The Expression Of MicroRNA-34c In Human Glioma And The Mechanism Of Its Effect On Glioma Cell Biological Characteristics

Glioma is the most common malignant tumor in the intracranial,accounting for78%of all the central nervous system malignant tumors,accounting for40%of primary intracranial tumor. The therapy of glioma isstill one of the most difficult problems in the field of Neurosurgery. At present,there is no effective method against glioma screening or early diagnosis.Although operation treatment method have great development in recent years,the result of glioma treatment is not satisfactory and the outcome is notoptimistic because of the rapid growth rate, unclear boundary and easilyrecurrent features. The median survival of glioblastoma multiforme is only12-15months, so it’s necessary to combine the operation therapy method andother auxiliary treatment in order to improve the outcome. In recent years, thediagnosis and treatment of the glioma have been explored at the molecularlevel. A variety of genes are related to the happen, development, and evolutionof glioma. Hence, looking for the gene associated with occurrence of gliomamay be the effective way to treat glioma.MiRNAs is an endogenous non-coding single-stranded RNAs. It caninhibit the degradation of target mRNA or translation process, and regulategene expression by complete or incomplete complementary binding with thetarget mRNA3’non-coding region. MiRNAs can participate in a series ofimportant biological behaviors, such as cell differentiation, cell proliferationand apoptosis and stress reaction. MiRNAs has been reported that it has theclose relation with human cancer occurrence and development, and plays apivotal role in the regulation of tumor angiogenesis. Therefore, experts havepredicted it’s more effective that miRNA as targets for tumor biologicaltherapy than coding molecules as molecular targets. MiRNAs will become anew bright spot in the field of biological treatment for tumor, and may provide a new platform for new drugs design and targeted therapy.It has been reported that miR-34is involved in the regulation of p53andNotch signaling pathway with the characteristics of the tumor suppressor. In amammal, miR-34family comprises three classes: miR-34a having theirtranscripts, miR-34b and miR-34c share a transcript. It is reported that lowexpression of miR-34a is in pancreatic cancer, osteosarcoma, breast andnon-small cell lung cancer. Also miR-34a is in a inactivation state in lungcancer, breast cancer, renal cancer, bladder cancer, pancreatic cancer andmelanoma cell lines. In malignant melanoma, colorectal cancer, oral squamouscell carcinoma, miR-34b/c performs inactive because of the CGP islandmethylation. The low expression of MiR-34a is correlate with high recurrencerate of non-small cell lung cancer, indicating that miR-34a can be used as anew target for non-small cell lung cancer prognosis. So far, all published datashow that inactivated miR-34is common in malignant tumors. However, theexpression of miR-34c, as a member of miR-34family, and the effect onglioma development has not been reported.In this study, we detected the expression of miR-34c-3p and miR-34c-5pin18formalin fixed paraffin embedded glioma specimens and5cases of braintissue samples by using quantitative PCR, in order to investigate expressionand clinical significance of miR-34c-3p and miR-34c-5p in human glioma.Next we tested cell proliferation, cell migration, cell invasion, cell cycle, andapoptosis after transfection in human glioma cell line U251and U87by usingLipofectamineTMRNAiMAX. Then we used the database to search thepossible downstream target genes of miR-34c-3p and miR-34c-5p, and findNotch2was the interesting common regulated target genes. Eventually, weused westenblot experimental methods and fluorescein reporter genetechnology to directly verify whether miRNA-34c was directly regulatedtarget genes Notch2. Further, it is desirable to find a feasible and effectivetherapeutic targets and provide a theoretical basis for the clinical applicationof gene therapy of glioma, provide a new idea for the treatment of tumors. Theexperiment is divided into three parts. The first part: The miRNA-34c expression in human gliomaObjective: To investigate miRNA-34c expression in human gliomatissuesMethods: We detected the miR-34c-3p and miR-34c-5p content in18cases of WHO grade II-IV class fresh glioma specimens and decompressionsurgery in patients with trauma or cerebral hemorrhage obtained five cases ofbrain tissue samples by Quantitative PCR. We compared the differentialexpression of miR-34c-3p and miR-34c-5p between different grade gliomasand normal brain tissue, and analysis the correlation of miR-34c-3p andmiR-34c-5p expression and the malignancy degree of glioma.Results: Compared with normal brain tissue, miR-34c-3p andmiR-34c-5p expression in gliomas were significantly reduced, and there was asignificant negative correlation between miR-34c-3p and miR-34c-5pexpression and the degree of malignancy (spearman correlation coefficient ofmiRNA-34c-3p=-0.856, P <0.001; miRNA-34c-5P spearman correlationcoefficient=-0.767, P <0.01).Conclusion: miRNA-34c expression significantly reduced in humanglioma cells, and there was a significant negative correlation betweenmiR-34c-3p and miR-34c-5p expression and the degree of malignancy.MiRNA-34c has important implications in the progression of glioma, and canbe used as an important indicator of glioma clinical classification.The second part: Increased miRNA-34c expression inhibits theproliferation and invasion of glioma cellsObjective: To study the effect of miRNA-34c on proliferation, migration,invasion, apoptosis and cell cycle in human glioma cell line U251and U87.Methods: After increased the expression of miRNA-34c in glioma cellline U251and U87by liposome, we tested validation transfection efficiencyby quantitative PCR, detected cell proliferation by MTS assay, cell migrationand invasion by Transwell chamber, apoptosis and cell cycle by flowcytometry.Results: In U87and U251cell lines, miRNA-34c-3p and miRNA-34c-5p expression was significantly lower than normal glial cells line HEB. ThemiRNA-34c-3p expression in transfection group was significantly higher thanthe non-transfected group (Normal group, P<0.05) and NC cells (P<0.05),miRNA-34c-5p also showed the same changes. In U251cells, cell viability intransfected with miR-34c-3p or miR-34c-5p group was significantly lowerthan the Normal group (P<0.05) and NC group (P<0.05) after48,72and96hours. However, the condition was different in U87cells. The cell survivalrate in miR-34c-3p transfection group was significantly lower than the Normalgroup (P<0.05) and NC group (P<0.05) after48,72and96hours; cellviability in miR-34c-5p transfection group was significantly lower than theNormal group (P<0.05) after24,48,72and96hours, while there was nosignificant difference (P>0.05) with the NC group. The migration to the lowerchamber of the number of cells in transfection group significantly reducedcompared with the Normal group and NC group in U251and U87cells byusing Transwell chambers. Transfected with miRNA-34c-3p ormiRNA-34c-5p in U251cells, and transfected with miRNA-34c-3p in U87cells can lead to cell cycle arrest in the S phase, reducing the cell number inG0/G1phase. In U251cells, apoptotic cells accounted for6.57%and6.3%inthe Normal group and NC group respectively, while apoptotic cells raised to28.49%and28.14%in transfected with miRNA-34c-3p or miRNA-34c-5p.However, apoptotic cell number increased only in transfected withmiRNA-34c-3p group (3.56%) in U87cells, Normal group, NC group andtransfected with miRNA-34c-5p were1.53%,1.68%and1.91%, respectively.Conclusion: Increased miRNA-34c expression inhibited the proliferation,migration and invasion in glioma, lead to the cell cycle arrest in the S and G2/M phase, induced cell apoptosis, and provided a reliable basis for gliomatargeted therapy. However, the effect of miRNA-34c-3p and miRNA-34c-5pwas different in different cell lines, suggesting that they may act through adifferent target or mechanism.The third part: The target genes and mechanism of miR-34c in gliomacells Objective: To explore the downstream target genes and the mechanism ofmiRNA-34c in glioma cellsMethods: We found the predicted targets by miRanda, PICTAR andTargetScan database screening, and found the gene we are interested in. Thenwe detect the expression of the gene in glioma cells by PCR and Westerntechnology. Finally, the luciferase reporter gene experimental was used toverify the interesting downstream target gene miRNA-34c.Results: Through a variety of databases screening, we focus theinteresting gene on Notch2. Quantitative PCR results showed that Notch2mRNA expression was significantly lower after transfection withmiRNA-34c-3p, compared with the Normal group and NC group (P <0.05),but transfected with miRNA-34c-5p did not change significantly. Western blotresults also showed that Notch2expression levels decreased by15%aftertransfected with miRNA-34c-3p in the U251and U87cells, compared to theNormal group and NC group. The Notch2expression levels did not changesignificantly in miRNA-34c-5p transfection group. In transfected clones3’UTR plasmid Notch2gene experiments, luciferase activity was significantlyreduced in miRNA-34c-3p group (P<0.05), compared with the control groupand NC group, demonstrating that miRNA-34c-3p can bind the Notch2gene3’UTR. While luciferase activity was significantly higher in miRNA-34c-3pInhibitor group, compared with the control group and NC Inhibitor group(P<0.05). In transfected clones mut Notch2-2gene3’UTR plasmidexperiments, there were no differences between miRNA-34c-3p group andcontrol group and NC group. In transfected clones Notch2gene3’UTRplasmid experiments and transfected clones mut Notch2-1gene3’UTRplasmid experiments, miRNA-34c-5p group was not differ with the controlgroup and NC group, demonstrating that miRNA-34c-5p can’t be combinedwith Notch2gene3’UTR.Conclusion: The Notch2expression was significantly reduced aftertransfected with miR-34c-3p in glioma cells, while transfected withmiR-34c-5p did not change significantly. We verified that Notch2was indeed the target gene of miR-34c-3p, but not miR-34c-5p, by using the luciferasereporter vector strategy.