KLF5抑制氧化应激状态下猪卵泡颗粒细胞中miR-370与miR-219a的转录

doi:10.11843/j.issn.0366-6964.2026.01.020

Abstract

Abstract:

The aim of this study was to investigate the expression pattern and regulatory mechanism of miR-370 and miR-219a in sow follicular granulosa cells （GCs） under oxidative stress， and provide theoretical basis for the use of endogenous small molecule regulators to inhibit oxidative stress and promote follicular development. In this study， primary sow GCs were used as experimental materials and an in vitro oxidative stress GC model was established using 150 μmol·L^-1 H₂O₂. The regulatory effect of oxidative stress on the transcription of miR-370 and miR-219a was analyzed by RNA-seq and RT-qPCR. Their conservation and expression distribution were detected by bioinformatics analysis and tissue expression profiling. FACS and ELISA were performed to detect the effect of two miRNAs on the oxidative stress and apoptosis of sow GCs. The overexpression plasmid of KLF5 was constructed and its expression efficiency was verified by WB. Furthermore， RT-qPCR， luciferase activity analysis and ChIP were conducted to identify the mechanism by which KLF5 inhibits the transcription of two miRNAs in sow GCs under oxidative stress. The results showed that， oxidative stress inhibits the transcription of miR-370 and miR-219a， resulting in down-regulation of their pri-， pre- and mature expression levels （P<0.01）. Characterization analysis revealed that both miRNAs are conserved and highly expressed in ovary. Correlation analysis revealed that their expression levels were significantly positively correlated with SOD activity （P<0.01）， significantly negatively correlated with MDA level （P<0.01）， suggesting that they have anti-oxidative capacity. Overexpression of two miRNAs effectively and synergistically alleviated the oxidative stress-induced ROS accumulation， low SOD activity， high MDA levels， and high apoptosis rate. Prediction analysis found that their promoters contain potential KLF5 binding sites， and overexpression of KLF5 significantly inhibited their transcription and promoter activities （P<0.01）， while had no effect on the luciferase activities of the reporter vectors containing mutant KLF5 binding sites. ChIP results confirmed that KLF5 directly bound to their promoters， and its expression and binding enrichment were elevated by oxidative stress. In summary， oxidative stress inhibits the transcription of antioxidant miRNAs （miR-370 and miR-219a） by inducing the expression and transcription factor activity of KLF5， which exacerbates the oxidative damage of sow GCs.

Key words: sow, follicular granulosa cells, oxidative stress, miRNA, KLF5

CLC Number:

S828.3

GUO Zhennan, LÜ Shizheng, XIAO Zongxian, WU Qiji, BAO Yujia, LI Qing, LI Qifa, LI Qiqi, DU Xing. KLF5 Inhibits the Transcription of miR-370 and miR-219a in Sow Follicular Granulosa Cells under Oxidative Stress[J]. Acta Veterinaria et Zootechnica Sinica, 2026, 57(1): 234-245.

Figures/Tables 5

Table 1

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References 47

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引物 Primer	引物序列（5′→3′） Sequence	片段长度/bp Length	用途 Usage
miR-370 stem-loop	CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGACCAGGTT	—	成熟miRNAs反转录
miR-219a stem-loop	CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGCGGGACGT	—
miR-7136 stem-loop	CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGGCTCCACA	—
miR-9860 stem-loop	CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGGCGGTCCA	—
Mature miR-370-F	GCCGAGGCCTGCTGGGGTGG	64	成熟miRNAs 定量PCR
Mature miR-219a-F	GCCGAGAGAGTTGAGTCTGG	64
Mature miR-7136-F	GCCGAGTCTGGTCCAGACAC	64
Mature miR-9860-F	GCCGAGTTGCCCGAGAGCT	64
Common miRNA-R	CTCAACTGGTGTCGTGGA	—
Pri-miR-370-F	TCTCGAATCAGATTGGTGGC	145	初级miRNAs 定量PCR
Pri-miR-370-R	CAGGCACTCGTGAGCTGTGTA	145
Pri-miR-219a-F	TCAGACATTCGGCCCCTTGT	140
Pri-miR-219a-R	CTCGAGAATTGCGTTTGGAC	140
Pre-miR-370-F	AAGACAGAGAAGCCAGGTCAC	75	前体miRNAs 定量PCR
Pre-miR-370-R	GACAGACCAGGTTCCACCCC	75
Pre-miR-219a-F	CCGCGGCTCTTGATTGTC	76
Pre-miR-219a-R	GCTCGGGACGTCCAGACT	76
miR-370 promoter-F	CGGGGTACCCGGACGAGCTAATGTATGCTGA	314	荧光素酶报告载体构建
miR-370 promoter-R	CCGCTCGAGTCTGTGATATCGCCGTGTCTATA	314
miR-219a promoter-F	CGGGGTACCCCACGGACTGAAGGAGAGGA	320
miR-219a promoter-R	CCGCTCGAGCACAAGGGGCCGAATGTCT	320
miR-370 promoter-F1	AACGACCCTCCGCTGCTG	150	染色质免疫沉淀
miR-370 promoter-R1	CTCCCACCGCCACCCCTG	150
miR-219a promoter-F2	TCCCCGTCCCCTTCCTT	200
miR-219a promoter-R2	CATGCGGAAGCAGGGAC	200

KLF5 Inhibits the Transcription of miR-370 and miR-219a in Sow Follicular Granulosa Cells under Oxidative Stress

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