miR-30d-5p靶向ATG5影响A549细胞增殖迁移和自噬的研究  

Mechanism of miR-30d-5p targeting ATG5on proliferation,migration and autophagy of A549cells

作　　者：张国臣 刘璐 唐敬翔 辛绍晨 崇江倩 尹崇高 ZHANG Guochen;LIU Lu;TANG Jingxiang;XIN Shaochen;CHONG Jiangqian;YIN Chonggao(Second Medical University in Shandong,Weifang,Shandong261053,China)

机构地区：[1]山东第二医科大学临床医学院,山东潍坊261053 [2]山东第二医科大学基础医学院,山东潍坊261053 [3]山东第二医科大学生命科学与技术学院,山东潍坊261053 [4]山东第二医科大学护理学院,山东潍坊261053

出　　处：《中华肿瘤防治杂志》2024年第18期1101-1107,共7页Chinese Journal of Cancer Prevention and Treatment

基　　金：山东省自然科学基金(ZR2022MH311);山东省高等学校省级大学生创新创业训练计划(S202110438173)。

摘　　要：目的研究miR-30d-5p对肺腺癌细胞A549增殖、迁移以及自噬能力的影响,为肺腺癌诊治提供新思路。方法从基因表达数据库(GEO)筛选肺腺癌差异表达miRNA;Starbase数据库查询miR-30d-5p在肺腺癌组织中表达情况;Kaplan-Meier网站查询miR-30d-5p对肺腺癌患者生存预后的影响;采用实时荧光定量聚合酶链式反应(qRT-PCR)检测非小细胞肺癌(NSCLC)患者癌组织及其癌旁组织、正常肺支气管上皮细胞BEAS-2B及肺腺癌细胞系中miR-30d-5p表达与质粒转染率;EdU细胞增殖实验、细胞划痕实验和Transwell迁移实验探究miR-30d-5p对肺腺癌A549细胞增殖和迁移能力的影响;通过生物信息学数据库预测与miR-30d-5p具有潜在结合关系的自噬蛋白5(ATG5);双荧光素酶实验验证miR-30d-5p与ATG5是否存在靶向结合位点;利用stubRFP-sensGFP-LC3慢病毒检测miR-30d-5p对自噬流速的影响。结果癌旁组织和癌组织miR-30d-5p表达水平分别为1.00±0.00和0.92±1.42,t=2.089,P=0.041。BEAS-2B、H1299和A549细胞中miR-30d-5p表达水平分别为1.00±0.00、0.40±0.15和0.13±0.03,F=78.070,P=0.001。EdU细胞增殖实验结果表明,miR-30d-5p mimics/A549组和con/A549组EdU细胞阳性率分别为(23.00±4.00)%和(38.00±2.00)%,t=5.281,P=0.006。细胞划痕实验结果显示,miR-30d-5p mimics/A549组细胞迁移能力(0.15±0.01)低于con/A549组(0.29±0.01),t=21.830,P=0.001。Transwell实验结果显示,miR-30d-5p mimics/A549组细胞穿过小室微孔膜的细胞数量为(165.33±5.51)个,少于con/A549组的(410.67±9.50)个,t=7.482,P=0.002。双荧光素酶实验结果显示,miR-30d-5p与ATG5之间存在靶向结合的序列。stubRFP-sensGFP-LC3慢病毒感染结果显示,与con/A549组相比,miR-30d-5p mimics/A549组自噬体数量增加,自噬体到自噬溶酶体的自噬流速增加;相较于miR-30d-5p mimics/A549组,miR-30d-5p mimics/ATG5组自噬体数量减少,自噬体到自噬溶酶体的自噬流速降低。结论miR-30d-5p通过靶向调控ATG5抑制肺腺癌AObjective To study the effects of miR-30d-5p on the proliferation,migration,and autophagy ability of lung adenocarcinoma cell line A549,providing new ideas for the subsequent diagnosis and treatment of lung adenocarcinoma.Method Screened differentially expressed miRNAs in lung adenocarcinoma from GEO database;Starbase database was used to query the expression of miR-30d-5p in lung adenocarcinoma;Kaplan-Meier website inquired about the influence of miR-30d-5p on the survival and prognosis of lung adenocarcinoma patients.Quantitative reverse transcription polymerase chain reaction(qRT PCR)was used to detect the expression and plasmid transfection rate of miR-30d-5p in cancer tissues and adjacent tissues of non-small cell lung cancer(NSCLC)patients,normal lung bronchial epithelial cells BEAS-2B,and lung adenocarcinoma cell lines.To explore the effects of miR-30d-5p on the proliferation and migration ability of lung adenocarcinoma A549cells by using EdU cell proliferation assay,cell scratch assay,and Transwell migration assay;Predicting autophagy associated protein 5(ATG5)with potential binding to miR-30d-5p through bioinformatics databases;Double luciferase assay was used to verify whether miR-30d-5p has a targeted binding site with ATG5;Using stubRFP-sensGFP-LC3lentivirus to detect the effect of miR-30d-5p on autophagy flow rate.Results The expression levels of miR-30d-5p in adjacent and cancerous tissues were 1.00±0.00and 0.92±1.42,t=2.089,P=0.041.The expression levels of miR-30d-5p in BEAS-2B,H1299,and A549cells were 1.00±0.00,0.40±0.15and 0.13±0.03,respectively,F=78.070,P=0.001.The EdU cell proliferation experiment results showed that the EdU positive cell rates in the miR-30d-5p mimetics/A549group and con/A549group were(23.00±4.00)%and(38.00±2.00)%,respectively,t=5.281,P=0.006.The results of cell scratch assay showed that the cell migration rate of miR-30d-5p mimics/A549 group(0.15±0.01)was lower than that of con/A549group(0.29±0.01),t=21.830,P=0.001.Transwell assay showed that the number of cells pass

关 键 词：miR-30d-5p 增殖 迁移 自噬 肺腺癌 自噬蛋白5 

分 类 号：R734.2[医药卫生—肿瘤]