敲除和恢复揭示了 PPARγ1 和 PPARγ2 在鸡脂肪形成中的不同功能作用,Journal of Agricultural and Food Chemistry

当前位置： X-MOL 学术 › J. Agric. Food Chem. › 论文详情

Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)

敲除和恢复揭示了 PPARγ1 和 PPARγ2 在鸡脂肪形成中的不同功能作用
Journal of Agricultural and Food Chemistry ( IF 5.7 ) Pub Date : 2022-11-16 , DOI: 10.1021/acs.jafc.2c05549
Yang Jing _{1,

2,

3} , Fang Mu _{1,

2,

3} , Xiaoxu Xing _{1,

2,

3} , Jiaxin Huang _{1,

2,

3} , Ming Lou _{1,

2,

3} , Haidong Xu _{1,

2,

3} , Bolin Ning _{1,

2,

3} , Yuqi Lou _{1,

2,

3} , Zhihui Gao _{1,

2,

3} , Haoyu Luo _{1,

2,

3} , Xiaohong Yan _{1,

2,

3} , Hui Li _{1,

2,

3} , Ning Wang _{1,

2,

3}

Affiliation

过氧化物酶体增殖物激活受体 γ (PPARγ) 是脂肪生成的主要调节因子，以两种亚型 PPARγ1 和 PPARγ2 表达。我们之前的慢病毒过表达研究表明，PPARγ1 和 PPARγ2 差异调节永生化鸡前脂肪细胞系 (ICP2) 的增殖、分化和凋亡。然而，我们不能排除 PPARγ 亚型的内源性表达可能损害我们的发现的可能性。在这项研究中，使用双 sgRNA 指导的 CRISPR/Cas9 系统，我们生成了PPAR γ ( PPAR γ ^–/– ) 和PPAR γ 2特异性敲除 ( PPAR γ 2 ^–/–) ICP2 细胞系并研究了PPAR γ ^–/–、PPAR γ 2 ^–/–和野生型 ICP2 细胞在增殖和分化方面的差异。EdU 增殖试验表明，PPAR γ 2特异性和PPAR γ 敲除都显着增加了增殖率。一致地，实时 RT-PCR 分析表明，PPAR γ 2特异性和PPAR γ 敲除都显着上调了增殖标记基因 PCNA 和细胞周期蛋白 D1 的表达。FACS 分析显示PPARγ基因敲除显着增加了在S期积累的细胞数量，并减少了在G ₁ /G ₀期积累的细胞数量。油红 O 染色和基因表达分析显示PPAR γ 2特异性和PPAR γ 敲除都显着降低了脂肪形成分化的能力。为了证实我们之前的发现，分别使用表达鸡 PPARγ1 (LV-PPARγ1) 和 PPARγ2 (LV-PPARγ2) 的慢病毒在PPAR γ ^{–/–细胞中恢复了 PPARγ1 和 PPARγ2 的表达。}随后的测定表明，恢复 PPARγ1 或 PPARγ2 的表达可抑制增殖并刺激细胞分化。PPAR γ ^–/–细胞。相比之下，PPARγ2 比 PPARγ1 具有更强的抗增殖和促脂肪形成作用。为了了解它们对PPAR γ ^–/–细胞分化产生不同影响的分子机制，我们在PPAR γ ^– /–中进行了 RNA-seq在分化 72 小时时恢复个体 PPARγ 亚型表达的细胞。转录组学分析表明，恢复 PPARγ1 表达导致的差异表达基因 (DEG) 远多于恢复 PPARγ2 表达。GO 和 KEGG 通路富集分析表明 PPARγ1 和 PPARγ2 在脂肪生成中具有不同和重叠的功能。总之，我们的结果清楚地表明 PPARγ1 和 PPARγ2 对鸡脂肪生成的影响不同。

"点击查看英文标题和摘要"

Knockout and Restoration Reveal Differential Functional Roles of PPARγ1 and PPARγ2 in Chicken Adipogenesis

Peroxisome proliferator-activated receptor γ (PPARγ) is the master regulator of adipogenesis and is expressed as two isoforms, PPARγ1 and PPARγ2. Our previous lentiviral overexpression study showed that PPARγ1 and PPARγ2 differentially regulated proliferation, differentiation, and apoptosis of the immortalized chicken preadipocyte cell line (ICP2). However, we cannot rule out the possibility that the endogenous expression of PPARγ isoforms may compromise our findings. In this study, using the dual sgRNA-directed CRISPR/Cas9 system, we generated PPARγ (PPARγ^–/–) and PPARγ2-specific knockout (PPARγ2^–/–) ICP2 cell lines and investigated the differences in proliferation and differentiation among PPARγ^–/–, PPARγ2^–/–, and wild-type ICP2 cells. EdU proliferation assay showed that both PPARγ2-specific and PPARγ knockouts significantly increased the proliferation rates. Consistently, real-time RT-PCR analysis showed that both PPARγ2-specific and PPARγ knockouts significantly upregulated the expression of proliferation marker genes PCNA and cyclinD1. FACS analysis revealed that PPARγ knockout significantly increased the number of cells accumulating in the S phase and decreased the number of cells accumulating in the G₁/G₀ phase. Oil Red O staining and gene expression analysis showed both PPARγ2-specific and PPARγ knockouts dramatically reduced capacity for adipogenic differentiation. To corroborate our previous findings, PPARγ1 and PPARγ2 expression were restored in PPARγ^–/– cells by using the lentiviruses expressing chicken PPARγ1 (LV-PPARγ1) and PPARγ2 (LV-PPARγ2), respectively. Subsequent assays showed that restoration of expression of either PPARγ1 or PPARγ2 suppressed proliferation and stimulated differentiation of the PPARγ^–/– cells. By comparison, PPARγ2 had stronger anti-proliferative and pro-adipogenic effects than PPARγ1. To understand the molecular mechanism underlying their differential effects on differentiation of the PPARγ^–/– cells, we performed RNA-seq in the PPARγ^–/– cells in which individual PPARγ isoform expression was restored at 72 h of differentiation. Transcriptomic analysis revealed that restoring PPARγ1 expression caused far more differentially expressed genes (DEGs) than restoring PPARγ2 expression. GO and KEGG pathway enrichment analyses indicated that PPARγ1 and PPARγ2 had distinct and overlapping functions in adipogenesis. Taken together, our results clearly indicate that PPARγ1 and PPARγ2 differentially impact chicken adipogenesis.

更新日期：2022-11-16

点击分享查看原文

点击收藏

阅读更多本刊新发论文本刊介绍/投稿指南