Solexa sequencing identification of conserved and novel microRNAs in backfat of Large White and Chinese Meishan pigs

Abstract

The domestic pig (Sus scrofa), an important species in animal production industry, is a right model for studying adipogenesis and fat deposition. In order to expand the repertoire of porcine miRNAs and further explore potential regulatory miRNAs which have influence on adipogenesis, high-throughput Solexa sequencing approach was adopted to identify miRNAs in backfat of Large White (lean type pig) and Meishan pigs (Chinese indigenous fatty pig). We identified 215 unique miRNAs comprising 75 known pre-miRNAs, of which 49 miRNA*s were first identified in our study, 73 miRNAs were overlapped in both libraries, and 140 were novelly predicted miRNAs, and 215 unique miRNAs were collectively corresponding to 235 independent genomic loci. Furthermore, we analyzed the sequence variations, seed edits and phylogenetic development of the miRNAs. 17 miRNAs were widely conserved from vertebrates to invertebrates, suggesting that these miRNAs may serve as potential evolutional biomarkers. 9 conserved miRNAs with significantly differential expressions were determined. The expression of miR-215, miR-135, miR-224 and miR-146b was higher in Large White pigs, opposite to the patterns shown by miR-1a, miR-133a, miR-122, miR-204 and miR-183. Almost all novel miRNAs could be considered pig-specific except ssc-miR-1343, miR-2320, miR-2326, miR-2411 and miR-2483 which had homologs in Bos taurus, among which ssc-miR-1343, miR-2320, miR-2411 and miR-2483 were validated in backfat tissue by stem-loop qPCR. Our results displayed a high level of concordance between the qPCR and Solexa sequencing method in 9 of 10 miRNAs comparisons except for miR-1a. Moreover, we found 2 miRNAs, miR-135 and miR-183, may exert impacts on porcine backfat development through WNT signaling pathway. In conclusion, our research develops porcine miRNAs and should be beneficial to study the adipogenesis and fat deposition of different pig breeds based on miRNAs.

Figure 1. The differentital expressions of porcine conversed miRNAs between Large White and Meishan were shown.

Each point in the figure represents a miRNA. Red points represent miRNAs with fold change>2, blue points represent miRNAs with 1/2

Figure 2. Four novel miRNAs were cloned and validated in porcine backfat tissue by stem-loop qPCR.

A. miR-1343, miR-2320, miR-2411 and miR-2483 were cloned in porcine backfat tissue of Large White and Meishan samples. M represents DNA size marker, DL700 and DL2000. Large White samples (lane 1–3) and Meishan (lane 4–6) samples. U6 snRNA was used as an internal control. A 3% agarose gel, stained by ethidium bromide, was run to indicate the cloned miRNA because of the small product size, and the 1.5% agarose gel was used to indicate U6. B. The four novel miRNAs were validated by stem-loop qPCR. All experiments were performed three times. The data shown in Y-axis were calculated using the expression values of 2 ^−ΔΔCt and expressed as means ± standard error. The significance of differences for the expression between samples was calculated by one-way ANOVA. The corresponding significance value (p) was listed above their respective columns. 0.01<p<0.05 was considered to be significant.

Figure 3. Validation of the miRNAs with significantly differential expressions using stem-loop qPCR method.

Expression levels of 6 miRNAs were detected by Real-time PCR. The number of biological replicates is three. The corresponding significance value (p) was shown above their respective columns. 0.01

Figure 4. The folding secondary structure of porcine mir-17-mir-20 cluster predicted computationally by RNAfold.

Folding secondary structure of porcine mir-17 cluster including four miRNAs (mir-17, mir-18, mir-19a and mir-20) and flanking sequences was predicted by RNAfold. The sequences from start to end represent pre-miRNAs.

Figure 5. Phylogenetic trees of mir-17-mir-20 cluster among different mammalian species were constructed using MEGA4.

Figure 6. Hypothesis of miR-135 in porcine adipogenesis.

A. 3′ UTR of APC was predicted as a potential target of miR-135 (position and GenBank No. are listed). B. Potential regulation pathway of miR-135 in porcine adipogenesis.

Figure 7. Potential miR-183-LRP6 interaction was predicted by algorithms RNA22, RNAhybrid, TargetScan, DIANA LAB and PicTar.

We found putative binding site in 3′ UTR of LRP6, and the predicted results were identical among five different algorithms.