Human RCC cell lines A498, 786-O, CAKI-1, ACHN, and GRC were purchased from KeyGEN Company (Nanjing, China). These cell lines are well-established models in RCC research and have been widely used in previous studies, enabling better comparison and validation of our results with the existing literature; they represent different subtypes of RCC, which helps us comprehensively understand the role of BNIP3 in various RCC phenotypes, and they also have different levels of endogenous BNIP3 expression. Cells were cultured in Roswell Park Memorial Institute (RPMI) medium using 1640 complete medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Cells were cultured under hypoxic conditions. Specifically, they were placed in a 3131 Thermo Forma Water-Jacketed CO₂ incubator and exposed to a gas mixture composed of 1% oxygen, 5% CO₂, and 94% N₂ for 48 hours.

PCR was employed to enhance the entire coding segment of BNIP3. The DNA segment was incorporated into the pcDNA3.1 vector (Invitrogen, USA) to produce pcDNA3.1-BNIP3 (pc-BNIP3). An unoccupied pcDNA3.1 vector was employed as a reference (pc-NC) for transfection objectives. Cell transfection was carried out using Lipofectamine^® 2000 transfection reagent (Invitrogen, USA). A498 and 786-O cells were seeded into 6 well plates and transfected when they reached a confluence of 70%-80%. To overexpress genes, pc-BNIP3 (2 μg/well) or pc-NC (2 μg/well) along with the transfection reagent (12 μL/well) were mixed separately in serum-free Opti-MEM for 5 minutes. The diluted plasmid and transfection reagent were then combined and incubated at 37°C for half an hour. The resulting mixture was added to the cell culture medium, followed by replacement with fresh RPMI 1640 after six hours of transfection. Culture continued for another two days thereafter.

A498 and 786-O cells were cultured under two different oxygen conditions: cell hypoxia conditions and normoxia conditions. In the cell hypoxia conditions, the gas composition in the incubator was set to 1% O₂, 5% CO₂, and 94% N₂. In the normoxia conditions, the gas composition was 21% O₂, 5% CO₂, and 74% N₂. A498 and 786-O cells were divided into 6 groups: OE-NC (pc-NC); OE-BNIP3 (pc-BNIP3); OE-NC Normoxia; OE-NC Hypoxia; OE-BNIP3 Normoxia; OE-BNIP3 Hypoxia. A498 and 786-O cells were subjected to an additional experiment involving the introduction of pc-BNIP3, either with or without 3-MA, an autophagy inhibitor, and cultured under hypoxia (OE-BNIP3 + 3-MA Normoxia and OE-BNIP3 + 3-MA Hypoxia groups). Each experiment was performed in triplicate.

The Trizol Total RNA Extraction Kit (9108, TaKaRa, Japan) was utilized to extract the total cellular RNA, followed by reverse transcription into cDNA using the Reverse Transcription Kit (2690S, TaKaRa, Japan). SYBR Premix Ex TaqTM II kit (RR037Q, TaKaRa, Japan) was used for real-time fluorescence quantification. The real-time qPCR system was performed in a 20 µL system under the conditions of 95°C for 30s pre-denaturation, 95°C for 5s, 55°C for 30s, 45 cycles, and 72°C for 30s extension. The comparative CT method (2^-ddCT method) was used to calculate BNIP3 and HIF-1α mRNA expression with β-actin as the internal reference control. Primers were designed using Primer 5.0 Fast software and the sequences are shown in Table 1 as follows.

Cell proliferation was quantified with the Cell CCK-8 kit (BS350B, Biosharp, Shanghai, China), following the manufacturer’s guidelines. The renal carcinoma cells that underwent transfection were plated and left to incubate for a duration of 24 hours. Following this, 10 µL of CCK-8 reagent was added to each well, extending the incubation for three hours. Absorbance at 450 nm was then measured using a microplate reader (ELx800, Buchi Instruments, USA).

Cells were transfected using a lentiviral vector overexpressing BNIP3. The BNIP3 overexpression lentiviral vector was purchased from GeneChem (Shanghai, China). The cell concentration was adjusted to 2 × 10⁵/mL 100 µL of cell suspension was inoculated into each well and the culture medium was replenished. The cells were mixed well and incubated until clones were visible. When clones first became visible, the culture medium was disposed of and the plate was rinsed with PBS. The cells were treated with a 4% volume of polymethanol for a duration of 30 minutes and subjected to staining using a solution containing 0.1% crystal violet for a period of 15 minutes. The plate was dried after removing the staining solution. Photographs were taken, images were recorded, the number of clones visible was counted, and the results were analyzed.

After trypsin digestion of transfected cells, a 1 × 10⁵ cells/mL concentration was achieved with the medium. Before the experiment, matrix gels were preincubated in Transwell chambers at 37°C for 3 hours. Post-solidification of the Matrigel, serum-free medium was replenished. A cell suspension was then added to Transwell chambers in a 24-well plate, with culture solution outside. The Transwell was washed with PBS after 24 hours of incubation. Fixation was performed using 4% paraformaldehyde, followed by crystal violet staining for 30 minutes. Six random visual fields were selected from each well, and the number of invasive cells was observed with a light microscopic camera system (DMI1, LEICA, Germany).

The A498 and 786-O cells in the logarithmic growth phase were inoculated into 12-well plates at a density of 1×10⁵ cells per well. Once the cells had adhered to the plate, transfection or intervention was carried out. After 48 hours, all cells from the 6-well plates were collected and washed with PBS to eliminate any background interference. The cells were then resuspended in 100 µl of 1×Binding Buffer and incubated with AnnexinV-FITC and PE-PI dual fluorescent probes (KGA1107, KeyGEN, China) for a duration of 15 minutes at room temperature under light protection. Subsequently, the samples were immediately analyzed using flow cytometry (Cytoflex, Beckman Coulter, USA) through the FITC/PI channel to determine the percentage of apoptotic cells.

The extraction of total protein from tissues and cells involved the use of lysis buffer, followed by a 30-minute incubation on ice to collect the cells. The supernatant was collected, and the protein concentration was determined by the BCA (P0009, Beyotime, Shanghai, China) method. A 30 μg protein sample was taken and denatured in a boiling water bath and uploaded, proteins were separated by 10% SDS-PAGE and transferred to a PVDF membrane (ISEQ00010, Sigma-Aldrich, USA), and blocked with 5% skimmed milk for 1h at room temperature. The BNIP3 antibody (1:1000, A19593, ABclonal, USA), HIF-1α antibody (1:1000, A7684, ABclonal, USA), caspase3 antibody (1:500, 9662, Cell Signaling Technology, USA), cleaved caspase3 antibody (1:1000, 9661, Cell Signaling Technology, USA), Bax antibody (1:1000, A0207, ABclonal, USA), LC3B antibody (1:1000, A19665, ABclonal, USA), p62 antibody (1:1000, A19700, ABclonal, USA), and β-actin antibody (1:50000, AC026, ABclonal, USA) were incubated at 4°C overnight. The membrane was washed 3 times with TBST, incubated with HRP-labeled secondary antibody (1:7000, S0001, Affbiotech, Jiangsu, China) for 1h at room temperature, washed 3 times with TBST, and developed dropwise with a chemiluminescent solution. Quantitative analysis was performed using Image-ProPlus 6.0 software.

Pretreated cells were lysed in a lysis buffer containing 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1% Triton X-100, 0.1 mM EDTA, 0.5 mM MgCl₂, and supplemented with 10% glycerol, protease inhibitor cocktail (Roche), phosphatase inhibitor cocktail (Roche), and 10 μM pervanadate (NEB). The cell lysates were incubated with Bc1–2 antibody (diluted at a ratio of 1:50; A0208, ABclonal, USA) for one hour at a temperature of 4°C followed by overnight incubation with Protein-A/G Sepharose beads (Abcam, UK) also at a temperature of 4°C. The agarose beads were then collected through centrifugation at a speed of 500 g for five minutes. Subsequently, the beads were thoroughly washed three times in the lysis buffer and heated to a temperature of 95°C for five minutes. Finally, proteins were probed using antibodies against Beclin1(1:30, A7353, ABclonal, USA) and BNIP3 (1:30, A19593, ABclonal, USA).

The cell crawl sections were stained. The sections were immersed in 5% film breaker for 10 min. After PBS washing, goat serum sealing solution was added and sealed for 20 minutes at room temperature. Primary antibodies (1:100, LC3B, 14600-1-AP, Proteintech, USA) were added and incubated overnight at 4°C. After washing again with PBS, the second antibody (1:100, FITC-labeled goat anti-rabbit, GB22303, Servicebio, Wuhan, China) was added and incubated for 30 min. After washing with PBS, 4’,6-diamidino-2-phenylindole (DAPI) was added and incubated. After a final wash with PBS, the slices were sealed with an anti-fluorescence attenuating sealer. The images were a3-MAuired using a microscopic camera system (OlyVIA, OLYMPUS, Tokyo, Japan). The fluorescence intensity and area of all images were measured using ImageJ6 (National Institutes of Health, Bethesda, MD, USA), and the mean fluorescence intensity of each image was calculated.

A498 and 786-O cells were cultured in 6-well plates, and a total of 1 × 10⁵ cells from each group underwent two washes with PBS. The cells were then loaded with probes, resulting in a suspension containing 5 mol/L DCFH-DA (MCE). After incubating for 30 minutes at 37°C in the absence of light, the cells were washed twice with PBS. The fluorescence intensity detected using the FITC channel was utilized to quantify the variations in ROS levels among different experimental groups.

We used Human MPTP ELISA KIT (ZC-56711, ZCIBIO Technology, Shanghai, China) to detect changes in mitochondrial MPTP concentration. The target antibody is immobilized on a 48-well microplate to form a solid phase carrier. Standard or sample solutions are added to the wells, and the target is connected to the immobilized antibody on the solid phase carrier. Then, horseradish peroxidase-labeled antibody is added, and the unbound antibody is washed away before adding substrate for color development. The absorbance (OD value) at 450 nm is measured using a microcoder (SpectraMAX Plus384, MOLECULAR DEVICES, Shanghai, China), and the sample concentration is calculated.

Twelve 6-week-old BALB/c nude mice (18~22 g) were used for subcutaneous tumor xenograft experiments. The animals in this study were purchased from Chengdu Dashuo Experimental Animal Co., Ltd. (Chengdu, China) (SCXK(Chuan)2019-031). BALB/c nude mice were randomly divided into two groups (OE-NC group and OE-BNIP3 group, n =6). In the subcutaneous carcinogenic experiment, the right axillas of nude mice in the OE-NC group and OE-BNIP3 group were subcutaneously injected with stably transfected pcDNA-NC 786-O and pcDNA-BNIP3 786-O cells. The density of 786-O cells was adjusted to 1.0 × 10⁶ cells/mL, and the right axillary side of nude mice was injected subcutaneously to construct tumorigenesis of nude mice for 30 days. Starting on day 20, the tumor volume was measured every 3 days for a total of 10 measurements. After 26 days, the mice were anesthetized (sodium pentobarbital, 100 mg/kg, 200-323-9, Sigma-Aldrich, St. Louis, MO, USA) and sacrificed via cervical dislocation, and all 12 subcutaneous tumors were isolated and weighed. Tumor tissue was then stored in a –80°C freezer for subsequent studies. The Ethics Committee of 363 Hospital (IRB number: 2022035) approved all experimental protocols conducted in this study.

After completing the experiment, the mice were sacrificed, and part of the tumor tissue was collected, fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned at 4 μm. After dewaxing and rehydration, the histological morphology was observed with hematoxylin-eosin (H&E, C0105M, Beyotime, Shanghai, China) staining.

Tumor tissue samples of mice in each group were fixed with 5% glutaraldehyde for up to 5 hours. After fixation, samples were washed three separate times using neutral phosphate buffer, each wash lasting 10 min intervals. Brain tissue samples were fixed with 0.1 mol/L osmic acid for 3 h and washed three times with phosphate buffer, every 10 min. Gradient dehydration was then performed at 50%, 70%, 80%, 90%, 95%, and 100% alcohol concentrations every 15 minutes. The tissue blocks were embedded in resin for ultrathin sectioning (50~70nm), and then stained with uranyl acetate (1261209, SPI), and the ultrastructure of cells and autophagy were observed under a transmission electron microscope.

TUNEL (Terminal deoxynucleotide transferase (TdT) dUTP notch terminal marker) was used to detect apoptosis in tumor tissue cells. Paraffin sections were dewaxed and hydrated with PBS 3 times. The remaining steps of the procedure are conducted by the TUNEL kit (1168479590, Roche Group). Cells with green nuclei under the light microscope were apoptotic positive cells. Three regions were randomly selected from each mouse brain tissue section, and the number of positive cells was used as the apoptotic index.

After the standard preparation of tumor tissue sections, antigen retrieval was performed using citrate buffers followed by treatment with 3% hydrogen peroxide to suppress endogenous peroxidase activity. Afterward, the sections were securely closed using serum derived from goats (AR1009, BOSTER) and incubated overnight with primary antibodies of BNIP3 (1:400, bs-4239R, Bioss, USA) and Ki67 (1:400, HA721115, HUABIO, Shanghai, China) at 4°C, followed by another round of PBS washing. A secondary antibody (1:100, GB22303, Servicebio, Shanghai, China) was added and incubated at 37°C for 30 min. Tissues were visualized using a DAB (36201ES03, YEASEN) staining solution, and hematoxylin restaining was performed before dehydration and sealing. Image analysis was performed using a digital trimoscope (BA210Digital, Meiji Technology Co., LTD., China) and a data image analysis system (Halo 101-WL-HALO-1, Indica Labs, USA).

The statistical analysis was conducted using GraphPad Prism 9.0 software. The mean () ± standard mean (SD) were used to represent the experimental statistics. To compare multiple groups, a one-way analysis of variance (ANOVA) was employed, and statistical analysis was performed post hoc by Tukey. The T-test was used for comparison between the two groups. P<0.05 was statistically significant.

Initially, we investigated the protein expression of BNIP3 and HIF-1α in renal cell carcinoma cell lines A498, 786-O, CAKI-1, ACHN, and GRC. As depicted in Figure 1A , elevated levels of BNIP3 and HIF-1α were observed in CAKI-1, ACHN, and GRC, whereas these proteins exhibited minimal expression in A498 and 786-O. Consequently, A498 and 786-O were chosen as the subjects of investigation. WB and RT PCR were used to detect the expression of BNIP3 protein and mRNA in A498 and 786-O cells with overexpression of BNIP3. As shown in Figures 1B, C , the level of BNIP3 protein expression was notably elevated in the OE BNIP3 group (BNIP3 expression group) compared to the OE-NC group (negative control group). Furthermore, BNIP3 mRNA was significantly increased in the OE BNIP3 group compared to the OE-NC group (P<0.01) ( Figure 1D ).

A498 and 786-O cells were transfected and divided into the OE-NC and OE-BNIP3 groups. According to the data presented in Figure 2A , there was a significant decrease in the proliferative activity of the OE-BNIP3 cells compared to that of the OE-NC group. We obtained comparable findings from the cell cloning assay, with the number of clones formed by A498 and 786-O cells in the OE-BNIP3 group being significantly less than that in the OE-NC group (P<0.01) ( Figures 2B, C ). Furthermore, the Transwell invasion assay indicated that the OE-BNIP3 group had a significantly lower number of A498 and 786-O cells crossing the Transwell compartment than the OE-NC group (P<0.01) ( Figures 2D, E ). Flow cytometry showed that OE-BNIP3 had increased A498 and 786-O cell death compared to OE-NC (P<0.01) ( Figures 2F, G ). In addition, OE-BNIP3 increased the expression of caspase3, cleaved caspase3, and Bax compared with OE-NC ( Figures 2H–K ). Based on the above results, overexpression of BNIP3 suppressed the proliferation and invasion and promoted apoptosis in renal cell carcinoma cells.

In the following step, our objective is to determine whether the overexpression of BNIP3 can definitively bind to BCL-2 and induce the dissociation of Beclin-1 from Bcl-2. By performing co-immunoprecipitation experiments, we successfully co-precipitated Bcl-2. Analysis using the Western Blot technique demonstrated that under hypoxic conditions, BNIP3 overexpression significantly increased the co-precipitation of Bcl-2 with BNIP3, while simultaneously reducing the co-precipitation of Bcl-2 with Beclin1 ( Figures 3A, B ). This indicates an enhanced binding capacity between BNIP3 and Bcl-2, and a weakened binding capacity between Bcl-2 and Beclin1. This change led to the dissociation of the Bcl-2/Beclin1 complex, thereby promoting the process of protective autophagy in RCC cells under hypoxic conditions.

To explore the effect of overexpression of BNIP3 on autophagy under hypoxia. LC3B is a key protein involved in autophagy and is crucial in regulating cellular processes and responses to stress. The results of immunofluorescence staining confirmed that A498 and 786-O cells exposed to hypoxia exhibited elevated levels of LC3B compared to those cultured under normoxic conditions. Moreover, additional treatment with OE-BNIP3 further enhanced the effects induced by hypoxia. ( Figures 4A, B ). In the context of TEM analysis, it was observed that the number of autophagosomes in A498 and 786-O cells exhibited an elevation upon undergoing hypoxia treatment. Notably, the augmentation in the count of autophagosomes was more pronounced in cells that had undergone BNIP3 overexpression ( Figure 4C ). Western blot analysis revealed that when compared to A498 and 786-O cells under normoxic conditions, the expression of p62 in 786-O cells under hypoxic conditions experienced a decrease, and the ratio of LC3-II to LC3-I exhibited an elevation in A498 and 786-O cells. However, the presence of BNIP3 overexpression contributed to the facilitation of these hypoxia-induced effects ( Figures 4D-F ). Under hypoxia conditions, ROS levels increased in A498 and 786-O cells, and overexpression of BNIP3 increased ROS levels compared to the OE-NC hypoxia and the OE-BNIP3 normoxia groups (P<0.01) ( Figures 4G, H ). Additionally, under hypoxic conditions, the extent of mitochondrial damage was exacerbated in A498 and 786-O cells, and this damage was further exacerbated upon transfection with BNIP3 overexpression ( Figure 4I ).

To explore the effect of autophagy inhibitor 3-MA combined with OE-BNIP3 on apoptosis of renal cell carcinoma cells by mediating autophagy under hypoxia conditions. Flow cytometry results showed that BNIP3 overexpression promoted apoptosis in A498 and 786-O cells under hypoxic conditions, and the addition of autophagy inhibitor 3-MA could inhibit this promotion and reduce apoptosis ( Figures 5A, B ). As shown in Figures 5C, D , ROS production is more likely to be promoted by BNIP3 overexpression in a hypoxic environment, and the autophagy inhibitor 3-MA can effectively inhibit this promoting effect. The findings from WB analysis indicated that the level of expression for autophagy-related regulator LC3-II/LC3-I increased and p62 protein decreased in the OE-BNIP3 hypoxic group. In contrast, the addition of autophagy regulator 3-MA reversed the expression of LC3-II/LC3-I and p62 proteins ( Figures 5E–G ). For the increase of the protein expression associated with programmed cell death cleaved caspase3 and Bax in the OE-BNIP3 hypoxic group, the addition of autophagy regulator 3-MA had a certain inhibitory effect, and the protein expression of cleaved caspase3 and Bax in the OE-BNIP3 + 3-MA hypoxic group decreased ( Figures 5E, H-J ). Based on the above results, it was found that BNIP3 overexpression promoted the apoptosis of renal cancer cells by mediating autophagy under hypoxic conditions.

After 26 days, the mice were euthanized under anesthesia via cervical dislocation, and all 12 subcutaneous tumors were removed and their weights were measured ( Figure 6A ). The tumor sizes in each group of mice were measured. The OE-NC group experienced a significantly faster increase in tumor volume ( Figure 6B ). Upon completion of the experiments, the dissected and isolated tumors were weighed. In comparison to the OE-NC group ( Figure 6C ), the OE-BNIP3 group showed notably reduced tumor volume. Meanwhile, the OE-BNIP3 group exhibited significantly reduced levels of Ki67 expression in tumor tissues compared to the OE-NC group ( Figures 6D, E ). The Tunel staining technique was employed to assess tumor cell apoptosis, revealing a significant increase in apoptosis within the OE-BNIP3 group ( Figures 6F, G ). In addition, the protein expression levels of LC3-II/LC3-I, caspase 3, cleaved caspase 3, and Bax were found to be significantly elevated in the OE-BNIP3 group compared to the OE-NC group. Conversely, the protein expression levels of p62 were observed to be lower in the OE-BNIP3 group as compared to the OE-NC group ( Figures 6H-J ). This part of the experiment showed that overexpression of BNIP3 can inhibit tumor growth in vivo and promote the occurrence of autophagy and apoptosis of tumor cells.