Integrated Analysis of miRNA and mRNA Expression Profiles Reveals Functional miRNA-Targets in Development Testes of Small Tail Han Sheep

Abstract

Small Tail Han Sheep is a highly valued local breed in China because of their precocity, perennial estrus, and high fecundity. The average annual lambing rate of ewes is as high as 180-270%, the semen of ram has characteristics of high yield, high density, and good motility. To reveal the key miRNAs and miRNA-targets underlying testis development and spermatogenesis in male Small Tail Han Sheep, integrated analysis of miRNA and mRNA expression profiles in 2-, 6-, and 12-month-old testes was performed by RNA-seq technology and bioinformatics methods. The results showed that total of 153 known sheep miRNAs and 2712 novel miRNAs were obtained in 2-,6 - and 12-month-old Small Tail Han Sheep testes; 5, 1, and 4 differentially expressed (DE) known sheep miRNAs, and 132, 105, and 24 DE novel miRNAs were identified in 2- vs. 6-, 6- vs. 12-, and 2- vs. 12-month-old testes, respectively. We combined miRNA results of this study and the mRNA results obtained in our previous study to predict the target mRNAs of DE known sheep miRNAs; 131, 10, and 15 target mRNAs of DE known sheep miRNAs and 76, 1, and 11 DE miRNA-targets were identified in the three groups, respectively. GO and KEGG analyses showed that: in 2- vs. 6-month-olds, the target genes of DE known sheep miRNAs were involved in 100 biological processes and 11 signaling pathways; in 6- vs. 12-month-olds, the target genes of DE known sheep miRNAs were involved in 4 biological processes; and in 2- vs. 12-month-olds, the target genes of DE known sheep miRNAs were involved in 17 biological processes and 4 signaling pathways. Three miR-target regulatory networks were constructed based on these DE miRNA-targets. The key miRNA-Targets involved in testis development and spermatogenesis were screened. 6 known sheep miRNAs and 6 novel miRNAs were selected to validate the accuracy of miRNA sequencing data by qRT-PCR. The binding sites of oar-miR-379-5p with WNT8A was validated by a dual luciferase reporter gene detection system.

Keywords: Small Tail Han Sheep; mRNAs; miRNAs; spermatogenesis; testis development.

Figure 1

Venn and series-cluster analyses of DE mRNAs and DE miRNAs in 2-, 6-, and 12-month-old testes. a. Venn diagram of DE mRNAs in 2- vs. 6-, 6- vs. 12-, and 2- vs. 12-month-old testes. b. Venn diagram of DE miRNAs in 2- vs. 6-, 6- vs. 12-, and 2- vs. 12-month-old testes. c. Eight profiles of DE mRNAs in 2-, 6-, and 12-month-old testes. d. Eight profiles of DE miRNAs in 2-, 6-, and 12-month-old testes. Profile number is shown at the top left corner of each square. Number of DE mRNAs or miRNAs grouped in each profile is shown at the bottom left corner of each square. Profiles are ordered based on the number of mRNAs or miRNAs assigned.

Figure 2

Top 15 BP terms of candidate target mRNAs. a. Two- vs. 6-month-old testes; b. 6- vs. 12-month-old testes; c. 2- vs. 12-month-old testes; P < 0.05.

Figure 3

Top 15 pathways of candidate target mRNAs. a. Two- vs. 6-month-old testes; b. 2- vs. 12-month-old testes; red terms represent P < 0.05.

Figure 4

miRNA-Target regulatory networks in 2- vs. 6-, 6- vs. 12-, and 2- vs. 12-month-old testes. a. Two- vs. 6-month-old testes; b. 6- vs. 12-month-old testes; c. 2- vs. 12-month-old testes.

Figure 5

qRT-PCR validation of RNA-seq data for miRNAs. a. RNA-seq data. Values were calculated and normalized by EB-Seq algorithm if the fold changes were >1.5 and FDR < 0.05. b. RT-PCR analysis of 6 novel miRNAs and 6 known miRNAs. The fold change in each time point was relative to 2 months. The expression values were calculated by the 2^−ΔΔCt method. Different capital letters represent extremely significant differences (P < 0.01); different letters indicate significant differences (P < 0.05), whereas the same letters indicate no significant differences (P > 0.05).

Figure 6

Detection of interactions between oar-miR-379-5p and WNT8A by dual luciferase reporter system. a. Binding site sequence of miRNA and target gene WNT8A. b. Structure of pmirGLO plasmid vector. c. Working principle of pmirGLO plasmid vector. The dual luciferase reporter gene was inserted the 3′UTR sequence of the target gene, which can bind with miRNA into the downstream of the luciferase gene in pmirGLO plasmid vector, the translation of luciferase from firefly will be inhibited and the luciferase activity will eventually decrease when endogenous miRNA or introduced exogenous miRNA are combined with the inserted 3′UTR sequence. d. Relative luciferase activity after transfecting oar-mir-3799-5p and WNT8A recombinant plasmid for 24h. e. Relative luciferase activity after transfecting oar-mir-3799-5p and WNT8A recombinant plasmid for 48h.