模拟微重力对大鼠甲状腺滤泡上皮细胞miRNA表达的影响  

Effects of simulated microgravity on miRNA expression in Fisher rat thyroid cell line

作　　者：闫如意 张倍宁 李考[3] 郑冬[4] 宋淑军[5] 吴继华[6] 司少艳[5] 周金莲[1,6] 崔彦[2] 张建中[6] YAN Ru-yi;ZHANG Bei-ning;LI Kao;ZHENG Dong;SONG Shu-jun;WU Ji-hua;SI Shao-yan;ZHOU Jin-lian;CUI Yan;ZHANG Jian-zhong(Department of Pathology,306 Clinical Hospital of Chinese PLA,Anhui Medical University,Beijing 100101,China;Department of General Surgery;Department of Respiratory Medicine;Department of Radiology;Special Medicine Laboratory Center;Department of Pathology,306 Hospital of PLA,Beijing 100101,China)

机构地区：[1]安徽医科大学解放军306临床学院病理科,北京100101 [2]解放军306医院普通外科,北京100101 [3]解放军306医院呼吸内科,北京100101 [4]解放军306医院放射科,北京100101 [5]解放军306医院特种医学实验研究中心,北京100101 [6]解放军306医院病理科,北京100101

出　　处：《解放军医学杂志》2018年第8期668-673,共6页Medical Journal of Chinese People's Liberation Army

基　　金：军队试验技术研究计划重点项目(SYFD1500128)

摘　　要：目的探讨微重力环境对大鼠甲状腺滤泡上皮细胞(FRTL-5)微小RNA(mi RNA)表达的影响。方法体外培养FRTL-5,随机分为模拟微重力(SMG)组和正常重力(NG)组,每组3个样本。SMG组釆用旋转细胞培养系统(RCCS)模拟微重力环境,NG组于培养箱静置培养,24h后提取样本总RNA,进行荧光标记和芯片杂交。采用Feature Extraction软件处理杂交图像提取原始数据,应用Genespring软件进行分位数标准化和后续处理,筛选差异表达显著的mi RNA,反转录实时定量PCR(RT-q PCR)验证芯片结果准确性。通过mi RNA靶基因预测数据库Target Scan和micro RNAorg对差异mi RNA进行靶基因预测,以其交集作为潜在调节靶基因,对预测靶基因分别进行基因本体(GO)和京都基因与基因组百科全书(KEGG)富集分析,判定差异mi RNA主控的生物学过程和信号通路。结果 mi RNA芯片检测发现,FRTL-5在模拟微重力环境下与正常重力环境下差异表达的mi RNA有81个,其中53个显著上调,28个显著下调,差异表达最显著的3条,包括显著下调的rno-let-7b-3p、rno-mi R-346(P<0.05),以及显著上调的rno-mi R-494-3p(P<0.05),对其芯片结果进行RT-q PCR验证,结果相一致。GO分析显示,差异表达的mi RNA与细胞老化、凋亡、对低氧的反应、转录等生物学过程相关;KEGG分析显示,差异表达的mi RNA与低氧诱导因子-1(HIF-1)信号通路、环磷酸鸟苷-蛋白激酶G(c GMP-PKG)信号通路、叉头框转录因子O(Fox O)信号通路及多种甲状腺激素调节通路相关。结论微重力环境下大鼠甲状腺滤泡上皮细胞相关mi RNA表达发生显著变化,基于芯片技术的mi RNA靶基因预测和功能富集分析可为甲状腺失重应激反应、损伤及修复措施的探讨提供理论参考。Objective To investigate the effects of simulated microgravity by rotary cell culture system （RCCS） on the expression profiles of miRNA in thyroid follicular epithelial cell line （FRTL-5）. Methods FRTL-5 cells were cultured in vitro and randomly divided into simulated microgravity group （SMG） and normal gravity group （NG）, with three samples in each group. The samples of two groups were collected at 24th hours of culture, and the total RNAs were extracted, labeled and hybridized in sequence. Feature Extraction software was used to collect the array images and get raw data which were analyzed by Genespring software. Differentially expressed miRNAs were identified through gene chip hybridization and then validated by RT-qPCR. The target genes of differentially expressed miRNAs were predicted by the databases of TargetScan and microRNAorg,and the intersections of databases were identified as potential regulatory target genes. Gene ontology （GO） and Kyoto encyclopedia of genes and genomes （KEGG） analysis were applied to determine the roles of these target gene. Results The miRNA chip hybridization showed that there were 81 differentially expressed miRNAs in FRTL-5 under simulated microgravity including 53 miRNAs up-regulated and 28 miRNAs down-regulated significantly （P〈0.05）. The most differentially expressed miRNAs were down-regulated rno-let-7b-3p and rno-miR-346 and up-regulated rno-miR-494-3p. The RT-qPCR showed a high concordance with the results of the miRNA chip hybridization. GO analysis showed that the differentially expressed miRNAs were related to the biological processes of aging, apoptosis, transcription and response to hypoxia. KEGG analysis showed that these target genes were related to the signal pathways of HIF- 1, cGMP-PKG, FoxO and a variety of thyroid hormone regulation pathways. Conclusions The simulated microgravity by RCCS could significantly affect the expression of miRNA in FRTL-5. The miRNA target gene prediction and functional enrichment analysis based on gene

关 键 词：模拟微重力 FRTL-5 MIRNA 芯片杂交 功能富集分析 

分 类 号：R857[医药卫生—航空、航天与航海医学]