Tissues and cell lines
The tumor tissues and paired para-cancer (PC) tissue samples (margins of tumor tissues 3 cm) were collected from 12 patients who underwent surgery for RC at the Second Affiliated Hospital of Xi'an Jiaotong University (Xi'an, China) from January to December 2019. The inclusion criteria was as follows: Patients who were diagnosed with renal cancer by abdominal CT and postoperative pathological examination; in line with the indications of surgical treatment; complete clinical data; patients and their family members were informed of this study and signed informed consent. The exclusion criteria was as follows: hypertension, diabetes mellitus, kidney disease, adrenal disease and hepatic and renal insufficiency; people with coagulation dysfunction; confused patients; poor compliance; women who were lactating or pregnant. The study included 9 male and 3 female patients, with a mean age of 54.7 years (range from 42 to 71 years). All of the included patients were informed of the study and signed an informed consent. Our study was approved by the Ethics Committee of the Second Affiliated Hospital of Xi'an Jiaotong University, Xi'an, China.
Human normal renal cells HK-2 and human RCC cell lines A498, GRC-1, 786-O and ACHN were purchased from the Cell Bank of the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. These cells were cultured in RPMI-1640 medium (Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) in an incubator containing 5% CO2 at 37°C.
Cell transfection
The cDNA of LAMP-2A and PKM2 were amplified using RNA extracted from 293T cells (Cell Bank of the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences). After the cells were cultured at 37°C to the logarithmic growth phase, the cells were inoculated into a 12-well plate, and the cell density was adjusted to 3×105 cells/well. A minimum of two independent shRNAs for LAMP-2A or PKM2 were designed, synthesized and packaged by Guangzhou RiboBio Co., Ltd. shRNAs were cloned into a pLKO.1 puro vector (Addgene, Inc.) according to the manufacturer's protocol. The shRNA sequences used are presented in Table I. The second generation of 80% confluent cells were transfected with lentiviral particles (MOI=16), with 15 µg pLKO.1-shRNA-LAMP-2A/PKM2 plasmid or a scrambled sequence using Lipofectamine 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) for 10 min at 20°C. A total of 72 h after transfection, the cells were harvested. The cells were divided into four groups and transfected with empty control vector (Control), sh-LAMP-2A vector [LAMP-2A(−)], sh-PKM2 vector [PKM2(−)], and sh-LAMP-2A + sh-PKM2 vectors [LAMP-2A(−) PKM2(−)].
Reverse transcription-quantitative (RT-q) PCR
RT-qPCR was performed using the SYBR-Green PCR Master Mix according to the manufacturer's instructions with an ABI 7300HT RT-PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) to determine the expression level of related mRNA. After extracting the total RNA from cells or tissue with the TRizol reagent (Invitrogen; Thermo Fisher Scientific, Inc.), the cDNA was synthesized by reverse transcription experiment using a reverse transcription synthesis kit (PrimeScript™ RT reagent kit; Takara Bio, Inc.) with Oligo dT primer, Prime Script Buffer and dNTP according to the manufacturer's instructions. GAPDH was used as an internal reference. The primer sequences used are presented in Table II. The thermocycling conditions were as follows: 95°C for 8 min, 1 cycle; 95°C for 25 sec, 64°C for 20 sec; 72°C for 20 sec, 10 cycles; 93°C for 25 sec, 60°C for 35 sec, and 72°C for 20 sec, 35 cycles. The relative expression level of related genes was calculated using the 2−ΔΔCq method (14). ΔΔCq=[Cq target gene (sample)-Cq GAPDH (sample)]-[Cq target gene (calibration sample)-Cq GAPDH (calibration sample)].
Western blot analysis
Cells or tissues were collected and lysed with RIPA cleavage buffer (Thermo Fisher Scientific, Inc.). The protein concentration was quantified with BCA reagent (Thermo Fisher Scientific, Inc.). Then, 250 µg proteins were separated by electrophoresis in 10% SDS-PAGE and transferred to nitrocellulose membranes (EMD Millipore). The membranes were blocked in 5% BSA at 37°C for 2 h and then incubated with a primary antibody at 4°C overnight. After washing with TBST (1% Tween-20), the membranes were incubated with a secondary antibody (goat anti-rabbit IgG HRP; 1:3,000 dilution; product code ab205718; Abcam) for 2 h at room temperature. The membranes were visualized with an ECL kit (Thermo Fisher Scientific, Inc.), and finally the band density on the membrane was scanned and analyzed using an image analyzer (model no. MSD910) with Biomaster analysis software (both from Beijing Maisiqi High Technology Co., Ltd.).
The following primary antibodies were used: anti-LAMP-2A (1:1,000 dilution; product code ab125068; Abcam), anti-PKM2 (1:1,000 dilution; product no. 4053; Cell Signaling Technology, Inc.), anti-proliferating cell nuclear antigen (PCNA) (1:1,000 dilution; product code ab92552), anti-hypoxia inducible factor (HIF)-1 (1:1,000 dilution; product code ab179483), anti-p21 (1:1,000 dilution; product code ab109199), anti-vascular endothelial growth factor (VEGF) (1:1,000 dilution; product code ab150375), anti-cleaved caspase-3 (1:1,000 dilution; product code ab32042), anti-Bcl-2 (1:1,000 dilution; product code ab32124), anti-Bax (1:1,000 dilution; product code ab182733) and anti-GAPDH (1:2,000 dilution; product code ab8245; all from Abcam).
Cell proliferation assay
The proliferation level of A498 cells was detected by CCK-8 kit (Beyotime Biotechnology), and the experiment was carried out according to the manufacturer's instructions. Briefly, the cells were inoculated in 96-well plates with an inoculation density of 5×103 cells/well. The cells were cultured at 37°C for 48 h and 10 µl of CCK-8 reagent was added to each well. After incubation for 1 h, the absorbance at 450 nm was measured with a microplate reader. The results were representative of three independent experiments.
Cell cycle and apoptosis analysis
After transfection, cells (1×105 per well) were collected, prepared into single-cell suspension, washed twice with PBS, and fixed with 70% ethanol at 4°C overnight. According to the manufacturer's instructions, 1 µl propidium iodide (PI; Thermo Fisher Scientific, Inc.) was added and placed in the dark at 37°C for 30 min. The cell cycle of each group was detected by flow cytometry (FACSCanto II; BD Biosciences) with FlowJo VX 10 software (FlowJo, LLC), and the proportion of cells in the G1, S and G2 phases was counted.
Cells were collected by the same method without ethanol fixation. A total of 2.5 µl Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) were added into the cell suspension according to the manufacturer's instructions, and placed in the dark at 37°C for 15 min. Cell apoptosis was detected by flow cytometry in each group, and early apoptotic cells, late apoptotic cells and dead cells were counted.
Transwell migration assay
Matrigel was diluted with 0.5% FBS in DMEM (Gibco; Thermo Fisher Scientific, Inc.) medium at 1:5 and seeded on the upper surface of the bottom membrane of a 24-mm Transwell chamber (8-µm pore size; Corning, Inc.) for 16 h at 37°C. Then the cells were incubated for 4 h at 37°C in an incubator with 5% CO2. Cells were further cultured in 5% FBS medium for 24 h at 37°C. After cell counting, the cells were seeded on the upper chamber at 3×104 cells/well. Transwell chambers were fixed with 4% methanol at room temperature for 30 min and stained with 0.1% crystal violet for 20 min at room temperature. A total of 700 µl of culture medium containing 20% calf serum (Gibco; Thermo Fisher Scientific, Inc.) was added in the lower chamber, and the cells were cultured in an incubator at 37°C in an atmosphere containing 5% CO2 for 72 h. Five fields were randomly selected under a low-power microscope (magnification ×10; Olympus Corporation) for each group to compare the differences in the number of transmembrane cells between the groups.
Immunofluorescence
Cells were fixed with 4% methanol at room temperature for 30 min and permeabilized with 0.1% Triton X-100 in PBS for 20 min. The cells were incubated with anti-PKM2 (1:1,000 dilution; product no. 4053; Cell Signaling Technology, Inc.) and anti-HSC70 (1:1,000 dilution; product code ab223356; Abcam) primary antibodies overnight at 4°C, and then incubated in fluorochrome-conjugated secondary antibodies goat anti-rabbit IgG (cat. no. A-31556) and donkey anti-mouse IgG (cat. no. Q22082; both 1:2,000 dilution; both Thermo Fisher Scientific, Inc.) for 2 h at room temperature. DAPI was used to counterstain the cell nuclei at 37°C for 30 min. Images were captured with a laser confocal microscope (Olympus Corporation) in five different fields for each sample.
Co-immunoprecipitation analysis
Approximately 5×106 cells were collected 48 h after transfection and lysed in 0.5 ml RIPA buffer (Beyotime Biotechnology) and centrifuged at 1,000 × g at 4°C for 10 min. Total cell proteins were extracted as Input controls. The precleared lysates (500 µg) were immunoprecipitated against the epitopes with 20 µl of protein A/G-agarose beads (Santa Cruz Biotechnology, Inc.) and incubated with anti-PKM2 antibody (1:1,000 dilution; product no. 4053; Cell Signaling Technology, Inc.) at 4°C overnight. The resultant mixtures were centrifuged at 250 × g at 4°C for 5 min and the supernatants were removed. The pellets were washed three times with PBS and western blotting was performed using an anti-HSC70 antibody (1:1,000 dilution; product code ab223356; Abcam) according to the manufacturer's instructions.
Statistical analysis
Each sample was assessed 3 times and all experiments were performed in triplicate. All data were statistically analyzed and plotted using GraphPad software 8 (GraphPad Software, Inc.). The data were expressed as the mean ± standard deviation (SD). The comparisons between two groups were performed by unpaired Student's t-test or Mann-Whitney U test. Comparisons among more than two groups were performed by ANOVA with Fisher's Least Significant Difference (LSD). P<0.05 was considered to indicate a statistically significant difference.