Oncogene Transcription Factor Creb Affect Glioma Cell Growth And Migration Through The Regulation Of Mirna Expression

Glioma is the most common and deadly brain cancer, and no very effective drug therapy has been developed so far. Studing the molecular mechanism of gliomageneis will be helpful to find out valuable drug targets and provide experimental evidence and new insights for target therapy. Recently, high-throughput screenings for dysregulated miRNAs by microRNA microarray were performed and the functions of these miRNAs in gliomagenesis have been charicatorized. However, the mechanism of the dysregulation of these oncogenic or tumor suppressive miRNAs remains unknown. That uncovering the reasons of the dysregulation of these miRNAs will be valuable for understanding the mechanism of gliomagenesis and finding out critical drug target or more effective treatment of therapy. As a highly-expressed transcription factor with critical functions in various cancers, CREB was charicatorized as an important oncogene involving in proliferation, survival and metastasis of tumor cells. However, the expression and the role of CREB in glioma deserve to be further investigated.Firstly, we showed that CREB is highly-expressed in glioma and promote the growth and survival of glioma cells. Western blotting was used to dectect the protein level of CREB in two normal brain tissues and fifteen glioma tissues (each grade of5cases) as well as four glioma cell lines (U87MG, T98G, A172and U251), and we found that the protein level of CREB is high in glioma tissues and cell lines. Utilizing real-time PCR method we detected the gene copy number of CREB in three glioma tissues and the four glioma cell lines and found that the CREB gene copy number is amplified in one case grade IV glioma tissue and the four glioma cell lines. Knocking-down CREB with specific siRNAs in glioma cell lines significantly inhibited the growth, survival and colony formation of glioma cells (U87MG, T98G and U251). The anchor-independent growth in soft agrose of T98G and U251but not U87MG cells was also inhibited by knocking-down CREB. Furthermore, the U87MG and U251cells with CREB knocked-down formed much smaller tumors when transplanted subcutaneously in nude mice compared with control cells. Interestingly, knocking-down CREB with adenovirus-delivered shcreb significantly inhibited the growth and survivl of T98G and U251but not the cervical carcinoma cell line HeLa and the normal human glial cell line HEB.Secondly, we showed that CREB could widespreadly modulate the expression of miRNAs in glioma cells and directly regulated mir-9and mir-23a. We knocked down CREB in T98G cells and found a widespread decrease of miRNAs. The results of ChIP-chip and ChIP-QPCR as well as luciferase reporter assays confirmed the directly regulation of mir-9and mir-23a by CREB.Next, we investigated the functions of mir-23a and mir-9in glioma cells and generally identified their targets. Knocking down mir-23a inhibited the growth and survival of glioma cells (U87MG, T98G and U251). Over-expressing mir-23a can partly restore the growth and survival of T98G and U251cells with CREB knocked-down. By bioinformatic prediction we found that three key tumor suppressors (FoXO3a,FoXO4and PTEN) could be potential targets of mir-23a, and the results of luciferase reporter assays confirmed the interaction between mir-23a and the3'UTR of the three genes. By detecting the protein level of the three tumor suppressors in glioma cell lines and the change of protein level of them after over-expressing or knocking-down mir-23a in glioma cells, we identified PTEN as a potential downstream target of mir-23a in glioma cells. Moreover, knocking-down mir-9inhibited the migration of glioma cells (U87MG, T98G and U251).We also characterized a migration-inhibiting role of CREB in glioma cells.At last, we showed that mir-9can directly target CREB by luciferase reporter assays and Western blotting, suggesting a potential negative feedback loop formed by mir-9and CREB.In summary, we showed that CREB is highly-expressed in glioma tissues and cell lines and promotes the growth and survival of glioma cells. As a downstream target of CREB, mir-23a enhances the growth and survival of glioma cells, possibly by targeting PTEN. Mir-9, the other target of CREB, can stimulate the migration of glioma cells and directly target CREB. The potential feedback loop consiting of mir-9and CREB might contribute to the balance of glioma cell growth and migration.