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. 2017 Jul 31;12(7):e0181897.
doi: 10.1371/journal.pone.0181897. eCollection 2017.

MicroRNA expression profiles differ between primary myofiber of lean and obese pig breeds

Dongting He  1 , Tiande Zou  1 , Xiangrong Gai  2 , Jideng Ma  3 , Mingzhou Li  3 , Zhiqing Huang  1 , Daiwen Chen  1
Affiliations

MicroRNA expression profiles differ between primary myofiber of lean and obese pig breeds

Dongting He et al. PLoS One. .

Abstract

MicroRNAs (miRNAs) are non-coding small miRNAs ~22 nucleotides in length and play a vital role in muscle development by binding to messenger RNAs (mRNAs). Large White (LW, a lean type pig) and Meishan pigs (MS, a Chinese indigenous obese breed) have significant postnatal phenotype differences in growth rate, muscle mass and meat quality, and these differences are programmed during prenatal muscle development. Little research shed light directly on the miRNA transcriptome difference in prenatal muscles between these two distinct pig breeds. Myofiber phenotypes of LW and MS were measured at developmental stages of 35, 55 and 90 days post-conception (dpc), which revealed that the myogenesis process is more intense in MS than in LW at 35 dpc. To investigate the role of miRNAs involved in regulating muscle development at earlier stages of myogenesis and decipher the miRNAs transcriptome difference between LW and MS, here, the miRNAomes of longissimus dorsi muscle collected at 35 dpc from female LW and MS were analyzed by deep sequencing. Overall, 1147 unique miRNAs comprising 434 known miRNAs, 239 conserved miRNAs and 474 candidate miRNAs were identified. Expression analysis of the 10 most abundant miRNAs in every library indicated that functional miRNAome may be a small amount and tend to be greater expressed. These sets of miRNA may play house keeping roles that were involved in myogenesis. A total of 87 miRNAs were significantly differentially expressed between LW and MS (reads > 1000, P < 0.05). Gene ontology (GO) and KEGG pathway enrichment analysis revealed that the differentially expressed miRNAs (DE miRNAs) were associated mainly with muscle contraction, WNT, mTOR, and MAPK signaling pathways. Some myogenesis related miRNAs (miR-133, miR-1, miR-206 and miR-148a) are highly abundant in MS, while other miRNAs (let-7 family, miR-214, miR-181) highly expressed in LW. In addition, the expression patterns of miRNAs (miR-1, -133, -206) at three prenatal stages (35, 55 and 90 dpc) were determined using qRT-PCR. Notably, ssc-miR-133 was significantly more highly expressed in LW pigs skeletal muscle at all prenatal stages compared with its expression in LW pigs skeletal muscle. Taken together, the main functional miRNAs during muscle development are different between lean and obese pig breeds. The present study adds new information to existing data on porcine miRNAs and will be helpful to investigate the dominant (main functional) muscle-related miRNAs sets in different pig breeds.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1
Comparison of myofiber density and diameter between Large White (LW) and Meishan (MS) fetuses (A, B) during skeletal muscle development at 35 to 90 dpc (E35, E55 and E90). The mRNA expression of Myogenesis-related genes in longissimus dorsi between LW and MS at 35 dpc (C). Values are mean ± SD, n = 4. *Mean values were significantly different, P<0.05.
Fig 2
Fig 2. Description of miRNAs in two pig breeds.
(A) Overview of the miRNA precursors including known, conserved and candidate miRNAs between Large White (LW) and Meishan (MS). (B) Venn charts indicate expression patterns of known, conserved and candidate miRNAs. (C) Distribution of read counts between LW and MS.
Fig 3
Fig 3. Hierarchical clustering analysis and heat map matrix of Pearson correlations of the reads counts of 1147 unique miRNAs between Large White (LW) and Meishan (MS).
LW1, LW2 and LW3 refer to the three biological replications of Large White pigs; MS1, MS2 and MS3 refer to the three biological replications of Meishan pigs. The color legend at top represents the correlation coefficient.
Fig 4
Fig 4. Top 10 unique miRNAs with the highest reads counts.
Fig 5
Fig 5. Hierarchical cluster analysis of the top 50 differentially expressed miRNAs (P < 0.05).
Normalized log (base 2) data was hierarchically clustered by miRNA reads counts and is plotted as a heat map. LW1, LW2 and LW3 refer to the three biological replications of Large White pigs; MS1, MS2 and MS3 refer to the three biological replications of Meishan pigs.
Fig 6
Fig 6. GO (A) and KEGG (B) pathways analysis of top 50 differentially expressed miRNAs.
Fig 7
Fig 7. Validation of sequenced results using RT-qPCR for eight representative DE miRNAs.
The RT-qPCR result for miRNAs fold change (FC) between LW and MS was calculated as FC = 2-ΔCt MS /2-ΔCt LW, and the sequenced result for miRNA log2FC expression levels was calculated by log2FC = log2(reads of LW/ reads of MS), The sequenced reads were normalized.
Fig 8
Fig 8. The expression levels of miR-133, miR-1 and miR-206 in Large White (LW) and Meishan (MS) pigs during skeletal muscle development at 35 to 90 dpc (E35, E55 and E90).
For each miRNA, the expression level of LW pigs at 35 dpc are given as a negative control and set at 1. Two-way ANOVA (n = 3 per breed per time point). ‘B’ and ‘T’ mean breed and time, respectively. Values are means ± SD. Different capital letters means significant difference (P<0.05).
Fig 9
Fig 9. The biological function of miR-133 during skeletal muscle development.
MyoD modulate muscle proliferation by regulating expression of miR-133.
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Grants and funding

The present study was supported by the National Program on Key Basic Research Project of China (2012CB12470; http://program.most.gov.cn/). DC and ZH received the funding. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.