| Glioblastoma multiforme (GBM), WHO grade IV astrocytoma, is the most common primary neoplasm of the central nervous system (CNS), which has the highest malignancy and mortality rates. The median survival of GBM patients,97%of whom dying in12months, is no more than3months from the time of diagnosis without effective treatment. It is impossible to remove the tumor aggressively with satisfaction for patients with surgical treatment, cause of the Invasive characters of malignant glioma cells. Even so, researchers keep working on methods of treatment all the times. In the recent decade, developments of microinvasive surgery and image-guided neurosurgery promote the total removal and clinical curative rate. Currently, the treatment of glioblastoma involving surgery, followed by radiation and chemotherapy, has made great improvement of overall survival with the2-year survival rate increasing from10.9%to27.2%,3-year survival rate increasing from4.4%to16%, compared to radiotherapy alone post-operation. Despite all above, it is the invasive nature of GBM still complicates surgical resection and restricts chemotherapeutic access contributing to gloomy outcomes.The migration of tumor cells is closely related to the tumors proliferation. Some researchers reported the "go or grow" hypothesis in early1996. Phenotypic transformation between proliferation and migration happened while tumor cells migrate from a niche to the better one. The alteration of cell phenotype is proposed to be a crucial mechanism during this invasive progession, and there have to be some of intracellular factors regulating this complex process. MicroRNAs (miRNAs,miRs) are endogenous single-stranded RNA molecules that act as key regulators of gene expression. It has been well established that miRs play critical roles in the development and progression of cancers and have been found to have positive or negative effects on cell proliferation, apoptosis, and invasion in various cancer cells by inhibiting target genes expression. Recently, it was demonstrated that miR-451is downregulated in migrating glioma cells. It is also indicated to be involved in proliferation and migration through LKB1-AMPK paythway.In the present study, we discussed functions of miR-451in glioma cells proliferation and migration, and its effect on mTOR, a major proliferation factor, and Racl, a crucial tumor invasion factor. We tried to reveal potential mechanism of miR-451in glioma cells.This study included two parts:The first part of this study focused on the expression and role of miR-451in glioma cells.40specimens of glioma with pathological grades WHO IV and6control brain tissues acquired from surgeries for temporal lobe epilepsy were collected to perform qRT-PCR analysis to examine the expression level of miR-451in tissues. We used synthetic miR-451mimics and miR-451inhibitor to increase and decrease the expression level of miR-451. qRT-PCR was performed to test the efficiency of the processes. MTT assay was performed to measure the effect of miR-451on cell proliferation. The wound-healing assay and transwell migration assay were conducted to evaluate the effect of miR-451on cell migration. The data demonstrated that the level of miR-451was (33.58±5.19)%in glioma tissues. Furthermore, MiR-451enhanced tumor cells proliferation but suppressed the migration.In the second part of this study, we tried to reveal the potential mechanism of miR-451on proliferation and migration in glioma cells. Part I. We transfected miR-451mimics and miR-451inhibitor in glioma cell lines U87, U251, and SNB19. Semi-quantitative assessment of p-AMPK, AMPK, Raptor, p-Raptor and Racl GTP-Racl expression levels were examined with western blot. Racl Activation Assay was recruited to analyze GTP-Racl expression level. The data showed that no statisitical difference of AMPK, Raptor and Racl were validated at protein level, while expressions of p-Raptor were elevated by miR-451and expressions of p-AMPK were declined as well as GTP-Racl. Part II. To validate the mechanism of miR-451in proliferation and migration of glioma cells, we employed a synthetic shRNA to knock down the AMPKal in glioma cell lines, establishing3new cell lines. qRT-PCR and western blot were performed to test the efficiency of RNAi. Then procedure of Part I was conducted with new cell lines. The data confirmed that the function of miR-451on proliferation and migration, as well as its effects on activation of AMPK, mTORCl and Racl, were attenuated without AMPK.Conclusion 1. The miR-451was downregulated in GBM tissues compared with control brain tissues. The balance of proliferation and migration in glioma cells is altered by miR-451expression.2. MiR-451regulated activation of mTORC1and Racl, which played key roles of proliferation and migration in glioma cells, through the activation of AMPK. |
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