能通过靶向miR-942-5p来发挥抑癌作用,LINC00472/miR-942-5p轴在NSCLC防治中有一定潜能。更多还原

【Abstract】 Objective To investigate the effect of long intergenic non-coding RNA 472( LINC00472) on the expression of microRNA-942-5 p( miR-942-5 p) and its effect on epithelial mesenchymal transition( EMT) of non-small cell lung cancer( NSCLC)cells. Methods GEPIA was used to analyze LINC00472 levels in 969 NSCLC tissues including 483 adenocarcinoma and 486 squamous cell carcinoma from TCGA database. Levels of LINC00472 in normal lung epithelial cell BEAS-2 B and NSCLC cells( NCIH157,NCI-H1975 and A549) were detected by real-time quantitative PCR( q PCR). LINC00472 overexpression plasmid pc DNA3. 1-LINC00472 was constructed and transfected into A549 cells by lipofectamine method. According to the transfection plasmids,A549 cells were divided into three groups: Blank control group without any plasmid transfection,Negative control group transfected with pc DNA3. 1 plasmid and Overexpression group transfected with pc DNA3. 1-LINC00472 plasmid. Proliferating vitality,wound healing rate and number of cells penetrating the membrane were detected by MTT colorimetry,scratch test and Transwell chamber test. Levels of EMT related factors including E-cadherin( E-cad),N-cadherin( N-cad) and Vimentin( Vim) were detected by q PCR and Western blotting. Lncbase was applied to predict the binding sequence of LINC00472 and miR-942-5 p,and their targeting relationship was verified by luciferase reporter gene experiment. Results GEPIA online analysis showed that LINC00472 level in NSCLC tissues was lower than that in normal tissues( P<0. 05). LINC00472 levels in NSCLC cells were lower than those in BEAS-2 B cells( P<0. 05),and A549 cells with the lowest LINC00472 level were selected for subsequent functional verification test. In Overexpression group,levels of LINC00472 and E-cad increased,while levels of miR-942-5 p,N-cad and Vim decreased( P<0. 05). The proliferation activity of Overexpression group was lower than other two groups at 48 and 72 h after transfection( P< 0. 05). The wound healing rate of Overexpression group was( 29. 148±3. 034) %,lower than( 84. 517 ± 8. 722) % of Blank control group and( 87. 479 ± 5. 672) % of Negative control group. The number of penetrating cells of Overexpression group was 144. 437±12. 178,lower than 240. 365±14. 156 of Blank control group and 224. 713±17. 805 of Negative control group( P<0. 05). Luciferase activity assay showed that miR-942-5 p mimics could inhibit the luciferase activity of LINC00472 wild type( P<0. 05),but had no effect on mutant( P>0. 05). There was no significant difference in the above indexes between the Blank control group and Negative control group( P> 0. 05). Conclusion LINC00472 was down-regulated in NSCLC tissues and cells. Enhanced LINC00472 expression can inhibit EMT,invasion and migration of NSCLC cells. It may play an anti-cancer role through miR-942-5 p,which has certain potential in the prevention and treatment of NSCLC.更多还原