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. 2022 Jan 28;23(3):1509.
doi: 10.3390/ijms23031509.

circSLC41A1 Resists Porcine Granulosa Cell Apoptosis and Follicular Atresia by Promoting SRSF1 through miR-9820-5p Sponging

Huiming Wang  1 , Yi Zhang  1 , Jinbi Zhang  2 , Xing Du  1 , Qifa Li  1 , Zengxiang Pan  1
Affiliations

circSLC41A1 Resists Porcine Granulosa Cell Apoptosis and Follicular Atresia by Promoting SRSF1 through miR-9820-5p Sponging

Huiming Wang et al. Int J Mol Sci. .

Abstract

Ovarian granulosa cell (GC) apoptosis is the major cause of follicular atresia. Regulation of non-coding RNAs (ncRNAs) was proved to be involved in regulatory mechanisms of GC apoptosis. circRNAs have been recognized to play important roles in cellular activity. However, the regulatory network of circRNAs in follicular atresia has not been fully validated. In this study, we report a new circRNA, circSLC41A1, which has higher expression in healthy follicles compared to atretic follicles, and confirm its circular structure using RNase R treatment. The resistant function of circSLC41A1 during GC apoptosis was detected by si-RNA transfection and the competitive binding of miR-9820-5p by circSLC41A1 and SRSF1 was detected with a dual-luciferase reporter assay and co-transfection of their inhibitors or siRNA. Additionally, we predicted the protein-coding potential of circSLC41A1 and analyzed the structure of circSLC41A1-134aa. Our study revealed that circSLC41A1 enhanced SRSF1 expression through competitive binding of miR-9820-5p and demonstrated a circSLC41A1-miR-9820-5p-SRSF1 regulatory axis in follicular GC apoptosis. The study adds to knowledge of the post-transcriptional regulation of follicular atresia and provides insight into the protein-coding function of circRNA.

Keywords: SRSF1; ceRNA; circRNA; circSLC41A1; follicular atresia; granulosa cell apoptosis; miR-9820-5p; non-coding RNA.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Identification and validation of circSLC41A1. (A) Sketch of the structure of circSLC41A1, which is generated from the SLC41A1 gene exon 2 via back splicing. (B) SLC41A1 mRNA and circSLC41A1 expression with and without RNase R digestion. Untreated GAPDH was used as an internal control. (C) Differential expression of circSLC41A1 in HFs and AFs detected by qRT-PCR (n = 8). (D) The cytoplasmic localization of SLC41A1 in GCs detected by FISH. SLC41A1 was labeled with red fluorescence, and the nuclei were stained by DAPI (blue). Scale bar: 20 μm. Data are expressed as the mean ± SEM, * p < 0.05, ** p < 0.01.
Figure 2
Figure 2
circSLC41A1 inhibits GCs apoptosis. (A) Expression of circSLC41A1 and its corresponding linear mRNA in GCs treated with siRNA control (NC) or si-circSLC41A1 RNA detected by qRT-PCR. (B) The apoptosis rate of GCs after circSLC41A1 knockdown examined by Annexin V-FITC/PI staining assay using flow cytometry at the top of panel B. (C) Protein levels of cleaved CASP3 after circSLC41A1 knockdown were analyzed by western blot. Data are expressed as the mean ± SEM of three experiments; * p < 0.05, *** p < 0.001.
Figure 3
Figure 3
circSLC41A1 is a possible sponge for miR-9820-5p. (A) Differentially expressed miR-9820-5p in HFs and AFs detected by qRT-PCR. (B) Negative correlation between circSLC41A1 and miR-9820-5p expression levels in individual follicles (n = 9). (C,D) Shift of GC apoptosis rates after miR-9820-5p mimics and inhibitor transfection detected by flow cytometry. (E,F) The binding of miR-9820-5p of circSLC41A1 was verified by dual-luciferase activity analysis. (G) The subcellular co-localization of circSLC41A1 (labeled by red fluorescence) and miR-9820-5p (labeled by green fluorescence) in GCs verified by FISH. The nuclei were stained by DAPI (blue). Scale bar: 20 μm. Data are expressed as the mean ± SEM of three experiments; * p < 0.05.
Figure 4
Figure 4
SRSF1 resists GC apoptosis and follicular atresia. (A) Venn diagram of the DEGs (differentially expressed genes) between HFs and AFs and miR-9820-5p target genes predicted by PITA, Miranda, and TargetSpy. (B) Differentially expressed SRSF1 in HFs and AFs detected by qRT-PCR. (C,D) Effective knockdown of SRSF1 by si-SRSF1 qRT-PCR and western blotting (WB). (E) The shift of GC apoptosis rates after SRSF1 knockdown was detected by flow cytometry. Data are expressed as the mean ± SEM of three experiments; * p < 0.05, ** p < 0.01.
Figure 5
Figure 5
miR-9820-5p promotes pGCs apoptosis by targeting SRSF1. (A,B) The binding of miR-9820-5p and SRSF1-WT was predicted by RNAhybrid and verified by dual-luciferase activity analysis. (C,D) SRSF1 mRNA and protein levels after transfection of miR-9820-5p mimics. (E,F) SRSF1 mRNA and protein levels after transfection of miR-9820-5p inhibitors. (G) The shift of GC apoptosis rates after co-transfection of miR-9820-5p inhibitors and si-SRSF1 detected by flow cytometry. Data are expressed as the mean ± SEM of three experiments; * p < 0.05, ** p < 0.01.
Figure 6
Figure 6
circSLC41A1 inhibits GCs apoptosis by upregulating SRSF1 through sponging miR-9820-5p. (A) The shift of SRSF1 mRNA levels after co-transfection of si-circSLC41A1 and miR-9820-5p inhibitors detected by qRT-PCR. (B) The shift of SRSF1 protein levels after co-transfection of the si-circSLC41A1 and miR-9820-5p inhibitors detected by WB. (C) Change of GC apoptosis rates after co-transfection of si-circSLC41A1 and miR-9820-5p inhibitors detected by flow cytometry. Data are expressed as the mean ± SEM of three experiments; * p < 0.05, ** p < 0.01.
Figure 7
Figure 7
Evaluation of the coding ability of circSLC41A1. (A) Upper panel: the putative open reading frame (ORF) in circSLC41A1. Note that the circSLC41A1 junction is inside the ORF1. Lower panel: the sequences of the putative ORF are shown in green, internal ribosomal entrance site (IRES) sequences are shown in orange, the potential m6A site is shown in red, and specific amino acid sequences of ORF1 are shown in purple. (B) Hydrophobicity analysis of circSLC41A1-134aa. The ordinate score greater than 0 indicates hydrophobicity and less than 0 indicates hydrophilicity. (C) The transmembrane domain prediction of circSLC41A1-134aa. The transmembrane domain is shown in red, the intramembranous region is shown in blue, and the extramembranous region is shown in fuchsia. (D) The tertiary structure prediction of circSLC41A1-134aa. α-helix, β-sheet, and a random coil are shown in red, blue, and yellow, respectively. (E) The ligand prediction of 134aa. The green segments represent ligands and red segments represent the binding domains.
Figure 8
Figure 8
Schematic diagram of circSLC41A1 functions in GC apoptosis. circSLC41A1 can function by competing with SRSF1 mRNA for miR-9820-5p interactions, thereby alleviating the post-transcriptional repression of SRSF1 and thus resisting GC apoptosis and follicular atresia. circSLC41A1 also had the potential to encode small circSLC41A1-134aa peptides.
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