Zhou Yan, Liu Jie, Lei Qiuxia, Han Haixia, Liu Wei, Cunwei Tang, Li Fuwei, Cao Dingguo
Institute of Poultry Science, Academy of Agricultural Sciences of Shandong Province, Jinan, 250023, Shandong, China.
Poultry Breeding Engineering Technology Center of Shandong Province, Jinan, 250023, Shandong, China.
G3 (Bethesda). 2020 Nov 5;10(11):4071-4081. doi: 10.1534/g3.120.401701.
As a class of transcription regulators, numerous miRNAs have been verified to participate in regulating ovary follicular development in chickens (). Previously we showed that gga-miR-135a-5p has significant differential expression between high and low-yield chicken ovaries, and the abundance of gga-miR-135a-5p is significantly higher in follicular theca cells than in granulosa cells. However, the exact role of gga-miR-135a-5p in chicken follicular theca cells is unclear. In this study, primary chicken follicular theca cells were isolated and then transfected with gga-miR-135a-5p inhibitor. Transcriptome sequencing was performed in chicken follicular theca cells with or without transfection. Differentially expressed genes (DEGs) were analyzed using bioinformatics. A dual-luciferase reporter assay was used to verify the target relationship between gga-miR-135a-5p and predicted targets within the DEGs. Compared with the normal chicken follicle theca cells, 953 up-regulated and 1060 down-regulated genes were detected in cells with gga-miR-135a-5p inhibited. The up-regulated genes were significantly enriched in Gene Ontology terms and pathways involved in cell proliferation and differentiation. In chicken follicular theca cells, Krüppel-like factor 4 (KLF4), ATPase phospholipid transporting 8A1 (ATP8A1), and Complexin-1 (CPLX1) were significantly up-regulated when the expression of gga-miR-135a-5p was inhibited. In addition, KLF4, ATP8A1, and CPLX1 confirmed as targets of gga-miR-135a-5p by using a dual-luciferase assay The results suggest that gga-mir-135a-5p may involve in proliferation and differentiation in chicken ovarian follicular theca cells by targeting KLF4, ATP8A1, and CPLX1.
作为一类转录调节因子，众多微小RNA（miRNA）已被证实参与调控鸡卵巢卵泡发育（）。先前我们发现，gga-miR-135a-5p在高产和低产鸡卵巢中存在显著差异表达，且在卵泡膜细胞中gga-miR-135a-5p的丰度显著高于颗粒细胞。然而，gga-miR-135a-5p在鸡卵泡膜细胞中的确切作用尚不清楚。在本研究中，分离出鸡原代卵泡膜细胞，然后用gga-miR-135a-5p抑制剂进行转染。对转染和未转染的鸡卵泡膜细胞进行转录组测序。利用生物信息学分析差异表达基因（DEG）。采用双荧光素酶报告基因检测法验证gga-miR-135a-5p与DEG中预测靶点之间的靶向关系。与正常鸡卵泡膜细胞相比，gga-miR-135a-5p受到抑制的细胞中检测到953个上调基因和1060个下调基因。上调基因在参与细胞增殖和分化的基因本体术语和通路中显著富集。在鸡卵泡膜细胞中，当gga-miR-135a-5p的表达受到抑制时，Krüppel样因子4（KLF4）、ATP酶磷脂转运8A1（ATP8A1）和突触结合蛋白-1（CPLX1）显著上调。此外，通过双荧光素酶检测证实KLF4、ATP8A1和CPLX1是gga-miR-135a-5p的靶点。结果表明，gga-mir-135a-5p可能通过靶向KLF4、ATP8A1和CPLX1参与鸡卵巢卵泡膜细胞的增殖和分化。
