| Channel catfish (Ictalurus spp.) is an economically important species in freshwater aquaculture around the world and occupies a prominent position in the aquaculture industry of the United States. To overcome the issues of rising cost of aquaculture and fierce market competition, catfish producers need to make profit by improving various performance traits in fish production, such as disease resistance, growth rate and feed conversion ratio. In recent years, intensive studies of this species, especially concerning disease control, nutritional requirements, immune mechanism and other aspects, have been performed using molecular techniques. However, little information is available concerning channel catfish microRNAs (miRNAs), which play important roles in the regulation of almost every biological process in eukaryotes. In the studies, we applied a comprehensive strategy to identify a batch of miRNAs in channel catfish. Then, we analyzed the characters, which includes the phenomenon of IsomiRs and the phylogeny of miRNA*, of the miRNAs identified in channel catfish. To preliminarily acquire the functions of these miRNAs, different expression characters of the selected miRNAs were studies in channel catfish. Meantime, a microtubule-associated protein RP/EB family member 1 (EB1) gene was predicted and validated to be the target of the ipu-miR-143 by using a luciferase reporter gene assay with HEK293 and CCK cell line in vitro.Catfish whole-genome sequencing is underway. However, the significant expansion of the channel catfish ESTs resources in recent years may be particularly useful as the sources of identifying miRNAs with the method of bioinformatics computing. In the present studies, we applied a bioinformatic strategy to identify 16 miRNAs which represent 12 miRNA families in channel catfish by searching both expressed sequence tags (ESTs) and genome survey sequences (GSS) databases. Three pairs of miRNAs including ipu-miR-9-3p and ipu-miR-9-5p, ipu-miR-126-3p and ipu-miR-126-5p,ipu-miR-142-3p and ipu-miR-142-5p were found in one precursor sequence respectively.To verify the predicted miRNAs, real-time PCR was used to profile the expressions of the 16 miRNAs with different tissues of channel catfish. All the miRNA candidates were detectable in 5 tissues except for ipu-miR-9-3p. Based on sequence complementarity between miRNAs and their mRNA targets, 75 potential targets for the 16 miRNAs were predicted. These potential target sequences are involved in immune regulation,transcriptional regulation, metabolism and many other biological functions.In the past few years, many conserved miRNAs have been discovered by traditional identification methods, such as bioinformatic approaches or direct cloning methods.However, non-conserved miRNAs are seldom identified by traditional methods, because they are often expressed at a lower level than the conserved miRNAs. In this study, a small-RNA cDNA library was constructed, and Solexa sequencing technology was used to perform high throughput sequencing of the library. To increase the coverage of channel catfish miRNAs, the library of small cDNAs was generated with pooled total RNAs from 10 tissues. A total of 14,919,026 raw reads, representing 161,288 unique sequences, were obtained from the library. The length distribution analysis showed that 54.8% of reads were 21-23 nucleotides (nt). After comparing the small RNA sequences with the RFam database, 4,542,396 reads that represent 25,538 unique sequences were mapped to the genome sequence of zebrafish to perform a distribution analysis and to screen for candidate miRNA genes. Allowing 1 mismatch between sequences, we finally identified 260 conserved miRNAs, which belong to 105 families. For the sequences that didn't match the known miRNAs, we compared the sequences with the genome of zebrafish to detect 26 novel miRNAs in channel catfish. To perform deep mining of the dataset, we also used channel catfish EST and GSS databases to identify more novel miRNAs, and then another 28 novel miRNAs have been found.Owing to financial restraints and a desire to obtain the whole miRNA transcriptome of channel catfish, the cDNA library of small RNAs was constructed with pooled total RNAs from different tissues. Therefore, we did not obtain tissue-specific expression profiles of the miRNAs through the high throughput Solexa sequencing. Quantifying miRNAs in different tissues is an important initial step to investigate the functions of miRNAs. Stem-loop qRT-PCR was applied to validate and profile the expression of the novel miRNAs in 10 tissues. Some novel miRNAs, such as ipu-miR-129b, ipu-miR-7562 and ipu-miR-7553, were expressed in all tissues examined. However, some novel miRNAs appear to be tissue specific. Ipu-miR-7575 is predominantly expressed in stomach. Ipu-miR-7147 and ipu-miR-203c are highly expressed in heart, but are relatively weakly expressed in other tissues. Except for ipu-novel-7556, ipu-novel-7562,ipu-miR-7553 and ipu-novel-129b, most of the miRNAs exhibited lower expressions in the tissues of skin and intestines (sites of pathogen exposure, which are involved in the specific host defense mechanisms in fish). To detect the expression patterns of these miRNAs during the embryonic development process in channel catfish, samples from 11 stages were sampled and analyzed using stem-loop qRT-PCR. Twenty-three miRNAs,which demonstrated significant different expressions in 11 stages, were detected in the embryonic development process of channel catfish (P < 0.05). These miRNAs are mostly highly expressed in the blastodisc forming stage, somite appear stage and pre-hatching stage. Most of the significant different expressed miRNAs exhibited lower expressions during the cleavage stage. Considering that miRNAs mainly play negative regulation in biological process in eukaryotes, we deduced that these miRNA expression patterns are associated with the regulation of special gene expression in the embryonic development process of channel catfish. Based on sequence complementarity between miRNAs and their mRNA targets, a total of 281 targets were predicted for the 45 novel miRNAs by searching for antisense hits in the reference RN A sequences of the channel catfish. These potential target sequences are involved in immune regulation, transcriptional regulation,metabolism and many other biological functions.Liver, the major organ for basal metabolism in fish, is involved in multiple physiological process which including metabolism, digestion, immunity and other biological functions. The expression patterns of the 45 novel miRNAs in the liver of channel catfish were detected using a stem-loop qRT-PCR at different time post infection of Aeromonas hydrophila. Clustering of the 45 miRNAs yielded 2 major clusters with specific expression profiles. In summary, infection of A. hydrophila induced the expressions of the 23 miRNAs in Cluster 1 (including ipu-miR-7147, ipu-miR-7547 and other miRNAs) at 2h post infection. At 8h, 24h and 48h post infection, most of the miRNAs in cluster 1 showed decreased expression patterns in liver of channel catfish.The expressions of 65.22% miRNAs in Clusterl were significantly decreased at least in one time point during the time of post infection from 8h to 48h (P<0.05). In Cluster 2,infection induced the expressions of the 22 miRNAs from 2h to 24h post of infection.Ipu-miR-24b, ipu-miR-7550 and ipu-miR-7567 showed significantly increased expressions at least in one time point from 2h to 24h post infection (P<0.05). However,most of the miRNAs in Cluster 2 were detected with decreased expression patterns at 48h post infection. Among the miRNAs with decreased expressions, significant changes were observed for ipu-miR-7568 and ipu-miR-7575 comparing with control groups(P<0.01). Overall, there were more up-regulated miRNAs than down-regulated miRNAs at 2h post infection. Meanwhile, much greater numbers of down-regulated miRNAs were detected at 24h and 48h post infection. The targets of the miRNAs that demonstrated significant expressions at least in one time point post infection were predicted with bioinformatics methods. The results showed that the targets are involved in immune regulation, stress response, metabolism and many other biological functions in channel catfish.Bioinformatic analyses indicated that the 3' untranslated region (3'-UTR) of the microtubule-associated protein rp/eb family member 1 gene (EB1) contains an ipu-miR-143 target site, which are perfectly complementary to the seed region (positions 2-8) of the mature ipu-miR-143 sequence. Therefore, we speculated that the EB1 gene might be act as a direct regulatory target of miR-143 in channel catfish. The pMIR-EB1 report vector containing ipu-miR-143 complementary sequence was constructed to investigate the target of ipu-miR-143 in channel catfish. Then, pMIR-EB1 and miR-143 mimics were co-transfected into HEK293 and CCK cells to detect the biological activity of ipu-miR-143. Comparing with the control groups, miR-143 mimics groups showed significantly lower levels of luciferase expressions in the two cell lines (P < 0.05). The luciferase activity of ipu-miR-143 inhibitor groups was significantly higher than that of the control groups in CCK cell line (P < 0.05). Nonetheless,there was no significant differences between the inhibitors and the control groups in HEK293 cell line (P=0.064).The results demonstrated that the EB1 gene should be a target gene of ipu-miR-143, and the results provides information for further research of post-transcriptional mechanisms of ipu-miR-143 in channel catfish.In conclusion, we firstly applied a comprehensive strategy to identify a batch of miRNAs in channel catfish. Moreover, preliminary functions of some selected miRNAs have been studied with different strategy. The discovery in this study of miRNA genes in the channel catfish genome will contribute to a better understanding of the roles miRNAs play in regulating diverse biological processes in fish and vertebrates. |
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