The Expression Of MTOR In Human Glioma And Their Significance

IntroductionBrain glioma is the most common primary central nervous system tumor,which accounts for about 50%to 60%of adult primary brain tumor.Surgical therapy is the most important in the treatment.Because of the tumor infiltrating growth,the majority of tumor and normal brain tissue have no clear boundaries.It is hardly to completely removed so it needs radiotherapy,chemotherapy,and other complementary therapies after operation.Even a comprehensive treatment of glioma,recurrence is almost inevitable for the biological characteristics of glioma.TOR(target of rapamycin, rapamycin target protein)was found firstly in mutant yeast TOR1-1 and TOR2-1 in 1991.In 1994,the mammals TOR gene was confirmed and cloned by Brown,and it has only one protein product which is mTOR(mammalian target of rapamycin,also known as FRAP,RAFT,RAPT).Human mTOR gene locates in 1p36.2,encoding a protein with the molecular weight of 280 kDa composed by 2549 amino acid residues.Research shows that mTOR plays a central control point role in cell proliferation,growth, differentiation.In addition,several studies confirm that it also plays an important role in the formation and development process of the tumor.This study by using reverse transcriptase polymerase chain reaction(RT-PCR),and the western blot method to study the expression of mTOR in malignant glioma and the relationship between the histological grades.Experimental materialSelect the surgical resection of brain tissue samples from 35 patients with glioma from December 2006 to June 2007 in the First Affiliated Hospital of China Medical University,including 21 cases of men,14 cases of women,age from 20 to 74 years old, on average age of 46 years old.According to the WHO(2001)on human glioma pathological classification methods,includeⅠgrade 6 cases,Ⅱgrade 15 cases,Ⅲgrade 9 cases,Ⅳgrade 5 cases.All patients were not receiving preoperative radiotherapy,chemotherapy or any other treatments likely to interfere with the biological characteristics of the tumor.Main Reagent:PCR primers(Dalian-biological company);DNA marker (Dalian-biotechnology company);nitrocellulose membrane(the Millipore Corporation,United States);rabbit anti-human mTOR antibody(Biochemicals company,United States);alkaline phosphatase-labeled goat anti-rabbit antibody (Beijing Zhongshan company);mouse anti-β-actin(Sigma company,United States).Major equipment:High-speed refrigerated centrifuge(Sigma 3C15K United States);PCR Instrument(Biometra UnioⅡtype,Germany);Electrophoresis System (Beijing June 1 Instrument Factory);UV spectrophotometer(UV3000,United Kingdom);automatically gel imaging and analysis system(Chemi Imager, USA);transfer electrophoresis bath(Beijing June 1 Instrument Factory).Experimental methods1.RT-PCRUsing Trizol reagent one-step method extracts Total RNA,UV spectrophotometer determinates the purity and quantitative.According to the mTOR andβ-actin mRNA sequence provided by the GenBank,design primers by Primer Premier 5.0 software applications.Reaction conditions:94℃for 5min,a cycle;94℃for 30 s,55℃for 30 s,72℃for 1min,35 cycles;72℃for 5min,finish amplification reaction.PCR products mixed with sample buffer,a 2%agarose gel electrophoresis,120 V,30min, after the end of electrophoresis,use gel imaging device to analyze image.2.Western blotExtract protein samples,determinate protein quantitative and adjust sample concentration,electrophoresis,transfer,use anti-mTOR and anti-β-actin antibody incubation,finally join the alkaline phosphatase-labeled secondary antibody,add color reagent then record the results.Finally use gel imaging devices to analyze image.3.Statistical analysisGel image analysis system for gray-scale analysis.The expression of mTOR gene is demonstrationed by(mTOR optical density value/β-actin absorbance value)×100%,and the results is demonstrationed by(?)±s.Applicate SPSS13.0 statistical analysis software to analyze.The two groups are compared with t-test,and the two samples rate are compared with the of X~2 test,and p＜0.05 with statistical significance.The experimental resultsThe results of this study show that:30 cases of human glioma samples have the mRNA and protein expression of mTOR,and the positive rate of mTOR from gradeⅠto gradeⅣhave no significant differences.With the degree of malignancy increased, the mRNA and protein expression of mTOR also increase.The mRNA and protein expression of mTOR mutually compared in theⅠ,Ⅱ,Ⅲ,Ⅳgrade,the differences are significant(p＜0.01).DiscusstionThe pathogenesis of glioma is very complex.A lot of basic researches expect to seek a breakthrough in the treatment of glioma from the level of gene.mTOR is found in recent years,which is a protein kinase family with very conservative on the evolution,and widely exists in various biological cells.Recently the study of its complex function provides a reasonable explanation of the tumor and the metabolic disease mechanism.Past studies show that the majority of tumor tissue have higher expression of mTOR,such as prostate cancer,breast cancer,liver cancer,parotid gland carcinoma,cholangiocarcinoma,leukemia and so on.Above results suggest that the high expression of mTOR has closely relation to tumor formation and cell proliferation active.The experimental results show that With the degree of malignancy increased, the mRNA and protein expression of mTOR also increase.The mRNA and protein expression of mTOR mutually compared in theⅠ,Ⅱ,Ⅲ,Ⅳgrade,the differences are significant(p＜0.01).Rapamycin and its derivatives as the mTOR specific inhibitor has aroused extensive concern in the treatment of cancer,which on prostate cancer, breast cancer,gastrointestinal stromal tumor therapy has entered the stageⅠandⅡstage clinical trials.Therefore in-depth study of the mTOR may help to enrich human glioma development mechanism,as well as for providing new ideas for the treatment of glioma.Conclusion1.There exists an up-regulation of mTOR expression in human glioma.2.The up-regulation of mTOR expression may be involved in the formation and development of glioma.3.With the degree of malignancy increased,the mRNA and protein expression of mTOR also increase.