ABSTRACT
Objective: To investigate the effects of DNAJ homolog subfamily B member 11 (DNAJB 11) gene-silencing on proliferation, cell cycle and apoptosis of human hepatocellular carcinoma cell line SMMC7721. Methods: The recombinant lentiviral vector pCDH-Puro/DNAJB11-shRNA carrying the specific shRNA targeting DNAJB 11 gene was established. The SMMC7721 cells were infected with high infective lentivirus pCDH-Puro/DNAJB11-shRNA. Then the proliferation of SMMC7721 cells was detected by CCK-8 method. The expression levels of DNAJB11, proliferating cell nuclear antigen (PCNA) and caspase-3 mRNAs and proteins in SMMC7721 cells were detected by real-time fluorescent quantitative PCR and Western blotting, respectively. The cell cycle distribution and the apoptosis rate of SMMC7721 cells were analyzed by FCM. Results: The pCDH-Puro/DNAJB11-shRNA was constructed successfully. The proliferation of SMMC7721 cells was significantly inhibited after infection with pCDH-Puro/DNAJB11- shRNA (P<0.05). In SMMC7721 cells infected with pCDH-Puro/DNAJB11-shRNA, the expressions of DNAJB11 mRNA and protein were silenced effectively (both P<0.05). After DNAJB 11 gene-silencing, the expressions of caspase-3 mRNA and protein in SMMC7721 cells were up-regulated (both P<0.05), while the expressions of PCNA mRNA and protein were down-regulated (both P<0.05). Furthermore, the cell cycle was arrested in G1 phase (P<0.01), and the apoptosis rate was significantly increased (P<0.01). Conclusion: The DNAJB 11 gene-silencing can effectively suppress the proliferation of SMMC7721 cells, and promote their apoptosis. These effects may be related to downregulation of PCNA expression and upregulation of caspase-3 expression in SMMC7721 cells.
ABSTRACT
Objective Transcription factor forkhead box L 2 (FOXL2) is a key regulator of granulosa cells (GCs) estrogen syn-thesis and function maintenance .However, the FOXL2 protein expres-sion and function regulation mechanism are unknown .We explored how DNAJB11 regulates estrogen synthesis of granulosa cells with immunoprecipitation , immunofluorescent staining and luciferase re-porter gene. Methods The expression and localization of DNAJB 11 was detected by immunohistochemistry staining in isolated mouse ovary tissues .we use immunoprecipitation , immunofluorescence staining and luciferase reporter gene assay to investigate the mechanism of DNAJB11, a member of the endoplasmic reticulum Hsp 40 /DnaJ family, regulating the estrogen synthesis in granulosa cells . Results DNAJB11 is expressed in the mouse ovary and granulosa cells .Follicle-stimulating hormone (FSH) promotes DNAJB11 ex-pression in a time and concentration dependent manner and induces endogenous DNAJB 11 protein translocation from the ER to the nu-cleus in KGN cells.Moreover, Adenovirus-mediated overexpression of DNAJB11 did not affect the proliferation of granulosa cells .How-ever, the concentration of estrogen in granulosa cells was affected by concentration -dependent and subcellular localization-dependent manner (10749±801.7 pg/mL vs 14217±1218.0 pg/mL P<0.01).Immunoprecipitation assay confirmed that DNAJB11 binds to FOXL2 in granulosa cells .When overexpressed in the nucleus of granulosa cells , DNAJB11 could significantly enhance the stability of FOXL2 (P<0.05) and promote FOXL2-mediated activity of Cyp19A1 promoter (P<0.01), while the expression of DNAJB11 in the nucleus increased the expression of Cyp 19A1 protein by 1.5 times ( P<0.05) . Conclusion These results demonstrate that DNAJB 11 was a new binding molecule of transcription factor FOXL 2 and regulated FOXL 2 protein stability and transcription activity .
ABSTRACT
Objective Transcription factor forkhead box L 2 (FOXL2) is a key regulator of granulosa cells (GCs) estrogen syn-thesis and function maintenance .However, the FOXL2 protein expres-sion and function regulation mechanism are unknown .We explored how DNAJB11 regulates estrogen synthesis of granulosa cells with immunoprecipitation , immunofluorescent staining and luciferase re-porter gene. Methods The expression and localization of DNAJB 11 was detected by immunohistochemistry staining in isolated mouse ovary tissues .we use immunoprecipitation , immunofluorescence staining and luciferase reporter gene assay to investigate the mechanism of DNAJB11, a member of the endoplasmic reticulum Hsp 40 /DnaJ family, regulating the estrogen synthesis in granulosa cells . Results DNAJB11 is expressed in the mouse ovary and granulosa cells .Follicle-stimulating hormone (FSH) promotes DNAJB11 ex-pression in a time and concentration dependent manner and induces endogenous DNAJB 11 protein translocation from the ER to the nu-cleus in KGN cells.Moreover, Adenovirus-mediated overexpression of DNAJB11 did not affect the proliferation of granulosa cells .How-ever, the concentration of estrogen in granulosa cells was affected by concentration -dependent and subcellular localization-dependent manner (10749±801.7 pg/mL vs 14217±1218.0 pg/mL P<0.01).Immunoprecipitation assay confirmed that DNAJB11 binds to FOXL2 in granulosa cells .When overexpressed in the nucleus of granulosa cells , DNAJB11 could significantly enhance the stability of FOXL2 (P<0.05) and promote FOXL2-mediated activity of Cyp19A1 promoter (P<0.01), while the expression of DNAJB11 in the nucleus increased the expression of Cyp 19A1 protein by 1.5 times ( P<0.05) . Conclusion These results demonstrate that DNAJB 11 was a new binding molecule of transcription factor FOXL 2 and regulated FOXL 2 protein stability and transcription activity .