Scientia Agricultura Sinica ›› 2024, Vol. 57 ›› Issue (5): 1000-1009.doi: 10.3864/j.issn.0578-1752.2024.05.014

• ANIMAL SCIENCE·VETERINARY SCIENCE • Previous Articles     Next Articles

miR-34a Induces Early Apoptosis of Porcine Ovarian Granulosa Cells by Activating lncRNA NORHA Transcription

WANG SiQi(), ZHOU ChunXue, LI YuQi, DU Xing, PAN ZengXiang, LI QiFa()   

  1. College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095
  • Received:2023-05-22 Accepted:2024-01-27 Online:2024-03-01 Published:2024-03-06
  • Contact: LI QiFa

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Abstract:

【Objective】 miR-34a has been shown to be a pro-apoptotic factor in porcine ovarian granulosa cells (GCs) in our previous study. The aim of this study was to explore the RNA activation mechanism by which miR-34a induced apoptosis in the nucleus, so as to provide a basis for uncovering the molecular mechanism of porcine follicular atresia, and to screen small nucleic acid regulator that regulated procine reproduction. 【Method】 Bioinformatics methods were used to analyze the characteristics of the porcine miR-34a, the binding sites of miR-34a to the promoter region of target lncRNA NORHA, and the binding capacity. The subcellular localization of miR-34a was analyzed in porcine GCs by using nucleocytoplasmic fractionation. The effects of miR-34a on the expression of NORHA, apoptosis marker genes BCL-2 and BAX in porcine GCs were determined by qPCR. Luciferase assay was used to verify the regulatory relationship of miR-34a on the transcriptional activity of the NORHA promoter. Chromatin immunoprecipitation (ChIP) was utilized to detect the enrichment of AGO2 and histone modifiers at the miRNA response element (MRE) in the NORHA promoter in porcine GCs by miR-34a. The regulatory effect of miR-34a on downstream factor FOXO1 expression and GC apoptosis via NORHA were analyzed by western blot and flow cytometry. 【Result】 The porcine miR-34a was an intergenic miRNA, and its sequence was highly conserved among mammals. Subcellular localization analysis showed that miR-34a was distributed in both nucleus and cytoplasm in porcine GCs. Bioinformatic analysis revealed that the MRE of miR-34a was located at -194 nt - -173 nt in the NORHA promoter, and the binding free energy of the two compounds was -23.9 kcal·mol-1. qPCR showed that miR-34a significantly upregulated NORHA expression in porcine GCs. Luciferase assay revealed that miR-34a significantly upregulated the activity of the reporter vector with NORHA promoter, whereas it had no significant effect on the activity of promoter with the mutated MRE of miR-34a. ChIP assay showed that miR-34a increased the enrichment of AGO2, a core member of RNA induced transcriptional activation (RITA) complex, and H3K4me3, a transcription activated histone modifier, while or decreased the enrichment of H3K9me3, a transcription repressed histone modifier at MRE motif of miR-34a in the NORHA promoter. Co-transfection assay showed that knockdown of NORHA significantly reversed the upregulation of FOXO1 protein level and early apoptosis rate caused by miR-34a overexpression, as well as the downregulation of the BCL-2/BAX ratio. 【Conclusion】 Nuclear miR-34a was an endogenous small activating RNA (saRNA) in porcine GCs, and might be involved in the initiation of porcine follicular atresia by inducing early apoptosis in porcine GCs through target activating the transcription of the pro-apoptotic lncRNA NORHA.

Key words: porcine GC apoptosis, miR-34a, saRNA, NORHA

Table 1

Information of oligonucleotides"

寡核苷酸
Oligonucleotides		 寡核苷酸序列
Oligonucleotide sequence (5′ to 3′)
mimics NC UUGUACUACACAAAAGUACUG
miR-34a mimics UGGCAGUGUCUUAGCUGGUUGU
NC-siRNA UUCUCCGAACGUGUCACGUTT ACGUGACACGUUCGGAGAATT
NORHA-siRNA GGAAAUAGCGAGUUGGGUUTT
AACCCAACUCGCUAUUUCCTT

Table 2

Information of primers"

名称
Name		 序列
Primer sequence (5′ to 3′)		 退火温度
Annealing temp (℃)		 产物长度
Product size (bp)
miR-34a F: CGGGCTGGCAGTGTCTTAGC 60 56
R: CAGCCACAAAAGAGCACAAT
U6 F: GCTTCGGCAGCACATATACT 60 86
R: TTCACGAATTTGCGTGTCAT
NORHA F: GCTGAACCTGGGGAGTGTCTA 60 176
R: TCTCTGTCCTTGTGGTGTCTGC
GAPDH F: GGACTCATGACCACGGTCCAT 60 163
R: TCAGATCCACAACCGACACGT
BAX F: CCGAAATGTTTGCTGACG 60 122
R: AGCCGATCTCGAAGGAAGT
BCL-2 F: TTCTTTGAGTTCGGTGGGG 60 195
R: CCAGGAGAAATCAAATAGAGGC
MRE-ChIP F: CTTCCTGCTCCTGCTGTC 54 130
R: GCCGTCGTCTGAAAGGAC
miR-34a茎环
miR-34a stem-loop		 CCTGTTGTCTCCAGCCACAAAAGAGCAC
AATATTTCAGGAGACAACAGGACAACCA

Fig. 1

Characteristics of pig miR-34a and its subcellular localization in GCs A. Genomic location of pig miR-34a. B. Sequence alignment of miR-34a in mammals. Red bases as seed sequences. C. Subcellular localization analysis of miR-34a in porcine GCs"

Fig. 2

miR-34a promotes NORHA transcription in porcine GCs A-B. The binding site (A) and MFE (B) between miR-34a and NORHA. C-D. The expression levels of miR-34a (C) and NORHA (D) in porcine GCs after transfection of miR-34a mimics. * P<0.05"

Fig. 3

miR-34a promotes the transcriptional activity of NORHA promoter A. Diagram of reporter vectors of NORHA promoter with wild type and muted MRE. pGL3-wt, wild type reporter vector. pGL3-mut, mutated reporter vector. B-C. Effects of miR-34a on the activity of NORHA promoter with wild type (B) and mutated (C) MRE. ** P<0.01. ns, P>0.05"

Fig. 4

miR-34a influences the enrichment of AGO2 and histone modification at MRE motif of the NORHA promoter in porcine GCs A. Schematic showed the location of primers for ChIP-PCR. F/R was the upstream and downstream primer. B. After transfection with miR-34a mimics for 48 h, ChIP assay was performed by using antibodies specific for AGO2, H3K4me3 and H3K9me3"

Fig. 5

miR-34a promotes FOXO1 expression in porcine GCs via NORHA (A) The effect of miR-34a mimics on FOXO1 protein levels in porcine GCs. (B) After miR-34a mimics and NORHA siRNA were co-transfected into porcine GCs for 48 h, FOXO1 protein levels were measured by western blot. * P<0.05, ** P<0.01"

Fig. 6

miR-34a induces apoptosis of porcine GCs via NORHA A. After co-transfection with both miR-34a mimics and NORHA-siRNA into GCs for 48 h, the apoptosis rate was determined by flow cytometry. B-D. After co-transfection with both miR-34a mimics and NORHA-siRNA into GCs for 24 h, the levels of BAX (B) and BCL-2 (C) were quantified, and BCL2/BAX ratio (D) were calculated. *, P<0.05; **, P<0.01; ***, P<0.001; ns, no significant"

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