The updated version of GEPIA2 was utilized to analyze the differential expression of UCP2 in liver cancer, perform survival analysis, and assess its expression across different clinical stages (gepia2.cancer-pku.cn/#analysis). GEPIA2, an upgraded version of GEPIA, integrates RNA-seq data from 9,736 tumors (The Cancer Genome Atlas, TCGA) and 8,587 normal (Gene-Tissue Expression, GTEx) samples processed through the UCSC Xena standardized pipeline (http://xena.ucsc.edu).

Human THLE-2 and Hep3B cell lines (Shanghai Fuheng Biotechnology Co., Ltd.), derived from normal human hepatocytes and HCC cells, respectively, were cultured in RPMI-1640 and Eagle's minimum essential medium supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin [all (Shanghai Xiaopeng Biotechnology Co., Ltd.)]. Cells were maintained at 37°C in a humidified incubator with 5% CO₂.

OSW-1 (HY-101213, CAS:145075-81-6) was purchased from MedChem Express. It was dissolved in dimethyl sulfoxide (Beijing Solarbio Science & Technology Co., Ltd.) to a concentration of 100 µg/ml and stored at −20°C.

Cell proliferation was monitored using a CCK-8 assay (Beijing Solarbio Science & Technology Co., Ltd.). The aforementioned cell lines in the logarithmic growth phase were seeded in a 96-well plate (1×10⁴/well) for 24 h. After treatment, CCK-8 reagent was added, followed by incubation at 37°C for 2 hours. Absorbance at 450 nm was measured using a microplate reader (Synergy Mx; BioTek; Agilent Technologies, Inc.). Data analysis was performed using SPSS (version 24.0; IBM Corp.) and samples were analyzed in quintuplicate.

Hep3B cells in the logarithmic growth phase were incubated with OSW-1 at concentrations of 0.0, 12.5, 25.0 and 50.0 ng/ml in a 6-well plate (5×10⁴/well) at a constant 37 °C for 24 h. JC-1 staining solution (Beyotime Institute of Biotechnology) was added and incubated at 37°C for 25 min in the dark. Cells were then washed twice with JC-1 staining buffer. Images were captured using an inverted fluorescence microscope (Discover Echo Inc.) and analyzed with ImageJ software (Version 1.54h, National Institutes of Health). Each sample was tested in triplicate.

The cell cycle was assessed using a cell cycle and apoptosis detection kit (cat. no. C1052; Beyotime Institute of Biotechnology). Following treatment aforementioned, single-cell suspensions were prepared and stained according to the manufacturer's instructions. Flow cytometry (FACSCalibur; BD Biosciences) was used to analyze (FlowJo; 10.6.2; BD) the cell cycle and triplicate experiments were performed.

According to the manufacturer's instructions (ROS Assay Kit; Beyotime Institute of Biotechnology), cells were loaded with 10 µM DCFH-DA (diluted 1:1,000 in extracellular solution) and incubated at 37°C for 20 min in the dark. After washing three times with extracellular solution to remove residual probe, ROS levels were assessed using a short-term stimulation method. Based on preliminary tests showing no detectable changes in ROS levels at lower concentrations, the drug concentration was increased 10-fold for experimental treatments. For positive controls, cells were stimulated with Rosup (diluted 1:1,000 in PBS) at 37°C for 20–30 min to induce ROS elevation. After digesting the Hep3B cells and preparing a single-cell suspension at a concentration of 1×10⁶/ml, dichlorodihydrofluorescein diacetate was added and the cells were incubated at 37°C. The probe was invested and mixed every 3–5 min to allow full contact with the cells. The non-probe and Rosup positive control groups were used as the reference. The Rosup group was stimulated for 20 min and the OSW-1 group for 2 h. Flow cytometry was used to observe changes in ROS levels and each sample was analyzed in triplicate.

Hep3B cells were treated with OSW-1 as aforementioned. RNA was extracted using the TRNzol Universal method (DP424; Tiangen Biotech Co., Ltd.). Reverse transcription was performed with the FastKing cDNA First Strand Synthesis Kit (KR116; Tiangen Biotech Co., Ltd.) under the following conditions: 42°C for 15 min followed by 95°C for 3 min. Quantitative PCR was performed using a Thermo 7300 Plus PCR instrument (Thermo Fisher Scientific) with the SuperReal Fluorescence Quantitative PreMix Enhanced kit (FP205; Tiangen Biotech Co., Ltd.). Thermocycling conditions consisted of an initial denaturation at 95°C for 15 min (1 cycle), followed by 40 cycles of 95°C for 10 sec and 60°C for 26 sec. Gene expression levels were analyzed by the 2^−ΔΔCq method (14). Quantification was performed according to the manufacturer's protocol in the kit manual. The primer sequences were as follows: UCP2 forward, 5′-GGAGGTGGTCGGAGATACCAA-3′ and reverse, 5′-ACAATGGCATTACGAGCAACAT-3′ and GAPDH forward, 5′-TCAAGGCTGAGAACGGGAAG-3′ and reverse, 5′-TGGACTCCACGACGTACTCA-3′.

Cells were treated as aforementioned and proteins were extracted using RIPA cell lysate (Beijing Solarbio Science & Technology Co., Ltd.) mixed with PMSF. A BCA protein assay kit (Beyotime Institute of Biotechnology) was used for protein quantification. The prepared protein samples (40 ug/lane) were separated by 12% SDS-PAGE (Beijing Solarbio Science & Technology Co., Ltd.) and transferred to nitrocellulose membranes (MilliporeSigma). The membrane was blocked in 5% skim milk at room temperature for 1 h and incubated overnight at 4°C with the following primary antibodies: BAX (1:1,000 dilution, catalogue no. WL01637; Wanleibio Co., Ltd.), Bcl-2 (1:1,000, WL01556; Wanleibio Co., Ltd.), Cleaved Caspase-3 (1:1,000, WL01992; Wanleibio Co., Ltd.), Caspase-3 (1:1,000, WL04004; Wanleibio Co., Ltd.), UCP2 (1:1,000, 11081-1-AP; Proteintech), and β-actin (1:1,000, AF7018; Affbiotech). The next day, the membrane was incubated with a fluorescent secondary antibody (IRDye 800CW goat anti-Rabbit; 1:20,000; LI-COR Biosciences) for room temperature 1 h in the dark, followed by visualization using a dual near-infrared fluorescent molecular imaging system. ImageJ was used to analyze the gray values of the bands.

All data are presented as the mean ± SD from three independent experimental repeats. All data were processed using SPSS 24.0 and analyzed using one-way analysis of variance followed by post hoc LSD test. Graphs were generated using the GraphPad Prism 6 software (Dotmatics). P<0.05 was considered to indicate a statistically significant difference.

The GEPIA database was used to assess differences in UCP2 expression between cancerous and normal human tissue. UCP2 expression was significantly higher in HCC compared with paired normal tissue (Fig. 1A). Compared with normal tissue in TCGA and GTEx data, the expression of UCP2 in liver cancer was significantly higher compared with that in normal tissues (P<0.05; Fig. 1B). Moreover, patients with high UCP2 expression demonstrated shorter overall survival compared with those with lower expression (Fig. 1C). No significant association was found between UCP2 expression and the clinical stage of HCC (Fig. 1D).

To evaluate the ability of OSW-1 to inhibit cell survival, a CCK-8 assay was used. OSW-1 had a significant cytotoxic effect on Hep3B cells; however, the viability of THLE-2 cells was significantly higher compared with Hep3B cells at the same concentration (P<0.05; Fig. 2). The half-maximal inhibitory concentration of OSW-1 against Hep3B cells was 73.97 ng/ml.

A decline in mitochondrial membrane potential is a hallmark of early apoptosis. To measure this, the membrane-permeable cationic dye JC-1 was used as a fluorescent probe. A decrease in mitochondrial membrane potential is indicated by the transition of JC-1 from red to green (15). OSW-1 significantly decreased the mitochondrial membrane potential in Hep3B cells in a dose-dependent manner compared with untreated controls (P<0.05; Fig. 3).

OSW-1 induced significant G2/M phase arrest and partial S phase accumulation in Hep3B cells, concurrent with reduced G0/G1 population. (P<0.05; Fig. 4).

OSW-1 markedly increased intracellular ROS levels (Fig. 5A). Compared with the control group, 125, 250, and 500 ng/ml OSW-1 treatments induced significant increases in M2 gated cells (%) (P<0.05; Fig. 5B).

To investigate how OSW-1 induces apoptosis in Hep3B cells, WB and RT-qPCR were performed to assess UCP2 protein and mRNA expression, respectively. RT-qPCR analysis demonstrated that the expression of UCP2 mRNA decreased in a dose-dependent manner with increasing OSW-1 concentration. Compared with the control group, 12.5, 25.00, and 50.00 ng/ml OSW-1 reduced UCP2 mRNA (P<0.05; Fig. 6B). Similarly, WB (Fig. 6C) demonstrated that the expression of UCP2 decreased with an increase in OSW-1 concentration (P<0.05). The protein expression of UCP2 decreased with longer treatment times at 12.5 ng/ml OSW-1 (Fig. 6A) and the 48- and 72-h groups were significantly decreased compared with the untreated control group (P<0.05). Compared with the control, Bcl-2 was downregulated, Bax was upregulated and the Bax/Bcl-2 ratio was significantly increased with OSW-1 treatment. In addition, cleaved-caspase3/caspase3 levels were significantly increased compared with the control (P<0.05).