Knockdown of CREB1 promotes apoptosis and decreases estradiol synthesis in mouse granulosa cells
Authors: Xiaohui Liu
Chengcheng Tan
Weixia Wang
Hongyan Lang
Yumin Zhao
Xintao Li
Pengju Zhang
Jun Wang
Xinghong Wu
Haiyan Liu
Journal: Biomedicine & Pharmacotherapy (2018)
Discipline: PHARMACOLOGY & TOXICOLOGY
Keywords: Apoptosis; CREB1; Estradiol synthesis; Granulosa cells; RNA interference.
Affiliations: Institute of Animal Sciences, Jilin Academy of Agricultural Sciences, #1363 Shengtai Street, Changchun 130124, Jilin Province, PR China., College of Animal Science and Technology, Jilin Agricultural University, 2888 Xincheng Street, Changchun 130118, Jilin Province, PR China., Institute of Animal Sciences, Jilin Academy of Agricultural Sciences, #1363 Shengtai Street, Changchun 130124, Jilin Province, PR China. Electronic address: .

Abstract:  Cyclic AMP response element-binding protein 1 (CREB1), a member of the CREB family, is known to be involved in follicular growth, ovulation, and ovarian disease. However, the physiological function of CREB1 in mouse granulosa cells (mGCs) remains lagerly unknown. The aim of this study was to determine the role of CREB1 in mGCs by knocking down CREB1 expression. CREB1 knock-down in mGCs at the mRNA and protein levels, was confirmed by quantitative real-time polymerase chain reaction and western blot. Results of enzyme linked immunosorbent assay revealed that CREB1 knockdown significantly decreased the concentrations of estradiol (E2) and progesterone (P4) in mGCs. Furthermore, the CREB1 knockdown in mGCs promoted cell proliferation and apoptosis, and arrested the cell cycle in S-phase. To elucidate the regulatory mechanism underlying the effects of CREB1 knockdown on steroid synthesis, cell cycle, and apoptosis, we measured the protein expression levels of several related genes in mGCs knocked down CREB1. When CREB1 was knocked down, the expression of Cyp1b1 and Cyp19a1, which encode steroidogenic enzymes, was down-regulated; the expression of the cell cycle factors CyclinA1, CyclinB1, and CyclinD2 were significantly decreased. Among apoptosis-related genes, Bcl-2 was down-regulated, whereas Bax and cleaved Caspase3 were upregulated. Moreover, CREB1 knockdown significantly decreased expression level of Has2, Ptgs2, and Igfbp4, which are essential genes for folliculogenesis in mGCs. Taken together, these findings suggested that CREB1 might be a key regulator of mGCs through regulating steroid synthesis, cell proliferation, cell cycle, apoptosis, and other regulators of folliculogenesis. 

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 *[left]* Fig 1B.  CREB1 protein expression level was measured in mGCs by western blot 48 h after transduction . *[upper right]* Fig 5C from [Song et al (2015)] .  Western blotting of EGFR protein expression in the HeLa cells after transfection with the miR-133a mimic or miR-Ctrl. *[lower right]* Fig 3C from [Zhang et al (2015)] .  The expression levels of MMP-2 and MMP-9 were determined by Western blot after being transfected with miR-133a or miR-NC . ![file] Other manifestations of the CREB1 band include Fig 5D from [Wang et al (2016)](http: www.ijcem.com files ijcem0017368.pdf),  IRS2 protein expression was determined in four ovarian cancer cell lines (SKOV3, A2780, OVCAR and HO-8910) and human ovarian surface epithelial cell line (HOSEpiC) . ![] From Fig 5B in Wang et al, here s that loading control again: ![file] , 
 *[left]* Fig 4A.  Cell apoptosis was determined in mGCs transduction with sh-CREB1 or sh-NC lentiviruses by flow cytometry . *[right]* Fig 2C from [Liu et al (2019)] .  Cell apoptosis was detected in A549 cells transfected with si-HOXA-AS2 or si-NC by flow cytometry assay. ![file] Quite different experiments, no authors in common, same Figure. ,