lncRNA FDNCR promotes apoptosis of granulosa cells by targeting the miR-543-3p/DCN/TGF-β signaling pathway in Hu sheep

Abstract

Long non-coding RNAs (lncRNAs) regulate the development of follicles and reproductive diseases, but the mechanisms by which lncRNAs regulate ovarian functions and fertility remain elusive. We profiled the expression of lncRNAs in ovarian tissues of Hu sheep with different prolificacy and identified 21,327 lncRNAs. Many of the lncRNAs were differentially expressed in different groups. We further characterized an lncRNA that was predominantly expressed in the ovaries of the low prolificacy Fec^B+ (LPB+) group and mainly present in granulosa cells (GCs), and the expression of this lncRNA decreased during follicular development, which we named follicular development-associated lncRNA (FDNCR). Next, we found that FDNCR directly binds miR-543-3p, and decorin (DCN) was identified as a target of miR-543-3p. FDNCR overexpression promoted GC apoptosis through increased expression of DCN, which could be attenuated by miR-543-3p. Furthermore, miR-543-3p increased and FDNCR reduced the expression of transforming growth factor-β (TGF-β) pathway-related genes, including TGF-β1 and inhibin beta A (INHBA), which were upregulated upon DCN silencing. Our results demonstrated that FDNCR sponges miR-543-3p in GCs and prevents miR-543-3p from binding to the DCN 3' UTR, resulting in DCN transactivation and TGF-β pathway inhibition and promotion of GC apoptosis in Hu sheep. These findings provide insights into the mechanisms underlying prolificacy in sheep.

Keywords: FDNCR; Hu sheep; apoptosis; granulosa cells; miR-543-3p.

Graphical abstract

Figure 1

Profile of lncRNA expression in the ovaries of Hu sheep and identification of DE lncRNAs (A) Workflow for the preparation and analysis of lncRNA libraries. (B) Identification of lncRNAs in the ovarian tissue of Hu sheep. (C) Classification of lncRNAs. (D) Circos plot showing the distribution of lncRNAs in different chromosomes. The outermost ring represents different chromosomes. From the outside toward the inside: sense-lncRNA (green), lincRNA (red), intronic-lncRNA (blue), and antisense-lncRNA (gray). (E) Boxplots showing the expression levels of lncRNAs and mRNAs. FPKM, fragments per kilobase of transcript per million mapped reads. (F) Length of lncRNAs and mRNAs. (G) Lengths of open readings frames of lncRNAs and mRNAs. (H–J) Volcano plot of DE lncRNAs in each group. FDR, false discovery rate; FC, fold change. Red indicates upregulated and green indicates downregulated. (K–M) Hierarchical clustering of DE lncRNAs in each group.

Figure 2

Identification and characterization of a candidate transcript in Hu sheep (A) Representative images from RACE (5′ RACE and 3′ RACE) and Sanger DNA sequencing. (B) The full-length RNA sequence of this transcript. (C) The coding potential of this transcript and other RNAs was predicted using three computational approaches (CPC, coding potential calculator; CNCI, coding-non-coding index; CPAT, coding potential assessment tool). (D) Schematic view of the chromosomal location of this transcript. (E and F) Expression levels of this transcript in each group using qRT-PCR (E) and RNA-seq (F). (G and H) Expression level of this transcript in different tissues of the LPBB group (G) and healthy follicles of various sizes (H) from Hu sheep. (I) Localization of the transcript in the ovaries was detected by FISH. GC, granulosa cell. Scale bars, 20 μm. RNA-seq data are presented as the log₁₀(FPKM+1) of each transcript. Values represent means ± SEM for three individuals. ∗p < 0.05.

Figure 3

FDNCR promotes Hu sheep GC apoptosis (A and B) mRNA expression of FDNCR in GCs in each group. (C–F) GC proliferation and apoptosis were detected using EdU (C and D), CCK8 (E), and flow cytometry (F). Scale bars, 50 μm. (G and H) mRNA (G) and protein (H) expression of cell cycle- and/or apoptosis-related genes in GCs in each group. Values represent means ± SEM for three individuals. ∗p < 0.05.

Figure 4

FDNCR acts as a ceRNA and sponges miR-543-3p in the GCs of Hu sheep (A and B) Subcellular localization of FDNCR was determined using qRT-PCR (A) and FISH (B). NC, negative control. Scale bars, 20 μm. (C) RNAhybrid and miRanda predicted that miRNA targets FDNCR. (D) Construction of the ceRNA network. (E and F) Expression levels of miR-543-3p in each group. Pearson’s correlation was determined between FDNCR and miR-543-3p. (G–J) Expression of miR-543-3p in the GCs in each group. (K) Schematic depicting the interactions of miR-543-3p with wild-type FDNCR (blue) and mutant FDNCR (green). Red nucleotides indicate the seed sequence of miR-543-3p. (L) The regulatory relationship between FDNCR and miR-543-3p was assessed using a dual-luciferase reporter gene assay. (M and N) Expression of FDNCR in the GCs in each group. (O) Association of FDNCR and miR-543-3p with AGO2 was investigated using the RIP assay. FDNCR and miR-543-3p expression was quantified by qRT-PCR. Values represent means ± SEM for three individuals. ∗p < 0.05.

Figure 5

miR-543-3p regulates GC proliferation and apoptosis and mediates FDNCR function (A) GC apoptosis was detected using flow cytometry. (B and C) mRNA (B) and protein (C) expression of apoptosis-related genes in the GCs in each group. (D–F) GC proliferation was detected using EdU (D and E) and CCK8 (F). Scale bars, 50 μm. (G) Expression of cell cycle-related genes in the GCs in each group. (H and I) Apoptosis (H) and expression of apoptosis-related genes (I) were detected using flow cytometry and qRT-PCR, respectively. Values represent means ± SEM for three individuals. ∗p < 0.05.

Figure 6

DCN is a direct target of miR-543-3p in the GCs of Hu sheep (A) miRNA-response elements (MREs) within the 3′ UTR of sheep DCN that enable the binding of miR-543-3p were predicted using RNAhybrid. mfe, minimum free energy. (B) Schematic depicting the interaction of miR-543-3p with wild-type (blue) and mutant DCN (green). Red nucleotides indicate the seed sequence of miR-543-3p. (C) The regulatory relationship between miR-543-3p and DCN was assessed using a dual-luciferase reporter gene assay. (D and E) Expression of DCN mRNA (D) and protein (E) was detected in each group. Values represent means ± SEM for three individuals. ∗p < 0.05.

Figure 7

miR-543-3p enhances GC proliferation by targeting DCN (A–D) DCN expression at mRNA and protein levels in the GCs in each group. (E–H) GC proliferation and apoptosis were detected using EdU (E and F), CCK8 (G), and flow cytometry (H). Scale bars, 50 μm. (I and J) mRNA (I) and protein (J) expression of cell cycle- and/or apoptosis-related genes in the GCs in each group. (K) GC apoptosis in each group was detected using flow cytometry. Values represent means ± SEM for three individuals. ∗p < 0.05.

Figure 8

FDNCR regulates DCN and TGF-β signaling pathways by affecting the levels of miR-543-3p in the GCs of Hu sheep (A) DCN expression in the ovaries in each group. Pearson’s correlation between FDNCR and miR-543-3p. (B and C) Expression of DCN in the GCs in each group. (D) Correlation coefficient between siRNA-NC and siRNA-DCN. (E) Volcano plot showing the DE genes between the siRNA-NC and siRNA-DCN groups. Red indicates upregulated, green indicates downregulated genes. (F) Hierarchical clustering showing DE genes between the siRNA-NC group and siRNA-DCN group. (G) Validation of TGF-β1 and INHBA mRNA expression in different groups. (H–J) mRNA or protein expression of TGF-β1 and INHBA in the GCs in each group. (K) Proposed model of FDNCR regulation of GCs state in Hu sheep. RNA-seq data are presented as the log₁₀(FPKM+1) of each transcript. Values represent means ± SEM for three individuals. ∗p < 0.05.