MicroRNA靶向调控OSTF1在氟中毒鱼鳃渗透调节中的作用机制

Fluoride is one of the main environmental pollutants that harms the health of organisms. High levels of fluoride in the fish ponds caused damages to fish. Our early results demonstrated that positive cells of OSTF1 were mainly located in epithelial cells of branchial filaments. With the increase of fluoride concentration, the protein expression of OSTF1 increased. However, it is unclear that the functional mechanisms of fluoride-induced expression of OSTF1 in the gills in common carp exposed to high fluoride. The proposed research intends to predict and verify microRNAs regulating OSTF1, analysis the correlation of candicate microRNAs and OSTF1, and study the effects of microRNA mimic/inhibitor on the osmotic regulation related factors in branchial cells, such as Na+/K+-ATPase, Osmotic regulation of transcription factors 1 (OSTF1), Cystic fibrosis transmembrane regulator (CFTR), Aquaporin 3(AQP-3), Sodium hydrogen exchanger1 (NHE-1) using bioinformatics, cell culture, transfection, gene silencing, real-time quantitative PCR, Western blotting and immunohistochemistry methods in fluoride-exposed gills or branchial cells based on their pollution situations in the water. Our study will clarify functional mechanism of microRNAs’ regulation to OSTF1 in osmotic adjustment of gills in common carp exposed to high fluoride. Our results could also reveal the mechanism of the effects of fluoride on osmotic regulation in fish from the molecular levels.

氟是对生物危害严重的主要环境污染物。部分养殖水体含氟量高，对鱼类产生毒害效应。申请者前期研究发现，OSTF1阳性细胞主要位于鳃丝上皮细胞，且随着氟暴露浓度的增加，OSTF1蛋白表达增加，但氟诱导OSTF1表达的调控机制并不清楚。本项目拟在前期研究基础上，模拟水氟污染，以氟暴露鱼鳃及鳃细胞为材料，利用生物信息学及细胞培养、转染、基因沉默、荧光定量PCR、免疫印迹和免疫组化等细胞生物学和分子生物学等技术，预测靶向调控OSTF1的microRNAs并进行验证，对候选microRNAs和OSTF1的相关性进行分析，并研究microRNA mimic/inhibitor对氟中毒鱼鳃细胞渗透调节因子Na+-K+-ATPase、CFTR、OSTF1、AQP-3和NHE-1基因和蛋白表达的影响，探讨microRNAs靶向调控OSTF1在氟中毒鱼鳃渗透调节中的作用，从分子水平揭示氟对鱼鳃渗透调节影响的机理。

氟是对生物危害严重的主要环境污染物。部分养殖水体含氟量高，对鱼类产生毒害效应。申请者前期研究发现，OSTF1阳性细胞主要位于鳃丝上皮细胞，且随着氟暴露浓度的增加，OSTF1蛋白表达增加，但氟诱导OSTF1表达的调控机制并不清楚。本项目拟在前期研究基础上，模拟水氟污染，以氟暴露鱼鳃及鳃细胞为材料，利用生物信息学及细胞培养、转染、基因沉默、荧光定量PCR、免疫印迹等细胞生物学和分子生物学等技术，预测靶向调控OSTF1的microRNAs并进行验证，对候选microRNAs和OSTF1的相关性进行分析，并研究microRNA mimic/inhibitor对氟中毒鱼鳃细胞渗透调节因子CFTR表达的影响。结果表明，低浓度组与对照组相比，共发现584个差异miRNA，其中322个上调，262个下调。高浓度组与对照组相比，共发现327个差异miRNA，其中219个上调，108个下调。低浓度组特有的差异micRNA共394个，而高浓度组特有的差异micRNA共137个。使用TargetScan预测OSTF1的miRNAs，找到调控OSTF1的miRNAs共36个。将调控OSTF1的miRNAs与通过差异micRNA分析得到的差异miRNAs进行比对，二者共有的miRNAs有7个，其中4个上调，3个下调。候选的miR-429和miR-30可以与OSTF1的3’UTR结合进而抑制其荧光素酶活性。miR-429和miR-30的表达与OSTF1的表达呈负相关。miR-429 mimic和miR-30 mimic转染后显著增加了miR-429和miR-30的表达量。过表达miR-429，明显降低了OSTF1的表达，显著改善氟中毒对鱼鳃细胞造成的损伤。MiR-429 inhibitor转染氟中毒鱼鳃细胞后，能明显抑制miR-429的表达，显著增加OSTF1的表达，同时加重氟中毒对鱼鳃细胞造成的损伤；MiR-30 inhibitor转染氟中毒鱼鳃细胞48h后，能显著抑制miR-30的表达，但抑制miR-30随增加OSTF1的表达，但差异不显著，氟中毒对鱼鳃细胞造成的损伤与对照组相比差异不显著。本研究结果探讨microRNAs靶向调控OSTF1在氟中毒鱼鳃渗透调节中的作用，从分子水平揭示氟对鱼鳃渗透调节影响的机理。