Page 152 - 《水产学报》2023年第1期

李晓晖，等 水产学报, 2023, 47(1): 019610

Analysis of miR-17a-5p and its target genes in
Hypophthalmichthys molitrix under hypoxia stress

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LI Xiaohui , FENG Cui , Wang Qiaoxin , ZOU Guiwei , LIANG Hongwei 1,2*
(1. Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan 430223, China;
2. Hubei Hongshan Laboratory, Huazhong Agricultural University, Wuhan 430070, China)
Abstract: Silver carp (Hypophthalmichthys molitrix) is an important freshwater economic fish in China ’s
aquaculture industry. In 2020, six species of freshwater farmed fish in China had production greater than a
million tons, of which the yield of silver carp was 3.812 9 million tons, ranking second. Silver carp is widely dis-
tributed in natural waters, and plays a major role in regulating the ecological environment. In the process of H.
molitrix culture, hypoxia has become an important factor restricting the increase of its yield. In previous studies,
miR-17a-5p was identified and differentially expressed in H. molitrix under hypoxia stress based on the small
RNA sequencing. The micro-ribonucleic acids (miRNAs), a class of endogenous and noncoding small RNA, play
an important role in biological resistance to environmental stresses. In order to explore the role of miR-17a-5p in
H. molitrix under hypoxia, sequence analysis and target gene prediction of miR-17a-5p was performed. The res-
ults showed that the miR17a-5p are highly conserved among species, and 381 target genes were identified, includ-
ing HIF-1α. Dual-luciferase reporter assay results demonstrated that when 293T cells were co-transfected with
miR17a-5p mimics and WT-pmirGLO-HIF-1α 3′UTR, the luciferase activity decreased, confirming HIF-1α to
be a target gene of miR17a-5p. This conclusion is in agreement with the observation of the increasing trend of HIF-
1α alongside the decreasing pattern of miR17a-5p under hypoxic stress in different tissue. KEGG pathway ana-
lysis revealed that the target genes were significantly enriched in Sulfur metabolism, mTOR signaling pathway and
Terpenoid backbone biosynthesis. The expression of 11 target genes involved in the above three signaling path-
ways under hypoxia in liver, brain, heart and gill were detected by quantitative real-time PCR. The results showed
that the overall trend of increase for the relative expressions of these genes, and upregulation was most pro-
nounced in liver. In this study, we analyzed the bioinformatics of miR-17a-5p, predicted and further identified its
target genes, and subjected H. molitrix to different degree of hypoxia stress, revealing that the downregulation of
miR-17a-5p expression in H. molitrix under hypoxia stress attenuates its inhibitory effect on its target genes. Our
finding provides a new idea for miR-17a-5p regulation of its target genes in H. molitrix under hypoxia stress, and
provides a reference for further analysis of the role of miRNAs in the process of hypoxia tolerance of fish.
Key words: Hypophthalmichthys molitrix; miR-17a-5p; hypoxia; dual-luciferase reporter assay; target genes; pathway

Corresponding authors: ZOU Guiwei. E-mail: zougw@yfi.ac.cn;
LIANG Hongwei. E-mail: lianghw@yfi.ac.cn

Funding projects: China Agriculture Research System (CARS-45); Foundation of Hubei Hongshan Laboratory
(2021hskf014); Funded Project for Fundamental Scientific Research Business Expenses of Chinese Academy of
Fishery Sciences (YFI202211, 2020TD33)

https://www.china-fishery.cn 中国水产学会主办 sponsored by China Society of Fisheries
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