Circ_UBE2C promotes proliferation and glycolysis of lung cancer cells by regulating miR-107/HK2 axis

Abstract

Objective To investigate the effects of circ_UBE2C on the proliferation and glycolysis of lung cancer cells and its mechanism of action. Methods The expression of circ_UBE2C, miR-107, and hexokinase 2 (HK2) in lung cancer tissues and normal lung tissues were analyzed based on the The Cancer Genome Atlas (TCGA) database. Kaplan-Meier survival curve was plotted to analyze the correlation between circ_UBE2C/miR-107/HK2 expression and survival of the lung cancer patients. qRT-PCR was used to detect the expression of circ_UBE2C and miR-107, and Western blotting was employed to measure the expression of HK2 and PCNA in human normal lung epithelial cells (BEAS-2B) and human lung cancer cell lines (A549, NCI-H460, and NCI-H1299). Then A549 and NCI-H1299 cells were divided into the blank group (NC), circ_UBE2C knockdown group (si-circ), HK2 knockdown group (si-HK2), simultaneous knockdown of miR-107+HK2 group (miR-inhibitor+si-HK2), and simultaneous knockdown of circ_UBE2C+miR-107 group (si-circ+miR-inhibitor). CCK-8 assay and colony formation assay were employed to measure the proliferation of above cell groups. The glucose uptake, lactate generation, and ATP production in the cells were measured by glucose uptake colorimetric assay kit, lactic acid detection kit, and ATP content determination kit, respectively. Seahorse XF glycolytic stress test and Seahorse XF cellular mitochondrial stress test were respectively performed to detect the extracellular acidification ratio (ECAR) and cellular oxygen consumption ratio (OCR). The targeting relationship between the circ_UBE2C and miR-107, and miR-107 and HK2 was validated by dual-luciferase reporter gene assay. Results Compared with normal lung tissues, the expression of circ_UBE2C and HK2 was up-regulated, while that of miR-107 was down-regulated in lung cancer tissues (P < 0.05). The expression of circ_UBE2C and HK2 was elevated, and that of miR-107 was reduced in A549 and NCI-H1299 cells when compared with BEAS-2B cells (P < 0.05). Survival analysis showed that the patients with high expression of circ_UBE2C and HK2 or low expression of miR-107 had shorter survival (P < 0.05). Compared with the NC group, both si-circ and si-HK2 resulted in significantly reduced proliferation, glucose uptake, lactate generation, ATP production, and ECAR and OCR values in A549 and NCI-H1299 cells (P < 0.05). Dual luciferase reporter gene assay confirmed that circ_UBE2C could directly bind to and negatively regulate miR-107 expression. HK2 was then identified as a downstream target of miR-107. Compared with the si-HK2 group, the proliferative ability and glycolysis level of A549 and NCI-H1299 cells were significantly increased in the miR-inhibitor+si-HK2 group and the si-circ+miR-inhibitor group. Conclusion Knockdown of circ_UBE2C significantly suppresses the proliferation and glycolysis of lung cancer cells via targeting up-regulation of miR-107 and then exerting inhibitory effect on HK2 expression.

Keywords

• lung cancer
• circ_ube2c/mir-107/hk2 axis
• warburg effect
• proliferation