miRNA-216 and miRNA-499 target cyb561d2 in zebrafish in response to fipronil exposure.
MicroRNA (miRNA) can regulate the expression of its target gene by mediating mRNA cleavage or by translational repression at a post-transcriptional level. Usually, one miRNA may regulate many genes as its targets, while one gene may also be targeted by many miRNAs. We previously demonstrated that cyb561d2, whose protein product is involved in cell defense, and chemical stress, is targeted by miR-155 in adult zebrafish (Danio rerio) when exposed to fipronil (5-amino-1-[2,6-dichloro-4-(trifluoromethyl) phenyl]-4-[(trifluoromethyl) sulphinyl]-1H-pyrazole-3-carbonitrile). Microcosm Targets prediction showed that the cyb561d2 gene is also highly possibly targeted by miR-194a, miR-216b, miR-429, and miR-499. These interactions need to be further validated experimentally. In this study, we evaluated the effects of fipronil on miR-194a, miR-216b, miR-429, miR-499 and cyb561d2 in zebrafish and investigated whether these four miRNAs could regulate the expression of cyb561d2 in both mRNA and protein levels. The expression of cyb561d2 was upregulated in both mRNA and protein level in a dose-dependent manner upon stimulation of fipronil, and miR-216b and miR-499 were downregulated concurrently, whereas there was no significant changes were observed in the expression level of miR-194a and miR-429. The dual luciferase report assay demonstrated that miR-216b and miR-499 interacted with cyb561d2 3'-untranslated regions (3'-UTR), miR-194a and miR-429 did not stimulate degradation of cyb561d2 mRNA. The expression of cyb561d2 was reduced in both mRNA and protein level when ZF4 cells were transfected with miR-499 mimic, whereas expression level of both mRNA and protein was increased when endogenous miR-499 was inhibited by transfection with miR-499 inhibitor. Likewise, the mRNA and protein level of cyb561d2 was affected by treatment with the mimics and the inhibitor of miR-216b. In contrast, when ZF4 cells were transfected with a mimic of miR-194a or miR-429, the expression of cyb561d2 mRNA was not significantly changed. As a result, cyb561d2 is targeted by miR-155, miR-216b and miR-499 upon fipronil exposure, and miR-194a and miR-429 can not target cyb561d2. The expression pattern of these 3 miRNAs presents novel fipronil responses that could be used as a toxicological biomarker.
College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou 310018, People's Republic of China.
Yongyong
Y
Zhou
Department of Applied Engineering, Zhejiang Economic & Trade Polytechnic, Hangzhou 310018, People's Republic of China.
Hannian
H
Huang
College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou 310018, People's Republic of China.
Kai
K
Zhang
College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou 310018, People's Republic of China.
Xianfeng
X
Ding
College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou 310018, People's Republic of China.
Longlue
L
Jia
Institute of Pesticide and Environmental Toxicology, Zhejiang University, Hangzhou 310029, People's Republic of China.
Liang
L
Yu
Institute of Pesticide and Environmental Toxicology, Zhejiang University, Hangzhou 310029, People's Republic of China.
Guonian
G
Zhu
College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou 310018, People's Republic of China. Electronic address: 603422689@qq.com.
Jiangfeng
J
Guo
D000818:Animals; D000977:Antiparasitic Agents / Q000633:toxicity; D002460:Cell Line; D003573:Cytochrome b Group / Q000235:genetics* / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007306:Insecticides / Q000633:toxicity; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D035683:MicroRNAs / Q000235:genetics*; D011720:Pyrazoles / Q000633:toxicity*; D012333:RNA, Messenger / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism; D015027:Zebrafish / Q000235:genetics / Q000378:metabolism; D029961:Zebrafish Proteins / Q000235:genetics* / Q000378:metabolism
D000977:Antiparasitic Agents; D003573:Cytochrome b Group; D007306:Insecticides; D008565:Membrane Proteins; D035683:MicroRNAs; D011720:Pyrazoles; D012333:RNA, Messenger; D025521:Tumor Suppressor Proteins; D029961:Zebrafish Proteins; C082360:fipronil
Fipronil; MicroRNA; Toxicity; Zebrafish; cyb561d2; miR-155; miR-216b; miR-499