Identification And Preliminary Application Of MicroRNAs Of Chinese Rare Minnow(Gobiocypris Rarus) In Aquatic Ecotoxicology

Micro RNAs(mi RNAs) is an abundant class of endogenous short(~22 nucleotides in length), single stranded non-protein-coding RNAs(nc RNAs). It is widely involved in cell proliferation, differentiation, apoptosis, aging and metabolism in animals, plants and other eukaryotes as well as in many physiological and biochemical processes in organisms. Studies have shown that the abnormal expression of mi RNAs was closely related to the occurrence of many human diseases. There were many literatures reported on the harm of PAHs to human health. But most studies were mainly focused on the high molecular weight PAHs, while environmental hazards of low molecular weight PAHs ignored.The Chinese rare minnow(Gobiocypris rarusis), a local small Cyprinidae fish, is extensively used as an experimental fish in China. As we know, identification of mi RNA genes in rare minnow was the first step to discover the biological function of mi RNAs, as well as to screen new molecule biomarkers for assessing the risk of environmental pollution in the future. However relevant biological data, especially mi RNA transcriptome profile, have not been well documented.The illunima high-throughput sequencing technology was applied to obtain the rare minnow mi RNAs biological information in this study. The toxicity effects of Phe(Phenanthrene) to adult rare minnow(~5 months) liver were examined with 30 days exposure experiments in different concentrations, which separately in 10 μg/L, 100 μg/L, and 1000 μg/L.The results in this study as follows:1. A pool of equal amounts of RNA obtained from 6 different adult rare minnow tissues(brain, eye, gill, liver, muscle and heart) was sequenced using illumina high-throughput sequencing technology. 26,930,553 raw reads, representing 2,118,439 unique high-quality reads, were obtained from the pooled small RNA library. 352 conserved rare minnow mi RNAs and 112 novel candidate mi RNAs were first discovered and characterized by using bioinformatics analysis.2. TUNEL and Hoechst 33342 staining were applied to the Phe group in liver of rare minnow. The staining results showed that the number of apoptosis of liver cells was increased accompanied by the increase of exposure concentration, of which the treatment of 10 μg/L and 100 μg/L groups significantly induced the apoptosis of liver cells. We also founded that the caspase 3 enzyme activity significantly increased in the treatment groups of 10 μg/L and 100 μg/L, but caspase 9 enzyme activity was significantly increased in the treatment of 1000 μg/L group only(P < 0.05). There were no significant increase observerd on caspase 8 enzyme activity in all three treatment groups. The former experimental results of apoptosis related enzyme of caspase 3, caspase 8 and caspase 9 activity, and histological staining, showed that the apoptosis of liver cells in rare minnow was induced by Phe via the intrinsic pathway mediated by caspase 9.3. The q PCR method was used to detect p53 signaling pathway in cell cycle and apoptosis related gene expressions in liver of rare minnow. The expression levels of cell cycle regulatory genes of p21 and gadd45α were significantly increased(P < 0.05) in each concentration group, which up-regulated more than 10 times in the treatment group of 10 μg/L and 100 μg/L when compared with the control group. There were no significant changes in the expression of anti-apoptotic gene of bcl-2 in 100 μg/L and 1000 μg/L, while it significantly increased in 10 μg/L concentration group(P < 0.05). Compared with the control group, the expression of pro-apoptotic gene bax was just opposite to bcl-2, which was significantly induced in high concentration group(100 μg/L and 1000 μg/L)(P < 0.05).4. The poly(A) plus tail method was used to detect the effect of Phe on mi R-17~92 cluster(mi R-17, mi R-18 a, mi R-19 a, mi R-20 a, mi R-19 b, and mi R-92) transcription levels of liver in rare minnow. The results showed that at 100 μg/L and 1000 μg/L treatment groups, the transcription levels of all members of the mi R-17~92 cluster were significantly up-regulated(P < 0.05) compared to the control group. At the lowest concentration group, only mi R-19 a and mi R-92 were increased significantly(P < 0.05). These results are consistent with the apoptosis of liver tissue staining, indicating that mi R-17~92 clusters was participated in regulation of liver apoptosis, which can be used as potential biomarkers for liver toxicity induced by low molecular weight PAHs-Phe.