摘要
目的探讨多壁碳纳米管（multi—walled carbon nanotubes，MWCNT）对人胸膜间皮细胞MeT-5A的细胞毒性及对细胞microRNAs（miRNAs）表达的影响。方法用乳酸脱氢酶活力检测法（LDH）检测不同浓度MWCNT染毒24h和同一浓度不同染毒时间细胞相对LDH活力改变；从课题组前期microRNA芯片结果中筛选间皮瘤细胞中差异表达miRNAs，并用实时荧光定量聚合酶链反应（real-time quantitative polymerase chain reaction，RT—qPCR）验证有显著改变的5个miRNAs的表达改变；MeT-5A细胞用10μg／cm。的MWCNT处理0、8、24、48、72h后，用RT—qPCR方法检测上述5个有明显改变的miRNAs的表达水平；用Targetscan和miRanda两种在线软件对差异表达miRNAs进行靶基因预测，最后用David6．7对靶基因进行基因本体论（GO）分析和全基因组及代谢途径数据库（KEGG）通路分析。实验数据录入SPSS17．0软件，使用单因素方差分析（one—wayANOVA）和Dunnett—T检验进行统计分析。结果MWCNT染毒MeT-5A24h后，与对照组比较，染毒剂量大于等于20μg／cm。时，细胞存活率降低，差异有统计学意义（P〈0．01）；利用高通量方法从肿瘤细胞系中筛选获得5个差异表达的miRNAs：hsa—miR-155表达上调，hsa．miR．30d-5p，hsa—miR-34c-5p，hsa-miR-28-5p和hsa—miR-324—5p的表达下调；用RT—qPCR方法进一步验证，两者结果一致。10μg／cm。MWCNT染毒不同时间后，5个miRNAs表达改变与芯片结果一致。我们对这些差异表达miRNAs进行靶基因预测和通路分析，发现AKAP13，CCND3，Twist，E—Cadherin等是其潜在的功能靶点，主要参与肿瘤坏死因子p信号通路，肿瘤信号通路，小细胞肺癌等信号通路。结论MWCNT可对MeT-5A造成细胞毒性作用，染毒可引起MeT-5A细胞miRNAs差异表达，差异表达的miRNAs可能参与肿瘤相关的信号通路。
Objective To explore the cytotoxicities of MWCNT to the mesothelialcells and screen the changes of microRNA profile after exposure to MWCNT. Methods A LDH method was used to test the cytotoxicities of MWCNT to MeT-5A cell lines. And then the differentially expressed miRNAs between mesothelioma cells and normal mesothelial cells were selected from previous work of research group. Among the significant expression changed miRNAs, 5 were verified by RT-qPCR in mesothelioma cells. The same five ones were further tested in MeT-5A cells exposed to 10 μg/cm2 MWCNT for 8, 24, 48, 72 h by RT-qPCR. Target genes of 5 miRNAs were predicted using Targetscan and miRanda softwares. David6.7 was used to perform GO enrichment and KEGG pathway analysis of target genes. All the data were analyzed by one-way ANOVA and Dunnett-T test in SPSS17.0. Results After 24 h exposure to MWCNT, cell proliferation was significantly suppressed at more than 20 μg/cm2 concentration. Among the differentially expressed miRNAs, 5 were chosen to further vestified, namely hsa-miR-155 （up-regulated） ,hsa-miR-30 d-5p, hsa-miR-34c-5p, hsa-miR-28-5p and hsa-miR-324-5p （down-regulated）, which were consistent with the miRNA array results. The 5 miRNAs also had the same expression changes in MeT-5A cells after exposure to 10 μg/cm2 MWCNT for different time periods. The potential target genes of the 5 miRNAs may be AKAPI3,CCND3, Twist and E-Cadherin, which mainly involved in TGF-15 signal pathway, small cell lung cancer, etc. Conclusion MWCNT could induce to MeT-5A eells, and also cause miRNA expression changes. The differential changed miRNAs may involve in cancer related signal pathways.
出处
《中华劳动卫生职业病杂志》
CAS
CSCD
2016年第7期531-534,共4页
Chinese Journal of Industrial Hygiene and Occupational Diseases
基金
国家自然科学基金资助项目（81302464,81402718,81202244,81502794）
浙江省科技计划项目（2013F20004）
浙江省医药卫生科技计划项目（2014RCA003,2015120487）
浙江省重大科技专项重点社会发展项目（2014C03030）
关键词
多壁碳纳米管
胸膜
微rRNA
间皮瘤
Multi-walled carbon nanotubes
Pleura
MicroRNAs
Mesothelioma
