Transcriptome Analysis of the Chicken Follicular Theca Cells with miR-135a-5p Suppressed

Abstract

As a class of transcription regulators, numerous miRNAs have been verified to participate in regulating ovary follicular development in chickens (Gallus gallus). Previously we showed that gga-miR-135a-5p has significant differential expression between high and low-yield chicken ovaries, and the abundance of gga-miR-135a-5p is significantly higher in follicular theca cells than in granulosa cells. However, the exact role of gga-miR-135a-5p in chicken follicular theca cells is unclear. In this study, primary chicken follicular theca cells were isolated and then transfected with gga-miR-135a-5p inhibitor. Transcriptome sequencing was performed in chicken follicular theca cells with or without transfection. Differentially expressed genes (DEGs) were analyzed using bioinformatics. A dual-luciferase reporter assay was used to verify the target relationship between gga-miR-135a-5p and predicted targets within the DEGs. Compared with the normal chicken follicle theca cells, 953 up-regulated and 1060 down-regulated genes were detected in cells with gga-miR-135a-5p inhibited. The up-regulated genes were significantly enriched in Gene Ontology terms and pathways involved in cell proliferation and differentiation. In chicken follicular theca cells, Krüppel-like factor 4 (KLF4), ATPase phospholipid transporting 8A1 (ATP8A1), and Complexin-1 (CPLX1) were significantly up-regulated when the expression of gga-miR-135a-5p was inhibited. In addition, KLF4, ATP8A1, and CPLX1 confirmed as targets of gga-miR-135a-5p by using a dual-luciferase assay in vitro The results suggest that gga-mir-135a-5p may involve in proliferation and differentiation in chicken ovarian follicular theca cells by targeting KLF4, ATP8A1, and CPLX1.

Keywords: Gallus gallus; gga-miR-135a-5p; ovarian theca cells; transcriptome sequencing.

Figure 1

The relative expression level of gga-miR-135a-5p in NG, TG and NC. ** P < 0.01. NG: follicular theca cells; TG: inhibitor transfected cells; NC: negative control.

Figure 2

The expression level of the top 22 up-regulated genes with the most significant differential expression greater than fourfold between cells with normal and inhibited expression of gga-miR-135a-5p.

Figure 3

Volcano plot of differentially expressed genes in cells with normal and inhibited expression of gga-miR-135a-5p. The X-axis represents log2 (FC) and Y -axis represents –log10 (FDR). The green dots indicate the down-regulated genes, the black dots indicate the genes with no significant differences, and the red dots indicate up-regulated genes.

Figure 4

The heatmap of differentially expressed genes across all samples via Illumina sequencing. The depth of the color represents the level of gene expression in samples. Normal group: T07 and T08; Transfected group: T10, T11, and T12.

Figure 5

GO annotation of differentially expressed genes,The y-axis on the left indicates the percentage of genes in each term, the x-axis indicates the enriched GO terms.

Figure 6

KEGG annotation of up- regulated differentially expressed genes.

Figure 7

Relative expression of 12 DEGs between cells with normal (NG) and inhibited (TG) expression of gga-miR-135a-5p.

Figure 8

gga-miR-135a suppresses the expression of KLF4, ATP8A1, and CPLX1 in 293T cells. **P < 0.01.