Zebrafish let-7b acts downstream of hypoxia-inducible factor-1α to assist in hypoxia-mediated cell proliferation and cell cycle regulation.
AIMS
Hypoxia-inducible factor-1α (HIF-1α) is a transcriptional regulator of cellular responses to hypoxic stress. MicroRNAs (miRNAs) play an essential role in hypoxia-mediated cellular responses. Previous studies have identified some let-7 family members as hypoxia-regulated miRNAs (HRMs). In the present study, we aimed to investigate whether zebrafish let-7b/7f contribute cellular hypoxic response in a Hif-1α-dependent manner.


MAIN METHODS
Stable suppression of zebrafish hif-1α was achieved by microinjection of an optimized short-hairpin RNA (shRNA) expression vector. Next-generation sequencing was conducted to characterize miRNA and mRNA expression profiles. MiRNA promoter analysis and target detection was performed by dual-luciferase assay. Quantitative real-time PCR (qRT-PCR) and western blot were used to determine the expression of let-7b/7f, Hif-1α and Foxh1. Proliferation of ZF4 cells was examined using Cell Counting Kit-8 (CCK-8) and cell cycle progression was analyzed by flow cytometry assay.


KEY FINDINGS
Correlation between 7 miRNAs and 76 putative targets was identified based on integrated analysis of miRNA-mRNA profiles. Let-7b and let-7f were further considered as potential HRMs, with let-7b further validated as Hif-1α up-regulated. In addition, Forkhead-box H1 (Foxh1) was confirmed as a bona fide downstream target of let-7b. Furthermore, overexpression of both let-7b and let-7f repressed cell proliferation through blocking cell cycle progression of the G1-S transition.


SIGNIFICANCE
Our findings for the first time suggest zebrafish let-7b acts downstream of Hif-1α to assist in hypoxia-mediated cell proliferation and cell cycle regulation at least in part through the downregulation of foxh1. We also identified 4 novel potential HIF-1α-regulated miRNAs in zebrafish.
['Huang|Chun-Xiao|CX|', 'Chen|Nan|N|', 'Wu|Xin-Jie|XJ|', 'He|Yan|Y|', 'Huang|Cui-Hong|CH|', 'Liu|Hong|H|', 'Wang|Wei-Min|WM|', 'Wang|Huan-Ling|HL|']
D000818:Animals D002453:Cell Cycle D049109:Cell Proliferation D020868:Gene Silencing D051795:Hypoxia-Inducible Factor 1, alpha Subunit D035683:MicroRNAs D012333:RNA, Messenger D014158:Transcription, Genetic D015027:Zebrafish
Proliferation Cell cycle Hif-1α Let-7b/7f shRNA foxh1 miRNA profile