Analysis And Identification Of MicroRNAs In Broiler Chicken On Chromium Metabolism

Chromium (Cr) is an essential trace element and has beneficial effect on protein synthesis, carbohydrate and lipid metabolism. The trivalent chromium ion (Cr3+) is known to be an active component of glucose tolerance factor (GTF), which increases the active of insulin and participates in the body’s metabolic functions. MicroRNAs (miRNAs) are considered to be a class of post-transcription single-stranded non-coding RNA molecules and many miRNAs are involved in protein metabolism and skeletal muscle differentiation. Up to now, little is known about miRNA expression profiles in chicken chromium metabolism. This study was aimed to screen the chromium dose that had effect on protein metabolism of skeletal muscle in broilers by animal experiment, and screened miRNAs related chicken chromium metabolism in order to obtain differential expression profiles of the known miRNAs used Solexa sequencing and real-time quantitative PCR technology. The results in this research were as follows:1.288Arbor Acres broilers (male) at the age of1d were randomly allotted to one of the four dietary treatments fed with the diets containing0mg/kg chromium picolinate (CrPic, control group),0.4mg/kg CrPic,2.0mg/kg CrPic and10.0mg/kg CrPic, respectively. Each treatment consisted of6replicates with12birds each. On d21and d42, all the birds and diets were weighed in replicate. On d21and d42,1bird from each replicate was randomly selected for blood sampling. The results showed that no significant difference was observed in the average daily gain, the average daily feed intake and the feed conversion rate among all treatments on d21(P>0.05). The level of10.0mg/kg CrPic affected the average daily feed intake (P<0.05) on d42. Serum total protein concentrations significantly increased in the2.0mg/kg CrPic group (P<0.05) and10.0mg/kg CrPic group (P<0.05) on d42. Besides, the supplementation of10.0mg/kg CrPic significantly decreased the levels of serum glucose (P<0.05) on d42and serum triglycerides (P<0.05) on d21and d42. Thus,10.0mg/kg CrPic increased the growth performance and protein synthesis of broilers to some extent.2. According to the results of the growth performance and protein metabolism indexs of broilers, two small RNA libraries (10mg/kg CrPic group and control group) were sequenced to gain further identification of miRNAs in broiler chickens from skeletal muscle tissues using the Illumina Solexa sequencing approach followed by computer analysis to produce over12.7and12.1million short sequence reads of the2groups respectively. The length distribution analysis showed a majority of the small RNAs were20～23nt in size and the sequences of22nt in size accounted for70%～80%in total small RNAs sequences. Analyses of the first nucleotide bias of the20～24nt miRNAs, we discovered uridine (U) was the dominant first nucleotide of the22nt miRNAs. The small RNA tags were annotated and matched tags from unannotated tags were got rid of by BLASTN searches against the Rfam and Genbank databases. We found miRNAs of the broiler skeletal muscles occupied more than80%of the total small RNAs. However, when we considered numbers of the unique sequence tags, only a relatively small fraction (1%～2%) was discovered. To gain insight into the efficiency of known miRNAs, we mapped the reads that could be aligned perfectly to the chicken genome onto the Gallus gallus miRNA precursor of miRBase (Release15.0) using SOAP program, we respectively identified198and179conserved miRNA/miRNA*between the2groups. Using Mireap software, we discovered28and17candidate novel miRNA/miRNA*. Among these miRNAs,57miRNAs were significantly differentially expressed.3. Among the57miRNAs,6up-regulated (gga-let-7b、gga-miR-103、 gga-miR-140*、gga-miR-181a、gga-miR-206and gga-miR-30d)and2down-regulated miRNAs (gga-miR-301b-3p and gga-miR-1682)were validated by real-time quantitative PCR. All the miRNAs showed differential expression (P<0.05), which was consistent with Solexa sequencing results.4. miRNA mainly interact with the target3’untranslated regions (3’UTR) of messenger RNAs by base pairing, which result in translational inhibition and/or degradation of target mRNA. On the basis of bioinformatics method, we forecasted the target genes of miRNAs identified by real-time quantitative PCR. The transcription factor EIF4E and insulin growth factor1(IGF1) were indicated as the target gene of gga-miR-206, insulin-like growth factor2mRNA-binding protein3(IGF2BP3) and phosphoinositide-3-kinase regulatory subunit3(PIK3R3) were indicated as the target gene of gga-miR-181a, PIK3R3was also indicated as the target gene of gga-miR-103, PI3K related kinase SMG1was indicated as the target gene of gga-miR-140*. The results indicated these predicted target genes combined with miRNAs might relate to the protein synthesis of the skeletal muscle in broilers.The results of this study showed organic chromium played a role in miRNA expression regulation of broiler skeletal muscle.10.0mg/kg CrPic had significant effect on expression profiles of the known miRNAs。This study enrich the chicken miRNA information and should provide new ideas and useful clues for investigating the molecular mechanism of organic chromium nutrition regulation.