Construction and expression of lentiviral vectors encoding recombinant mouse CREBZF in NIH 3T3 cells

Chen, Fenglei; Lin, Peng; Li, Xiao; Sun, Jie; Zhang, Zhe; Wang, Jianxiao; Wang, Aihua; Jin, Ya

doi:10.1016/j.plasmid.2014.08.004

Cited by 19 publications

(10 citation statements)

References 24 publications

(26 reference statements)

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“…[25]. Lentivirus vectors encoding Luman shRNA (shLuman) and a non-silencing negative control (shRNA-negative) were constructed by our group.…”

Section: Methodsmentioning

confidence: 99%

Knockdown of CREB3/Luman by shRNA in Mouse Granulosa Cells Results in Decreased Estradiol and Progesterone Synthesis and Promotes Cell Proliferation

Zhao

Wang

et al. 2016

PLoS ONE

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Luman (also known as LZIP or CREB3) is a transcription factor and a member of the cAMP responsive element-binding (CREB) family proteins. Although Luman has been detected in apoptotic granulosa cells and disorganized atretic bodies, the physiological function of Luman in follicular development has not been reported. Our objective is to determine the role of Luman in folliculogenesis by knocking down Luman expression in mouse GCs (granulosa cells) using shRNA. Luman expression was successfully knocked down in mouse GCs at the mRNA and protein level, as confirmed by real-time quantitative PCR, western blot and immunofluorescence staining, respectively. Knockdown of Luman significantly decreased the concentrations of estradiol (E2) and progesterone (P4) in cell culture medium. Furthermore, Luman knockdown promoted cell proliferation but had no effect on cell apoptosis. To elucidate the regulatory mechanism underlying the effects of Luman knockdown on steroid synthesis and cell cycle, we measured the mRNA and protein expression levels of several related genes. The expression of Star, Cyp19a1, and Cyp1b1, which encode steroidogenic enzymes, was down-regulated, while that of Cyp11a1 and Runx2, which also encode steroidogenic enzymes, was up-regulated. The expression of the cell cycle factors Cyclin A1, Cyclin B1, Cyclin D2, and Cyclin E was significantly up-regulated. Among apoptosis-related genes, only Bcl-2 was down-regulated, while Caspase 3, Bax and p53 were not significantly affected, suggesting that Luman knockdown may regulate cell cycle activity and hormone secretion at the transcriptional and translational level in mouse GCs. The expression of two important genes associated with folliculogenesis in mouse GCs, Has2 and Ptgs2, were also significantly altered by Luman knockdown. In conclusion, the findings of this study indicate that Luman regulates mouse GCs modulation of steroid synthesis, cell cycle activity and other regulators of folliculogenesis.

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“…[25]. Lentivirus vectors encoding Luman shRNA (shLuman) and a non-silencing negative control (shRNA-negative) were constructed by our group.…”

Section: Methodsmentioning

confidence: 99%

Knockdown of CREB3/Luman by shRNA in Mouse Granulosa Cells Results in Decreased Estradiol and Progesterone Synthesis and Promotes Cell Proliferation

Zhao

Wang

et al. 2016

PLoS ONE

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“…Construction of ESAQP5-shRNA recombinant lentiviral vectors was performed as described previously [ 20 ]. Briefly, before transfection, 5×10 3 HEK 293T cells (human embryonic kidney cells expressing the large simian virus 40 T-antigen) were seeded into each well.…”

Section: Methodsmentioning

confidence: 99%

Aquaporin 5 Plays a Role in Estrogen-Induced Ectopic Implantation of Endometrial Stromal Cells in Endometriosis

et al. 2015

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Aquaporin 5 (AQP5) participates in the migration of endometrial cells. Elucidation of the molecular mechanisms associated with AQP5-mediated, migration of endometrial cells may contribute to a better understanding of endometriosis. Our objectives included identifying the estrogen-response element (ERE) in the promoter region of the AQP5 gene, and, investigating the effects of AQP5 on ectopic implantation of endometrial cells. Luciferase reporter assays and electrophoretic mobility shift assay (EMSA) identified the ERE-like motif in the promoter region of the AQP5 gene. After blocking and up-regulating estradiol (E2) levels, we analysed the expression of AQP5 in endometrial stromal (ES) cells. After blocking E2 /or phosphatidylinositol 3 kinase(PI3K), we analysed the role of AQP5 in signaling pathways. We constructed an AQP5, shRNA, lentiviral vector to knock out the AQP5 gene in ES cells. After knock-out of the AQP5 gene, we studied the role of AQP5 in cell invasion, proliferation, and the formation of ectopic endometrial implants in female mice. We identified an estrogen-response element in the promoter region of the AQP5 gene. Estradiol (E2) increased AQP5 expression in a dose-dependent fashion, that was blocked by ICI182,780(an estrogen receptor inhibitor). E2 activated PI3K /protein kinase B(AKT) pathway (PI3K/AKT), that, in turn, increased AQP5 expression. LY294002(PI3K inhibitor) attenuated estrogen-enhanced, AQP5 expression. Knock-out of the AQP5 gene with AQP5 shRNA lentiviral vector significantly inhibited E2-enhanced invasion, proliferation of ES cells and formation of ectopic implants. Estrogen induces AQP5 expression by activating ERE in the promoter region of the AQP5gene, activates the PI3K/AKT pathway, and, promotes endometrial cell invasion and proliferation. These results provide new insights into some of the mechanisms that may underpin the development of deposits of ectopic endometrium.

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“…Short hairpin RNA-PRL-3 lentiviruses were packaged according to Chen and colleagues (20). We further generated lentivirus vectors encoding shRNA-PRL-3 and shRNA-control.…”

Section: Transfections Of Ht29 With Shrna Lentivirusesmentioning

confidence: 99%

CCL26 Participates in the PRL-3–Induced Promotion of Colorectal Cancer Invasion by Stimulating Tumor-Associated Macrophage Infiltration

Lan

Wei

Zeng

et al. 2018

Molecular Cancer Therapeutics

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Both phosphatase of regenerating liver-3 (PRL-3) and tumor-associated macrophages (TAM) influence cancer progression. Whether PRL-3 plays a critical role in colorectal cancer invasion and metastasis by inducing TAM infiltration remains unclear. In the current study, we investigated the effects of chemokine ligand 26 (CCL26) on TAM infiltration and colorectal cancer invasion and the underlying mechanism in colorectal cancer cells by overexpressing or silencing PRL-3. We found that PRL-3 upregulated CCL26 expression correlatively and participated in cell migration, according to the results of gene ontology analysis. In addition, IHC analysis results indicated that the PRL-3 and CCL26 levels were positively correlated and elevated in stage III and IV colorectal cancer tissues and were associated with a worse prognosis in colorectal cancer patients. Furthermore, we demonstrated that CCL26 induced TAM infiltration by CCL26 binding to the CCR3 receptor. When LoVo-P and HT29-C cells were cocultured with TAMs, CCL26 binding to the CCR3 receptor enhanced the invasiveness of LoVo-P and HT29-C cells by mobilizing intracellular Caof TAMs to increase the expression of IL6 and IL8. In addition, IHC results indicated that protein levels of CCR3 and TAMs counts were higher in stage III and IV colorectal cancer tissues and correlated with CCL26. Moreover, similar results were observed using mice injected with LoVo-P and HT29-C cells. These data indicate that PRL-3 may represent a potential prognostic marker that promotes colorectal cancer invasion and metastasis by upregulating CCL26 to induce TAM infiltration..

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Construction and expression of lentiviral vectors encoding recombinant mouse CREBZF in NIH 3T3 cells

Cited by 19 publications

References 24 publications

Knockdown of CREB3/Luman by shRNA in Mouse Granulosa Cells Results in Decreased Estradiol and Progesterone Synthesis and Promotes Cell Proliferation

Knockdown of CREB3/Luman by shRNA in Mouse Granulosa Cells Results in Decreased Estradiol and Progesterone Synthesis and Promotes Cell Proliferation

Aquaporin 5 Plays a Role in Estrogen-Induced Ectopic Implantation of Endometrial Stromal Cells in Endometriosis

CCL26 Participates in the PRL-3–Induced Promotion of Colorectal Cancer Invasion by Stimulating Tumor-Associated Macrophage Infiltration

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