MiR-455-3p Regulates Glioma Cell Proliferation By Targeting PAX6

Objective: Glioma is a seriously progressive disease that needs effective therapeutic approaches.Glioma development is always related to changes in the expression level of regulators involved in cell cycle.Abnormal expression of miRNAs is reported related to tumor progression,and up-regulation or down-regulation of miRNA leads to oncogenic and suppressive effects in tumors involved in lung cancer,gastric cancer,breast cancer,colorectal cancer,and even glioblastoma.The purpose of this study is to: 1.Detecting the expression of miR-455-3p in glioma tissues;2.Finding the role of miR-455-3p in the development of glioma;3.Exploring the underlying mechanism of action of miR-455-3p in the development of glioma.Methods: 1.Collected tissue samples from 39 patients with glioma(They were not treated with radiotherapy and chemotherapy before surgery),grouped according to pathology and clinical stage.Quantitative real-time polymerase chain reaction(qRT-PCR)was used to determining the expression level of miR-455-3p in glioma tissues;2.Multiple glioma cell lines(SW1783,A172,U87,U251 and H4)and Normal human astrocytes(NHA)to detect the expression of miR-455-3p in glioma cell lines and normal human astrocytes by qRT-PCR;3.Overexpression or inhibition of miR-455-3p by transfection,using qRT-PCR to detect the efficiency of transfection;4.Using MTT to detect the proliferation of glioma cells after overexpression or inhibition of miR-455-3p;5.Using bioinformatics analysis to predicting the target gene of miR-455-3p,and confirmed that PAX6 is the target gene of miR-455-3p by dual luciferase reporter assay;6.Verifying that miR-455-3p regulates PAX6 through functional recovery experiments;7.Using qRT-PCR and Western blotting to detect the expression of cell cycle regulators in the abnormal expression of miR-455-3p and PAX6.Results: 1.The expression level of miR-455-3p was higher in patients with clinical grade IV than that was in grade II patients(P < 0.05);2.The expression level of miR-455-3p was significantly higher in glioma cell lines than that of normal astrocytes(P < 0.05).3.Overexpression of miR-455-3p in cells increased the proliferation of glioma cells and interfered with cell cycle process(P < 0.01).Notably,endogenous miR-455-3p silencing significantly prevented glioma cell proliferation by regulating cell cycle progression(P < 0.05).4.Bioinformatics analysis and luciferase reporter gene assay results showed that PAX6 is the target gene of miR-455-3p;5.PAX6 regulates cell cycle,PAX6 silencing selectively regulates p21 expression(P < 0.01).At the same time,re-introduction of PAX6 expression vector in glioma cells rescued the tumor-promoting effect of miR-455-3p overexpression;6.miR-455-3p regulates selective p53 expression in cell cycle pathway by targeting PAX6 in glioma,which in affects the proliferation of glioma cells.Conclusion: 1.miR-455-3p is highly expressed in glioma tissues and cells;2.miR-455-3p could promotes cell proliferation of glioma;3.PAX6 is the target gene of miR-455-3p;4.miR-455-3p selectively regulates the cell cycle pathway of glioma via PAX6.These results indicate,for the first time,that high expression of miR-455-3p plays an important role in the development of glioma by targeting PAX6.In addition,these results demonstrate that miR-455-3p may be a potential therapeutic target for gliomas via regulation of cell cycle elements at the transcriptional level.