DF-1 cells were transfected with miR-146b-3p mimics or inhibitor, and then the expression levels of miR-146b-3p and thegene were detected after 48 h. The results show that overexpression of miR-146b-3p downregulatedmRNA expression ( Figure 6 A), and that the inhibition of endogenous miR-146b-3p increasedmRNA expression ( Figure 6 B). We constructed two dual-luciferase reporters with the wild-type or mutant ofinserted at the 3′ end of the firefly luciferase gene ( Figure 6 C). Dual-luciferase reporters assay showed that miR-146b-3p significantly reduced the firefly luciferase activity of the wild-type GHR reporter (< 0.05) compared with no-insert control ( Figure 6 C). Furthermore, when miR-146b-3p was co-transfected with the mutant reporter, the firefly luciferase activity was only slightly decreased (> 0.05) compared with the no-insert control. The expression of miR-146b-3p andwere detected in our sequencing samples, and the results indicate that their expression reveals a negative relationship ( Figure 7 ). The expression of miR-146b-3p was higher (< 0.05) in chickens with low body weight than those with high body weight in both WRR and XH chickens ( Figure 7 B), while the expression ofwas higher in chickens with high body weight than those with low body weight ( Figure 7 C). These results indicated that the predicted site ofis a target of miR-146b-3p.

For the 22 highly differentially expressed miRNAs ( Table 4 ), 3151 potential targets were obtained by both miRanda [ 30 ] and RNAhybrid [ 31 ]. The further GO analysis for these targets showed that 192 biological process categories were significantly enriched (< 0.05). The important growth-related GO terms are listed in Table 5 , and are involved in regulation of growth, cell growth, muscle cell differentiation and development, and the transforming growth factor beta receptor signaling pathway. These GO terms contained 87 target genes, and their interactions were predicted using database of STRING 9.1 [ 32 ]. Two possible regulatory networks of interactions among miRNAs and their targets were constructed ( Figure 5 ). A total of 20 miRNAs and 34 targets ( Table 4 ) were involved in the two networks presented. Most of these 20 miRNAs have rarely been identified for their functional role in growth or muscle development. However, a number of their target genes have been reported to play a key role in regulation of growth, such asand. [ 33 ]. These miRNAs could participate in the regulation of growth through their target genes. For instance, gga-miR-34c has six targets in the present network, includingand; gga-miR-146b-3p was predicted to targetand; gga-miR-223 was predicted to targetand; and the predicted target gene of gga-miR-9-5p was

Figure 4. qRT-PCR validation of four differentially expressed miRNAs in all four comparisons. ( A ) WRRh vs . XHh; ( B ) WRRh vs. WRRl; ( C ) WRRl vs. XHl; ( D ) XHh vs. XHl. qRT-PCR reactions were run in triplicates and presented as means ± S.E.M. The Student’s t -test was used to compare expression levels among different groups. * p < 0.05; ** p < 0.01. WRRh vs. WRRl indicated the comparison between the two-tail samples of Recessive White Rock; XHh vs. XHl indicated the comparison between the two-tail samples of Xinhua Chickens; WRRh vs. XHh indicated the comparison between the groups of Recessive White Rock and Xinhua Chickens with high body weight; WRRl vs. XHl indicated the comparison between the groups of Recessive White Rock and Xinhua Chickens with low body weight.

Figure 4. qRT-PCR validation of four differentially expressed miRNAs in all four comparisons. ( A ) WRRh vs . XHh; ( B ) WRRh vs. WRRl; ( C ) WRRl vs. XHl; ( D ) XHh vs. XHl. qRT-PCR reactions were run in triplicates and presented as means ± S.E.M. The Student’s t -test was used to compare expression levels among different groups. * p < 0.05; ** p < 0.01. WRRh vs. WRRl indicated the comparison between the two-tail samples of Recessive White Rock; XHh vs. XHl indicated the comparison between the two-tail samples of Xinhua Chickens; WRRh vs. XHh indicated the comparison between the groups of Recessive White Rock and Xinhua Chickens with high body weight; WRRl vs. XHl indicated the comparison between the groups of Recessive White Rock and Xinhua Chickens with low body weight.

Differentially expressed miRNAs were identified by DEGseq analysis (fold change > 1.5 or < 0.66;-value < 0.05;-value < 0.01), as a result, 200, 279, 257 and 297 miRNAs were detected in four comparisons of WRRhWRRl, WRRhXHh, WRRlXHl and XHhXHl, respectively. Multiple comparisons revealed 80 miRNAs within breeds (WRRhWRRl and XHhXHl), and 110 miRNAs between breeds (WRRhXHh and WRRlXHl). The details of differentially expressed miRNAs are shown in Table S3 in supplementary materials . Among them, moreover, 26 miRNAs (including 10 known miRNAs of miR-122, miR-1329-3p, miR-1587, miR-1736-3p, miR-1769-3p, miR-1769-5p, miR-1773-5p, miR-205a, miR-31 and miR-375) were found in all four comparisons ( Table 3 ). Furthermore, we focused on the miRNAs that were both abundant (read counts > 1000) and highly differentially expressed (fold change > 2 or < 0.5;-value < 0.05;-value < 0.01) in our comparisons, and found that 22 miRNAs met the standards ( Table 4 ).

The abundance of the miRNAs could reflect differences in the roles of these miRNAs in the regulation of growth. The top 20 abundant known miRNAs in our libraries are listed in Table 2 . The most abundant miRNAs is the gga-miR-133 family, which includes gga-miR-133a, gga-miR-133b and gga-miR-133c. The let-7 family was also expressed abundantly in the breast muscle libraries, five of them (gga-let-7a, gga-let-7c, gga-let-7f, gga-let-7j and gga-let-7k) are in the list of the top 20 abundant miRNAs. We found that putative novel miRNAs were less abundant than known miRNAs. There were 12 putative novel miRNAs and 131 known miRNAs in the libraries which have read counts greater than 1000 ( Table S2 in supplementary materials ). The predicted secondary structures of the two most abundant novel miRNAs are shown in Figure S2 . Comparing the chicken miRNAs sequences, heterogeneity at the 5′ and/or 3′ ends of miRNAs was observed ( Figure S3 ). These variations of miRNAs from their miRBase reference sequences are referred to as isomiRs [ 29 ]. We found that many miRNAs have various isoforms in chicken breast muscle libraries, and some miRNAs have more than one highly abundant isoform (e.g., gga-let-7c, gga-miR-205a and gga-miR-223). In addition to a few miRNAs (e.g., gga-let-7c), the most abundant isoforms are identical to the reference in miRBase. The most highly expressed miRNAs appear to have a greater number of different isoforms, e.g., 26 isoforms of gga-let-7c were identified.

Then, the clean reads were assembled by groups, giving rise to 577,820, 513,726, 494,539, and 625,063 unique sequences for WRRh, WRRl, XHh, and XHl, respectively ( Table 1 ). The unique small RNA reads were mapped to the chromosome by blasting with the chicken genome. Results showed that over 70% of the reads could be perfectly mapped to the chicken genome. Moreover, they were mainly located at chromosome 1 (29.25%), 2 (7.96%), 3 (6.71%), 4 (4.97%), 5 (4.71%) and 7 (9.59%) ( Figure 2 ). Finally, the type and number of sRNA were searched using Rfam databases (rRNA, tRNA, sn/snoRNA, miRNAs, other noncoding RNA). The unique sequences were categorized into seven groups; 71.0% of them were defined as miRNAs, 14% were unmatched, and 15% were other known categories of identified small RNA including rRNA, tRNA, snRNA, snoRNA, Figure 3 ).

In this study, three pooled breast muscle tissues for each group of WRRh (the group of Recessive White Rock with high body weight), WRRl (the group of Recessive White Rock with low body weight), XHh (the group of Xinhua Chickens with high body weight) and XHl (the group of Xinhua Chickens with low body weight) were sequenced simultaneously by Illumina Solexa sequencing. All sequencing data were submitted to the NCBI GEO database with the accession number GSE62971 [ 27 ]. A range of 22,080,436 to 16,410,221 raw reads for the four groups was obtained. Firstly, low-quality reads and meaningless reads were filtered out. The quality data for RNA samples are shown in Figure S1 . A total of 20,424,161, 18,160,137, 14,806,039 and 17,295,270 clean reads was obtained for WRRh, WRRl, XHh and XHl, respectively ( Table 1 ). The size distribution of clean reads was assessed for all four groups. Small RNA sequence length was mainly concentrated at 21–24 nt, and the length of 22 nt was the maximum size ( Figure 1 ).

3. Discussion

34, SRF (serum response factor) [ GHR [ Small RNA sequencing can uncover miRNAs expression at an overall level; it has become a useful tool for identifying functional miRNAs. Many miRNAs have been identified as being associated with animal growth performance by high-throughput sequencing [ 26 35 ]. In this study, we firstly reported the miRNAs expression profiles of chicken breast muscle between fast-growing and slow-growing chicken breeds. The sequence analysis showed that the main size of small RNAs in chicken breast muscle was 21–24, and 22 nt is the predominant size. This result was consistent with the known 19–24 nt range for miRNAs, and previous studies in chicken skeletal muscle and ovary also have similar findings [ 36 37 ]. In our sequencing libraries, a total of 921 miRNAs were detected, including 733 known miRNAs and 188 novel miRNAs. The three miRNAs of the gga-miR-133 family (gga-miR-133a, gga-miR-133b and gga-miR-133c) were the most abundant miRNAs in our breast muscle libraries. Previous studies showed that miR-133a was specifically expressed in muscle and regulates the process of skeletal muscle proliferation [ 38 ]. Overexpression of miR-133 could inhibit myoblast differentiation, but promotes myoblast proliferation by targeting(serum response factor) [ 39 ]. The other abundantly expressed miRNAs family in our libraries was gga-let-7, which also was reported to have abundant expression in chicken skeletal muscle [ 40 ]. The family of let-7 miRNAs has been shown to play vital roles in mediating cell proliferation and differentiation, in particular, gga-let-7b has demonstrated a role in growth regulation through targeting 26 ]. Of the top 20 abundantly expressed miRNAs identified, some, such as miR-133, miR-10b, miR-26a, miR-30e and gga-miR-30a were also abundantly expressed in chicken somites [ 41 ].

38,42, p -value < 0.05; q -value < 0.01), which also have abundant expression (read counts > 1000) were found in our comparisons. Among these miRNAs, miR-21 was found to be highly expressed in many species, and associated with cardiac disease and a wide variety of human cancers [ p < 0.05) for these targets, including many growth-related biological processes, such as regulation of growth, cell growth, muscle cell differentiation and development, and transforming growth factor beta receptor signaling pathway. miRNAs have an important role in muscle tissues during embryonic development and miR-133, miR-1 and miR-206 are crucial regulatory factors during myogenesis [ 19 43 ]. In the present study, gga-miR-133 was expressed most highly in our samples, which indicated that it might also play a key role in chicken postnatal stages. Furthermore, miR-206 was also abundantly expressed in our samples, while miR-1 was not. These results suggest that some miRNAs might regulate muscle tissues during all development process, whereas some might play a key role only during a special muscle development stage. Differentially expressed miRNAs were confirmed by qPCR analysis. The good correlation between the two methods indicated that deep sequencing results were credible. A total of 22 highly differentially expressed miRNAs (fold change > 2 or < 0.5;-value < 0.05;-value < 0.01), which also have abundant expression (read counts > 1000) were found in our comparisons. Among these miRNAs, miR-21 was found to be highly expressed in many species, and associated with cardiac disease and a wide variety of human cancers [ 44 45 ]. In this study, miR-21 was upregulated in low-body weight of both WRR and XH chickens. Different expression levels of miR-21 were found in chicken skeletal muscle of different growth periods [ 26 ], and in skeletal muscle between broiler and layer [ 37 ]. One recent study showed that miR-21 can inhibit cell proliferation [ 46 ], suggesting that it might be a negative regulatory factor for chicken growth. These highly differentially expressed miRNAs may play a key role in chicken growth traits, and could be used as candidate genes for further study. However, with the exception of miR-21, most of these miRNAs were little known in terms of growth. It is known that the effects of miRNAs are mainly through regulating the expression of target genes [ 13 ]. Therefore, target gene prediction, and annotation of their biological function is useful to predict miRNA function. In this study, we used two different methods to predict the targets for the differently expressed miRNAs, and removed the different targets to reduce false-positive predictions. A total of 3151 consensus targets were obtained for the 22 highly differentially expressed miRNAs. The GO result showed that 192 biological process categories were significantly enriched (< 0.05) for these targets, including many growth-related biological processes, such as regulation of growth, cell growth, muscle cell differentiation and development, and transforming growth factor beta receptor signaling pathway.