INTRODUCTION

Clear-cell renal cell carcinoma (ccRCC) refers to a subtype of renal cancer. This cancer originates from the proximal tubular epithelium of the kidney and is characterized by clear cytoplasm rich in glycogen and lipids.[1,2] ccRCC is highly heterogeneous and invasive and exhibits varying clinical features that complicate treatment. Furthermore, some patients may present with metastases at the time of diagnosis.[3,4] The occurrence and mortality rates of ccRCC have gradually increased. Meanwhile, various treatments for ccRCC, such as targeted therapies that inhibit vascular endothelial growth factor and its receptors, tyrosine kinase inhibitors, mechanistic target of rapamycin complex 1, and immunotherapies, have been introduced. However, surgery remains the primary treatment option. Furthermore, some patients experience relapse following surgical intervention.[5-7] Therefore, finding new potential therapeutic targets for ccRCC is important to improve patient survival.

Genes related to metabolic pathways contribute to the development of ccRCC.[8,9] Normally, glucose generates pyruvate through glycolysis, and pyruvate generates acetylcoenzyme, which enters the tricarboxylic acid cycle. A large amount of adenosine–triphosphate is produced through mitochondrial oxidative phosphorylation to provide energy for cells, and tumor cells use the glycolysis pathway to provide energy, which allows them to rapidly proliferate and spread under hypoxia conditions. This metabolic change process is called aerobic glycolysis and is particularly pronounced in ccRCC. Thus, ccRCC is also known as a metabolic disease.[10-12] Many researchers, aiming at this property of ccRCC, search for targets for its early diagnosis and treatment by studying changes in the glycolysis level, such as 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 overexpression, which promotes the glycolysis and proliferation of renal cell cancer cells,[13] and knockdown of spectrin beta, non-erythrocytic 1, which accelerates the development of ccRCC through glutamic-pyruvic transaminase 2-dependent glycolysis.[14]

Ferroptosis is a type of cell death separate from autophagy and necroptosis. This phenomenon has potential anticancer effects and plays an important role in tumor growth and treatment.[15] Ferroptosis regulatory factors are upregulated in RCC.[16,17] Deficiency in SUV39H1 inhibits the growth of ccRCC by inducing ferroptosis.[18] In addition, mutations in the von Hippel–Lindau (VHL) gene frequently occur in ccRCC. VHL mutations can activate hypoxia-inducible factor-α (HIF-α); in addition, the regulation of intracellular iron levels is involved in the VHL/HIF-α pathway.[19] The iron content of ccRCC cells exceeds that of normal renal tissues, although it decreases with disease progression.[20] The inhibition of HIF-α can induce ferroptosis in renal cancer cells.[21] As a novel type of cell death, ferroptosis holds great application prospect in the treatment of ccRCC.

Solute carrier family 6 member 3 (SLC6A3) is a dopamine transport protein predominantly expressed in dopaminergic neurons of the central nervous system. With advancements in omics and experimental techniques, studies have revealed that SLC6A3 is considerably elevated in various cancers. In conventional ameloblastoma, the SLC6A3 gene is markedly upregulated and associated with cell proliferation and invasion.[22] In gastric cancer, SLC6A3 is highly expressed in serum and tissue, which suggests its potential value for diagnosis and monitoring progression.[23] In addition, SLC6A3 has been identified as a biomarker for screening various anticancer compounds.[24] Targeting SLC6A3 can inhibit cell viability in hepatocellular carcinoma.[25] Research has also confirmed that SLC6A3 expression is elevated in ccRCC patients and may be a biomarker for its diagnosis.[26,27] However, the specific regulatory mechanisms of SLC6A3 remain unclear. This research investigated the effects of SLC6A3 on ccRCC and analyzed the potential molecular mechanisms involved.

MATERIAL AND METHODS Cell culture

Renal tubular epithelial cell lines HK-2 (iCell-h096) and four ccRCC cell lines, including OS-RC-2 (iCell-h166), Caki-1 (iCell-h033), 769-P (iCell-h236), and 786-O (iCell-h235), were obtained from Cellverse Co., Ltd. (Shanghai, China). The cells were cultured in Roswell Park Memorial Institute-1640 (31800-022, Gibco, USA) supplemented with 10% fetal bovine serum (FBS, 100–500, Thermo Fisher, USA) and 1% penicillin–streptomycin (15140-122, Gibco, USA) at 37°C and 5% carbon dioxide (CO2), and authenticated by short tandem repeat analysis. The test of mycoplasma contamination was confirmed negative.

Overexpression and knockdown of SLC6A3

Construction of a lentivirus for SLC6A3 overexpression and knockdown was acquired by Sangon Biotech Co., Ltd. (Shanghai, China). The sequences are shown in Supplementary Materials. The sh-SLC6A3 vectors were transfected into OS-RC-2 cells and SLC6A3 overexpression (OE-SLC6A3) vectors into 786-O cells. The entire transfection process was executed with meticulous attention and strictly followed the protocol recommended by the manufacturer. The multiplicity of infection of 786-O cells was 20, and that of OS-RC-2 cells was 10. After 24 h, puromycin (60210ES25, Asen Corporation, Shanghai, China) was added to select stable cell lines.

Tumor formation experiment on nude mice

For the establishment of a xenograft model, 40 aged 4–6 weeks BALB/c nude mice (18–22g) were selected. They were kept in a controlled environment with a light/dark cycle of 12/12 h (40% ± 5% humidity and 20 ± 2℃ temperature) and provided with standard food and water. After 1-week acclimation feeding, these mice were randomly allocated into four groups (n = 10/group): Control of SLC6A3 overexpression (OE-NC), OE-SLC6A3, control of SLC6A3 knockdown (sh-NC), and SLC6A3 knockdown (sh-SLC6A3).

Cells exhibiting logarithmic growth from each experimental group were prepared as a 5 × 107/mL cell suspension and mixed 1:1 with Matrigel. Then, a 0.20 mL mixture (containing 1 × 107 cells/mouse) was subcutaneously injected into nude mice. Following tumor formation, the longest and shortest diameters of tumors were measured using a Vernier caliper. The formula V = (longest diameter × shortest diameter2)/2 was used to calculate the volume, with mean values from each group utilized to plot the growth curves. On completion of the experiment after 18 days, the mice were euthanized through intraperitoneal injection of pentobarbital sodium (110 mg/kg, P3761, Sigma, Merck, USA), and tumors were completely harvested for measurement of tumor mass and volume. This study received approval from the ethics committee, and all animal procedures complied with the Guidelines for Ethical Review of Laboratory Animal Welfare.

Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA was extracted using TRNzol Universal (DP424, TIANGEN, Beijing, China). The RNA was transcribed to complementary DNA (cDNA) using a FastQuant cDNA Synthesis Kit (KR116, TIANGEN, Beijing, China). Specific amplification primers were designed based on the target gene sequence and synthesized by Beijing Dingguo Changsheng Biotechnology Co., LTD. The primer sequences were as follows: SLC6A3-F: 5’-TTGTAGACGCACCTGCTGAG-3’, SLC6A3-R: 5’-TATTGATGTGGCACGCACCT-3’, β-actin-F: 5’-ACACTGTGCCCATCTACG-3’, and β-actin-R: 5’-TGTCACGCACGATTTCC-3’. qRT-PCR was carried out using SuperReal PreMix Plus (SYBR Green) (FP205, TIANGEN, Beijing, China). The entire procedure adhered strictly to the detailed protocols provided by the manufacturer. To precisely determine the relative expression levels of messenger RNA (mRNA), we adopted the 2−ΔΔCt method, with β-actin as the internal reference for normalization.

Western Blot (WB)

Total protein samples from cells and tissues were extracted using radioimmunoprecipitation assay (P0013B, Beyotime, Shanghai, China) and quantified using a bicinchoninic acid protein assay kit (P0010S, Beyotime, Shanghai, China). Protein samples were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and subsequently transferred onto polyvinylidene fluoride (IPVH00010, Millipore, USA) membranes. The membranes were blocked with 5% blocking solution for 60 min, followed by incubation with primary antibodies overnight at 4°C, including anti-SLC6A3 (HA500007; 1:1000; HUABIO, China), anti-β-actin (66009-1-Ig; 1:20000; PROTEINTECH, China), anti-glucose transporter 1 (GLUT1) (ET1601-10; 1:5000; HUABIO, China), anti-pyruvate kinase M2 (PKM2) (R1603-5; R1603-5; HUABIO, China), anti-hexokinase 2 (HK2) (66974-1-Ig; 1:5000; PROTEINTECH, China), anti-acyl-CoA synthetase long-chain family member 4 (ACSL4) (ET7111-43; 1:1000; HUABIO, China), anti-glutathione peroxidase 4 (GPX4) (ET1706-45; 1:5000; HUABIO, China), anti-ferritin heavy chain 1 (FTH1) (ET1610-78; 1:2000; HUABIO, China), and anti-solute carrier family 7 member 11 (SLC7A11) (HA721868; 1:1000; HUABIO, China). Following the washing step with phosphate-buffered saline with Tween 20, the membranes underwent incubation with secondary antibodies conjugated to horseradish peroxidase, including goat anti-rabbit immunoglobulin G (IgG) H&L (ZB-2301; 1:2000; ZSGB-BIO) and goat anti-mouse IgG H&L (ZB-2305; 1:2000; ZSGB-BIO). This incubation was allowed to proceed at constant room temperature for 60 min. The chemiluminescent substrate mixture (P5755103, Thermo Fisher, USA) was uniformly applied to the membrane and visualized. Densitometric analysis was performed using ImageJ software V1.52a (National Institutes of Health).

Cell viability

Cell viability was tested using a cell counting kit-8 (CCK-8) (MA0218-5, Meilunbio, Dalian, China). The digested cells were then resuspended in medium and inoculated into 96-well plates (07-6096, Biolglx, USA) with a density of 5 × 103 cells/well. A total of 10 µL CCK-8 solution was added to each well for incubation after 24, 48, and 72 h. Absorbance (optical density [OD]) was detected at 450 nm using a microplate reader (CMax Plus, MOLECULAR DEVICES, China), and the cell proliferation curve was drawn based on the detected ODs to calculate the cell proliferation rate.

Flow cytometry

Cell apoptosis was detected by Annexin V-fluorescein isothiocyanate Apoptosis Detection Kit (AP101, AULTI SCIENCES, Hangzhou, China), and the levels of intracellular reactive oxygen species (ROS) were assessed using a ROS Assay Kit (Beyotime, S0033, China). 2',7'-Dichlorodihydrofluorescein diacetate (DCFH-DA) (S0033S, Beyotime, Shanghai, China) was diluted in serum-free culture medium (CM15019, MACGENE, Beijing, China) at a ratio of 1:1000. After digestion with trypsin, centrifugation was performed to collect the cells, and the diluted DCFH-DA was added at 37°C for 20 min. Then, the sample was washed thrice with a serum-free culture medium to ensure the complete removal of any DCFH-DA that had not been internalized. After they were collected, the cells were resuspended for flow cytometry analysis (CytoFLEX, BECKMAN, USA).

Transwell assay

Transwell invasion chambers (3422, Corning, NY, USA) were assembled in 24-well plates (07-6024, Biolglx, USA). Matrigel matrix was diluted with serum-free medium and coated onto the upper chambers (80 µL/well), followed by 4 h incubation at 37°C. After trypsinization, the cells were resuspended in the culture medium. After cell counting, the suspensions were adjusted to 2.5 × 105 cells/mL. The lower chambers were loaded with 500 µl medium containing 10% FBS, and 300 µL of cell suspension was added to the upper chambers. After 24 h incubation at 37°C, the cells were rinsed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde for 30 min, and washed thrice with PBS. Staining was performed using 0.1% crystal violet solution (G1064, Solarbio, Beijing, China) for 10 min. Images were acquired through fluorescence microscopy.

Terminal deoxynucleotidyl transferase media dUTP nick end labeling (TUNEL)

Apoptosis was analyzed through TUNEL assay (11684817910, Roche, Basel, Switzerland). Paraffin-embedded tissue sections were processed through deparaffinization, rehydration, and antigen retrieval procedures. Subsequently, 50 µL of the prepared TUNEL reaction mixture was added and incubated at 37°C for 2 h. Following incubation, the slides were washed thrice with PBS, counterstained with 4',6-diamidino-2-phenylindole (G1012, Servicebio, Wuhan, China), and placed in the dark (37°C, 60 min). After washing with PBS, the slides were mounted with an antifade mounting medium and imaged under fluorescence microscopy (Eclipse C1, Nikon, Japan).

Extracellular acidification rate (ECAR) measurement

The cells were cultured in a Seahorse XF microplate and incubated overnight. The sensor cartridge was equilibrated with calibration buffer (37°C, CO2 free), and the assay medium (XF Base Medium, 2 mM glutamine, pH 7.4) was preheated to 37°C. Glucose (10 Mm, G8270), oligomycin (1 µM, 75351), and 2-DG (50 Mm, D8375) were purchased from Sigma-Aldrich, USA and loaded into designated injection ports. Following medium replacement, cells were incubated (37°C, 45 min) to stabilize. Real-time ECARs were monitored using a Seahorse XFe24 Analyzer (S7801B, Agilent, Beijing, China), and glycolysis parameters were derived using automated software analysis.

Biochemical analysis

The concentrations of lactate, glucose, glutathione (GSH), malondialdehyde (MDA), and iron levels were determined using biochemical kits (A019-2-1, A154-1-1, A006-2, A003-1-2, and A039-2-1, respectively, Nanjing Jiancheng Bioengineering Institute, China). Superoxide dismutase (SOD) and human 4-hydroxynonenal (4-HNE) were determined using biochemical kits (A001-3 and JYM2007Hu, Wuhan Jiyinmei Biotech, China). All experiments were performed in accordance with the manufacturers’ instructions.

Statistical analysis

GraphPad Prism (version 9.0, San Diego, CA, USA) was utilized for plotting. Experimental data are expressed as the mean ± standard deviation. Independent samples t-tests were used for comparisons between the two groups. Statistical significance was defined as P < 0.05.

RESULTS SLC6A3 is highly expressed in ccRCC

To explore the expression pattern of SLC6A3 in ccRCC, we examined the SLC6A3 mRNA and protein levels in various cell lines (HK-2, Caki-1, OS-RC-2, 786-O, and 769-P). As shown in Figure 1a-c, compared with HK-2, SLC6A3 was up-regulated to varying degrees in ccRCC cells. In addition, the SLC6A3 level was higher in the OS-RC-2 cell line (P < 0.001) and lower in the 786-O cell line (P < 0.01).

Figure 1: SLC6A3 is highly expressed in ccRCC. (a) Relative mRNA levels of SLC6A3 in HK-2 and four ccRCC cell lines obtained during qRT-PCR. (b and c) WB analysis of SLC6A3 in HK-2 and four ccRCC cell lines. n = 6; ✶P < 0.05, ✶✶P < 0.01, ✶✶✶P < 0.001. SLC6A3: Solute carrier family 6 member 3, ccRCC: Clear-cell renal cell carcinoma, mRNA: Messenger RNA, qRT-PCR: Quantitative real-time polymerase chain reaction, WB: Western blot.

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SLC6A3 promotes proliferation and invasion of ccRCC cells in vitro

For the investigation of the effect of SLC6A3 on ccRCC, the 786-O cell line with low SLC6A3 expression was selected for overexpression and the OS-RC-2 cell line with high SLC6A3 expression for knockdown. The sh-2 group, which had the highest overall efficiency, was selected for subsequent experiments (P < 0.001) [Figure 2a-f]. Cell viability was measured through CCK-8 assay. The results show that the overexpression of SLC6A3 considerably enhanced the cell viability of 786-O cells (P < 0.001), and the knockdown of SLC6A3 greatly weakened the viability of OS-RC-2 cells (P < 0.001) [Figure 2g and h]. Transwell assay showed that overexpression of SLC6A3 increased the invasive ability of 786-O cells (P < 0.01), and its knockout reduced that of the OS-RC-2 cell line (P < 0.001) [Figure 2i and j]. In addition, cell apoptosis in each group was detected, and the results unveil that compared with the OE-NC, the proportion of apoptosis was substantially reduced in the OE-SLC6A3 group (P < 0.001), and it was markedly increased in the sh-SLC6A3 group compared with sh-NC (P < 0.001) [Figure 2k and l]. The above results indicate that SLC6A3 can promote the proliferation and invasion of ccRCC cells.

Figure 2: SLC6A3 enhanced the proliferation and invasion of ccRCC cells in vitro. (a-c) Overexpression efficiency of SLC6A3 was detected through qRT-PCR and WB. (d-f) Knockdown efficiency of SLC6A3 was detected through qRT-PCR and WB. (g and h) Proliferative ability was tested by CCK-8 assay. (i and j) Invasive ability was measured using Transwell assay. Staining was performed using 0.1% crystal violet solution (G1064, Solarbio, Beijing, China) for 10 min (scale bar: 100 µm, magnification, 100×). (k and l) Cell apoptosis rates were examined through flow cytometry. n = 6; ✶✶P < 0.01, ✶✶✶P < 0.001. SLC6A3: Solute carrier family 6 member 3, ccRCC: Clear-cell renal cell carcinoma, qRT-PCR: Quantitative real-time polymerase chain reaction, WB: Western blot, CCK-8: Cell counting kit-8.

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Overexpression of SLC6A3 promoted the glycolysis of ccRCC cells

After the overexpression of SLC6A3 in 786-O cells, we observed an increase in the ECAR, whereas the knockdown of SLC6A3 resulted in its decrease [Figure 3a and b]. Biochemistry assay results indicate that the overexpression of SLC6A3 enhanced glucose consumption (P < 0.001) and lactate production (P < 0.01), and SLC6A3 knockout resulted in glucose consumption (P < 0.001) and lactate production (P < 0.01) decrease [Figure 3c-f]. In addition, we detected the expressions of glycolytic-related proteins GLUT1, HK2, and PKM2. The results show that GLUT1 (P < 0.05), HK2 (P < 0.01), and PKM2 (P < 0.01) protein expressions were increased markedly in 786-O cells after SLC6A3 overexpression [Figure 3g and h], and they were considerably reduced when SLC6A3 was down-regulated in OS-RC-2 cells (P < 0.05) [Figure 3i and j]. These findings suggest that SLC6A3 enhanced the glycolysis of ccRCC cells.

Figure 3: Overexpression of SLC6A3 promoted the glycolysis of ccRCC cells. (a and b) ECAR was detected using a Seahorse XFe/XF24 Analyzer. (c and d) Lactate concentrations in each group were quantified using lactic acid assay kits. (e and f) Glucose concentrations in each group were quantified using glucose kits. (g-j) WB analysis was performed to assess the expression of GLUT1, HK2, and PKM2 proteins. n = 6; ✶P < 0.05, ✶✶P < 0.01, ✶✶✶P < 0.001. SLC6A3: Solute carrier family 6 member 3, ccRCC: Clear-cell renal cell carcinoma, ECAR: Extracellular acidification rate, GLUT1: Glucose transporter 1, PKM2: Pyruvate kinase M2, WB: Western blot.

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Overexpression of SLC6A3 inhibited ferroptosis in ccRCC cells

To further investigate the molecular mechanism by which SLC6A3 promotes the progression of ccRCC, we evaluated the correlation indices related to ferroptosis following the overexpression and knockdown of SLC6A3. The results [Figure 4a] indicate that the overexpression of SLC6A3 led to notable decreases in the total iron, MDA, and 4-HNE levels and the significantly increased contents of SOD and GSH (P < 0.001). Conversely, the knockdown of SLC6A3 resulted in tremendous increases in the total iron, MDA, and 4-HNE levels, alongside the greatly reduced SOD and GSH levels (P < 0.001). Flow cytometry analyses revealed that the overexpression of SLC6A3 corresponded to decreased ROS levels, whereas its knockdown was associated with increased ROS levels (P < 0.001) [Figure 4b and c, respectively]. Furthermore, WB analysis was conducted to evaluate the expressions of ferroptosis-related proteins, which corroborated the biochemical findings. Specifically, the overexpressed SLC6A3 led to the decreased expressions of ACSL4 proteins (P < 0.01) and increased expressions of GPX4, FTH1, and SLC7A11 (P < 0.05), and SLC6A3 knockdown resulted in the opposite results (P < 0.05) [Figure 4d and e]. These findings suggest that the overexpression of SLC6A3 inhibits ferroptosis in ccRCC cells, whereas the knockdown of SLC6A3 promotes ferroptosis.

Figure 4: Overexpression of SLC6A3 inhibited ferroptosis in ccRCC cells. (a) Levels of total iron, MDA, 4-HNE, SOD, and GSH were detected using biochemical kits. (b and c) ROS levels was quantified through flow cytometry. (d and e) WB analysis was performed to quantify the expression levels of ACSL4, GPX4, FTH1, and SLC7A11 proteins. n = 6; ✶P < 0.05, ✶✶P < 0.01, ✶✶✶P < 0.001. SLC6A3: Solute carrier family 6 member 3, ccRCC: Clear-cell renal cell carcinoma, WB: Western blot, MDA: Malondialdehyde, 4-HNE: Human 4-hydroxynonenal, SOD: Superoxide dismutase, GSH: Glutathione, ROS: Reactive oxygen species, ACSL4: Acyl-CoA synthetase long-chain family member 4, GPX4: Glutathione peroxidase 4, FTH1: Ferritin heavy chain 1, SLC7A11: Solute carrier family 7 member 11.

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SLC6A3 promotes ccRCC progression in vivo

A xenograft model was used to validate the oncogenic effects of SLC6A3 in vivo. The 786-O cells were transfected with OE-NC and OE-SLC6A3, and the OS-RC-2 cells were transfected with sh-NC and sh-SLC6A3. When SLC6A3 was overexpressed, the tumor volume and weight were notably greater than those in the OE-NC group (P < 0.001). Conversely, SLC6A3 knockdown resulted in a notable shrinkage in tumor volume and weight (P < 0.001) [Figure 5a-f]. WB analysis demonstrated the significantly elevated expression of SLC6A3 in the OE-SLC6A3 group (P < 0.001), whereas it was reduced in the sh-SLC6A3 group (P < 0.05) [Figure 5g and h]. In addition, TUNEL assays revealed that silencing of SLC6A3 expression considerably elevated the apoptosis rate, whereas its overexpression led to a notable reduction in apoptosis levels (P < 0.01) [Figure 5i and j]. These findings imply that SLC6A3 promotes tumor progression in vivo.

Figure 5: SLC6A3 promotes ccRCC growth in vivo. (a-c) Changes in tumor morphology, size, and volume after SLC6A3 overexpression. (d-f) Changes in tumor morphology, size, and volume after SLC6A3 knockdown. (g and h) WB analysis of SLC6A3 in tumor tissues after SLC6A3 overexpression and knockdown. (i and j) Apoptotic cells were assessed through TUNEL assay after SLC6A3 overexpression and knockdown (scale bar: 50 µm, magnification, 200×). n = 10; ✶P < 0.05, ✶✶P < 0.01, ✶✶✶P < 0.001. SLC6A3: Solute carrier family 6 member 3, ccRCC: Clear-cell renal cell carcinoma, WB: Western blot.

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SLC6A3 promotes the development of ccRCC by enhancing the glycolysis of renal cancer cells and inhibiting ferroptosis

To further verify the function of SLC6A3, we assessed the glycolytic and ferroptosis-related indicators after the overexpression and knockdown of SLC6A3 in vivo. The results are shown in Figures 6a and b. Compared with the OE-NC group, the OE-SLC6A3 group exhibited a marked reduction in glucose concentration alongside a pronounced elevation in lactate production (P < 0.001). Conversely, the sh-SLC6A3 group exhibited a marked elevation in glucose concentration and a notable reduction in lactate production relative to the sh-NC group (P < 0.001). WB analysis indicated that SLC6A3 overexpression resulted in increased expressions of glycolysis-related proteins GLUT1 (P < 0.05), HK2 (P < 0.001), and PKM2 (P < 0.05), and its knockdown resulted in the reduced expressions of these glycolysis-related proteins GLUT1 (P < 0.001), HK2 (P < 0.05), and PKM2 (P < 0.001) [Figure 6c and d]. Next, we evaluated ferroptosis-related indicators, which were consistent with the results observed in vitro. As shown in Figure 6e, the OE-SLC6A3 group exhibited substantially lower levels of total iron (P < 0.01), MDA (P < 0.001), and 4-HNE (P < 0.001) but remarkably higher levels of GSH (P < 0.001) and SOD (P < 0.01). By contrast, the knockdown of SLC6A3 produced the opposite results in the levels of total iron (P < 0.001), MDA (P < 0.01), 4-HNE (P < 0.001), GSH (P < 0.001), and SOD (P < 0.01). Similarly, the expression of ferroptosis-related protein ACSL4 (P < 0.01), FTH1 (P < 0.05), GPX4 (P < 0.001), and SLC7A11 (P < 0.01) was considerably decreased after SLC6A3 overexpression, and ACSL4 (P < 0.05), FTH1 (P < 0.05), GPX4 (P < 0.05), and SLC7A11 (P < 0.01) notably increased after SLC6A3 knockdown [Figure 6f and g].

Figure 6: SLC6A3 promotes the development of ccRCC by enhancing the glycolysis of renal cancer cells and inhibiting ferroptosis (a) Lactate levels in each group were quantified using Lactic Acid assay kits. (b) Glucose levels in each group were quantified using glucose kits. (c and d) Expression levels of GLUT1, HK2, and PKM2 proteins were detected via WB. (e) Measurement of 4-HNE, GSH, MDA, SOD, and total iron levels in different experimental groups. (f and g) WB analysis of ferroptosis-associated proteins ACSL4, FTH1, GPX4, and SLC7A11 in different experimental groups. n = 10; ✶P < 0.05, ✶✶P < 0.01, ✶✶✶P < 0.001. SLC6A3: Solute carrier family 6 member 3, ccRCC: Clear-cell renal cell carcinoma, WB: Western blot, GLUT1: Glucose transporter 1, PKM2: Pyruvate kinase M2, MDA: Malondialdehyde, 4-HNE: Human 4-hydroxynonenal, SOD: Superoxide dismutase, GSH: Glutathione, ACSL4: Acyl-CoA synthetase long-chain family member 4, GPX4: Glutathione peroxidase 4, FTH1: Ferritin heavy chain 1, SLC7A11: Solute carrier family 7 member 11.

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DISCUSSION

ccRCC, one of the most common malignant tumors of the kidney, has been a focal point in the research of its pathogenic mechanisms and effective treatment strategies. This study investigates the potential molecular mechanisms by which SLC6A3 promotes the progression of ccRCC both in vivo and in vitro through its overexpression and knockdown. The findings provide insights into the potential application of SLC6A3 in renal cancer treatment.

SLC6A3, a dopamine transporter associated with neurological disorders, plays important roles in cell differentiation, developmental regulation, and tumor formation.[28] Previous researchers have reported that SLC6A3 is highly expressed in ccRCC while exhibiting lower expression levels in normal renal tissue and other malignant tumors, and make SLC6A3 a biomarker for ccRCC patients.[26,27] However, further research on its mechanism is lacking. Consistent with these findings, we observed varying degrees of high SLC6A3 expressions in ccRCC cell lines. Moreover, our in vivo and in vitro experiments demonstrated that the overexpression of SLC6A3 promoted the proliferative activity and invasion of ccRCC cells, reduced apoptosis, and enhanced tumor growth. Conversely, its knockdown yielded opposing effects, which indicates that SLC6A3 is involved in renal cancer progression. Prior research has identified scoulerine as a potential therapeutic agent targeting SLC6A3 for RCC treatment,[29] which suggests the development of SLC6A3-targeted approaches.

SLC6A3 is also a target gene of HIFs; it responds to decreased oxygen levels in glioblastoma.[30] The characteristic feature of ccRCC is the inactivation of the VHL gene, which reduces the degradation of HIF and leads to a pseudohypoxic state.[31] Hypoxia is involved in multiple processes in tumor development.[32] HIF activates pyruvate dehydrogenase, catalyzes the conversion of pyruvate to lactate, and promotes glycolysis.[33] To further investigate the molecular mechanisms by which SLC6A3 promotes ccRCC progression, we assessed its glycolytic activity. Our results indicate that SLC6A3 overexpression led to increased ECAR, elevated glucose consumption, and enhanced lactate production along with upregulated glycolysis-related proteins. Conversely, the knockdown of SLC6A3 produced the opposite effects, which demonstrates that SLC6A3 facilitates the development of ccRCC by enhancing glycolytic activity.

In addition, the VHL/HIF pathway is closely involved in ferroptosis, which plays a critical function in the progression of ccRCC.[20] In this study, the overexpression of SLC6A3 resulted in decreases in the amounts of oxidative stress markers, ROS levels, iron levels, and expressions of the ferroptosis-promoting factor ACSL4, and expressions of ferroptosis-inhibiting factors GPX4, FTH1, and SLC7A11 increased. These outcomes suggest that SLC6A3 expression may help tumor cells evade ferroptosis, which promotes tumor survival and dissemination. Conversely, the knockdown of SLC6A3 produced the opposite effects. These findings were corroborated by in vivo experiments, which enhanced the credibility of our results.

The outcomes of in vivo and in vitro experiments indicate that SLC6A3 plays a crucial role in ccRCC. The relationship among SLC6A3, tumor cell glycolysis, and ferroptosis suggests the potential for developing SLC6A3-targeted therapies. By reducing SLC6A3 expression in ccRCC cells, we can diminish glycolytic activity and promote ferroptosis, which would inhibit tumor cell proliferation and invasion and restrict tumor adaptability and growth. These findings highlight the potential of SLC6A3 as an effective therapeutic strategy for ccRCC treatment.

SUMMARY

This study demonstrated that SLC6A3 is considerably upregulated in ccRCC and plays a key role in tumor metabolism and progression by enhancing glycolysis and inhibiting ferroptosis. Targeting SLC6A3 may provide a promising therapeutic approach for ccRCC treatment.

AVAILABILITY OF DATA AND MATERIALS

The data that support the findings of this study are available from the corresponding author on reasonable request.

ABBREVIATIONS

4-HNE: Human 4-hydroxynonenal

ACSL4: Acyl-CoA synthetase long-chain family member 4

CCK-8: Cell counting kit-8.

ccRCC: Clear-cell renal cell carcinoma

ECAR: Extracellular acidification rate

FTH1: Ferritin heavy chain 1

GLUT1: Glucose transporter 1

GPX4: Glutathione peroxidase 4

GSH: Glutathione

MDA: Malondialdehyde

mRNA: Messenger RNA

PKM2: Pyruvate kinase M2

qRT-PCR: Quantitative real-time polymerase chain reaction

ROS: Reactive oxygen species

SLC6A3: Solute carrier family 6 member 3

SLC7A11: Solute carrier family 7 member 11

SOD: Superoxide dismutase

WB: Western blot

AUTHOR CONTRIBUTIONS

HJZ and CZL: Designed the research study; HJZ, YJJ, and CZL: Performed the research; and HJZ and CZL: Collected and analyzed the data. HJZ, YJJ, and CZL: Involved in drafting the manuscript and all authors have been involved in revising it critically for important intellectual content. All authors give final approval of the version to be published. All authors have participated sufficiently in the work to take public responsibility for appropriate portions of the content and agreed to be accountable for all aspects of the work in ensuring that questions related to its accuracy or integrity. All authors meet ICMJE authorship requirements.