Leptin is an adipokine minimally known for its activities or underlying mechanisms in cholangiocarcinoma. The present study explored the effects of leptin on the epithelial-mesenchymal transition (EMT) and pro-angiogenic capability of cholangiocarcinoma cells, and investigated the underlying mechanisms. Cholangiocarcinoma cells were treated with leptin, and their migration and invasion rates were investigated using Transwell assays. Furthermore, conditioned medium was collected from cholangiocarcinoma cells following leptin treatment and applied to human umbilical vein endothelial cells to assess tube formation. The expression of EMT and pro-angiogenic factors was examined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analyses. Mechanistically, the function of pyruvate kinase muscle isozyme M2 (PKM2) was assessed in leptin-induced phenotypes using siRNA targeting PKM2 (si-PKM2). Bioinformatics screening and luciferase reporter assays were used to reveal microRNA (miR)-122 as the potential mediator between leptin and PKM2. Finally, the associations between leptin and miR-122 or PKM2 levels in patients with cholangiocarcinoma were assessed by ELISA and RT-qPCR. Leptin significantly increased the EMT and pro-angiogenic capability of cholangiocarcinoma cells, visibly inhibited endogenous miR-122 expression, and upregulated PKM2. Furthermore, si-PKM2 inhibited leptin-induced migration, invasion, EMT-associated marker expression levels and the pro-angiogenic capability in cholangiocarcinoma cells. In addition, miR-122 negatively regulated the expression of PKM2. When applied together with leptin, miR-122 was sufficient to reverse the multiple malignancy-promoting effects of leptin. Consistently, the serum leptin level positively correlated with that of PKM2, but negatively with that of miR-122 in patients with cholangiocarcinoma. Leptin, by downregulating miR-122 and elevating PKM2 expression, acts as a pleiotropic pro-malignancy cytokine for cholangiocarcinoma. Therefore, increasing miR-122 expression and inhibiting PKM2 may be future approaches for cholangiocarcinoma treatment.
Glycyrrhizin (GA) is the most essential active ingredient in licorice root, and has a wide range of biological and pharmacological activities. The present study aimed to conduct a detailed analysis of the effects of GA on liver cancer (LC) cell proliferation and the Warburg effect. Hexokinase-2 (HK2) is a glycolytic enzyme that catalyzes the Warburg effect. To this end, the LC HepG2 cell line was transfected with small interfering RNA-HK2 or pCDNA3.1-HK2, followed by GA treatment. A Cell Counting Kit-8 assay and EdU staining were employed to evaluate the proliferation rate of LC cells. The expression levels of HK2 and the phosphorylation level of AKT were measured by reverse transcription-quantitative PCR and western blotting, respectively. Furthermore, the glucose uptake capacity and lactic acid content were assessed by kits, and the glycolysis level was evaluated by assessing the extracellular acidification rate (ECAR) and the oxygen consumption rate (OCR). A pronounced increase in the OCR, and decreases in the cell proliferation, glucose uptake capacity, lactic acid content, ECAR and HK2 expression were detected in LC cells subjected to GA treatment or HK2-knockdown. Conversely, overexpression of HK2 reversed these trends, indicating that glycyrrhizin may inhibit LC cell proliferation and the Warburg effect through suppression of HK2. In addition, it was revealed that the PI3K/AKT signaling pathway was associated with LC cell proliferation and the Warburg effect; notably, treatment of LC cells with the AKT agonist SC79 induced elevation of the ECAR, cell proliferation, glucose uptake capacity, lactic acid content, phosphorylated-AKT and HK2 expression, and suppressed the OCR. In conclusion, GA may inhibit the Warburg effect and cell proliferation in LC by suppressing HK2 through blockade of the PI3K/AKT signaling pathway.
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