Cui Zhifu, Liu Lingbin, Kwame Amevor Felix, Zhu Qing, Wang Yan, Li Diyan, Shu Gang, Tian Yaofu, Zhao Xiaoling
Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu, China.
College of Animal Science and Technology, Southwest University, Chongqing, China.
Front Cell Dev Biol. 2020 Nov 5;8:580072. doi: 10.3389/fcell.2020.580072. eCollection 2020.
Chicken atrophic ovaries have decreased volume and are indicative of ovarian failure, presence of a tumor, or interrupted ovarian blood supply. Ovarian tumor is accompanied by an increase in follicular atresia, granulosa cell (GC) apoptosis, and autophagy. In a previous study, we found using high throughput sequencing that miR-204 is highly expressed in chicken atrophic ovaries. Thus, in the present study, we further investigated its function in GC apoptosis and autophagy. We found that overexpression of miR-204 reduced mRNA and protein levels of proliferation-related genes and increased apoptosis-related genes. Cell counting kit-8 (CCK-8), 5-ethynyl-2-deoxyuridine (EdU), and flow cytometry assays revealed that miR-204 inhibited GC proliferation and promoted apoptosis. Furthermore, we confirmed with reporter gene assays that () was directly targeted by miR-204. FOXK2, as a downstream regulator of phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signal pathways, promoted GC proliferation and inhibited apoptosis. Subsequently, we observed that miR-204 was involved in GC autophagy by targeting (). The luciferase activities of the two binding sites of TRPM3 were decreased in response to treatment with a miR-204 mimic, and the autophagic flux was increased after miR-204 inhibition. However, overexpression of miR-204 had opposite results in autophagosomes and autolysosomes. miR-204 inhibits GC autophagy by suppressing the protein expression of TRPM3/AMP-activated protein kinase (AMPK)/ULK signaling pathway components. Inhibition of miR-204 enhanced autophagy by accumulating and degrading the protein levels of LC3-II (Microtubule Associated Protein Light Chain 3B) and p62 (Protein of 62 kDa), respectively, whereas miR-204 overexpression was associated with contrary results. Immunofluorescence staining showed that there was a significant reduction in the fluorescent intensity of LC3B, whereas p62 protein was increased after TRPM3 silencing. Collectively, our results indicate that miR-204 is highly expressed in chicken atrophic ovaries, which promotes GC apoptosis repressing FOXK2 through the PI3K/AKT/mTOR pathway and inhibits autophagy by impeding the TRPM3/AMPK/ULK pathway.
鸡萎缩性卵巢体积减小，提示卵巢功能衰竭、存在肿瘤或卵巢血液供应中断。卵巢肿瘤伴随着卵泡闭锁增加、颗粒细胞（GC）凋亡和自噬。在先前的一项研究中，我们通过高通量测序发现miR-204在鸡萎缩性卵巢中高表达。因此，在本研究中，我们进一步研究了其在GC凋亡和自噬中的功能。我们发现miR-204的过表达降低了增殖相关基因的mRNA和蛋白质水平，并增加了凋亡相关基因的表达。细胞计数试剂盒-8（CCK-8）、5-乙炔基-2'-脱氧尿苷（EdU）和流式细胞术分析表明，miR-204抑制GC增殖并促进凋亡。此外，我们通过报告基因分析证实（）是miR-204的直接靶点。FOXK2作为磷脂酰肌醇3-激酶（PI3K）/蛋白激酶B（AKT）/雷帕霉素哺乳动物靶蛋白（mTOR）信号通路的下游调节因子，促进GC增殖并抑制凋亡。随后，我们观察到miR-204通过靶向（）参与GC自噬。用miR-204模拟物处理后，瞬时受体电位阳离子通道亚家族M成员3（TRPM3）两个结合位点的荧光素酶活性降低，miR-204抑制后自噬通量增加。然而，miR-204的过表达在自噬体和自溶酶体中产生相反的结果。miR-204通过抑制TRPM3/腺苷酸活化蛋白激酶（AMPK）/unc-51样激酶1（ULK）信号通路的蛋白表达来抑制GC自噬。抑制miR-204分别通过积累和降解微管相关蛋白轻链3B（LC3-II）和62 kDa蛋白（p62）的蛋白水平来增强自噬，而miR-204过表达则产生相反的结果。免疫荧光染色显示，TRPM3沉默后，LC3B的荧光强度显著降低，而p62蛋白增加。总的来说，我们的结果表明，miR-204在鸡萎缩性卵巢中高表达，通过PI3K/AKT/mTOR途径抑制FOXK2来促进GC凋亡，并通过阻碍TRPM3/AMPK/ULK途径抑制自噬。
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