Application of miR-9-5p in targeting UBE4B in regulating hypoxia-induced transcription factor 1α ubiquitination-mediated Warburg effect in glioma cells
ZHANG Jingjing YALIKUN Nu’er’aihemaitijiang DU Peng
Department of Neurosurgery, the Second Affiliated Hospital of Xinjiang Medical University, Xinjiang Uygur Autonomous Region, Urumqi 830063, China
Abstract：Objective To explore the regulation and mechanism of miR-9-5p on Waburg effect in glioma. Methods Thirty patient’s glioma and paracancerous tissue samples diagnosed from May 2018 to October 2020 in the Second Affiliated Hospital of Xinjiang Medical University were selected. A control group, a miR-9-5p overexpression group, and a miR-9-5p inhibitor group were established. Quantitative real-time PCR was used to detect the expression of miR-9-5p and its correlation with the expression of glycolysis-related genes. Construction of ectopic tumor models and in vitro cell experiments, using quantitative real-time PCR, Western blot, glucose uptake analysis, lactic acid production detection and cell viability detection to determine the impact of miR-9-5p on glioma and Warburg effect, while the dual luciferase reporter gene was used to detect the targeting relationship between miR-9-5p and UBE4B. Results The expressions of Hypoxia inducible factor-1α(HIF1α) protein, glucose transporter 1 (Glut1), human hexokinase-2 (HK2), lactate dehydrogenase (LDHA) and vascular endothelial growth factor (VEGF) in the miR-9-5p overexpression group were significantly higher than those in the control group, and the differences were statistically significant (P ＜ 0.05); while the mRNA level of HIF1α was not statistically significant (P ＞ 0.05). The expressions of HIF1α protein, Glut1, HK2, LDHA and VEGF in the miR-9-5p inhibitor group were significantly lower than those in the control group, and the differences were statistically significant (P ＜ 0.05); while the mRNA levels of HIF1α were not statistically significant (P ＞ 0.05). Compared with the control group, the miR-9-5p overexpression group significantly enhanced metabolism and metastasis (P ＜ 0.05), while the miR-9-5p inhibitor group significantly reduced glucose uptake and lactate production in U251 cells (P < 0.05). The cell viability of the miR-9-5p overexpression group was significantly higher than that of the control group after treatment for 24 h and 48 h, and the differences were statistically significant (P ＜ 0.05); compared with the control group, the cell viability of the miR-9-5p inhibitor group decreased significantly at 24 h and 48 h, and the differences were statistically significant (P ＜ 0.05). The tumor volume and wet weight of the miR-9-5p overexpression group were significantly higher than those of the control group, and the differences were statistically significant (P ＜ 0.05). The protein level of UBE4B in the miR-9-5p overexpression group was lower than that in the control group, and the difference was statistically significant (P < 0.05); the expression of HIF1α and glycolysis-related genes (Glut1, HK2 and LDHA) was significantly higher than that of the control group, and the difference was statistically significant (P ＜ 0.05). Conclusion miR-9-5p can regulate the Warburg effect of gliomas and promote the occurrence and development of gliomas through the UBE4B/HIF1α pathway.
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