Study On The Expression Of MiR-138 In Gliomas And Its Malignant Biological Function And Molecular Mechanism

Gliomas are the most frequent primary tumours in the central nervous system and a major cause of death in patients with intracranial tumours.Its fatality rate ranks the third among all kinds of tumours,after lung cancer and pancreatic cancer.The occurrence and development of glioma is a very complex biological process.The cause of glioma is still unknown,and may be related to head trauma,viral infection,endocrine,metabolic disorders and molecular genetics.In addition to traditional therapies such as surgery,radiotherapy and drug therapy,some new therapies such as immunotherapy,stem cell therapy,gene therapy and electric field therapy have emerged and are being used to treat glioma.Unfortunately,the effectiveness of these treatments remains low.Therefore,it is urgent for us to deeply understand the molecular mechanism of the occurrence and development of glioma,so as to find new molecular targets for more effective diagnosis and treatment of glioma.MicroRNAs(miRNAs) constitute a class of single-stranded noncoding RNAs that play an important role in the translation of tumour genes and the regulation of downstream proteins.It has been reported that more than 60% of protein translation is regulated by miRNAs.Convincing evidence has confirmed that miRNAs regulate a majority of cellular processes related to the biological behavior of tumors,including cell proliferation,apoptosis,differentiation and metastasis.Dysregulation of miRNA expression has been found in various types of human cancers.Compelling evidence has suggested that miRNAs are novel modulators of tumor progression and new targets for tumor therapy in GBM.In particular,miR-138 showed low expression in a variety of tumors and inhibited the growth and metastasis of tumor cells,indicating that miR-138 is closely related to the occurrence and development of tumors,but its role in glioma and its molecular mechanism have not been reported.In this study,we investigated the potential involvement of miR-138 in glioma.We examined the expression level of miR-138 in human glioma cells and tissues and tested its effects on cell growth,apoptosis,and invasion.In addition,we also explored the underlying mechanism of miR-138 functions in glioma.Our study will provide a better understanding of glioma pathogenesis,Part 1 Expression of miR-138 in glioma specimens and cell linesObjective:To investigate the expression of miR-138 in glioma tissues and cell lines,andanalyze the correlation between its expression level and clinicopathological grades and prognosis in glioma patients.Methods: Expression of miR-138 in 48 human glioma specimens and 12 normal brain specimens and 4 cell lines was detected by q RT-PCR.The correlation between miR-138 expression and clinicopathologic features was analyzed in 48 human glioma specimens.Results: The q RT-PCR results showed that the miR-138 expression in the glioma pecimens was significantly lower than that in the normal brain specimens.The high-grade glioma specimens(WHO grades III-IV)exhibited a lower expression of miR-138 than the low-grade glioma specimens(WHO grades I-II).Mi R-138 expression was associated with the pathological grade.The patients with high expression levels of miR-138 had significantly prolonged OS compared with those with low miR-138 expression levels by a Kaplan-Meier survival analysis.In addition,q RT-PCR results showed showed lower levels of miR-138 expression in U87 and U251 cells compared to A172 and U133 cells.Conclusions: The miR-138 expression was frequently downregulated in clinical glioma tissues and cell lines,and was closely related to the clinical pathological grade and survival of patients.These results suggest that miR-138 may play an important role in the development of gliomas.Part 2 Effects of miR-138 on glioma cell proliferation,apoptosis and invasionObjective: To investigate the biological function of miR-138 in glioma in vitro,find miR-138 as a new therapeutic target for glioma and provide experimental basis for future treatment of glioma by intervening miR-138.Methods: The U87 and U251 cells were transfected with miR-138 mimics or miR-NC.Cell viability was measured by an MTT assay.Cell apoptosis rates were measured by flow cytometry.Cell invasion was measured by Transwell invasion assay.Results:Glioma cells after transfection of miR-138 mimics showed significantly increased levels of miR-138 in these cells.MTT assay showed that overexpression of miR-138 significantly suppressed the proliferation of U87 and U251 cells when compared to their corresponding controls.miR-138 overexpression was also found to decrease cell invasion in U87 and U251 cells determined by Transwell invasion assay.Furthermore,flow cytometry indicated that the apoptotic rate was significantly increased in miR-138 mimics transfectants compared to the control cells.Conclusions miR-138 is a potential tumor suppressor gene in gliomas.Part 3 miR-138 regulates glioma cell apoptosis and invasion by modulating AKT/m TOR pathway through CREB1Objective: To screen and identify the target gene of miR-138,and clarify the mechanism of miR-138 in glioma cells.To provide new ideas and directions for the diagnosis and treatment of gliomas.Methods: 1)To predict miR-138 target genes by using bioinformatics software.Dual-luciferase reporter detection was used to verify miR-138 directly regulated the target gene CREB1.QRT-PCR and western blotting were used to examine the m RNA and protein expression of CREB1 respectively in glioma cells transfected with miR-138 mimics.Expression of CREB1 in 48 human glioma specimens and 12 normal brain specimens was detected by q RT-PCR.The correlation between CREB1 m RNA and miR-138 expression was analyzed in 48 human glioma specimens.2)To down-regulate CREB1 expression in glioma cells,U87 and U251 cells were transfected with CREB1 si RNA.Then the effects on cell proliferation,apoptosis and invasion of glioma cells were examined.U87 and U251 cells were co-transfected with miR-138 mimics and CREB1 expression vector or control vector.The changes of biological function of glioma cells were observed.3)The activation of the AKT/m TOR signalling pathway and the expression of Bcl-2 and MMP-2 in response to miR-138 and CREB1 expression changes were detected by Western blotting.Results:1)By using the bioinformatics algorithms,we speculated that CREB1 is a potential target of miR-138.The transfection of the reporter vectors containing the putative target sequence resulted in a significant decrease in the relative luciferase activity compared to that in the 293 cells co-transfected with the mutant sequence and miR-138.The miR-138 transfection significantly decreased both the m RNA and protein expression levels of CREB1.The q RT-PCR results revealed that the m RNA expression level of CREB1 was significantly increased in the glioma samples compared to that in the normal brain samples.The Spearman correlation analysis of the m RNA expression levels demonstrated that CREB1 expression was inversely associated with miR-138 expression.2)The U87 and U251 cells exhibited significantly higher expression levels of CREB1 m RNA and protein than the A172 and U133 cells.The Western blotting analysis results revealed that CREB1 protein expression was significantly suppressed in both cell types following transfection of si RNA targeting CREB1.The down regulation of CREB1 significantly suppressed viability and invasionin in the U87 and U251 cells.The downregulation of CREB1 promoted apoptosis in the U87 and U251 cells.The rescue experiments revealed that the upregulation of CREB1 restored the viability,apoptosis and invasion of the miR-138 mimics-transfected glioma cells.3)We found a dramatic decrease in p-AKT and p-m TOR expression in the U87 and U251 cells transfected with the miR-138 mimics,while the levels of total AKT and total m TOR did not differ between the mimics-transfected and miR-NC-transfected groups.The levels of the anti-apoptotic protein Bcl-2 and the invasion-associated protein MMP-2 were decreased in the miR-138-transfected U87 and U251 cells.CREB1 overexpression activated the AKT/m TOR signalling pathway and enhanced Bcl-2 and MMP-2 expression.The upregulation of CREB1 dramatically abrogated the decreases in AKT/m TOR activity and Bcl-2 ? MMP-2 expression in the cells overexpressing miR-138.The CREB1 transfection rescued the inhibitory effect of miR-138 on MMP-2 expression in the U87 and U251 cells.Conclusions: miR-138 likely mediates apoptosis and invasion in glioma cells by targeting CREB1,thus suppressing MMP-2 and Bcl-2 expression and AKT/m TOR signalling pathway activation.miR-138 may be a potential target for the treatment of gliomas.