| Glioblastoma multiforme(GBM) is the most common primacy malignant brain tumors.Although the research on glioma have made geat progress in the past few years,prognosis of patients with malignant glioma remains poor. GBM is a kind disease with abnormal multigenes and many signaling pathways and its pathogenesis is still not very clear. Better understanding the molecular mechanism of gliomagenesis will certainly lead to the development of novel targeted therapies.Recent studies have found leucyl-tRNA synthetase(LARS) possesses non-canonical activity in addition to its canonical activity,may be a tumor-related gene.However,it is rarely reported in glioma. MicroRNAs(miRs) are a class of short noncoding RNAs which act as posttranscriptional regulators of gene expression. Studies have showed that miRs are frequently deregulated in a variety of tumors including glioma and play an oncogenic or tumor suppressor role.Currently it is not clear whether miRs can influence on LARS gene expression.The main purpose of this research is to investigate LARS gene expression in malignant glioma and impact on the biological behavior of glioma cells as well as the relationship between miR-451and LARS gene expression,hope that the findings are helpful to provide clues for research on glioma pathogenesis and new treatment strategies.In the first part of this study,we detected LARS gene expression in9malignant glioma cell lines and1case of normal brain tissue by real-time PCR and western blot assay.The results of two assays consistently reveal that LARS is over-expressed in9malignant glioma cell lines compared with normal brain tissue(p<0.05).Subsequently by using immunohistochemical method to detect LARS expression in tissue microarray containing75cases with glioma and5cases normal brain tissue adjacent to tumors,we found that LARS expressed significantly higher in glioma tissue than in normal tissue(p<0.05).Although LARS expression level was positivly correlated to glioma grades,degree of correlation was not high(r=0.283,p<0.05) and the differences between low grade(Ⅰ/Ⅱ) and high grade(Ⅲ/Ⅳ) was also not obvious. These results implied that LARS might have a role in glioma development and progression.In the second part,we studies the biological role of LARS over-expression in glioma cell lines U251and SNB19detected by siRNA targeted knock-down analysis and its possible molecular mechanism.First of all,LARS expression following LARS siRNA(LARS siR) transfection was detected by real-time PCR and western blot techniques.The results showed that LARS siR successfully inhibited LARS expression level of RNA and protein in U251and SNB19. Then we analyzed biological behavior of glioma cell lines U251and SNB19following LARS expression knocked down by MTT,cell plate clone formation,flow cytometry analysis,wound healing assay,transwell assay,annexin V assay and western blot.The findings showed that knocking down LARS expression inhibited the growth of malignant glioma cell, blocked the cell cycle in G0/G1phase, reduced the ability of cell migration, lowered the activity of mammalian target of rapamycin complex1(mTORC1) signaling pathways, but had no obvious effect on cell early apoptosis.Therefore,we concluded that LARS may play the role of oncogene in glioma and mTORC1signaling pathway maybe involve in this role.LARS is expected to become a new gene target for the treatment of gliomas.In the third part,we mainly explored relation between miR-451and LARS using synthetic oligonucleotides miR-451mimics and inhibitor.The results showed that miR-451mimics and inhibitor could effectively up-regulate and down-regulate miR-451expression in glioma cells respectively.After transfecting miR-451mimics into glioma cell lines,LARS protein level was down-regulated detected by western blot;in contrast,LARS protein level was up-regulated after transfecting miR-451inhibitor.We constructed dual luciferase plasmid for LARS3’-UTR and then performed dual luciferase reporter assay. The findings revealed that LARS was the direct targeting gene of miR-451.In conclusion, from the present studies, we identify that LARS is up-regulated in gliomas and to some extent its expression is positively correlated with tumor grade. Inhibition of LARS represses the growth, migration and invasion of glioma without apoptosis in vitro. MiR-451can affect LARS expression by repressing LARS mRNA translation.The findings may provide new clues and strategies for treatment of gliomas,which is worth to further study. |
PDF Full Text Request