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

摘要/Abstract

摘要：

旨在探究猪卵泡颗粒细胞中miR-370和miR-219a响应氧化应激的表达模式及调控机制，为研发内源性小分子调节物抑制氧化应激、促进卵泡发育提供理论依据。本研究以原代猪卵泡颗粒细胞为试验材料，利用150 μmol·L^-1 H₂O₂构建体外氧化应激模型。结合RNA-seq与RT-qPCR试验明确氧化应激对miR-370和miR-219a转录的调控作用。基于生物信息学分析和组织表达谱构建鉴定二者保守性和组织表达分布。利用FACS和ELISA试验鉴定二者对颗粒细胞氧化应激和凋亡的影响。构建猪KLF5表达载体，通过WB试验鉴定表达效率，进一步结合RT-qPCR、荧光素酶活性检测和ChIP等试验鉴定KLF5抑制氧化应激状态下颗粒细胞中miR-370和miR-219a转录的机制。结果表明，氧化应激抑制miR-370和miR-219a转录，导致二者初体、前体和成熟表达水平均极显著下调（P<0.01）。特征分析发现二者高度保守且在卵巢组织高表达。相关性分析发现二者表达水平与SOD活性呈极显著正相关（P<0.01），与MDA水平呈极显著负相关（P<0.01），提示二者具有抗氧化功能。过表达二者可有效缓解氧化应激引起的ROS累积、低SOD活性、高MDA水平和高凋亡率，且二者功能存在协同效应。预测发现二者启动子区存在转录因子KLF5结合位点，过表达KLF5极显著抑制二者转录和启动子活性（P<0.01），但对结合位点突变型报告载体的荧光素酶活性并无显著影响。进一步通过ChIP证实KLF5与二者启动子区直接结合，且氧化应激可提高其结合丰度。综上所述，氧化应激通过促进KLF5的表达与转录因子活性抑制具有抗氧化功能的miR-370和miR-219a转录，加剧颗粒细胞氧化损伤。

关键词: 猪, 卵泡颗粒细胞, 氧化应激, miRNA, KLF5

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

中图分类号:

S828.3

郭震楠, 吕世政, 萧宗贤, 吴启吉, 包雨加, 李清, 李齐发, 李琦琦, 杜星. 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[J]. 畜牧兽医学报, 2026, 57(1): 234-245.

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.

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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