Decreased CUL4B Expression Inhibits Malignant Proliferation Of Glioma In Vitro And In Vivo

Gliomas are the most common primary tumors affecting the adult central nervous system (CNS), characterized by high morbidity and mortality as well as a high recurrence rate. Gliomas are presumed to arise from mature glia or neural stem cells and diffusely infiltrate the surrounding tissues, making surgical resection very difficult. Survival from gliomas depends on the tumor type and grades of malignancy. According to the World Health Organization (WHO) standards, gliomas are classified into four malignant grades (I, II, III, IV). The most lethal is grade IV glioblastoma (GBM), with a five-year survival rate of less than 10% due to difficulties in complete resection and the low sensitivity to radiotherapy and chemotherapy. Thus, the treatment of gliomas remains one of the most disappointing challenges in modern oncology. Early in the 20th century, scientists proposed glioma as a molecular disease of genetic defect, and can be solved by molecular means. Currently on molecular targeted therapy of glioma has attracted attention. CUL4B is a component of CRL4B (Cullin4B-Ring E3 ligase complex). Cullin 4B is involved in Ubiquitin-proteasome pathway, bearing the role of specific recognition of substrates. Ubiquitination is important as one of Post-translational modification of proteins. In vivo, the protein is is ubiquitinated by catalysis of the enzyme, then enter into the proteasome for degradation. Ubiquitination process Play an important role in Protein homeostasis about synthesis and degradation. Ubiquitination process is involved in the cell cycle of the body, Growth, reproduction and other physiological activities. Furthermore, numerous studies indicate that CUL4B plays an important role in DNA methylation, proteolysis and tumor formation. Moreover, related research suggests that Cullin4B plays an important role in the development of brain. However, less studies is related for the role of CUL4B in the human brain tumors, including gliomas. Forasmuch, we Conducted a preliminary study about the role of CUL4B in glioma. We want to find A new therapeutic target for gliomas and provide a new strategy in the targeted therapy.First of all, this study describes the expression levels of CUL4B in glioma and normal brain tissue. Afterward, we cultured glioma cell lines U87 and U251 in vitro experiments, and knockdown of CUL4B in glioblastoma cells by lentivirus-mediated shRNA. We detected whether lentivirus successfully transfected U87 and U251 glioma cells by western blot technique, resulting in decreased expression of CUL4B. In addition to, we also completed the study of CUL4B on cell growth and colony formation by CCK-8 cell proliferation assay and crystal violet staining. We monitored the influence of CUL4B on cell cycle by flow cytometry monitoring. We studied the function of CUL4B during cell migration. We find that knockdown of CUL4B effect on tumor growth in vivo experiments. Meanwhile, this paper relate to effects of CUL4B knockdown on regulators of cell cycle and migration.Experiment 1 Expression of CUL4B in glioma tissues1.1 Expression of CUL4B in glioma and adjacent brain tissuesTo explore the role of CUL4B in human glioma, we firstly evaluated its expression in 15 paired glioma and adjacent brain tissues by qPCR. The mRNA levels of CUL4B in 2/3 glioma tissues were increased by over 3 folds, as compared to matched normal tissues. These results indicated that CUL4B was highly expressed in glioma tissues, and might be associated with the development of glioma.Experiment 2 To investigate the mechanism for CUL4B2.1 Knockdown of CUL4B in glioblastoma cells by lentivirus-mediated shRNAU87 and U251 cells were transduced with shRNA-expressing lentivirus (Lv-shCon or Lv-shCUL4B), respectively, GFP expression was observed by fluorescent microscopy four days post-infection. Over 80% of cells expressed GFP in Lv-shCon and Lv-shCUL4B groups, indicating a successful infection rate in both cell lines. The inhibitory effects of Lv-shCUL4B on endogenous CUL4B expression in both cell lines were examined by qPCR and western blotting. The mRNA levels of CUL4B were significantly reduced in Lv-shCUL4B groups, by 77.4% in U87 cells and 77.8% in U251 cells, in contrast to uninfected (Con) groups. However, there was no significant difference between Lv-shCon and Con groups. Immunoblot also verified the down-regulation of CUL4B expression in both cell lines at protein levels. Therefore, lentivirus-delivered shRNA could specifically deplete endogenous CUL4B expression in glioblastoma cells.2.2 Effects of CUL4B knockdown on cell proliferation and colony formationTo evaluate the effect of CUL4B on glioma cell proliferation, CCK-8 assays were performed in U87 and U251 cells after lentivirus infection. The proliferation rates were significantly reduced in Lv-shCUL4B groups, particularly by 63.7% in U87 cells and 45.5% in U251 cells on day 5, in contrast to Con groups. These results indicated that knockdown of CUL4B could strongly decrease the proliferation of glioblastoma cells.Moreover, colony formation assays were performed in U87 and U251 cells after lentivirus infection, and representative photographs of the colonies per well with crystal violet staining were presented as. The average number of total colonies was nearly 150 in Con group or Lv-shCon group, while there was less than 30 colonies in Lv-shCUL4B group in U87 cells. Besides, the average number of total colonies was about 200 in Con group or Lv-shCon group, while there was less than 40 colonies in Lv-shCUL4B group in U251 cells. These results indicated that knockdown of CUL4B could strongly disrupt the colony formation of glioblastoma cells.2.3 Effects of CUL4B knockdown on cell cycle controlTo test the mechanism by which CUL4B modulating cell proliferation, flow cytometry assays were applied to determine the cell cycle distributions in U87 and U251 cells. As shown in Figure 4B, the percentage of U87 cells in G1 phase was increased from approximately 50.0% or 52.0% in Con group or Lv-shCon group to 82.0% in Lv-shCUL4B group. In contrast, the percentage of U87 cells in S phase was decreased from about 40.0% or 38.0% in Con group or Lv-shCon group to 10.0% in Lv-shCUL4B group. Similarly, the percentage of U251 cells was increased in G1 phase and decreased in S phase in response to CUL4B knockdown. These results suggested that knockdown of CUL4B could inhibit glioblastoma cell proliferation by inducing G1 phase cell cycle arrest.2.4 Effects of CUL4B knockdown on cell migrationFurthermore, we detected the effect of CUL4B on glioblastoma cell migration using the Transwell chambers. Representative images of U87 and U251 cells that migrated to the lower surface were presented as. The average number of migrated U87 cells in Lv-shCUL4B group was 65, which was much fewer than 224 in Con group and 210 in Lv-shCon group (p<0.01). In similar, the number of migrated U251 cells was significantly reduced by CUL4B knockdown (p<0.01). These results indicated that knockdown of CUL4B could alleviate migration of glioblastoma cells.2.5 Effects of CUL4B knockdown on tumor growth in vivoThe human xenograft nude mouse models of glioma were successfully developed using lentivirus-infected U87 or U251 cells. As the time of implantation prolonged, the tumor volume in each group showed a progressive increase, but the growth rates in Lv-shCUL4B groups were significantly slower than in Lv-shCon groups. Compared with Lv-shCUL4B groups, the tumor weights in Lv-shCUL4B groups were reduced by 57.7% in U87 cells-implanted mice and 75.4% in U251 cells-implanted mice (p<0.01). These results further verified that knockdown of CUL4B could potently suppress glioma growth in vitro and in vivo.2.6 Effects of CUL4B knockdown on regulators of cell cycle and migrationTo survey the molecular mechanism underlying CUL4B-mediated cell growth and migration, the expression alterations of some regulators associated with cell cycle and motility were detected in U87 and U251 cells by western blotting. As shown in Figure 6, the expression levels of Cyclin D1 and MMP-9 were obviously down-regulated, while p16 (INK4A) and PTEN were up-regulated, in Lv-shCUL4B groups compared with Con and Lv-shCon groups. These results indicated that CUL4B might play an essential role in glioma growth and metastasis by modifying the expression patterns of regulatory proteins.To investigate whether CUL4B is involved in glioma tumorigenesis, endogenous CUL4B expression was depleted in glioblastoma cell lines U87 and U251 by RNA interference (RNAi). Knockdown of CUL4B by shRNA-expressing lentivirus significantly decreased cell proliferation and colony formation. Moreover, knockdown of CUL4B caused G1 phase cell cycle arrest in both cell lines via down-regulation of Cyclin D1 and up-regulation of p16. The expression of the tumor suppressor PTEN was increased in response to CUL4B knockdown. Furthermore, CUL4B depletion was able to alleviate in vivo tumorigenesis in glioma xenograft nude mice. Additionally, knockdown of CUL4B could also impede cell migration via suppression of MMP-9. Therefore, knockdown of CUL4B is likely to provide a novel alternative to targeted therapy of glioma and deserves further investigation.