长链非编码RNA在镉毒作用中的表达谱筛选及功能研究

Recent studies have shown that long noncoding RNAs (lncRNA) are known to play important regulatory roles in DNA damage, DNA damage, DNA repair genes expression , DNA methylation and histone acetylation.We previously established a model system of morphological cell transformation with Cadmium chloride (CdCl2) in human bronchial epithelial cells (16HBE) and found that Cadmium can produce genotoxic and mutagenic events, arrest cell growth ,obviously cause apoptosis and DNA damage, inhibit DNA repair gene expression and epigenetic changes. However, It is not known whether the DNA damage and repair, epigenetic changes regulated by lncRNA in Cadmium toxicity.In the current study, our arm is to identify abnormal expression profiling of lncRNA via lncRNA microarray in 16HBE cells induced by cadmium. Bioinformatic analysis of predicted target genes(gene ontology, pathway and network analysis) will be done for further research. The abnormal expression of lncRNA will be tested in human bronchial epithelial cells line induced by cadmium, rat model of chronic Cadmium exposure, and Cadmium exposed workers.The main regulation function of lncRNA to cell cycle, cell apoptosis, DNA damage, DNA repair genes expression , DNA methylation and histone acetylation will be verificated by Technology of over expression and RNA interference. We also deal with the Cd-transformed 16HBE cells at 35th passage and tumorigenic cells by 5-aza-Dc and TSA, and then to explore the interaction of lncRNA and other epogenetic mechanisms in Cadmium toxicity. It will be positive significance to seek the lncRNAs as the early biological markers of Cadmium toxicity and their functions , to provide new ideas of the mechanisms in Cadmium toxicity.

研究显示：长链非编码RNA(lncRNA)与DNA损伤与修复、DNA甲基化及组蛋白乙酰化有关。项目申请人在前期研究发现，镉能引起DNA损伤与DNA修复基因表达下调，并引起异常甲基化和乙酰化。但其机制是否与lncRNA的调控有关？未见报道。本项目拟通过高通量的LncRNA芯片和生物信息学技术筛选出镉恶性转化人支气管上皮细胞（16HBE）中LncRNA差异表达谱，通过镉作业人群进行验证，并进一步运用LncRNA过表达技术和LncRNA沉默技术探索和验证镉毒作用中LincRNA在细胞周期，细胞凋亡、DNA损伤、DNA修复和DNA甲基化和组蛋白乙酰化中的调控功能;经过表遗传逆转,探索LncRNA和甲基化和乙酰化的互调控作用。本项目将为寻找LncRNA作为镉毒早期生物标志物的研究奠定基础，为探讨镉毒性和致癌机制研究提供新的思路，对阐明LncRNA在镉毒性中的的调控功能有积极的意义。

项目的背景: 近年研究显示,长链非编码RNA(lncRNA)在生物的生理和病理生理过程中起到重要的调控作用。然而，lncRNA在镉毒性机制中的作用如何，未见报道。主要研究内容：筛选和鉴定镉恶性转化人支气管上皮细胞（16HBE）中的LncRNA差异表达谱。探索在镉恶性16HBE细胞中LncRNA对DNA损伤与修复、细胞周期，凋亡、迁移与侵袭的调控功能及与甲基化和组蛋白乙酰化的互调控作用，并验证LncRNA作为镉毒性新生物标志物的作用。主要结果：1.在16HBE细胞中检测出21409个LncRNAs，与非转化16HBE细胞比较,镉恶性转化16HBE第35代细胞中有369个lncRNA表达上调,有90个lncRNA表达下调(≥2.0倍,P<0.05)。2.qPCR检测在表达谱中高表达的10个lncRNAs的表达情况，发现这些LncRNA在镉恶性转化细胞中均异常高表达(P<0.05)，与芯片结果吻合。3.在镉恶性转化16HBE中，LncRNAENST00000414355能调控 DNA损伤、修复；ENST00000446135能调控DNA损伤和细胞凋亡；MALAT1能调控细胞增殖、细胞凋亡、细胞迁移与侵袭过程。它们均能调控相关靶基因的表达水平。4.检测镉作业一线工人的血镉、尿镉和尿β2-MG的含量，检测血中LncRNAENST00000414355和MALAT1等LncRNA的表达。结果显示： LncRNAENST00000414355和MALAT1的表达与工人的血镉、尿镉和尿β2-MG的含量呈正相关关系。表明LncRNAENST00000414355和MALAT1可以考虑作为镉毒性的新生物标志物。5.采用甲基化试剂(5-aza-2’-dc)和组蛋白去乙酰化酶抑制剂（TSA）分别处理16HBE细胞和镉恶性转化16HBE细胞，用qPCR检测LncRNA（ENST00000414355、ENST00000446135）表达量。结果显示，两种细胞被5-aza-2’-dc和TSA处理后，在某些时点中LncRNA-ENST00000414355EN和EST00000446135的表达明显上调，但无时间反应关系。科学意义：本项目为阐明镉毒性中LncRNA的调控机制和寻找LncRNA新生物标志物的研究奠定了基础；为进一步阐明镉毒性机制提供了新的思路。