Wang Wenwen, Teng Jun, Han Xu, Zhang Shen, Zhang Qin, Tang Hui
Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Shandong Agricultural University, Taian, Shandong 271018, China.
Anim Biosci. 2021 Jun;34(6):957-966. doi: 10.5713/ajas.20.0392. Epub 2020 Oct 13.
Ovarian follicular development, which dependent on the proliferation and differentiation of granulosa cells (GCs), is a complex biological process in which miRNA plays an important role. Our previous study showed that miR-458b-5p is associated with ovarian follicular development in chicken. The detailed function and molecular mechanism of miR-458b-5p in GCs is unclear.
The luciferase reporter assay was used to verify the targeting relationship between miR-458b-5p and catenin beta-1 (CTNNB1), which is an important transcriptional regulatory factor of the Wnt/β-catenin pathway. The cell counting kit-8 (CCK-8) assay, flow cytometry with propidium iodide (PI) and annexin V-fluorescein isothiocyanate (FITC) labeling were applied to explore the effect of miR-458b-5p on proliferation, cell cycle and apoptosis of chicken GCs. Quantitative real-time polymerase chain reaction and Western blot were used to detect the mRNA and protein expression levels.
We demonstrated that the expression of miR-458b-5p and CTNNB1 showed the opposite relationship in GCs and theca cells of hierarchical follicles. The luciferase reporter assay confirmed that CTNNB1 is the direct target of miR-458b-5p. Using CCK-8 assay and flow cytometry with PI and Annexin V-FITC labeling, we observed that transfection with the miR-458b-5p mimics significantly reduced proliferation and has no effects on apoptosis of chicken GCs. In addition, miR-458b-5p decreased the mRNA and protein expression of CD44 molecule and matrix metallopeptidase 7, which are the downstream effectors of CTNNB1 in Wnt/β-Catenin pathway and play functional roles in cell proliferation.
Taken together, the data indicate that miR-458b-5p regulates ovarian GCs proliferation through Wnt/β-catenin signaling pathway by targeting CTNNB1, suggesting that miR-458b-5p and its target gene CTNNB1 may potentially play a role in chicken ovarian follicular development.
卵巢卵泡发育是一个复杂的生物学过程，依赖于颗粒细胞（GCs）的增殖和分化，其中miRNA发挥着重要作用。我们之前的研究表明，miR-458b-5p与鸡的卵巢卵泡发育有关。miR-458b-5p在GCs中的具体功能和分子机制尚不清楚。
采用荧光素酶报告基因检测法验证miR-458b-5p与连环蛋白β-1（CTNNB1）之间的靶向关系，CTNNB1是Wnt/β-连环蛋白通路的重要转录调节因子。应用细胞计数试剂盒-8（CCK-8）检测法、碘化丙啶（PI）和膜联蛋白V-异硫氰酸荧光素（FITC）标记的流式细胞术，探讨miR-458b-5p对鸡GCs增殖、细胞周期和凋亡的影响。采用定量实时聚合酶链反应和蛋白质免疫印迹法检测mRNA和蛋白质表达水平。
我们证明，在分级卵泡的GCs和卵泡膜细胞中，miR-458b-5p和CTNNB1的表达呈相反关系。荧光素酶报告基因检测证实CTNNB1是miR-458b-5p的直接靶点。使用CCK-8检测法以及PI和膜联蛋白V-FITC标记的流式细胞术，我们观察到转染miR-458b-5p模拟物显著降低了鸡GCs的增殖，且对其凋亡无影响。此外，miR-458b-5p降低了CD44分子和基质金属肽酶7的mRNA和蛋白质表达，它们是Wnt/β-连环蛋白通路中CTNNB1的下游效应分子，在细胞增殖中发挥功能作用。
综上所述，数据表明miR-458b-5p通过靶向CTNNB1调控Wnt/β-连环蛋白信号通路，从而调节卵巢GCs的增殖，提示miR-458b-5p及其靶基因CTNNB1可能在鸡卵巢卵泡发育中发挥作用。
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