2020 年第 2 期 第 15 卷

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 microRNA-375/short stature homobox 2 axis on gemcitabine sensitivity of lung cancer cells

作者：张继东1荣朝1胡娟2孙绪举3

英文作者：

单位：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

英文单位：

关键词：肺癌；吉西他滨；敏感性；微小RNA-375；矮小同源盒基因

英文关键词：

• 摘要：

• 【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4.405 μmol/L。4.405 μmol/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 mRNA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。

• 【Abstract】Objective    To explore the regulation mechanism of microRNA-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 of β-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 4.405 μmol/L. After treatment with 4.405 μmol/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 mRNA 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-regulation of miR-375 inhibits SHOX2 activating Wnt/β-catenin signaling pathway and increases the sensitivity of lung cancer A549 cells to gemcitabine.