The Expression Of CREB In Glioma And Influence In The Biological Characteristics Of Tumor Cells

Objective:To study the expression of CREB in the glioma tissues, and to investigate its role in the process of human brain glioma cells U251 after knockout CREB by CRISPR/Cas9.Methods:1. TISSUE1) Different grades of samples of human glioma tissues were collected and IHC?RT-PCR?Western blot were performed to detect the expression of CREB.2.CELL1)U251 were transfected with the CRISPR/Cas9 and stable tranfected cells were established. Western-blot,Cruiser? are performed to detect the expression of CREB.2)The effect on the U251-KO cells proliferative capacity:CCK-8 is used to detect the change in cell proliferation rate after knockout of CREB.To investigate the expression of Cyclin D1 in the U251-KO cells by Western blot.3)The effect on the U251-KO cells migration, invasion capability;:Transwell chamber is used to detect the change in cell migration, invasion capability after knockout of CREB.To investigate the expression of E-cadherin in the U251-KO cells by Western blot.4) the effect on the U251-KO cells apoptosis, and on the ERK-CREB and PI3/Akt-CREB of two important phosphorylation pathway after the knockout of CREB by CRISPR/CAS9: To investigate the expression of bcl-2?bax?p ERK?p Akt in the U251-KO cells by Western blot.3.NUDE MICE1) The establishment of the nude mice model by U251 human glioma.: BALB/C-nu nude mice as an animal model for the study, nude mice were randomly grouped and injected with U251?U251-NT and U251-KO, respectively. To investigate the expression of CREB in the Tumor tissue from nude mice by Western blot.Results:(1)Over-expression of CREB is detected in different differentiation degree of glioma, The expression of CREB was positively correlated with the different degrees,(2)m RNA and protein of CREB in U251-KO was significantly decreased by CRISPR/Cas9(3)The proliferation, migration and invasion capability of U251 after knockout CREB is reduced and the level of cells apoptosis is increased.The expression of protein in the signal pathway of phosphorylation was also significantly changed.(4) The tumorigenesis capability in nude mice of U251 knockout CREB by CRISPR/CAS9 is significantly reduced.Conclusion:Over-expression of CREB is detected in different differentiation degree of glioma, The expression of CREB was positively correlated with the different degrees.The proliferation, migration, invasion and tumorigenesis capability of U251 after knockout CREB by CRISPR/CAS9 is significantly reduced.the level of cells apoptosis is increased.Therefore, CREB might have an important regulatory effect on the progress of glioma.