The circular RNA aplacirc_13267 upregulates duck granulosa cell apoptosis by the apla‐miR‐1‐13/THBS1 signaling pathway

Abstract

Follicle development is a key factor that determines the reproductive performance of poultry. The existing evidence suggests that circular RNAs (circRNAs) play an important role in a variety of biological processes, especially in posttranscriptional regulation, but the regulatory mechanism of circRNAs in duck follicle development has rarely been reported. To better explore the molecular mechanism of follicle development in ducks, we sequenced and analyzed the follicular circRNAs; 4,204 circRNAs were predicted in the duck follicles. Fourteen circRNAs were differentially expressed between the white follicles and yellow follicles. The results of our studies showed that aplacirc_013267 promoted cell apoptosis in duck GCs. Moreover, a bioinformatics prediction analysis demonstrated that aplacirc_013267 was involved in a circRNA‐miRNA‐mRNA coexpression network and was observed to sponge two follicle‐related miRNAs by a luciferase activity assay. Furthermore, we found that overexpression of aplacirc_013267 significantly increased thrombospondin‐1 (THBS1) expression and downregulated granulosa cell apoptosis. The mechanistic study showed that aplacirc_013267 directly binds to and inhibits apla‐mir‐1‐13; then, aplacirc_013267 increases the expression of THBS1 and upregulates granulosa cell apoptosis. Taken together, our findings demonstrate that circRNAs have potential effects in duck ovarian follicles and that circRNAs may represent a new avenue to understand follicular development.

CircRNA Expression in Chicken Granulosa Cells Illuminated with Red Light

“…A total of 2,468 circRNAs were identi ed in GCs of SYFs, which is more than the number in liver of ALV-J-resistant chickens (1,800) [32] but less than the number in spleen of ALV-J-infected chickens (4,254) [33], possibly due to tissue-speci c factors. In total, 11,642 circRNAs of three types of GCs (SYF, F1, and F6) of chicken [23] and 4,204 circRNAs of two types of GCs (SYFs and large white follicles (LWFs)) were identi ed for duck follicles [22], both considerably more than identi ed herein, which indicates that the number of circRNAs may be closely correlated with the number of types of follicles. We found that the most common lengths for circRNAs were 200−300 nt and > 2,000 nt, and Shen et al (2019) similarly found that the majority of circRNAs were 200−300 nt, but lengths > 1400 nt were not mentioned [23].…”

“…2). Wu et al (2020) previously identi ed 14 DE circRNAs of GCs between LWFs and SYFs [22], while Shen et al (2019) identi ed 219 DE circRNAs of GCs between SYFs and F6 [23], indicating that the number of DE circRNAs is closely related to the contract group. Moreover, host genes of DE circRNAs were enriched in ovarian steroidogenesis, as well as MAPK signalling, prolactin signalling and PI3K-Akt signalling pathways.…”

“…Previous studies have shown that chr5:13061358|13064218 binds to miR-27b-3p and participates in follicular development of chicks [23], and aplacirc_013267 directly binds to and inhibits miR-1-13 in duck ovarian follicles [22]. In the present study, we found that circRNA_0320 and circRNA_0185 were differentially expressed between RL and WL groups, and a number of miRNAs were predicted to interact with these circRNAs, including miR-27b-3p, miR-143-3p and miR-142-3p ( Fig.…”

“…PCR products for cDNA and genomic DNA were analysed by agarose gel electrophoresis and backsplicing sites of circRNAs were con rmed by Sanger sequencing at Sango Biotech Co. Ltd. (Shanghai, China). Primer sequences were designed according to previous studies [22][23], and primers for circRNAs and mRNAs are listed in Additional le 6. RT-qPCR was performed using a LightCycler 480 II real-time PCR Instrument (Roche, Switzerland) using a 10 μL reaction mixture containing 1 μL of cDNA, 5 μL of 2× PerfectStart Green qPCR SuperMix, 0.2 μL of forward primer, 0.2 μL of reverse primer, and 3.6 μL of nuclease-free water.…”

“…Endogenous circRNA molecules can act as e cient miRNA sponges, adding to a growing repertoire of known regulatory functions in gene expression [19][20], and they also modulate pre-mRNA alternative splicing, and possess protein-coding capacity [21]. Wu et al (2020) found that host genes of circRNAs in GCs of duck preovulatory follicles are enriched in vital pathways related to oocyte development and cell proliferation [22]. Shen et al (2019) identi ed the key gene RalGPS2 which is produced in three isoforms of circRNAs in GCs of chicken follicles [23].…”