Transcriptome Analysis Reveals Key miRNA-mRNA Pathways in Ovarian Tissues of Yunshang Black Goats With Different Kidding Numbers

Abstract

The granulosa cell (GC) is the basic functional unit of follicles, and it is important for promoting follicle growth and sex hormones, as well as growth factor secretion in the process of reproduction. A variety of factors influence granulocyte proliferation, yet there are still many gaps to be filled in target and non-coding RNA regulation. In our study, the differentially expressed (DE) mRNAs and miRNAs were detected by using RNA-seq, and we constructed a mRNA-miRNA network related to goat prolificacy. Then, the goat primary GCs were isolated from the follicle for the function validation of candidate genes and their regulator miRNAs. A total of 2,968 DE mRNAs and 99 DE miRNAs were identified in the high- and low-prolificacy goat by RNA-seq, of which there were 1,553 upregulated and 1,415 downregulated mRNAs, and 80 upregulated and 19 downregulated miRNAs, respectively. JAK3 was identified as highly expressed in the low-prolificacy goats (3 times higher than high-prolificacy goats), and the integrated analysis showed that chi-miR-493-3p was a potential regulator of JAK3. The analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) showed that JAK3 was involved in the PI3K-Akt signaling pathway, the Jak-STAT signaling pathway, and signaling pathways regulating pluripotency of stem cells. In particular, the PI3K-Akt signaling pathway was a typical pathway for cell proliferation, differentiation, apoptosis, and migration. We found that the chi-miR-493-3p targets JAK3 directly via RT-qPCR, dual fluorescence assays, and Western blot. Furthermore, the expression of JAK3 was significantly decreased by the chi-miR-493-3p mimic and increased by the chi-miR-493-3p inhibitor. The CCK-8 assay showed that overexpression of JAK3 promoted cell proliferation, while inhibiting JAK3 had the opposite effect. The expression of cell proliferation markers CDK4 and cyclin D2 also showed the same results. Moreover, the enzyme-linked immunosorbent assay showed that steroid hormones E₂ and PROG were increased by overexpressing JAK3 and decreased by inhibiting JAK3. Therefore, our results identified a chi-miR-439-3p-JAK3 regulatory pathway, which provided a new insight into the GC proliferation and prolificacy of goat.

Keywords: JAK3; chi-miR-493-3p; goat; reproduction trait; steroid hormone secretion.

Figure 1

Overall miRNA and mRNA prolife in goat ovary by RNA-seq. (A, C) were the differentially expressed mRNA cluster and Volcano diagram, respectively; (B, D) were the differentially expressed miRNA cluster and Volcano diagram, respectively.

Figure 2

The DE miRNA–mRNA interaction network in the comparison. Red color: upregulated; Green color: downregulated.

Figure 3

(A) The top 20 GO terms enriched in the comparison. (B) The top 20 KEGG pathways enriched in the comparison. (C) The protein–protein interaction (PPI) network of differentially expressed proteins from DEM–DEG pairs in the comparison.

Figure 4

Verification of differential gene expressions. RT-qPCR quantifies 10 DEM–DEG pairs in high (H)- and low (L)-prolificacy goat ovary. RPL19 is used as an internal control. Values are expressed as mean ± SD of n = 3. *p < 0.05, **p < 0.01.

Figure 5

JAK3 promoted the granulosa cell proliferation. (A) The protein of JAK3 in goat ovary. H represents the high-prolificacy goats; L represents the lowprolificacy goats. (B) The phylogenetic tree analysis of JAK3. (C, D) The mRNA expressions of JAK3, cyclin D1, cyclin D2, and CDK4 in granulosa cells transfected with pcDNA3.1-JAK3/pcDNA3.1-NC or siRNA-JAK3/siRNA-NC for 48 h are quantified using RT-qPCR. RPL19 is used as an internal control. (E) The protein expressions of JAK3, cyclin D2, and CDK4 in granulosa cells transfected with pcDNA3.1-JAK3/pcDNA3.1-NC or siRNA-JAK3/siRNA-NC for 48 h are quantified using WB. GAPDH is used as an internal control. Values are expressed as mean ± SD of n = 3. *p < 0.05, **p < 0.01.

Figure 6

chi-miR-493-3p specifically targets JAK3 in goat granulosa cells. (A) Target sites for chi-miR-493-3p in the JAK3 3’-UTR and the construction of the luciferase expression vector (Luc) fused with the JAK3 3’-UTR. JAK3-3’UTR-WT represents the Luc reporter vector with the wild-type JAK3 3’-UTR; JAK3-3’UTR-MUT represents the Luc reporter vector with the mutation at the chi-miR-493-3p site in JAK3 3’-UTR. After HEK293T cells transfected chi-miR-493-3p mimic or mimics NC with WT-/MUT-Luc reporter vectors for 48 h, the relative luciferase activities are measured. The (B) mRNA expressions of JAK3, cyclin D1, cyclin D2, and CDK4 in granulosa cells transfected with chi-miR-493-3p-mimic/-inhibitor or mimic/inhibitor NC for 48 h are quantified using RT-qPCR. RPL19 is used as an internal control. (C) The CCK8 assay analysis in granulosa cells transfected with chi-miR-493-3p-mimic/-inhibitor or mimic/inhibitor NC for 48 h. (D) The protein expressions of JAK3, cyclin D2, and CDK4 in granulosa cells transfected with chi-miR-493-3p-mimic/-inhibitor or mimic/inhibitor NC for 48 h are quantified using WB. GAPDH is used as an internal control. Values are expressed as mean ± SD of n = 3. *p < 0.05, **p < 0.01.

Figure 7

JAK3 promotes steroid hormone synthesis. Goat granulosa cells are transfected with pcDNA3.1-JAK3 or pcDNA3.1-NC (A), and siRNA-JAK3 or siRNA-NC (B). After 24 h, cell-free supernatants are collected and estrogen (E₂) and progesterone (PROG) secretions are measured in 50-μl supernatants using ELISA kits. Values are expressed as mean ± SD of n = 3. *p < 0.05, **p < 0.01.