EF-1delta在镉毒性作用中的非编码RNA调控机制与镉暴露人群验证

The applicant previously found that mouse translation elongation factor (EF-1delta) is a novel cadmium-responsive proto-oncogene. Recently the applicant also found that this gene expression was up-regulated in cadmium -transformed cells, -poisoned rats and -exposed population,however its toxic mechanism remains unclear. Our related research did not show any mutation in translation region and aberrant methylation in promoter of EF-1delta, but gave hints of ncRNA regulation for the gene. The project will start with high-throughput microarray and bioinformatics intending to screen the miRNA and lncRNA that are related to regulation profile of EF-1delta and expressed differently in cadmium toxic effects, and then explore the role and mode of miRNA and lncRNA regulation of EF-1delta at different stages of cadmium malignant transformed 16HBE cells using gene sequencing, ncRNA ovre-expression and interference, RNA pulldown as well as c-KLAN technique. Furthermore, the EF-1delta knockout cells model will be created to demonstrate the ability of cadmium malignantly transform cells, reveal the relationships among the EF-1delta, cells malignant transformation and ncRNA regulation. Finaly, with field investigation, the peripheral blood samples will be checked for ncRNA regulation mechanism of EF-1delta from cadmium-exposed population. This research will effectively clarify the important molecular mechanisms of EF-1delta toxic effects in cadmium-induced human cells malignant transformation and moreover the mechanism will be validated in cadmium-exposed population, which is very important to population health benefits.

申请者之前已发现和鉴定了鼠翻译延伸因子(EF-1delta)为新的镉应答原癌基因，最近又发现该基因在镉转化细胞、镉中毒大鼠和镉暴露人群中表达上调但其机制尚不清楚。相关的探索未见此基因翻译区突变和启动子甲基化情况，却发现与非编码RNA(ncRNA)调控相关。本项目拟通过高通量芯片和生物信息学技术筛选出镉毒作用中差异表达而且与EF-1delta相关的miRNA和lncRNA；再运用测序、过表达、表达沉黙、RNA pulldown和c-KLAN等技术探索EF-1delta在镉诱发细胞恶性转化中的miRNA和lncRNA调控功能和模式；进一步创建EF-1delta缺陷细胞模型以论证该基因与细胞恶变及ncRNA调控之间的关系；最后通过现场调研，验证镉暴露人群外周血EF-1delta的ncRNA调控机制。本研究将有力揭示和阐明EF-1delta在镉毒作用中的重要分子机制，对人群健康效益具有重要意义。

镉是人类危害最大的金属毒物之一，然而镉毒作用和致癌的分子机制至今仍未完全阐明。有研究表明环境应答基因在反映重金属对人类健康损害方面具有重要作用，但从镉相关应答基因(EF-1delta或EF-1δ)来探讨非编码RNA(ncRNA)调控机制，国内外未见报道。本项目运用一系列分子生物学方法，开展EF-1δ在镉毒作用中的ncRNA调控机制与镉暴露人群研究。结果如下：筛选出镉恶性转化不同阶段16HBE细胞中miRNA、lncRNA和mRNA差异表达谱；构建了与EF-1δ调控相关的lncRNA和miRNA网络图；干扰EF-1δ后发现AKT、Bax、BCL2、E-Cadherin、Caspase3、EGFR、FOXC2、STAT3、TGF-β1、VIMENTIN基因表达在镉处理组有明显改变；沉默EF-1δ表达的镉恶性转化细胞迁移力下降和凋亡率增加，提示EF-1δ对于镉暴露细胞生存活性是必要因素；发现miRNAs Hsa-miR-130a-3p对镉转化细胞株具有抑制增殖作用，而miRNAs Hsa-miR-301a-3p有促进增殖作用；进一步的研究发现EF-1δ的差异表达可受上述2个miRNAs调控，也与甲基化相关，初步阐明镉毒作用EF-1δ差异表达在ncRNA途径的调控机制。镉作业人群的调研表明，尿镉、血镉和β2 -微球蛋白含量明显超标，其外周血EF-1δ表达水平随着血镉、尿镉、β2 -微球蛋白含量的增加而升高，具有明显的剂量依赖关系；结合镉暴露大鼠和镉职业人群，发现在镉低毒、中毒和高毒组的大鼠肺和肾组织中上述2个miRNAs表达逐渐降低，并初步观察了miRNA在镉职业人群外周血中对EF-1δ因子调控的影响，为揭示和阐明EF-1δ在镉毒作用中的重要分子机制提供了科学依据。我们的研究成果在国内外期刊和会议上发表论文9篇，其中SCI收录论文5篇；培养博士和硕士毕业生4名、年青教师等4名。