| ObjectiveHuman gliomas are the most common tumor that arises in the central nervous system. Its incidence is 35.12%-61.10% in intracranial tumor. To date, the major clinical therapy methods are operation, radiotherapy and chemical therapy. As nearly half of gliomas are histologically malignant and most of gliomas are infiltrative growth, therapy of gliomas is one of the most formidable challenges in human malignancies. Therefore, studies of the molecular biology of the tumor are key issues to getting insight into the mechanisms underlying the pathogenesis of glioma and, in turn, leading to effective therapeutics finally. Many researchers have recently focused on gene therapy of tumor. So therapy target genes are considerable value for the therapy of glioma.PDCD4 (programmed cell death 4) is a new cell-apoptosis related gene. The research on animal and human tumor has been recently shown that it may be a new tumor suppressor gene and play an important role in tumor generation and development. It could inhibit the growth of tumor cells by inhibition of the gene transcription and translation. How about the status of PDCD4 expression and what effect of PDCD4 is on the progression of gliomas? And whether it could be the target gene for gene therapy of gliomas? These questions remain unknown.In present study, we analysed the relation between PDCD4 expression and tumor development from three main points:1. The expression state and clinical significance of PDCD4 in human gliomas.2. The relation between the methylation of PDCD4 5'CpG island and loss of PDCD4 mRNA.3. Exogenous PDCD4 expression and effect of PDCD4 on apoptosis, cell cycle and colony formation in glioma cell lines.Methods1. The expression status and clinical significance of PDCD4 in human gliomas.(1) A total of 91 glioma specimens including 30 frozen and 61 paraffin-embedded tissues were obtained from patients aged between 30 and 60 years (median = 40 years) who underwent operations at the Department of Neurosurgery in Qilu Hospital of Shandong University from October 2003 to March 2006. The pathological diagnosis was made according to the lately World Health Organization (WHO) criteria for gliomas. None of the patients studied had received adjuvant immunosuppressive treatments such as radiotherapy or chemotherapy prior to surgery in order to eliminate their effects on gene expression. Three normal nervous tissue samples were excised from tumor-adjacent sites.(2) The PDCD4 mRNA and protein expression in 91 cases of glioma specimens, 3 normal tissue specimens and glioma cell line cells were detected by RT-PCR, Western blot and immunohistochemistry.(3) The relationship between the PDCD4 exprssion and the pathological characteristics was analyzed by statistics. Cumulative survival time was calculated by the Kaplan-Meier method and analyzed by the log-rank test, p < 0.05 was considered statistically significant. Multivariate survival analysis was performed using the Cox regression model for all 84 astrocytomas. The calculations were performed using the SPSS statistical software.2. The relation between the methylation of PDCD4 5'CpG island and loss of PDCD4 mRNA.(1) Genomic DNAs were isolated from microdissected tissue samples and cultured cells using the phenol: chloroform extraction method. 1 ug of genomic DNA was treated with sodium bisulfite in a CpGenomeTM Fast DNA Modification kit. The DNA methylation status of the PDCD4 regulatory sequences was determined in glioma samples and cell lines by Methylation-Specific PCR (MSP).(2) To further detect the DNA methylation status of PDCD4, we use 5-aza-2' -deoxycytidine to treat the glioma cell lines, U251 and U87. To detect the restoration of PDCD4 expression by blocking methylation in glioma cells, RNA, DNA, and protein were extracted for reverse transcription-PCR, methylation analysis, and protein analysis. (3) We analysed that the relation between the methylation of PDCD4 5'CpG island and the loss of PDCD4 mRNA by Pearson's coefficient.3. Exogenous PDCD4 expression and effect of PDCD4 on apoptosis, cell cycle and colony formation in glioma cell lines.(1) To determine the effect of PDCD4 on the growth and survival of glioma cells, a recombinant plasmid carrying the full-length PDCD4 cDNA was transfected into PDCD4-nagative U251 and U87 glioma cells. The analysis of PDCD4 expression after transfection was by RT-PCR and Western blot.(2) The morphology detection of the glioma cell lines after PDCD4 transfection by fluorescence inverted microscope.(3) After U251 and U87 cell lines were transfected with PDCD4 vector, the apoptosis and cell cycle progression were analyzed by flow cytometry.(4) After U251 and U87 cell lines were transfected with PDCD4 vector, the colony formation were analyzed by Colony Formation Assay.Results1. The expression status and clinical significance of PDCD4 in human gliomas.(1) Lacked expression of PDCD4 at mRNA and protein levels in glioma cell lines.We firstly found that U251 and U87 cell lines derived from glioma lacked PDCD4 expression at mRNA and protein levels detected by semi-quantitative RT-PCR and Western blot. It suggested that the PDCD4 may play role in gliomas.(2) Lacked or decreased PDCD4 expression in primary glioma samples.To explore further role of PDCD4 in gliomas, PDCD4 expression at mRNA and protein levels in primary gliomas was also detected by semi-quantitative RT-PCR, Western blot and immunohistochemistry. All of three normal brain tissues adjacent to tumor lesion expressed high level of PDCD4 mRNA, whereas the most of gliomas expressed reduced or lacked PDCD4 mRNA. In 30 glioma specimens, 14 cases (47%, 14/30) lost PDCD4 mRNA expression and in other 16 glioma samples, 12 cases (40%, 12/30) decreased PDCD4 mRNA expression, compared with tissues adjacent to tumor. In 30 cases of the gliomas, 23 cases (77%) exhibited no relevant PDCD4 protein compared with normal brain tissues adjacent to tumor, which showed strong PDCD4 expression by western blot. In addition, 6 cases desreased PDCD4 expression in other 7 cases which PDCD4 expressed.Taken together, 82% (75 of 91) of glioma tissues we examined to date lacked detectable PDCD4 protein expression by immunohistochemistry, indicating that loss of PDCD4 expression is a frequent event in primary gliomas.(3) PDCD4 expression serves as an important prognostic biomarker for patients with gliomas. And the loss of PDCD4 in gliomas had no signification relationships with tumor grade or histological type.To detect the prognostic value of PDCD4 expression in primary gliomas, we studied a large cohort of patients with the disease. No significant correlation between PDCD4 expression and age, histological type or pathological grade was detected. However, there appeared to be a tendency for PDCD4 expression to be associated with gender. The expression of PDCD4 in gliomas from male patients was significantly higher than that from female patients (p = 0.0245). More importantly, the expression of PDCD4 significantly correlated with the long-term survival rate of patients. The survival rate of patients with PDCD4-expressing gliomas was significantly higher than that of patients with PDCD4-negative gliomas of grade III or higher (p = 0.0013). The difference for patients with low grade of gliomas (grade I-II) was not statistically significant. Thus, PDCD4 expression in primary gliomas can serve as an important biomarker for prognosis. To examine whether the PDCD4 silencing was an independent unfavorable prognostic factor for patients, we performed a multivariant Cox regression analysis, including gender, age, histological types, grade, and PDCD4 expression (Supplementary Table). The level of PDCD4 expression could significantly predict the patient outcome independent of other clinicopathologic variables for disease-specific survival (relative risk, 0.063; 95% confidence interval, 0.014-0.276,p < 0.0001).2. The relation between the methylation of PDCD4 5'CpG island and loss of PDCD4 mRNA.The previous results suggested that frequent loss of PDCD4 expression existed in gliomas. Loss of PDCD4 expression also existed in other types, such as lung cancer, pancreatic cancer and hepatoma. However, to date the reasons of loss of PDCD4 expression were unknown. So we based on the above research and analysed the relation between PDCD4 5'CpG island and PDCD4 mRNA expression.(1) The methylation of PDCD4 5 'CpG island was found in glioma cell lines.Promoter hypermethylation is a common epigenetic mechanism implicated in the silencing of tumor suppressor genes in human cancers. A typical CpG island in the upstream of the translation start codon (ATG) of the PDCD4 gene was found using the MethPrimer software (http://www.urogene.org/methprimer/index1.html), indicating that PDCD4 may be vulnerable to methylation-mediated silencing. To test this hypothesis, genomic DNAs from glioma cell lines were purified and used as templates for methylation-specific PCR after bisulfite modification. Significant methylation of the PDCD4 promoter was detected in both of the PDCD4-negative cell lines, U251 and U87, but not in the PDCD4-positive normal glial tissue.(2) The methylation of PDCD4 5 'CpG island was found in primary gliomas. Similarly, of the 30 primary glioma tissues tested, 14 (47%) of them showed clearPDCD4 CpG island methylation whereas no methylation was detected in the three normal brain samples. Of 14 methylation samples, 5 samples were exhaustive methylation and 9 cases were partial methylation. To confirm the specificity of the methylation-specific PCR, the products of MSP for the part of primary gliomas were inserted into a T-A clone vector and sequenced. Multiple methylated sites were found under the methylation condition but not under the unmethylation condition.(3) CpG island methylation of the PDCD4 promoter is associated with loss of gene expression in the glioma cell lines and primary glioma tissues.We next examined the relationship between PDCD4 promoter methylation and PDCD4 gene silencing. All normal brain tissues or gliomas which expressed PDCD4 showed no PDCD4 promoter methylation. In contrast, giloma cell lines and the majority of glioma tissues which expressed no PDCD4 at mRNA level but showed clear PDCD4 promoter methylation. The correlation between PDCD4 promoter methylation and PDCD4 gene silencing at mRNA level is statistically significant (r= -1.000,p<0.0001) by Pearson's coefficient.(4) Blocking methylation in glioma cells effectively restores PDCD4 expression.To determine whether CpG island methylation of the PDCD4 promoter causes the loss of PDCD4 expression, glioma cell lines that had the PDCD4 promoter methylation (U251 and U87) were exposed to increasing concentrations of the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine. The methyltransferase inhibitor significantly decreased the methylation. Remarkably, blocking methylation resulted in a substantial restoration of PDCD4 expression at both mRNA and protein levels by RT-PCR and Western blot. The restored PDCD4 expression in glioma cells was also detected by immunocytochemistry. These results establish that PDCD4 gene silencing at mRNA level in glioma cells was directly caused by CpG island methylation.3. Exogenous PDCD4 expression and effect of PDCD4 on apoptosis, cell cycle and colony formation in glioma cell lines.To further determine effect on tumor progress of PDCD4 expression, we restored PDCD4 expression in glioma cell lines by transfection of exogenous PDCD4 and analyzed its effect on tumor growth, apoptosis, cell cycle and colony formation.(1) Restoring PDCD4 expression in glioma cells inhibits their growth.To determine the effect of PDCD4 on the growth and survival of glioma cells, a recombinant pDsRed plasmid carrying the full-length PDCD4 cDNA was transfected into PDCD4-nagative U251 and U87 glioma cells. Forty-eight hours later, both the fluorencent marker and PDCD4 were detected in the glioma cells. Importantly, PDCD4 expression in these cells significantly inhibited their growth, compared with untreated control and mock-transfected groups (p< 0.05).(2) Restoring PDCD4 expression in glioma cells prevents their colony formation.Glioma cells, unlike normal glia cells, have the tendency to form colonies in the culture. Remarkably, PDCD4-transfection significantly diminished the capacity of these cells to form colonies (86.47%±1.10). Specifically, compared with the mock control, the expression of PDCD4 caused reduction in the number of colonies formed by U251 cells (p<0.01). And PDCD4-transfection significantly diminished the capacity of U87 cells to form colonies (32.68%±0.34) compared with the mock control (p<0.01). Thus, PDCD4 expression reduces the growth and colony formation capacity of glioma cells.(3) Restoring PDCD4 expression in glioma cells inhibits their cell cycle.To further determine the mechanism of inhibition of tumor growth, we analysed the effect on cell cycle of PDCD4 expression. Cell cycle analysis further revealed that PDCD4 expression significantly reduced the number of cells in the G2/M phase of the cell cycle. PDCD4-transfection significantly diminished the G2/M phase of the cell cycle of U251 cells (6.23%±0.91) compared with the mock control (10.6%±1.65) or negative control (12.6%±1.91) (p<0.05). However, PDCD4-transfection significantly upregulated the S phase of the cell cycle of U251 cells (27.9 %±1.38) compared with the mock control (25%±0.96) or negative control (18.1%±1.25) (p<0.05).(4) Restoring PDCD4 expression in glioma cells induces their apoptosis.After PDCD4 transfection for 48 h, Annexin V-FITC/PI analysis clearly showed that PDCD4-transfected glioma cells underwent more apoptosis (12%) than the mock control (5.3%) or negative control (0.1%) (p < 0.05). Furthermore, TUNEL analysis also clearly showed that PDCD4-transfected glioma cells appeared conspicuous nuclear fragmentation or nuclear condense and underwent more apoptosis than themock control or negative control (p < 0.05).Conclusion1. PDCD4 is frequently silenced in primary human glioma tissues.We firstly found that loss of PDCD4 expression is a frequent event in human glioma. PDCD4 expression serves as an important prognostic biomarker for patients with gliomas with grade III or higher. And the level of PDCD4 expression could significantly predict the patient outcome independent of other clinicopathologic variables for disease-specific survival2. 5'CpG island methylation is the direct reason of PDCD4 frequent silenced in glioma cell lines and primary human glioma tissues at mRNA level.PDCD4 5'CpG island methylation of the PDCD4 promoter is associated with loss of gene expression in the glioma cell lines and primary glioma tissues. Blocking methylation in glioma cells effectively restores PDCD4 expression at both mRNA and protein levels by DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine.3. PDCD4 could distinctly inhibit the growth and colony formation capacity of glioma cells in vitro.Exogenous PDCD4 expression in glioma cells inhibits their growth and prevents their colony formation. The mechanism might be the interference of cell cycle and induction of apoptosis.Originality1. We demonstrate, for the first time, that loss of PDCD4 expression is a frequent event in human glioma and that the expression of PDCD4 significantly correlated with the long-term survival rate of patients. And we found, for the first time, that restoration PDCD4 expression in glioma cells inhibits their growth, induces their apoptosis and inhibit cell cycle.2. Though many researchers found that loss of PDCD4 expression existed in other type of tumor, the reasons were unknown to date. We demonstrated from epigenetic point, for the first time, that 5' CpG island methylation of PDCD4 is the direct reason of the silencing of mRNA expression in glioma cell lines and glioma tissues.Limitations of this study1. The loss or absence of PDCD4 expression may be a key factor to the tumorigenesis of glioma. But the phenomenon that PDCD4 could induce the redifferentiation of the glioma cells and the precise mechanism of induction needs to be investigated further.2. The mechanism of decreased PDCD4 expression in gliomas needs further to be studied.3. The mechanism of signal transduction for PDCD4 to inhibit the growth of glioma cells need further to be demonstrated.
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