Mir-199 Modulates Wnt/Beta-Catenin Signaling Pathway To Involve In The Progression Of Brain Stem Glioma

Objective: To explore the mechanism of miR-199 involving in targeted modulation of Wnt/beta-catenin signaling pathway in glioma cells and the potential value of miR-199 acting as a therapeutic target as well as a prognosis sign of malignant glioma, by analyzing the expression difference of miR-199 in different levels of brain stem glioma. Methods: microRNA expression profile obtained from previous researches on different levels of brain stem glioma was used to analyze and screen differentially expressed microRNA. 28 cases of brainstem glioma specimen and 8 cases of normal brain tissue specimen that collected from March 2013 to March 2015 in the Department of Neurosurgery of our hospital were selected in this study to detect the expression of Wnt-2, mir-199a-5p and beta-catenin in different levels of brainstem glioma tissues and normal brain tissues by using qRT-PCR, Western Blot and immunohistochemistry method. We also analyzed the correlation between miR-199a-5p and Wnt-2 as well as the relationship of clinical pathology between miR-199a-5p and brainstem glioma. The results of luciferase assay proved that miR-199a-5p target-modulated Wnt2. We infected U251 cells with miR-199a-5p over-expressed lentivirus, and CCK-8, flow cytometry and Transwell assay verified that MiR-199a-5p involved in the modulation of proliferation, apoptosis and invasiveness of U251 cells through target inhibition of Wnt/beta-catenin signaling pathway, and confirmed the role of miR-199/Wnt2/beta-catenin signaling axis in the progression of brain stem glioma. Results: qRT-PCR detection showed that the expression of miR-199a-5p in normal brain tissues and low-grade brainstem glioma tissues was significantly higher than that of the high-grade brainstem glioma tissues (P < 0.01, P < 0.05). Western Blot assay showed that the expression of Wnt2 and beta-catenin in normal brain tissues and low-grade brainstem glioma tissues was significantly lower than that in the high-grade brainstem glioma tissues (P < 0.01, P < 0.01); immunohistochemistry results showed that Wnt2 expressed in Cytoplasm and membrane, and the expression of Wnt2 in tissues of normal brainstem and low-grade brainstem glioma was significantly lower than that in the high-grade brainstem glioma tissues (P < 0.01, P < 0.01). Luciferase assay showed that miR-199a-5p mimic significantly inhibited the activity of Renilla luciferase in 293 cells (P < 0.05), which confirmed that miR-199a-5p target-inhibited the expression of Wnt2. The CCK-8 growth curve showed that proliferation of U251 cells was significantly inhibited after the over-expression of miR-199a-5p (P < 0.01). Flow cytometry showed that miR-199a-5p promoted the apoptosis of U251 cells. Transwell assay confirmed that miR-199a-5p had significantly inhibited the invasion of U251 cells. Conclusion: Decreased expression of miR-199a-5p in brainstem glioma resulted in abnormal activation of Wnt2/beta-catenin signaling pathway, and the expression level of miR-199a-5p in brainstem gliomas was negatively correlated with the degree of tumor malignancy. For brainstem gliomas, miR-199a-5p can be used as a prognostic target and is promising to become a potential target for the treatment of tumors.