A Mutation in Endogenous saRNA miR-23a Influences Granulosa Cells Response to Oxidative Stress

Abstract

Phenotypes are the result of the interaction between the gene and the environment, so the response of individuals with different genotypes to an environment is variable. Here, we reported that a mutation in miR-23a influences granulosa cells (GCs) response to oxidative stress, a common mechanism of environmental factors affecting female reproduction. We showed that nuclear miR-23a is a pro-apoptotic miRNA in porcine GCs through the activation of the transcription and function of NORHA, a long non-coding RNA (lncRNA) induces GC apoptosis and responses to oxidative stress. Mechanistically, miR-23a acts as an endogenous small activating RNA (saRNA) to alter histone modifications of the NORHA promoter through the direct binding to its core promoter. A C > T mutation was identified at −398 nt of the miR-23a core promoter, which created a novel binding site for the transcription factor SMAD4 and recruited the transcription repressor SMAD4 to inhibit miR-23a transcription and function in GCs. Notably, g.−398C > T mutation in the miR-23a promoter reduced GCs response to oxidative stress. In addition, g.−398C > T mutation was significantly associated with sow fertility traits. In short, our findings preliminarily revealed the genetic basis of individual differences in the response to oxidative stress from the perspective of a single mutation and identified miR-23a as a candidate gene for the environmental adaptation to oxidative stress.

Keywords: GC apoptosis; NORHA-FoxO1 pathway; miR-23a; mutation; oxidative stress; saRNA.

Figure 1

miR-23a induces pGC apoptosis. (A) Multiple alignments of the mature sequences of miR-23a in mammals. The bases marked in red are the seed sequence. (B) The prediction of potential targets for miR-23a was performed using TarBase (blue), TargtScan (red), and miRDB (green). (C,D) After transfection with miR-23a mimics, miR-23a levels were examined by qRT-PCR at 24 h (C), and the apoptosis rate was examined by fluorescence-activated cell sorting (FACS) at 48 h (D) in pGCs. (E,F) After transfection with the miR-23a inhibitor, miR-23a levels were examined by qRT-PCR at 24 h (E), and the apoptosis rate was examined by FACS at 48 h (F) in pGCs. Data are expressed as the mean ± S.E.M., and the significance was tested using a t-test. (* p < 0.05, *** p < 0.001).

Figure 1

miR-23a induces pGC apoptosis. (A) Multiple alignments of the mature sequences of miR-23a in mammals. The bases marked in red are the seed sequence. (B) The prediction of potential targets for miR-23a was performed using TarBase (blue), TargtScan (red), and miRDB (green). (C,D) After transfection with miR-23a mimics, miR-23a levels were examined by qRT-PCR at 24 h (C), and the apoptosis rate was examined by fluorescence-activated cell sorting (FACS) at 48 h (D) in pGCs. (E,F) After transfection with the miR-23a inhibitor, miR-23a levels were examined by qRT-PCR at 24 h (E), and the apoptosis rate was examined by FACS at 48 h (F) in pGCs. Data are expressed as the mean ± S.E.M., and the significance was tested using a t-test. (* p < 0.05, *** p < 0.001).

Figure 2

miR-23a induces pGC apoptosis by activating NORHA expression. (A) Subcellular localization analysis of miR-23a by nucleocytoplasmic separation. U6 is a reference for the nucleus (blue) and GAPDH is a reference for the cytoplasm (red). (B,C) Potential binding of miR-23a to the lncRNA NORHA promoter. (B) An MRE of miR-23a was located in the NORHA promoter. (C) The minimum free energy (MFE) was predicted by RNAhybrid. (D,E) After transfection with miR-23a mimics (D) or the miR-23a inhibitor (E), NORHA levels were examined by qRT-PCR at 24 h and 48 h in pGCs. (F) After co-transfection with the miR-23a inhibitor and pcDNA3.1-NORHA, the pGC apoptosis rate was examined by FACS at 48 h. Data are expressed as the mean ± S.E.M., and significance was tested using a t-test (D,E) and a one-way ANOVA (F) (ns, not significant, * p < 0.05, ** p < 0.01).

Figure 2

miR-23a induces pGC apoptosis by activating NORHA expression. (A) Subcellular localization analysis of miR-23a by nucleocytoplasmic separation. U6 is a reference for the nucleus (blue) and GAPDH is a reference for the cytoplasm (red). (B,C) Potential binding of miR-23a to the lncRNA NORHA promoter. (B) An MRE of miR-23a was located in the NORHA promoter. (C) The minimum free energy (MFE) was predicted by RNAhybrid. (D,E) After transfection with miR-23a mimics (D) or the miR-23a inhibitor (E), NORHA levels were examined by qRT-PCR at 24 h and 48 h in pGCs. (F) After co-transfection with the miR-23a inhibitor and pcDNA3.1-NORHA, the pGC apoptosis rate was examined by FACS at 48 h. Data are expressed as the mean ± S.E.M., and significance was tested using a t-test (D,E) and a one-way ANOVA (F) (ns, not significant, * p < 0.05, ** p < 0.01).

Figure 3

miR-23a enhances NORHA promoter activity by altering histone modification of the MRE motif. (A) Vector diagram of the wild type (wt) and mutated type (mt) of the MRE motif in the NORHA promoter. The red base is the mutation site. TSS, transcription start site. (B,C) Luciferase activity was detected in pGCs after being co-transfected with miR-23a mimics and a reporter vector containing the MRE-wt (B) or the MRE-mut (C). (D) ChIP assays were performed using an AGO2-specific antibody. The image contains a schematic diagram of the porcine NORHA gene structure (top) and ChIP images (bottom). F1/R1 and F2/R2 are the primers used to amplify the MRE site and X site, respectively. The X site is an intronic region of NORHA that does not contain the MRE of miR-23a. M, DNA marker. (E) ChIP assays were performed using H3K9me2- and H3K9me3-specific antibodies. Data are expressed as the mean ± S.E.M., and significance was tested using a t-test (ns, not significant, ** p < 0.01).

Figure 4

The SNV g.−398C > T affects miR-23a promoter activity by recruiting the transcription factor SMAD4. (A) A point variant C > T was detected at −398 nt in the miR-23a promoter. (B) Identification of the core promoter region of miR-23a. The image contains a schematic representation of the four deletion constructions (left) and their luciferase activity (right). The first base of pre-miR-23a was taken as +1. (C) Schematic showing the constructions of pGL3-C and pGL3-T. (D) Reporter activity assays. pGL3-C and pGL3-T were transfected into pGCs; luciferase activity was detected. (E) miR-23a levels were determined in pGCs with different genotypes for the SNV g.−398C > T. GCs were isolated from the ovaries of sows with genotype CC (n = 3) and genotype TT (n = 3). (F) Prediction of binding sites for transcription factors in the miR-23a promoter with allele C and allele T by JASPAR. (G) Effects of transcription factors SMAD4 and NR2C1 on the activity of the miR-23a promoter with allele C and allele T. (H) ChIP assay. The image contains a schematic diagram of the porcine miR-23a (top) and ChIP images (bottom). F/R are the primers used to amplify a DNA fragment containing the SMAD4 binding element (SBE) motif. Data are expressed as the mean ± S.E.M., and significance was tested using a t-test (B,E) and a one-way ANOVA (D,G) (ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 5

SMAD4 inhibits the expression and function of miR-23a. (A) After transfection with pcDNA3.1-SMAD4, miR-23a levels were detected in pGCs with genotypes CC and TT for the SNV g.−398C > T. (B,C) NORHA levels (B) and the apoptosis rate (C) in pGCs after co-transfection with pcDNA3.1-SMAD4 and miR-23a mimics at 48 h. Data are expressed as the mean ± S.E.M., and significance was tested using a t-test (A) and a one-way ANOVA (B,C) (ns, not significant, * p < 0.05, ** p < 0.01).

Figure 5

SMAD4 inhibits the expression and function of miR-23a. (A) After transfection with pcDNA3.1-SMAD4, miR-23a levels were detected in pGCs with genotypes CC and TT for the SNV g.−398C > T. (B,C) NORHA levels (B) and the apoptosis rate (C) in pGCs after co-transfection with pcDNA3.1-SMAD4 and miR-23a mimics at 48 h. Data are expressed as the mean ± S.E.M., and significance was tested using a t-test (A) and a one-way ANOVA (B,C) (ns, not significant, * p < 0.05, ** p < 0.01).

Figure 6

The miR-23a SNV g.−398C > T affects the response of pGCs to oxidative stress. (A) After transfection with miR-23a mimics, FoxO1 protein levels were detected by western blotting in pGCs. (B,C) NORHA and FoxO1 levels were determined in pGCs from the ovaries of sows with genotype CC (n = 3) and genotype TT (n = 3). (D) The apoptosis rate was detected by FACS in pGCs at 2 h after being treated with 200 μM H₂O₂. Data are expressed as the mean ± S.E.M., and significance was tested using a t-test. (* p < 0.05, ** p < 0.01).

Figure 7

The SNV g. −398C > T within the miR-23a core promoter is associated with sow feritility traits. (A) Peak plot of g.−398C > T. (B) Genotype frequency of the SNV g.−398C > T in a Large White sow population (n = 346). (C) Allele frequency of the SNV g.−398C > T in a Large White sow population (n = 346). (D) Association analysis between the SNV g.−398C > T and TNB of sows for multiparities. Data are expressed as the least-squares mean ± S.E.M., n = 346, and significance was tested using a GLM program. Significant differences between groups (p < 0.05) are indicated by different lowercase letters, and insignificant differences (p > 0.05) are indicated by the same letter.

Figure 8

Working model. The SNV g.−398C > T for sow fertility traits in the miR-23a promoter mediated the regulation of NORHA expression by affecting miR-23a expression, which in turn affected pGCs apoptosis and the pGCs response to oxidative stress.