微小RNA-375/矮小同源盒基因轴参与调控肺癌细胞吉西他滨敏感性的机制研究  

Regulation mechanism of micro RNA-375/short stature homobox 2 axis on gemcitabine sensitivity of lung cancer cells

作　　者：张继东[1] 荣朝 胡娟[2] 孙绪举[3] Zhang Jidong;Rong Chao;Hu Juan;Sun Xuju(Department of Pharmacy,Hospital of Huazhong University of Science and Technology,Wuhan 430074,China;Department of Surgery,Hospital of Huazhong University of Science and Technology,Wuhan 430074,China;Department of Pharmacy,Liyuan Hospital Affiliated to Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430074,China)

机构地区：[1]华中科技大学医院药剂科,武汉430074 [2]华中科技大学医院外科,武汉430074 [3]华中科技大学同济医学院附属梨园医院药剂科,武汉430074

出　　处：《中国医药》2020年第2期226-230,共5页China Medicine

基　　金：国家自然科学基金(81803018)。

摘　　要：目的探讨微小RNA-375(miR-375)靶向矮小同源盒基因(SHOX2)对肺癌细胞吉西他滨敏感性的影响。方法体外培养非小细胞肺腺癌细胞株A549细胞,作为空白对照组;转染miR-375模拟物质粒或空质粒至A549细胞,作为miR-375过表达组和阴性对照组。采用噻唑蓝法和Annexin V/PI双染法检测吉西他滨对各组细胞增殖和凋亡的影响。采用双荧光素酶报告基因方法验证miR-375与SHOX2的靶向关系。应用逆转录-聚合酶链反应法检测各组细胞中miR-375和SHOX2基因的表达量;采用蛋白质印迹法检测各组细胞中SHOX2蛋白的表达量。采用荧光免疫检测法检测各组细胞β-连环蛋白表达情况。结果miR-375过表达组miR-375表达量明显高于空白对照组和阴性对照组[(1.45±0.11)比(1.00±0.05)、(0.97±0.11)](均P<0.05);且经不同浓度吉西他滨作用48 h后,miR-375过表达组细胞增殖抑制率明显高于空白对照组和阴性对照组(P<0.05);吉西他滨对miR-375过表达组A549细胞的半数抑制浓度为4.405μmol/L。4.405μmol/L吉西他滨作用24 h后,miR-375过表达组细胞凋亡率明显高于空白对照组和阴性对照组[(28±4)%比(12±4)%、(14±4)%](P<0.05)。经双荧光素酶报告基因分析,SHOX2是miR-375的下游靶基因。miR-375过表达组SHOX2 m RNA和蛋白表达量均明显低于空白对照组和阴性对照组[(0.39±0.08)比(1.00±0.07)、(1.02±0.09);(0.42±0.07)比(0.97±0.10)、(1.05±0.12)](均P<0.05)。与空白对照组和阴性对照组相比,miR-375过表达组细胞中β-连环蛋白表达量和进入细胞核的量明显减少。结论上调miR-375可抑制SHOX2激活Wnt/β-连环蛋白信号通路,从而增加A549细胞株对吉西他滨的敏感性。Objective To explore the regulation mechanism of micro RNA-375(miR-375)/short stature homobox 2(SHOX2)axis on gemcitabine sensitivity of lung cancer cells.Methods Non-small cell lung adenocarcinoma cell line A549 was cultured and transfected with miR-375 mimics plasmid(miR-375 overexpression group)or empty plasmid(negative control group);A549 cells without transfection were blank control group.Cell proliferation and apoptosis affecting by gemcitabine were tested by methyl thiazolyl tetrazolium assay and Annexin V/PI staining.Relation between miR-375 and SHOX2 was verified by double-luciferase reporter gene assay.Gene expressions of miR-375 and SHOX2 were detected by reverse transcription polymerase chain reaction.Protein expression of SHOX2 was detected by western blotting.Expression ofβ-catenin was detected by fluorescence immunoassay.Results Expression of miR-375 after transfected with miR-375 mimics plasmid significantly increased as compared with blank control and negative control groups[(1.45±0.11)vs(1.00±0.05),(0.97±0.11)](both P<0.05).Cell proliferation inhibition rate in miR-375 overexpression group was significantly higher than that in blank control and negative control groups(both P<0.05).The half maximal inhibitory concentration of gemcitabine on miR-375 overexpressed cells was 4.405μmol/L.After treatment with 4.405μmol/L gemcitabine,cell apoptosis rate in miR-375 overexpression group was significantly higher than that in blank control and negative control groups[(28±4)%vs(12±4)%,(14±4)%](both P<0.05).Double-luciferase reporter gene assay indicated that SHOX2 might be a target of miR-375.SHOX2 m RNA and protein expressions in miR-375 overexpression group were significantly lower than those in blank control and negative control groups[(0.39±0.08)vs(1.00±0.07),(1.02±0.09);(0.42±0.07)vs(0.97±0.10),(1.05±0.12)](all P<0.05).Besides,β-catenin expressions in miR-375 overexpressed cells and nuclei significantly decreased as compared with blank control and negative control groups.Conclusion Up-r

关 键 词：肺癌 吉西他滨 敏感性 微小RNA-375 矮小同源盒基因 

分 类 号：R734.2[医药卫生—肿瘤]