| Background:Gene function abnormalities involve two main mechanisms, that is genetic mechanism and epigenetic mechanism. As far as genetic mechanism is concerned, dozens of genes are involved in DNA damage repair to maintain genomic stability through different pathways. The genetic alterations of these genes may affect the function of their proteins and lead to diseases or cancers. Many studies have been limited by real-time quantitative PCR's ability to quantify only one gene at a time in a single RNA sample. TaqMan low-density array is able to determine the expression of multiple, user-defined gene clusters simultaneously with high sensitivity.The Human Epigenome Project was started formally after human genome project was accomplished completely. Epigenomics is widely thought to play the other half of a decisive role in the aetiology of virtually all human pathologies which controls the "open" and "close" of human genes in the world. Methylation is the main part of epigenomics, and constitutes so far missing link between genetics, disease and the environment. It promises to significantly advance our ability to understand and diagnose human malignant disease by identifying and interpreting DNA methylation patterns of human genes.Objective:To study the two main mechanisms of gene function abnormalities, that is genetic mechanism and epigenetic mechanism.1. As far as genetic mechanism is concerned, to identify specific therapeutic targets and prognostic indicators and to offer more information about clinical outcome associated with pathogenesis and prognosis of astrocytoma through TaqMan low-density array analysis of DNA repair genes.2. As far as epigenetic mechanism is concerned, to study the expression of the methylation status and their mRNA levels of tumor suppressor genes, their correlation with various clinical characteristics, and the relationship of the methylation status with their mRNA levels in human astrocytoma. To study the effects of demethylation agent on the methylation status and their mRNA levels of tumor suppressor genes as well as on apoptosis in tumor cells.Methods:1. We collected 88 primary astrocytomas (23 of grade II, 20 of grade III, and 45 of grade IV, according to the WHO Grading System) and 10 normal brain tissues. All the patients were followed up for 5 years after operation. 2. We choose primers and probes of 27 genes and GAPDH, a mandatory control online and configure the Micro Fluidic Card. And the 27 genes belong to five DNA repair pathways: MGMT in direct repair; MPG, MUTYH, NTHL1, OGG1, SMUG1, UNG, XRCC1 in base excision repair; ERCC1, ERCC2, ERCC3, ERCC4, NUDT1 in nucleotide excision repair; MLH1,MLH3, MSH2, MSH3, MSH6, PMS2 in mismatch repair; and MRE11A, MUS81, RAD50, RAD51, RAD52, XRCC3, XRCC4, XRCC5 in double-strand break repair. 3. We applied TaqMan low-density array to investigate the mRNA expressions of the 27 DNA repair genes and the control gene in 40 primary astrocytomas (10 of grade II, 10 of grade III, and 20 of grade IV) and 10 normal brain tissues among them by 7900HT sequence detection system. In addition, correlation of their mRNA levels with clinical characteristics was also analyzed. Real-time quantitative RT-PCR data were quantified using the SDS 2.2 software package. Gene expression values were conducted with the formulaΔCt and 2-ΔΔCt methods. 4. We analyzed the correlation of their mRNA levels with clinical characteristics in 40 primary astrocytomas and 10 normal brain tissues to determine their roles in astrocytomas, including age and gender. 5. The survival curves were calculated by the Kaplan-Meier method and the log-rank test was used to evaluate the correlation of their mRNA levels with prognosis of astrocytoma.6. The methylation status of promoter regions of LATS1, LATS2, PCDH-γ-A11, SLC5A8 and TMS1/ASC was studied by a methylation specific PCR (MSP) in these 88 primary astrocytomas, 10 normal brain tissues. 7. The mRNA expression levels of five tumor suppressor genes were determined by conventional RT-PCR and SYBGreen Real-time PCR assay in 30 primary glioma (10 of grade II, 10 of grade III, and 10 of grade IV) and 10 normal brain tissues among them. Relationship of methylation status of five genes with their mRNA levels and correlation of the methylation status and their mRNA levels with various clinical parameters were analyzed. 8. The demethylating agent, 5-Aza-2'-deoxycytidine (5-Aza-dC), was used to treat U251 and SHG-44 glioma cells, and then five genes' methylation status and mRNA expression levels were measured by RT-PCR and MSP. Cells were stained with Propidium iodide (PI), and were analyzed by flow cytometry (FCM) to determine the apoptosis rates after the treatment of 5-Aza-dC. Results:1. Analyzed by the 2-ΔΔCt method among these 27 genes, the expressions of 13 genes including ERCC1, ERCC2, ERCC3, ERCC4, MGMT, MLH1, MLH3, NTHL1, OGG1, RAD50, SMUG1, XRCC4, XRCC5 were significantly (P < 0.01) lower in astrocytoma at grade II, III and IV as compared to normal brain tissues. Among the 13 genes, the expression of MLH3 was significantly (P < 0.05) lower in grade III compared to grade II astrocytoma, and the expression of ERCC4 was significantly (P < 0.01) lower in grade IV compared to grade II astrocytoma. Meanwhile, the expressions of MSH2, MSH6, NUDT1 and XRCC3 were significantly (P < 0.01) lower in grade II and III astrocytoma. The expressions of MRE11A and MUS81 were significantly (P < 0.01) lower in grade III and IV astrocytoma. The expression of PMS2, RAD52 and XRCC1 were only significantly (P < 0.01) lower in grade III astrocytoma; UNG was only significantly (P < 0.01) lower in grade II astrocytoma. We did not find significant change for the expression of MPG, MSH3, MUTHY and RAD51 in any grade of astrocytoma (P > 0.05).2. There was no significant difference in the expressions of 27 genes between 29 male and 11 female primary astrocytoma patients (P>0.05). Also, there was no statistically significant (P>0.05) difference in the expressions of 27 genes among the astrocytoma at different age stage (age<40, 4060).3. The decrease expressions of eight genes were significantly (P < 0.05) associated with a poor prognosis, including ERCC3, ERCC4, MLH3, MRE11A, NTHL1, RAD50, XRCC4 and XRCC5.4. The promoter hypermethylation frequencies of LATS1, LATS2, PCDH-γ-A11, SLC5A8 and TMS1/ASC were 63.66%, 71.50%, 86.36%, 70.45% and 57.95% in 88 astrocytomas. But no methylation of LATS1, LATS2, SLC5A8 and TMS1/ASC promoter was detected, and the methylation frequency of PCDH-γ-A11 was only 20% in the 10 normal brain tissues. There were significant (P<0.05) differences in the methylation frequencies of five genes in grade II, III, IV of astrocytoma compared to normal brain tissues, partially among the astrocytomas at different pathological grade, including those of the SLC5A8 gene in grade II compared with grade IV (P<0.05). There were no significant differences between sex and age factors for LATS1, LATS2 and PCDH-γ-A11 (P > 0.05). But there were significant (P < 0.05) differences in those of SLC5A8 and TMS1/ASC between age<40 and age>60, 40< age < 60 and age> 60.5. The mRNA expressions of LATS1, LATS2, PCDH-γ-A11, SLC5A8 and TMS1/ASC were detected significantly decreased in different pathological grade astrocytoma compared to normal brain tissues(P < 0.05). There were significant differences in the methylation frequencies of SLC5A8 in grade II compared with grade IV, and TMS1/ASC in grade III, IV compared with grade II (P<0.05). There were no significant differences between sex and age factors for LATS1, LATS2 and PCDH-γ-A11 (P>0.05). But there were significant (P<0.05) differences in those of SLC5A8 between different ages and TMS1/ASC between age<40 and age>60,40< age<60 and age>60.6. And the mRNA levels of LATS1, LATS2, PCDH-γ-A11, SLC5A8 and TMS1/ASC in astrocytoma with hypermethylation were significantly (P<0.05) lower than those without methylation.7. LATS1, LATS2, PCDH-γ-A11, SLC5A8 and TMS1/ASC were hypermethylated in U251 and SHG-44 cell lines by MSP. By the 5μM 5-aza-dC treatment for 72h, both incomplete demethylation of the promoter CpG island and activation of transcription of five genes to high levels were achieved. The apoptotic rates after 24h, 48h and 72h treatment of U251 cells by 5-Aza-dC(5μM) were as follows: 8.99%, 24.40% and 46.75% and those of SHG-44 cells by 5-Aza-dC(5μM) were 13.85%, 32.35% and 62.50% which were higher than DMSO treated groups.Conclusions:1. The down-regulation of some DNA repair genes including ERCC1, ERCC2, ERCC3, ERCC4, MGMT, MLH1, MLH3, NTHL1, OGG1, RAD50, SMUG1, XRCC4 and XRCC5 may be associated with pathogenesis and poor prognosis of astrocytoma. TaqMan low-density array is an effective multivariate technique to examine the expression of DNA repair genes in astrocytomas, which can be applied to identify tumor-specific genes.2. The mRNA expressions of tumor suppressor genes LATS1, LATS2. PCDH-γ-A11, SLC5A8 and TMS1/ASC were down-regulated in astrocytoma by hypermethylation of the promoter. Hypermethylation of the promoter regions of tumor suppressor genes might play an important role in the anti-apoptosis and the maglinant development of astrocytoma. Demethylation agent may be a useful treatment for astrocytoma drug therapy.
|
PDF Full Text Request