The Study On P53-dependent Oncogene Wip1 Roles In Mechanism Of Human Malignant Brain Glioma Cells Proliferation

Objective Patients with gliomas often have mutations in the p53 gene. Wild-type p53-induced phosphatase 1 (Wip1 or PPM1D) encodes a negative regulator of p53. Wip1 over-expression inhibits p53 function and reduces selection for p53 mutations during cancer progression. Wip1 may have oncogenic function. To clarify the correlation of Wip1 with p53/p14ARF pathway disruption in glioma, the expression of Wip1 and p53/p14ARF pathway alterations have been investigated.Methods Tumor samples of 52 patients with primary glioams and 8 samples of normal brain tissues were examined for p53 mutations, p14ARF expression, and Wip1 expression. Direct sequencing of region from exons 5 to 8 of the p53 gene was performed on the genomic DNA in each tumor. The DNA methylation states of the CpG islands of the p14ARF gene were determined by methylation-specific PCR. All tumor specimens were analyzed for expression of Wip1 by real-time quantitative RCR, western blot and immunohistochemical staining.Results Disruption of the p53/p14ARF pathway was detected in 57.7%(30 in 52) of samples from patients with gliomas. Among the 22 cases without p53 and p14ARF alterations,11 (50%) had Wip1 mRNA over-expression. In tumors with p53 or p14ARF disruptions, Wip1 mRNA was over-expressed in only 1 case out of 30 (3.3%). Higher levels of Wip1 were associated with wild-type p53 but not with lower levels of expression of p14ARF or aberrant promoter hypermethylation of the p14ARF gene.Conclusion Wip1 is selectively over-expressed in tumors without alterations in p53 or p14ARF.Wip1 may inhibit the P53/p14ARF pathway.Objective To construct the lentiviral expression vector for RNA interference (RNAi) of Wip1 gene in glioma cells and select the optimal target sequence of Wip1 gene that is the most effective for RNAi.Methods Three double-stranded oligo DNAs were designed and synthesized according to the sequence of Wip1 gene and cloned into the pFU-GW-iRNA vector. After verification of the positive clones by PCR and sequence analysis, the verified plasmids were transfected into 293 T cells, the lenti virus was produced and the titer of virus was determined. The lentivirus was used to infect the glioma cell lines U251 and U-87MG. Real-time quantitative PCR and Western blot were performed to determine Wip1 expression levels in the virus infected glioma cells and the optimal interfering target was selected.Results Three recombinant lentiviral vector expressing shRNAs against Wip1 gene were obtained and confirmed by DNA sequencing. The titer of virus was 3-8×10 8TU/ml. Wip1 mRNA and protein expressions in U251 and U-87MG cells after infected with lentiviral vector were decreased remarkably. The mRNA expression in U251 cells was 36.3%,32.9% and 23.8% respectively, compared with the control group. And in U-87MG was 48.8%,36.7% and 23.7%. Western blot showed that Wip1 protein expression decreased 7 d after the infection significantly.Conclusion The lentiviral shRNA expression vector targeting human Wip1 gene capable of stable Wip1 gene silencing in glioma cells has been successfully constructed, which provides a basis for further study of mechanisms that Wip1 gene acts for malignant proliferation of glioma cells.Objective To detect the roles that Wip1 plays in the malignant growth of glioma cells and explore the possible mechanisms.Methods Glioma cells U251 and U-87MG were infected with Wip1 RNAi lentiviral vector. The proliferation activities of cells with Wip1 silencing were detected by cell counting kit-8 and clone formation experiments. Cell apoptosis was determined by flow cytometry AnnexinⅤ-APC/PI double labeling method and Hoechst 33258 staining. Cell cycle distributions were analyzed by flow cytometry PI staining. Cell invasiveness in glioma cells was determined by Transwell invasion assay. Real-time quantitative PCR was used to identify the mRNA expressions of differentially expressed genes after Wip1 silencing. Western blot was performed to detect the expressions of passageway proteins that Wipl may effect in. SPSS 13.0 software was used to analyze the significant difference.Results The expressions of Wip1gene in glioma cells were silenced effectively. Cells with Wip1 silencing, especially U-87MG cells, had reduced proliferation ability compared with mock cells. The number of apoptotic cells determined at 4 d after infection with Wip1 RNAi-R3 and NC lentiviral were not of the difference between them. At 7 d and 10 d, both U-87MG and U251 cells after Wip1 silencing showed significantly increased apoptotic cells compared with the mock cells. And the amount of apoptotic cells increased as the time periods extend. The apoptotic cells accounted for 52.2% and 67.1% of all the U-87MG cells with Wip1 silencing, while accounting for 26.1% and 38.2% in U251 cells. And U-87MG cells were more apoptotic than U251 cells 10 days after the RNAi-R3 lentiviral infection (P<0.05). Both U-87MG and U251 cells after Wip1 silencing had undergone extensive DNA strand breakage and nuclei-shrunk, characteristic changes of apoptosis. No significant changes of cell cycle distributions analyzed by flow cytometry were detected in U251 cells after Wip1 silencing. But increased S phase and decreased G1 and G2 phases were detected in U-87MG 10 d after the infection. U-87MG cells showed decreased invasiveness after Wip1 silencing. After Wip1 silencing, PIK3R1 gene mRNA expression was upregulated, while CDKN2A and p14ARF were downregulated in U251 cells. But in U-87MG cells, they all showed increased tendencies. Western blot showed that protein expression of p38MAPK in both U251 and U-87MG cells was increased, but expression of Akt was downregulated. Expression of p-Akt(Ser473)was only increased in U-87MG cells. The expressions of p53 and p-p53 (Ser 15) were both upregulated in U-87MG cell but remained unchanged in U251 cells.Conclusion Wip1 plays a important role in proliferation, apoptosis and invasiveness in glioma cells, and the effects are of much relevance with the fact that Wip1 could inhibit the function of p53.