| Megalobrama amblycephala, commonly known as wuchang bream, belonging to Megalobrama, Cyprinidae, Cypriniformes, Cultrinae, has been favored for its wide feeding habits, fast growing, low production cost and high survival rate, which was one of the most important freshwater commercial fishes exist only in China.MicroRNAs(miRNAs) belong to one of the endogenous non-coding small RNAs widely, exist in eukaryote for regulating gene expression at transcription level and involve in nearly all known biological processes, including growth, development, proliferation, differentiation, apoptosis and tumor development etc. A large number of studies have shown that certain hypoxia-regulated microRNAs(HRMs) participate in hypoxic response process. However, limited information is vailable to date in fish hypoxia adaptation of HRMs. In the present study, we analysed the sequence characterization and homology of teleost-specific miR-462/731 cluster and their spatio-temporal expression distribution. In addition, three hypoxia-related HRE(hypoxia response element) motifs upstream of mi R-462/731 cluster were constructed into PGL3-HRE vector and then the promoter activity was analyzed under hypoxia. Our study aimed to further understand the regulation mechanism about the miRNAs in fish hypoxia adaption. The main results of this study were as follows: 1) The mature sequence analysis of teleost-specific miR-462/731 clusterThe mature sequence of miR-462/731 of M. amblycephala was amplified. Multiple sequence alignment of the above sequence and other ten teleost miR-462/731 mature sequences and miR-191/425 sequence of human showed highly conservation of the seed sequences. Then the phylogenetic analysis revealed miR-462 and miR-731 were evolutionarily conservative among teleost fishes, and gathered into two different branches. For miR-462 sequences, C.idellus and M. amblycephala were gathered into a small branch, and together with other fishes gathered for a big branch; while in the tree of miR-731 sequence, M. amblycephala formed into a small branch with Danio rerio, C. carpio, C. idellus. No matter mi R-462 or miR-731, M. amblycephala was the most conservative in evolution with C. idellus. 2) Bioinformatic analysis of the teleost miR-462/731 upstream sequenceThe potential promoter sequence(-2500 bp) of mi R-462/731 of M.amblycephala was amplified, and some cis-elements including 3 HRE motifs, TATA box, HRE, ISRE, PU.1 and so on were found in the promoter of M. amblycephala and other nine teleost of the miR-462/731. Besides, genome location analysis showed all thses teleost miR-462/731 were located between impdh2 and dalrd3. 3) Expression analysis of M. amblycephala miR-462/731 under normoxia and hypoxiaThe real-time quantification PCR of M. amblycephala miR-462/731 showed that both miR-462 and mi R-731 were the most abundant in neural stage, whereas presented low abundant during early stage of embryonic development, and then with a trend of rising first then falling, suggesting that miR-462/731 may play an important role in the middle and late periods of embryonic development of M. amblycephala. The spatio expression analysis of M. amblycephala miR-462/731 exhibited the most abundant expression in gill, and low expression in blood and liver, which explained the significant role of miR-462/731 involved in modulating oxygen exchange in gill. Under hypoxia, both mi R-462 and miR-731 exhibited up-regulation in blood, and miR-731 also exhibited up-regulation in liver and spleen, while in other tissues showed down-regulation. Futher expression analysis of M. amblycephala miR-462/731 was carried out in vitro in MAF cells by simulating hypoxia conditions with CoCl2, and showed both miRNAs reached the highest expression level 48 h after 100 μM CoCl2 treatment.4) Construction of reporter plasmids of miR-462/731 promoter and analysis of their bioactivityThree recombinants including three HRE motifs were constructed into dual luciferase reporters respectively, and the luciferase activity analysis revealed that the luciferase activity of PGL3-1HRE, PGL3-2HRE and PGL3-3HRE were significantly increased, and was the highest for PGL3-1HR, indicating that this promoter deletion PGL3-1HRE(-2105 bp to-1765 bp) contains a minimal core promoter region. Treatment with 100 μM CoCl2 on the transfected recombinant vector showed that only the luciferase activity of PGL3-1HRE was significantly increased. As a result, the promoter deletion PGL3-1HRE may contain core promoter region, furthermore, may play important function in hypoxia response. 5) The effect of M. amblycephala miR-462/731 on cell proliferation and cell cycleMiR-462 and miR-731 mimics were transfected to the MAF cells respectively, and the cell proliferation and cell cycle were analyzed. The results showed that miR-462/731 inhibited cell proliferation and cell cycle on G2 phase. |
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