硫丹诱导miR-22表达变化与人内皮细胞损伤和功能失调的关联

Endosulfan is a class of persistent organic pollutants characterized by persistence, toxicity and bioaccumulation in organisms. Endosulfan has been implicated increasingly in a variety of human diseases due to its potential carcinogenicity, mutagenicity and endocrine disruptor effects. Recent studies about the molecular mechanism of human diseases induced by endosulfan are mostly limited on protein-coding genes, but it is still unknown whether and how non-coding miRNA genes are involved in epigenetic control of gene expression when exposure to endosulfan. Therefore, it becomes a key scientific problem that urgently needs to be recognized. It is known that miR-22 is a small non-coding RNA, functions in regulating protein expression at post-transcription level. It plays a critical role in many cellular processes such as cell growth, apoptosis and cellular senescence. Our preliminary study found that endosulfan induced cytotoxicity, cell cycle arrest, senescence and apoptosis in human umbilical vein endothelial cells. Interestingly, miR-22 was specifically upregulated by endosulfan. In this project, we will clarify the dose-effect of endosulfan on miR-22 expression. Based on it, we will observe the characteristic effect of miR-22 on biological functions in human endothelial cells. Further, we will analyze complicate network of miR-22 specific target genes regulated by miR-22 in endothelial cells and explain the relationship between altered miR-22 expression and endosulfan-induced dysfunction of endothelial cells. Our study will reveal the molecular mechanism of dysfunction induced by endosulfan in human diseases from miRNA levels, providing new insight of miRNAs that are used as power biomarker for diagnosis and prevention of human diseases caused by endosulfan.

硫丹是一类具有持久性、高毒性和生物富集特征的持久性有机污染物，由于其潜在的致癌致突变和内分泌干扰素作用，与人类许多疾病的发生有关。目前对于硫丹致病的分子机制研究仍多限于蛋白编码基因, 而非蛋白编码基因miRNA基因是否参与和如何调控硫丹导致基因表达失调，是一个急需认识的关键科学问题。miR-22是一种在转录后水平调控蛋白表达的小分子RNA，参与增殖，老化及凋亡等多种细胞学过程。我们前期的研究发现硫丹引起人脐静脉内皮细胞毒性、细胞周期阻滞、老化和凋亡，并特异性诱导了miR-22表达上调。因此本课题将在明确不同剂量的硫丹对miR-22表达变化的规律的基础上，进一步揭示miR-22对内皮细胞生物学功能的影响及对内皮细胞中特异性靶基因的调控作用，阐述miR-22变化与硫丹所诱导的内皮细胞损伤的关联，从miRNAs水平揭示硫丹引起人内皮细胞功能失调的分子机制，为硫丹致病的诊断和预防提供生物标志物。

硫丹是毒性最强的有机氯农药之一，属于持久性有机污染物，其生物毒性考虑与硫丹对细胞的直接损伤和对细胞功能的影响密不可分。已有研究显示硫丹具有多种细胞毒性，但其对内皮细胞的毒性研究尚未见报道。本研究着重探讨了硫丹对人脐静脉内皮细胞（HUVEC-C）的毒性和对内皮细胞功能的影响。目前取得的研究结果发现硫丹能够引起血管内皮细胞毒性， IC50为62.8μM。低剂量（20μM）硫丹暴露破坏了细胞微丝骨架，可能影响血管内皮屏障作用，在较高剂量下（40μM和60μM）硫丹通过线粒体介导的内源性凋亡信号通路引起细胞凋亡，而在高剂量下（60μM）硫丹通过CDK6/pRb信号通路引起细胞周期G1期阻滞，体现了硫丹暴露引起人血管内皮细胞毒性剂量效应的特征。随着硫丹暴露剂量的增加，炎症因子IL-6和IL-8分泌水平和表达水平升高。这些变化提示硫丹暴露能够引起血管内皮细胞功能失调。值得一提的是硫丹能够特异性诱导miR-22表达升高，呈剂量依赖性关系，而miR-22过表达可以导致血管内皮细胞发生细胞凋亡和炎症因子IL-6表达升高。利用生物信息学方法预测和筛选miR-22的靶基因，经荧光素酶报告载体系统验证了CDK6, Lamininγ-1, SRF和STAG2是miR-22靶基因，而硫丹能够引起CDK6, Lamininγ-1, SRF表达明显降低，但不影响STAG2的表达。利用RNAi的方法在HUVEC-C细胞中进行靶基因的siRNA转染实验，结果显示这些基因可能与细胞周期、骨架重构和细胞生存等有关，参与了miR-22介导的硫丹所致的血管内皮细胞功能失调。综上所述、本研究明确了不同剂量硫丹暴露引起人血管内皮细胞毒性效应的特征和对miR-22表达的影响，验证了miR-22靶基因CDK6、Lamininγ-1和SRF在硫丹暴露下的表达变化，推测这些基因可能参与了miR-22介导的硫丹致血管内皮细胞损伤和功能失调的转录后调控机制。血管内皮细胞功能失调与人类疾病的发生密切相关，本研究揭示了硫丹诱导miR-22表达变化与人血管内皮细胞损伤和功能失调的关联，为miR-22作为潜在的生物标志物用于硫丹致病的诊断和预防提供了重要的依据。