Huang Chun-Xiao, Chen Nan, Wu Xin-Jie, He Yan, Huang Cui-Hong, Liu Hong, Wang Wei-Min, Wang Huan-Ling
Key Lab of Freshwater Animal Breeding, Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Ministry of Education, College of Fishery, Huazhong Agricultural University, Wuhan, China.
Key Lab of Freshwater Animal Breeding, Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Ministry of Education, College of Fishery, Huazhong Agricultural University, Wuhan, China; Freshwater Aquaculture Collaborative Innovation Center of Hubei Province, Wuhan, China.
Life Sci. 2017 Feb 15;171:21-29. doi: 10.1016/j.lfs.2017.01.005. Epub 2017 Jan 8.
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.
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.
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.
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.
缺氧诱导因子-1α（HIF-1α）是细胞对缺氧应激反应的转录调节因子。微小RNA（miRNA）在缺氧介导的细胞反应中起重要作用。先前的研究已将一些let-7家族成员鉴定为缺氧调节的miRNA（HRM）。在本研究中，我们旨在研究斑马鱼let-7b/7f是否以Hif-1α依赖的方式参与细胞缺氧反应。
通过显微注射优化的短发夹RNA（shRNA）表达载体实现斑马鱼hif-1α的稳定抑制。进行下一代测序以表征miRNA和mRNA表达谱。通过双荧光素酶测定进行miRNA启动子分析和靶标检测。使用细胞计数试剂盒-8（CCK-8）检测ZF4细胞的增殖，并通过流式细胞术分析细胞周期进程。
基于miRNA-mRNA谱的综合分析，确定了7种miRNA与76个推定靶标之间的相关性。Let-7b和let-7f被进一步视为潜在的HRM，其中let-7b进一步被验证为Hif-1α上调。此外，叉头框H1（Foxh1）被确认为let-7b的真正下游靶标。此外，let-7b和let-7f的过表达均通过阻断G1-S期转变的细胞周期进程来抑制细胞增殖。
我们的研究结果首次表明，斑马鱼let-7b至少部分通过下调foxh1在Hif-1α下游发挥作用，以协助缺氧介导的细胞增殖和细胞周期调节。我们还在斑马鱼中鉴定了4种新的潜在HIF-1α调节的miRNA。
