Study On The Correlation Between DNA Double Strand Breaks Repair Genes In Non-homologous End Joining Pathway And Human Glioma

PartⅠ: The mRNA expression of NHEJ pathway repair genes in human gliomaObjective: To detect the mRNA expression of five NHEJ pathway DNA repair genes LIG4,XRCC4,XRCC5,XRCC6 and XRCC7 in glioma tissues.Methods: Total RNA were extracted from 50 human glioma tissue specimens (including 30 cases of astrocytic tumors, 10 cases of oligodendroglioma and 10 cases of edpendymoma) and 10 traumatic brain tissue specimens and reversely transcribed into cDNA. The five target genes LIG4,XRCC4,XRCC5,XRCC6 and XRCC7 were amplified using TaqMan real time PCR and quantitation analysis of these NHEJ repair genes expression in glioma were made.Results: Compared with normal control brain tissues, the mRNA levels of five DNA repair genes in NHEJ pathway LIG4,XRCC4,XRCC5,XRCC6 and XRCC7 were all significantly decreased in glioma tissues (p＜0.05). And the expression levels of LIG4,XRCC4,XRCC5 were negative corelated to the malignance of glioma, while that of XRCC6,XRCC7 were positive correlated to the malignace of glioma.Conclusions: The five DNA repair genes: LIG4,XRCC4,XRCC5,XRCC6 and XRCC7 of NHEJ pathway which were down-regulated in human glioma tissue might play an important role in the induce and development of glioma. PartⅡ: The protein expression of NHEJ pathway repair genes in human gliomaObjective: To detect the related proteins expression of these five NHEJ repair genes LIG4,XRCC4,XRCC5,XRCC6 and XRCC7 using high throughput tissue microarrays and Western blot, and to further confirm the expression of these DNA repair genes down-regulated in glioma and provide more evidence for further gene polymorphism research.Methods: 300 glioma paraffin wax specimens were used which including 214 cases of astrocytic tumors (WHOⅠ39 cases, WHOⅡ58 cases, WHOⅢ57 cases, WHOⅣ60 cases), 49 cases of oligodendroglioma and 37 cases of ependymoma in the period of Jan. 2004 to Dec. 2005 from the Department of Neurosurgery of Huashan Hospital. Tissue microarrys were made to analyze the prtotein expression of five NHEJ pathway repair genes in glioma using immunohistochemical methods. Futher more, 16 glioma tissue specimens and 8 normal control brain tissue specimens were used to assay related proteins quantitively by Western blot.Results: High throughput immunohistochemical tissue arrays showed that all the NHEJ repair proteins encoded by corresponding genes were down-regulated expressed in all pathological types of gliomas compared with peri-tumor tissues (p＜0.05). To analyze the expressions in every pathological type of glioma respectively, only LIG4,XRCC4,XRCC6,XRCC7 proteins expressed differently in astrocytic tumors between tumor and peritumor (p＜0.05). But no statistical difference was found in oligodendroglioma and ependymoma. What's more, comparing these three pathological types of glioma, only the positive ratio and positive degree of XRCC7 were statistically different among astrocytic tumors, oligodendrogliomas and ependymomas (p＜0.05). And the Western blot results demonstrated that the protein levels of these NHEJ repair genes were all much lower than that of the normal controls. Conclusions: The results of high throughput immunohistochemical assay and Western blot, which were corresponding to that of real time RT-PCR in PartⅠ, further demonstrated that the five NHEJ repair genes were all down-regulated in glioma, and suggested that the NHEJ repair mechanism might play an important role in the pathogenesis of glioma.PartⅢ: Study on the association between tagging single nucleotide polymorphisms (SNPs) of NHEJ repair genes and the genetic susceptibility of gliomaObjective: To explore whether tagging single nucleotide polymorphisms (tSNPs) of the NHEJ repair genes are associated with the genetic susceptibility of glioma.Methods: 22 tagging single nucleotide polymorphism (tSNP) of XRCC5, XRCC6 and XRCC7 were investigated using a haplotype-based approach in 771 anticoagulated blood samples of glioma patients and in 752 that of healthy controls. All the tSNPs were genotyped by Allelic-specific PCR (AS-PCR) with the TaqMan universal PCR master mix and detected by ABI Prism 7900 sequence detector (PE Applied Biosystems). TaqMan MGB probes were used for allelic discrimination. SNPs, haplotype and linkage disequilibrium studies were performed by using the Case-control and Transmission Disquilibrium Test (TDT) approach.Results: (a) in single locus analysis, the genetic susceptibility of glioma was significantly associated with one XRCC6 tSNP (rs132771, P=0.044) and three XRCC5 tSNPs (rs828704, rs3770502 and rs9288516, P=0.005, 0.042 and 0.003, respectively); (b) in multi locus haplotype analysis, the genetic susceptibility of glioma was significantly associated with one protective XRCC5 haplotype " CAGTT " (odds ratio 0.60, 95% confidence interval 0.43 to 0.85); and (c) the positive gene-gene interactions of XRCC5, XRCC6 and XRCC7 on the genetic susceptibility of glioma were also detected using the multifactor-dimensionality reduction (MDR) method.Conclusions: Tagging SNPs of XRCCS, XRCC6 and XRCC7 genes involved in the DNA double strand breaks NHEJ repair pathway might contribute to the genetic susceptibility of glioma and play an important role in the etiology of glioma.