Lnc MSTRG 4701.7 targets miR-1786/RORa to competitively regulate proliferation and apoptosis in chicken follicular granulosa cells

Abstract

Background: Follicular development in chickens is a highly coordinated and complex process. While the hypothalamic-pituitary-gonadal axis plays a central regulatory role in this process, the role of long noncoding RNAs (LncRNAs) remains unclear. Here, we aimed to identify key differentially expressed LncRNAs and explore their regulatory roles in follicular development, particularly focusing on the MSTRG.4701.7-miR-1786-RORa axis, to provide insights for improving fertility in low-fertility chicken varieties.

Methods: Intact follicular tissues were collected from Jilin Black chickens (low-fertility group) and Lohmann Brown Laying hens (high-fertility group) at each stage of ovarian development for transcriptome sequencing to identify key differentially expressed LncRNAs for follow-up analyses. Bioinformatics analysis was also performed to determine the role of the MSTRG.4701.7-miR-1786-RORa regulatory axis, and to clarify whether that MSTRG 4701.7 targets miR-1786/RORa to competitively regulate the proliferation and differentiation of chicken follicular granulosa cells (GCs). We also knocked down and overexpressed key genes, MSTRG.4701.7 and miR-1786 and thereafter, determined changes in the mRNA and protein expression levels of axial terminal mRNA-RORa.

Results: MSTRG.4701.7 promotes the apoptosis of chicken follicular GCs, while miR-1786 reverses this phenomenon. RORa was also identified as a functional target of miR-1786 in GCs, and as a competitive endogenous RNA, MSTRG.4701.7 regulated RORa expression by sponging miR-1786, thereby playing a regulatory role in GCs. Additionally, interfering with MSTRG.4701.7 expression significantly downregulated RORa mRNA and protein expression levels, while interfering with miR-1786 showed the opposite effect.

Conclusion: MSTRG.4701.7 plays a critical role in follicular development in chickens by influencing GC proliferation, differentiation, and apoptosis. The resent results provide useful molecular evidence for elucidating the genetic mechanism underlying ovarian follicle development associated with egg production in chicken.

Keywords: apoptosis; chicken follicular granulosa cells; egg production; mRNA expression; proliferation.

Figure 1

Differential LncRNAs co-expressed in JB and LB at different stages of follicular development. (A) Number of genes with negative potential encoded by each LncRNA based on three databases (the PLEK, CNCI, and PFAM databases). (B,C) Differentially expressed LncRNAs during the LYF and SYF stages of follicular development. (D,E) Differentially expressed LncRNAs during LWF and LYF stages of follicular development. (F,G) Differentially expressed LncRNAs during LWF and SYF stages of follicular development. JB, Jilin Black Chicken; LB, Lohmann Brown laying hens; LWF, large white follicles; SYF, small yellow follicles; LYF, large yellow follicles.

Figure 2

MSTRG.4701.7 functions as a molecular sponge for miR-1786. (A) Predicted binding site of miRNA-1786 in MSTRG.4701.7. (B) Changes in MSTRG.4701.7 expression level after miR-1786overexpression or knockdown. (C) Wild-type and mutant plasmid constructs of MSTRG.4701.7. (D) Luciferase activities in HEK293T cells co-transfected with miR-1786 mimics or NC controls and reporter vectors containing MSTRG.4701.7 wild-type or mutant binding sites. All experiments were performed in triplicates. Data are expressed as mean ± standard deviation (SD) of three experiments; *p < 0.05, ** p < 0.01. PC stands for positive control.

Figure 3

Overexpression of MSTRG.4701.7 promotes apoptosis and inhibits GC proliferation. (A,B) mRNA expression levels of PCNA and CCND1. (C,D) mRNA expression levels of BCL-2 and Caspase3. (E,F) Overexpression of MSTRG.4701.7 with miR-1786 to measure GC apoptotic rate. All experiments were performed in triplicates and data are expressed as mean ± SD of triplicate experiments; *p < 0.05; **p < 0.01. GCs, granulosa cells.

Figure 4

RORa is a functional target of miR-1786 in follicular GCs. (A) Predicted binding of miR-1786 and RORa. (B) Prediction of miR-1786 and RORa binding site and vector construction. (C) Dual-luciferase activity assay for the targeting relationship between miR-1786 and RORa. (D) Changes in RORa mRNA expression level following miR-1786overexpression. (E) Changes in RORa protein expression level after miR-1786 overexpression. Data are expressed as mean ± SD of triplicate experiments; *p < 0.05; **p < 0.01. GCs, granulosa cells. PC stands for positive control.

Figure 5

MSTRG.4701.7 regulates RORa expression by acting as a ceRNA for miR-1786. (A) Changes in RORa mRNA expression level after MSTRG.4701.7 co-transfection with miR-1786 interference. (B) Changes in RORa protein expression level after MSTRG.4701.7 co-transfection with miR-1786 interference. (C) Changes in RORa mRNA expression level after MSTRG.4701.7 co-transfection with miR-1786 overexpression plasmid. (D) Changes in RORa protein expression level after MSTRG.4701.7 co-transfection with miR-1786 overexpression plasmid. Data are expressed as mean ± SD of triplicate experiments; *p < 0.05; **p < 0.01.

Figure 6

Interference with RORa can promote cell proliferation and inhibit cell apoptosis. (A) BCL2 mRNA expression level; (B) CCND1 mRNA expression level; (C) PCNA mRNA expression level; (D) STAR mRNA expression level; (E) Caspase3 mRNA expression level; (F) mRNA expression level of CYP11A1. The results were expressed as the mean ± standard deviation of the three experiments. *p < 0.05; **p < 0.01.

Figure 7

Overexpression of RORa can promote apoptosis and inhibit cell proliferation. (A) The expression level of BCL2 mRNA; (B) CCND1 mRNA expression level; (C) PCNA mRNA expression level; (D) STAR mRNA expression level; (E) Caspase3 mRNA expression level; (F) mRNA expression level of CYP11A1. The results were expressed as the mean ± standard deviation of the three experiments. *p < 0.05; **p < 0.01.