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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.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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