Data collection, preprocessing, and bioinformatics analysis
Microarray datasets GSE36895, GSE53757, GSE68417, GSE66270, GSE16441, GSE781, and GSE634 were downloaded from the GEO database (http://www.ncbi.nlm.nih.gov/geo/). Each dataset was normalized and analysed using the Limma package, and transcriptional data from TCGA (https://portal.gdc.cancer.gov/) were used with the EdgeR package. Only differentially expressed genes (DEGs) with |log2 fold change| > 1 and P value < 0.05 in each microarray between ccRCC tissues and normal samples were included in this study for further analysis.
ccRCC specimens and patients
Thirty-five pairs of ccRCC specimens between tumor and adjacent normal samples were collected from patients diagnosed with ccRCC at Renmin Hospital of Wuhan University (Hubei, China). Fresh tissues obtained during surgery were stored in liquid nitrogen for subsequent RNA extraction and western blots. This study was approved by the Research Ethics Committee of Renmin Hospital of Wuhan University. Informed consent was obtained from all patients (No. 20190420).
Cell lines and reagents
The normal human renal cell line HK2 and human renal cancer cell lines ACHN, 786-O, 769P and Caki-1 were purchased from the China Centre for Type Culture Collection (Wuhan, China) and The American Type Culture Collection (USA). All cell lines were cultured based according to the manufacturers’ instructions. The expression lentivirus of PLCL1, small interfering RNA (siRNA) of PLCL1 (siRNA1 and siRNA2 target sequences: CCGGCCAAATTCTCGCATT, GCGCAAATACAAAGGGCAT), expression lentivirus of DEPP and corresponding controls were purchased from GeneChem (Shanghai, China) and Genecreate (Wuhan, China), respectively. The autophagy inhibitor 3-methyladenine (3-MA) and the autophagy inducer rapamycin were purchased from MedChemExpress (Shanghai, China).
Cell infection and transfection
ACHN, 786-O and 769P cell lines were cultured in complete medium with 10% foetal bovine serum (FBS, ScienCell, USA) and 1% penicillin–streptomycin (Gibco, USA) and maintained in humidified conditions of 5% CO2 at 37°C overnight. Then, ACHN, 786-O, and 769P cells were transfected with lentivirus, siRNA and corresponding controls using Lipofectamine 3000 (Invitrogen, USA) in complete medium with polybrene (Sigma, USA) for 24 h according to the manufacturers’ protocols. Then, the cells were harvested to extract total RNA and protein to verify the transfection efficiency using real-time (RT)–qPCR and western blotting.
Real-time quantitative PCR
Total RNA was extracted from ccRCC samples and cells using TRIzol reagent (Invitrogen, USA), and the purity and concentration were assessed using a NanoDrop ultramicro ultraviolet spectrophotometer (NanoDrop, USA). Then, these analytes were reverse transcribed into cDNA using the PrimeScript RT Reagent Kit with gDNA Eraser (Takara, Japan). RT–qPCR was conducted using SYBR Green mix (Thermo Fisher Scientific, USA). The primer sequences were as follows:
PLCL1: Forward, 5’-GTTGTTCTGGATGATGACTAC-3’;
Reverse, 5’-CTATGGCTTCTCGTAATGGA − 3’;
DEPP: forward, 5’-CTCTGTGAAGCCAGGATG-3’;
Reverse, 5’- AGGTGCGAGTAGAGTGTT-3’;
GAPDH: Forward, 5′-AATCCCATCACCATCTTCCAG-3′;
Reverse, 5′-GAGCCCCAGCCTTCTCCAT-3′.
Western blots
Western blotting analysis was performed as previously described [18]. Briefly, cellular protein was lysed in lysis buffer (Servicebio, China), and the concentration of protein was determined using a BCA Protein Assay Kit (Thermo Fisher Scientific, USA). Then, 50 µg of each sample was separated using 10% SDS–PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. After blocking in 5% milk, the membranes were incubated with primary antibodies (Table S1) overnight. Finally, the membranes were incubated with secondary antibody (LI-COR Biosciences, USA) and imaged using a two-colour infrared imaging system (Odyssey, USA).
Immunohistochemistry and immunofluorescence staining
Four-millimetre-thick sections of paraffin-embedded ccRCC patient and mouse tissues were stained with haematoxylin and eosin. Immunohistochemistry (IHC) staining was conducted according to the manufacturers’ protocols. Briefly, after deparaffinization, each slide was incubated with primary antibodies (Table S1) at 4°C overnight followed by incubation with HRP-conjugated secondary antibody for 30 minutes. Finally, each specimen was stained with 3,3-diaminobenzidine tetrahydrochloride (Maixin, China). For immunofluorescence staining, each slide was incubated with Alexa Fluor secondary antibody (Cell Signaling Technology, USA), and the nuclei of each slide were stained with DAPI for 5 min. Images of each slide were acquired using a microscope (Olympus, Japan).
Cell proliferation assay
A Cell Counting Kit-8 (CCK8, Japan) was used to measure the proliferation rate of ACHN, 786-O and 769P cells according to the manufacturer’s protocols. Briefly, cells in 100 µl of 10% FBS medium were seeded into 96-well plates and incubated with CCK8 reagent for 1 h at 37°C. Then, the absorbance of the cells was measured at 450 nm.
Wound healing and transwell assays
ACHN, 786-O and 769P cells were seeded into 6-well plates. When the cells reached 80% confluence, a 200 µl yellow pipette tip was used to create a wound, and the cells were subsequently incubated in serum-free medium. Images were acquired 0 and 24 h after wounding. For the transwell and invasion assays, a density of 6×104 cells was seeded into the upper chamber of each well in Matrigel (Becton, Dickinson and Company, USA) and incubated in serum-free medium. After 24 h, the cells that had invaded the membrane were stained with 0.05% crystal violet.
Flow cytometry analysis of apoptosis
ACHN, 786-O and 769P cells were infected with lentivirus and siRNA. After 24 h, the cells were collected and stained with Annexin V-FITC and propidium iodide (PI) and analysed using flow cytometry according to the manufacturer’s instructions (BD Biosciences, USA).
High‑throughput RNA sequencing
First, 2 µg of total RNA from 786-O cells with stable overexpression and vector were utilized for r-stranded RNA sequencing library preparation using the KCTM Stranded mRNA Library Prep Kit for Illumina (NO. DR08402, Seqhealth, China) following the manufacturer’s instructions as previously described [19, 20]. Briefly, after assessing the material using a Nanodrop spectrophotometer (Thermo Fisher Scientific, USA) and confirmation by 1.5% agarose gel electrophoresis, 200- to 500-bp PCR products were cleansed, quantified, and sequenced using a HiSeq X10 sequencer (Illumina). Then, the raw sequencing data of each sample were screened using Trimmomatic (version: 0.36) and mapped to the human genome (GRCh38.p13) using Tophat (version 2.1.0). The gene expression in each sample was determined using Cufflinks (version 2.21). Pathway enrichment results with a P value < 0.05 were obtained using the Database for Annotation, Visualization and Integrated Discovery (DAVID) version 6.8 (https://david.ncifcrf.gov).
Transmission electron microscopy
Cells were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) and then postfixed in 1% phosphate-buffered osmium tetroxide at pH 7.4. Subsequently, all specimens were dehydrated, embedded in epoxy resin, sectioned and double-stained with lead citrate and uranyl acetate. Finally, each section was imaged using transmission electron microscopy (Hitachi, Japan).
Adenoviral transfection
Adenovirus with human GFP-mRFP-LC3 was purchased from HanBio (Shanghai, China) to detect autophagosomes and autolysosomes in ACHN, 786-O and 769P cells according to the manufacturer’s protocols. Briefly, cells were seeded into confocal culture dishes and transfected with the adenovirus for 24 h. After washing with phosphate-buffered saline, autophagic flux was imaged with a confocal microscope (Olympus, Japan).
Immunoblot and coimmunoprecipitation
The coimmunoprecipitation (CoIP) reagent of the Pierce™ c-Myc-Tag Magnetic IP/Co-IP Kit (Thermo Fisher, USA) was utilized to examine the interaction based on the manufacturer’s protocols. Briefly, 5 µg of PLCL1 antibody (Abcam, USA), anti-myc antibody and IgG were covalently crosslinked with cell lysates at 4 ℃ overnight. The immune complex was then incubated with Sepharose protein A/G magnetic beads for 1 hour, and the unbound immune complexes were removed. The bound immune complexes were eluted using a low-pH elution buffer and analysed by immunoblotting.
Orthotopic and xenograft tumor model
All experimental procedures were approved by the ethical committee of Institutional Animal Care and Treatment Committee of Renmin Hospital of Wuhan University. Orthotopic and xenograft tumor models were constructed in male Balb/c nude mice (20–25 g) purchased from the Charles River Animal Technology Company (Beijing, China). Briefly, to establish an orthotopic renal tumor model, 786-O cells stably overexpressing PLCL1 and vectors were injected directly into the kidneys of mice after exposing the right kidney using a 1 ml syringe. After 28 days, all mice were scanned using positron emission computed tomography (TransPET Discoverist 180, Wuhan, China) and sacrificed for further study. To construct the xenograft tumor model, 786-O cells stably overexpressing PLCL1 and vectors were directly injected into the right armpit. Then, the mice were sacrificed, and the tumors were removed for further assessment 28 days later. Tumor volume in each group of mice (n = 5) was calculated as (length × width2)/2.
Statistical analysis
GraphPad Prism 7 (CA, USA) was applied to analyse differences between two groups (Student’s t test) and multiple groups (one-way ANOVA). The relationship between PLCL1 and DEPP was examined using Pearson correlation. Univariate and multivariate analyses were performed to determine the relationship between the expression of DEGs and patient survival time, and the Kaplan–Meier method was utilized to analyse the overall survival of patients in TCGA using R software (Version 3.6.1). Data in our study are shown as the mean ± error, and all experiments were completed three times. P values less than 0.05 were considered significant.