title: Knockdown of CEBPβ by RNAi in porcine granulosa cells resulted in S phase cell cycle arrest and decreased progesterone and estradiol synthesis.
abstract: Cultured ovarian granulosa cells (GCs) are essential models to study molecular mechanisms of gene regulation during folliculogenesis. CCAAT enhancer binding proteins β (CEBPβ) has been identified in the ovary and is critical for follicular growth, ovulation and luteinization in mice. In the present study, hormonal treatment indicated that luteinizing hormone (LH) and exogenous human chorionic gonadotropins (hCG) significantly increased the expression of CEBPβ in porcine GCs. By RNAi-Ready pSIREN-RetroQ-ZsGreen Vector mediated recombinant pshRNA vectors, CEBPβ gene was successfully knocked down in porcine GCs, confirmed by mRNA and protein level analyzed by real time PCR and western blot, respectively. We further found that knockdown of CEBPβ significantly increased the expression of p-ERK1/2. Furthermore, CEBPβ knockdown arrested the GCs at S phase of cell cycle, but had no effects on cell apoptosis. More importantly, it markedly down regulated the concentration of estradiol (E2) and progesterone (P4) in the culture medium. To uncover the regulatory mechanism of CEBPβ knockdown on cell cycle and steroids synthesis, we found that the mRNA expression of bcl-2 (anti-apoptosis), StAR and Runx2 (steroid hormone synthesis) was up-regulated, while genes related to apoptosis (Caspase-3 and p53), hormonal synthesis (CYP11A1) and cell cycle (cyclinA1, cyclinB1, cyclinD1) were down-regulated, suggesting that knockdown of CEBPβ may inhibit apoptosis, regulate cell cycle and hormone secretions at the transcriptional level in porcine GCs. Furthermore, knockdown of CEBPβ significantly increased the expression of PTGS2 and decreased the expression of IGFBP4, Has2 and PTGFR which are important for folliculogenesis in porcine GCs. In conclusion, this study reveals that CEBPβ is a key regulator of porcine GCs through modulation of cell cycle, apoptosis, steroid synthesis, and other regulators of folliculogenesis.
authors.affiliation: Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Education Ministry of China, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan 430070, Hubei, PR China.
authors.forename: Yan-Hong
authors.initials: YH
authors.lastname: Zhen
authors.affiliation: Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Education Ministry of China, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan 430070, Hubei, PR China.
authors.forename: Li
authors.initials: L
authors.lastname: Wang
authors.affiliation: Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Education Ministry of China, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan 430070, Hubei, PR China.
authors.forename: Hasan
authors.initials: H
authors.lastname: Riaz
authors.affiliation: Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Education Ministry of China, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan 430070, Hubei, PR China.
authors.forename: Jia-Bin
authors.initials: JB
authors.lastname: Wu
authors.affiliation: Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Education Ministry of China, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan 430070, Hubei, PR China.
authors.forename: Yi-Feng
authors.initials: YF
authors.lastname: Yuan
authors.affiliation: College of Animal Science and Technology/College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, PR China.
authors.forename: Li
authors.initials: L
authors.lastname: Han
authors.affiliation: Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Education Ministry of China, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan 430070, Hubei, PR China.
authors.forename: Yan-Ling
authors.initials: YL
authors.lastname: Wang
authors.affiliation: Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Education Ministry of China, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan 430070, Hubei, PR China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Education Ministry of China, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan 430070, Hubei, PR China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Dan
authors.affiliation: Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Education Ministry of China, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan 430070, Hubei, PR China. Electronic address: lijunhuo@yahoo.com.
authors.forename: Li-Jun
authors.initials: LJ
authors.lastname: Huo
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D059447:Cell Cycle Checkpoints / Q000187:drug effects / Q000502:physiology*; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000378:metabolism; D006063:Chorionic Gonadotropin / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D004958:Estradiol / Q000378:metabolism*; D005260:Female; D005786:Gene Expression Regulation; D006107:Granulosa Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D034461:Luteinization / Q000187:drug effects; D007986:Luteinizing Hormone / Q000494:pharmacology; D010060:Ovulation / Q000187:drug effects; D011374:Progesterone / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D012102:Reproductive Control Agents / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016196:S Phase / Q000187:drug effects / Q000502:physiology*; D013552:Swine
chemical_list: D022782:CCAAT-Enhancer-Binding Protein-beta; D006063:Chorionic Gonadotropin; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D012102:Reproductive Control Agents; D011374:Progesterone; D004958:Estradiol; D007986:Luteinizing Hormone; D002786:Cholesterol Side-Chain Cleavage Enzyme
keywords: CEBPβ; Gene regulation; Granulosa cells; Porcine; RNA interference