The Research On The Expression Of CAGE Gene And Its Mechanism In The Intracranial Tumors

AbstractGlioma is the most commonly seen primary intracerebral tumor (35.26% -60.96% ). As a kind of malignant tumor , glioma is hard to be treated . It usually can not be completely evacuated and radiotherapy has been proved to be ineffective. Besides, chemotherapeutic agents are not likely to pass through BBB (blood brain barrier) easily. So relapse of glioma occurs frequently and the incidence is rising gradually . Therefore it is called for new diagnostic and therapeutic methods for glioma. One of the new methods is called cancer immunother-apy.The cancer - specific antigen is the premise to get the needed specific vaccine for cancer immunotherapy. Cancer/testis antigens (CTAs) are characterized by their restricted expression pattern, which is that CTAs are expressed in testis and occasionally in caner tissues , but not in normal tissues. Testis has no HLA molecules and CTAS expressed in it does not induce immunal response but CTAs in cancer tissues can induce specific immunological response It is then a-greed that CTAs have the remarkable property to be the ideal antigen, in cancer patients and due to the restricted expression pattern they are considered promising targets for caner vaccines Because of the heterogeneous expression of CTAs in different kinds of cancers the identification of the characteristics of expressed CTAs is helpful for the production of vaccines against different kinds of cancers.Epigenetic inheritance means the inheritable changes of gene expression from unchanged DNA sequence , the methylation of DNA has always been studied with the most efforts. And great achievement has been obtained . It was discovered that the methylation of 5'and CpG insular modulates gene expression invertebrate animas, The expression of almost all house - keeping genes and half tissue - specially expression genes is controlled by CpG insular methylation. It is now considered DNA methylation is one of the important mechanisms that control gene expression lots of experiments have proved that DNA methylation plays an important rote in control of certain part of CTA expression.Cancer - associated gene(CAGE)is a cancer/Testis antigen encoding gene located in XP22 It was discovered by Cho. et al in 2002 when he screened testis cDNA library with gene in highly expressed in gastrioma (89% ) cervical carcinoma (100% ) and hepatic cancer (80% ) , the expression rates in lung cancer and kidney cancer are 50% and 40% respectively. But no CAGE expression is detected in leukemia, pancreas cancer, breast cancer, and prostate cancer and melanoma cell lines. Until now there has been no report concerning CAGE expression in brain tumors. In this study the method of PCR and heimquantative RT - PCR were used to measure the expression of CAGE in normal tissues and brain tumors in order to explore the role of CAGE in brain tumor's occurrence . Methylation specific PCR was used to detect methylation degree of CAGE promoter in brain tumor for the aim of discovering whether there in any correlation between promoter methylation and CAGE expression CAGE antigen in serum was examined by westernblot to explore the feasibility of CAGE being used to screening.Methods Materials and1. MaterialsAll brain tumor samples were from the Department of Neurosurgery the 1st Clinical Hospital of CMU there were 63 gliomas ( male 38, female 25 ). Among the 63 gliomas 19 were grade I, 16 grade II , 15 grade, 13 gradeIV- There were also 35 meningiomas ( male 20, female 15 ). All patients received neither chemotherapy nor radiotherapy. Five normal brain samples were taken from none brain tumor people during biopsy. Other 17 normal tissue samples including 5 skeletal muscle samples, 1 liver sample, 1 kidney sample, 1 stomach sample , 2 spleen samples were from the Emergency and General surgery Departments ofthe same hospital. All samples were stored at - 7QX. right after being taken. In addition, 16 serum samples from glioma patients and 20 normal serum samples were also taken and stored at 4CC.2. Total RNA extractionTotal RNA was extracted with frizol and the concentration and purity were measured the quality of RNA was checked with agar gel electrophoresis which was pretreated with 1% formaldehyde. The extracted RNA was stored at -10X.3. Synthesis of cDNAcDNA was reversely transcripted from total RNA with reverse transcription kit ( promega A 3500).4. PCR amplificationSpecific CAGE primer ranging from cDNA077 - 382 bp was designed with primer 3 ,The amplified segment was 306 bp.GAPDH serves as internal control ( Gene Bank: ID: Bc026907) whose amplified segment was 267 bp. The production of PCR was put into 1% agar gel and was observed in ultraviolet light.5. Hemi - quantative RT - PCR.Brain tumor cDNA was diluted with TE to 1/4 and 1/8 of original concentration, And then RT -PCR was repeated with the diluted cDNA sample. The greyness was examined with Fluorchem V2.0 stand Alone. The ratio of CAGE/ GAPDH represents CAGE expression amount. The greyness ratio of each sample was the mean value of the sample of 3 different concentrations.6. Statistical analysis7. DNA extractionDNA was extracted with satuation salting out method.8. Treatmnt of DNA sampleThe extracted DNA sample underwent restriction, annihilation and treatment of sodium bisulfite . Then sodium bisulfite was cleaned out with wizard DNA clean - up kit. The purified DNA was collected for methylation specific a-nalysis.9. Methylation specific PCRThe primer was designed with meth - primer from the area where there were plenty of CpG in CAGE promoter The amplified production was 180bp for methy-lation specific amplification and was 176 bp for non - methylation specific amplification.10 . Induction of CAGE protein expressionthe sense phase AGECBL 21 ( DE3 ) of precombinant PET28 - a( + ) -CAGE carrier and 0.4mM IPTG were induced express at different temperature of 20X,, 25^ , 30T1, 33 Tl ,37^ lml induced bacteria solution was taken on the 1st, 2nd, 3rd, 4th hour during induction expression for SDS - PAGE electrophore-sis to decide the best induction temperature and time. Then the bacteria was cultured at the best condition.11. Extraction and purification of proteinThe cDNA carrying bacteria were split with splitting buffer to get the protein which was then purified with Co + containing resin affinity chromatography. . Then the concentration of protein was examined with Bradford and SDS - PAGE methods.12. CAGE protein underwent Western bloUThe induced and uninduced protein products underwent SDS - PAGE elec-trophoresis. Then the protein was transmitted to a new membrane and was hybridized with His - tag monoclonal antibody for later developing and observation.13. Serum CAGE antigen underwent Western blotThe purified CAGE protein underwent SDS - PAGE electrophoresis, the protein was transmitted to a new membrane and was hybridized with patients'ser-um for later developing and observation.Results1. CAGE expression in normal tissuesThe result of RT - PCR showed CAGE expressed only in testis and in other 7 normal tissues there was no CAGE mRNA.2. CAGE expression in brain tumorsFour meningiomas (11.43%) presented positive CAGE expression while57 gliomas(90. 48% ) presented CAGE expression there was significant difference of CAGE expression in the 2 kinds of tumors (x2 =59. 825, P <0.001).3. CAGE expression in glioma of different pathological gradesCAGE expression in grade I glioma in 0. 5 0. 09 the amount of internal control, grade HO. 85 ±0. 20, grade I 1. 85 ±0. 33, grade 1V2. 68 ±0. 44, which suggested CAGE expression was in positive correlation with malignant degree of glioma.4. CAGE promoter methylation in normal tissueResult of methylation specific PCR showed that methylation amplification production presented in normal brains .5. CAGE promoter methylation in meningioma and glioma28 meningiomas presented positive methylation amplification and 3 presented positive nor — meth ylation amplification. 4 samples presented positive amplification in both methylation and non - methylation while for glioma samples the amplification results in the 3 condi tions were 4, 50 ,9 respectively.6. Induced expression and purification of CAGE proteinThe result of SDS — PAGE electrophoresis showed that the best conditions for the highest amount of CAGE expression was as following : induction temperature of 33c€ , induction time of 4 hours, 0.4mM of IPTG. In this condition the protein presented as a single band in affinity choromtography and the concentration of the protein was 0.5mg/ml.7. CAGE protein examined by Western blotThe split solution of recombinant expression carrier pET28 - ( + ) - CAGE present as a single band at 58KD during electrophoresis, which is in consistent with calculation result. But the split solution of control group did not present the hybridization band.8. Examination of hybridization signal of patients'serumWestern blot was conducted to detect the hybridization signal of patients'serum against purified CAGE antigen . Tow out of 19 glioma patients'serum samples showed positive signal In the tow positive serum samples there was CAGE expression.Conclusion1. CAGE is a cancer/testis antigen which expresses only in testis and not in other normal tissues.2. Expression of CAGE in meningoma and glioma is statistically different. And the expression amount of CAGE is in positive correlation with glioma's grade.3. The methylation of the promoter area in glioma and meningioma of positive CAGE expression is less than of negative CAGE expression. Therefore the expression of CAGE is in connection with the less methylation of promoter.4. The serum CAGE antigen is less in the glioma samples which presented positive gene expression so it does not serve as a independent serum lable.