Using Bioinformatics Method, Impact Of Id2 On Chemosensitivity In Human Glioblastoma

Glioblastoma (GBM) is the most common malignant tumor of the central nervous system and accounts for approximately 45～55% of gliomas. The mean survival of GBM is less than one year. As GBM is highly invasive and prone to angiogenesis, complete surgical resection is difficult. Chemotherapy is therefore an important adjuvant treatment for GBM patients by killing residual tumor cells and prolonging the survival duration of patients who have undergone partial tumor resection in particular. GBM is not satisfactory for improve in survival time and quality of life after chemotherapy. Resistance to chemotherapeutic drugs is an important reason for low efficiency. The survival duration of GBM patients with similar ages and KPS scores varies greatly after the same treatment. The reason may be due to different tumor moleculogy in these patients.Gene microarrays were used to study tumor and drug activity-related genes,3 glioma gene chips have been made in our lab, the greatest difficulty is how to make scientific analyses of the huge amounts of complex data obtained and determine the main responsible genes. The present study made use of bioinformatics method to analyze the data obtained from microarrays in combination with the complete data about survival of GBM patients in an attempt to investigate CCNU sensitivity-related genes in GBM patients. We believe that using these genes to assess the prognosis of GBM patients would be more accurate than conventional histopathology, and more helpful in working out more specific and individualized targeted treatment regimens according to genetic characteristics of individual patients.Through the bioinformatics analysis,29 core genes related to glioblastoma survival time and chemotherapy sensitivity were screened out. Among of them, inhibitors of differentiation 2 (Id2) become the object to next test. Id2 is a member of family of expressed transcription factor of cell differentiation and proliferation, belonging to helix-loop-helix (HLH) protein family members. Id2 has been demonstrated to control cell cycle, cell proliferation, to promote embryo and organ formation, to inhibit cell differentiation, induce apoptosis, to maintain cell survival and promote blood vessel formation, and play an important biological role in tumorigenesis. At present, research on the Id2 in glioma tumorigenesis and biological behavior was few reported.This study was designed to detect the expression of Id2 in various pathological grade brain gliomas and malignant glioma cell lines, to construct Id2 gene RNA interference lentiviral vector. To knock-down Id2 through siRNA interference in U87 cell lines, change of sensitivity to chemotherapeutic drugs Me-CCNU, VM26, TMZ is to be detected. The research would show great promise of individualized treatment programs and a new molecular target to GBM. This study is divided into four parts: the first part is to use bioinformatics method to investigate the genes related to chemosensitivity in human glioblastoma, the second part is to investigate the expression of Id2 in astrocytic tumors and glioma cell lines, the the third part is to construct and identificate lentiviral vector of RNAi of Id2 gene, and last part is to knock-down expression of Id2 in U87 cell line by lentivirus-mediated RNAi and to observe its effect on Me-CCNU, VM26, TMZ chemosensitivity.PartⅠUsing Bioinformatics Method to Investigate the Genes related to Chemosensitivity in Human GlioblastomaObjective:In this part of study, bioinformatics method was used to analyze data of cDNA microarray to investigate the genes related to survival time and drug sensitivity in GBM.Methods:Biostar and Affymetrix Microarray of GBM patients were analyzed, and clinical data of these patients in the microarray were perfected through long-term follow-up study. According to survival time of 12 months, the samples were divided into two groups. Differential expression genes between the long- and short- survival groups were picked out, GO-analysis and pathway-analysis of the differential expression genes were performed. Drug-related signal transduction networks were constructed. The methods combined three steps before were used to screen core genes that influenced chemosensitivity.Results:In 2003 Biostar microarray,2007 Biostar microarray and 2007 Affymetrix microarrays, there were 429,74,2018 differentiate genes that influenced survival duration of GBM respectively. They mainly participated in 108 gene functions and 94 signaling transduction pathways, based on which 29 core genes that influenced chemosensitivity of GBM to Chemistry drug were obtained.Conclusion:With complete clinical information, mass data of cDNA microaray can be further analyzed by using bioinformatics method.29 genes were found to predict the clinical outcomes and have a significant influence on chemosensitivity in GBM. This study will provide a foundation not only for the further functional research but also for prediction of prognosis and fulfillment of personalization chemotherapy in GBM.PartⅡExpression of Id2 in Astrocytic Tumors and Glioma Cell LinesObjective:To investigate expression level of Id2 mRNA and protein in normal brain tissues, astrocytomas of different grades and U87, U251 and SHG-44 Glioma Cell Lines.Methods:The expression levels of Id2 mRNA were evaluated by real-time quantitative PCR in 28 astrocytic tumors,4 normal brain tissue samples and U87, U251 and SHG-44 Glioma Cell Lines. By using immunohistochemistry, expression levels of Id2 protein were assessed in 56 astrocytic tumors of different pathological grades and 4 normal brain controls. By using Western blot, expression levels of Id2 protein were assessed in 28 astrocytic tumors of different pathological grades and 4 normal brain controls.Results:Quantitative real time PCR analysis demonstrated elevated expression levels of Id2/β-actin in high-grade astrocytomas versus low-grade (59.71±43.82vs10.24±8.46; P=0.000) and gliomas vs normal brain tissues(43.53±35.32 vs 1.89±1.16; P=0.000). It also demonstrated elevated expression levels of Id2/β-actin in U87, U251 and SHG-44 glioma cells versus astrocyte cell line(33.77±21.54,30.20±20.13 vs 1.00±1.57;P=0.000). Further, Id2 immunoreactivity was predominantly detected in the cytoplasm of tumor cells, whereas no positive staining for Id2 was observed in normal brain tissues. Statistical analysis showed Id2 increase in high-grade astrocytomas versus low-grade tumors (10.53±5.79% vs 4.232.85%; P=0.000) and gliomas vs normal controls (7.90±5.70% vs 0.0±0.0%; P=0.000). Western blot analysis showed elevated expression levels of Id2 in high-grade astrocytomas versus low-grade (1.17±0.84 vs 0.33±0.18; P<0.05) and gliomas vs normal brain tissues (0.61±0.48 vs 0.09±0.07; P<0.01), and elevated protein expression levels of Id2 in U87, U251 and SHG-44 glioma cells versus astrocyte cell line(1.25±0.89vs 0.49±0.35, P<0.05).Conclusion:Id2 is present in different grade gliomas and its expression levels significantly increase at both mRNA and protein versus normal brain tissues. There existed significant difference in both mRNA and protein leves of Id2 between high-grade astrocytomas and low-grade. Id2 is also present in glioma cell lines and its expression levels significantly increase at both mRNA and protein levels versus astrocyte cell line. These data suggest that overexpression of Id2 may serve as one important molecular mechanism that underlies the development and progression of astrocytic tumors.PartⅢConstruction and Identification of Lentiviral Vector of RNA Interference of Id2 GeneObjective:To construct a lentiviral vector of RNA interference(RNAi)of human Id2 gene.Methods:Four shRNA sequences targeting human Id2 gene were designed and then the complementary DNA containing both sense and antisense Oligo DNA of the targeting sequence was designed, synthesized and cloned into the pGCSIL-GFP vector, to construct a lentivirus vector which expressed short hairpin RNA (shRNA), and it was identified by PCR and DNA sequencing.Results:PCR identification and DNA sequencing demonstrated that insertion of oligonucleotide of the lentivirus RNAi vector containing human Id2 shRNA was effective.Conclusion:The high efficient lentivirus RNAi vector of Id2 was constructed successfully as the basis for further study of its influence on chemosensitivity in GBM.PartⅣSuppression of Brain Glioma Cells Expression of Id2 by Lentivirus-mediated RNAi and its Effect on Me-CCNU, VM26, TMZ ChemosensitivityObjective:To investigate the Me-CCNU, VM26, TMZ chemosensitivity change in U87 glioma cell after Id2 inhibition by lentivirus-mediated RNA interference. Methods:Lentiviral vectors of Id2 siRNA was transfected into brain glioma cell line U87, the expression of Id2 was determined by western blot. The effects of drugs on proliferation of RNAi, negative and control cells were assessed by MTT assay.Results:The expression level of Id2 protein was knocked down by Id2 siRNA as indicated by western blotting analysis. MTT assay showed the inhibition rate of U87 glioma cells cultured in Me-CCNU, VM26, TMZ was significantly higher in Id2-shRNA group (39.75%±3.62%,40.6%±7%,20.03%±2.56%) compared with that of vector control (22.24%±6.77%,16.73%±5.44%,8.4%±2.26%, p<0.05).Conclusion:RNAi vectors of Id2 could effectively suppress its exprssion in U87 cell line, and enhance the sensitivity of glioma cells to Me-CCNU, VM26 and TMZ. Given the great impact on sensitivity to chemotherapeutics drugs of GBM, knock-down of Id2 expression may therefore represent a potentially individualized treatment strategy against GBM.