| Many research detected that post-translational modification of proteins is observed in all known living organisms, and it is the chemistry modification to new synthetic polypeptide chains or proteins. Post-translational modification might regulate many functions of proteins (such as location, stability and specificity) and life. There are many phenomenon of post-translational modifications, such as phosphorylation, acetylation, methylation, ubiquitylation, sumoylation, lipidation and so on. Thirty years ago, many studies showed that protein methylation is the most common type of post-transcription modification in eukaryocyte cells, and it contains arginine methylation and lysine methylation. Argnine methylation is mainly catalyzed by protein arginine methyltransferase (PRMT), which widely spread among nucleus, cytoplasma and organelle. PRMTs take part in different process of cell growth, such as post-transcriptional regulation, RNA metabolism process, DNA damage and so on. PRMT1is the first cloned protein among protein arginine methyltransferase family, and it was reported that PRMT1is thought to contribute to as much as85%of all cellular PRMT activity. The expression of PRMT1was prevalence, and could be identified in almostly every mammals'organs. As reported, PRMT1was highly expressed in many kinds of tumor cell lines and tumor tissues, such as bladder cancer, gastric carcinoma, lung cancer, leukocythemia, testis carcinoma, esophagus cancer and pancreatic carcinoma, and it was proposed that PRMT1play important roles in cell cycle, RNA processing and DNA duplication. All of these showed that PRMT1might involve in human tumorgenesis.Glioma is the most common one of brain tumor and with high-malignancy. It occurs from neuroectodermal, and its incidence accounts for40%-50%of brain tumor. According to the beginning, glioma could be divided to astrocytoma, glioblastoma, medulloblastoma, ependymoma, spongiocytoma and so on. On the basis of pathology biology behavior of glioma, WHO proposed that glioma could be divided into I to IV grade. Due to the high-malignancy of glioblastoma, it was pointed into IV grade. Although the technologies of surgery and radio treatment have made great process, the treatment of glioma, especially high-malignancy of glioblastoma, was an annoying problem troubled with medical treatment. The statistic research showed that even if the patients of gliomblastoma were treated by the therapeutic alliance, which combined operation with radio treatment and chemotherapy, the middle survival only are two years after diagnose. So it is more important to study the pathogenesis mechanism of glioma, and to find the right drug target to promote the effect of treatment and prolong the survival time. The study of methylation in gliom is concentrated on DNA methylation and its influence, but the research on arginine methylation in glioma is little concerned. In our research, we utilized Western blotting, and found that the protein expression of PRMT1were much higher in4types of glioma cell lines (T98G, U87MG, A172and U251) than in normal brain tissues. Also among17human glioma tissue samples (5samples were Grade II,7samples were Grade III and5samples were Grade IV), the expression of PRMT1were increased in13samples, compared with normal brain tissues. Then by Real-time PCR, we detected the mRNA relative expression of PRMT1in glioma cells and normal human astroglia cell Heb, and the data showed that PRMT1was much lower in Heb than glioma cells. Futhermore, by immunohistochemistry on one Grade II glioma patient, the results showed that the expression of PRMT1in glioma tissue area was higher than the tissue adjacent to glioma. From the data of RNA level and protein level of PRMT1were much higher in glioma cells or glioma tissues, and PRMT1is one kind of protein arginine methytransferase. It indicated that the higher expression of PRMT1might effect the level of methylation of target protein, and the changes of methylation level would take part in the growth of glioma. Based on these proposal, we did celluar functional study to discuss the impact of PRMT1to the growth of glioma, and used mass spectrum to screen the candidate targets of PRMT1.First of all, the sequences of specifical StealthTM RNAi directed at PRMT1were synthetized and transfected with four types of glioma cells. Then by MTT, flow cytometer, TUNEL test, scratch test, Transwell test and nude mouse xenografts experiment, we detected the influence on glioma cell proliferation, apoptosis and migratory after knocking down PRMT1. It indicated that knock down of PRMT1would slow down the speed of glioma cell proliferation; increase the ratio of apoptosis cells; block the G1phase to S phase in cell cycle; but not affect the cell migration. After that, the specifical antibodies, which were directed at asymmetric arginine dimethylated proteins or asymmetric dimethylated Histone H4arginine site3, were utilized to detect the glioma cells and normal brain tissues. The data indicated that the level of asymmetric arginine dimethylation was mostly higher in glioma cells, and also the level of dimethylated Histone H4R3was increased. Their tendencies were as same as the expression of PRMT1. After knock down of PRMT1, the level of asymmetric arginine dimethylation was apparently decreased. It showed that PRMT1might regulate the glioma cell growth through asymmetric dimethylating to arginine sites of substrates. Additionally, by co-immunoprecipitation, western blotting and silver staining, we got some bands, on which some proteins might be combined with PRMT1and contained asymmetric dimethylated arginines. By mass spectrum, there were twenty six candidate proteins selected. Among them, there were6candidate proteins, which ion cores were greater than38, and the bioinformation web predicted that SEC23IP, ANKHD1-EIF4EBP3protein and1phosphatidylinositol3phosphate5kinase isoform2might contain the methylated arginine sites. |
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