MiR-134-3p targets HMOX1 to inhibit ferroptosis in granulosa cells of sheep follicles

Abstract

Background: The intricate interplay of gene expression within ovarian granulosa cells (GCs) is not fully understood. This study aimed to investigate the miRNA regulatory mechanisms of ferroptosis during the process of follicle development in lamb GCs.

Methods: Employing transcriptome sequencing, we compared differentially expressed mRNAs (DE-mRNAs) and miRNAs (DE-miRNAs) in GCs from lambs treated with follicle-stimulating hormone (FL) to untreated controls (CL). We further screened differentially expressed ferroptosis-related genes and identified potential miRNA regulatory factors. The expression patterns of HMOX1 and miRNAs in GCs were validated using qRT‒PCR and Western blotting. Additionally, we investigated the regulatory effect of oar-miR-134-3p on HMOX1 and its function in ferroptosis through cell transfection and erastin treatment.

Results: We identified a total of 4,184 DE-mRNAs and 304 DE-miRNAs. The DE-mRNAs were mainly enriched in ferroptosis, insulin resistance, and the cell cycle. Specifically, we focused on the differential expression of ferroptosis-related genes. Notably, the ferroptosis-related genes HMOX1 and SLC3A2, modulated by DE-miRNAs, were markedly suppressed in FLs. Experimental validation revealed that HMOX1 was significantly downregulated in FL and large follicles, while oar-miR-134-3p was significantly upregulated compared to that in the CLs. HMOX1 expression was regulated by the targeting effect of oar-miR-134-3p. Functional assays further revealed that modulation of oar-miR-134-3p influenced HMOX1 expression and altered cellular responses to ferroptosis induction by erastin.

Conclusion: This study suggested that oar-miR-134-3p and HMOX1 may be one of the pathways regulating ferroptosis in GCs. This finding provides new clues to understanding the development and regulatory process of follicles.

Keywords: Ferroptosis; Granulosa cells; Oar-miR-134-3p; Sheep.

Fig. 1

Graphical abstract of this study

Fig. 2

Identification of DE-mRNAs and enrichment analysis in GCs. A Volcano map of differentially expressed mRNAs in GCs between CLs and FLs. B Heatmap of differentially expressed mRNAs in GCs between CLs and FLs. C GO functions of differentially expressed mRNAs in enrichment results. BP, biological processes; CC, cellular components; MF, molecular function. D KEGG pathways of differentially expressed mRNAs in the enrichment results

Fig. 3

Construction of the miRNA-regulated network for ferroptosis-related genes. A Volcano map of differentially expressed miRNAs in GCs between CLs and FLs. B Heatmap of differentially expressed miRNAs in GCs between CLs and FLs. C Differentially expressed ferroptosis-related genes in GCs between CLs and FLs. D Regulatory network of DE-miRNAs to differentially expressed ferroptosis-related genes. E The expression of miRNAs that regulate ferroptosis-related genes in GCs between CLs and FLs. * p < 0.05, ** p < 0.01, *** p < 0.001

Fig. 4

Analysis of ferroptosis-related miRNAs and mRNAs in the regulatory network. A The mRNA expression of genes in ferroptosis-related regulatory networks detected by RT‒qPCR in CL and FL. B Relative expression of HMOX1 detected by Western blot in CLs and FLs. Original blots are presented in Supplementary Fig. 2A. C The mRNA expression of HMOX1 and oar-miR-134-3p detected by RT‒qPCR in GCs from large follicles and small follicles. D Relative expression of HMOX1 detected by Western blot in GCs from large follicles and small follicles. Original blots are presented in Supplementary Fig. 2B. *P < 0.05, **P < 0.01. ns, not significant. CLs, control lambs; FLs, lambs treated with FSH

Fig. 5

oar-miR-134-3p regulated HMOX1 expression. A The mRNA expression of oar-miR-134-3p determined by RT‒qPCR in GCs transfected with oar-miR-134-3p mimics, inhibitor, and NC. B The mRNA expression of HMOX1 determined by RT‒qPCR in GCs transfected with oar-miR-134-3p mimics, inhibitor, and NC. C Relative expression of HMOX1 detected by Western blot in GCs transfected with oar-miR-134-3p mimics, inhibitor, and NC. Original blots are presented in Supplementary Fig. 3. D Luciferase activities in cells transfected with a luciferase plasmid containing the wild-type (WT) or mutated (MUT) HMOX1 3′ UTR and either the oar-miR-134-3p mimic or a negative control (NC). *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 6

oar-miR-134-3p promotes cell proliferation by inhibiting ferroptosis. A The mRNA expression of HMOX1 determined by RT‒qPCR in GCs subjected to different treatments. B Relative expression of HMOX1 determined by Western blots in different treatments on GCs. Original blots are presented in Supplementary Fig. 4. C MDA levels in different treatments on GCs. D GSH levels in different treatments on GCs. E Cell proliferation measured with a CCK-8 kit in different treatment groups of GCs. **P < 0.01, ***P < 0.001. GC, granulosa cell; mimics, oar-miR-134-3p mimics; inhibitor, oar-miR-134-3p inhibitor

Fig. 7

ROS and mitochondrial membrane potential regulated by ferroptosis and oar-miR-134-3p in GCs. A ROS production in different treatment groups of GCs stained with DHE. B Fluorescence image and ratio of red/green fluorescence in different treatment groups of GCs stained with JC-1. ***P < 0.001