Data source
UALCAN (http://ualcan.path.uab.edu/) database provides gene transcriptome data for TCGA[16]. We used UALCAN to obtain the C1QBP and ALDH9A1 mRNA expression in urinary system tumors and normal tissues, while comparing ALDH9A1 mRNA expression in different tumor subgroups, which were defined by the gleason score.
GEPIA2 (http://gepia2.cancer-pku.cn/) is an updated version of GEPIA for analyzing the RNA sequencing expression data of tumors and normal samples from TCGA and GTEx projects[17]. We used GEPIA2 to analyze the effect of C1QBP and ALDH9A1 mRNA expression on the survival of PCa patients.
UCSC Xena (http://xena.ucsc.edu/) contains a variety of database data such as TCGA, IGGC, GTEX, etc, and support online analysis of TCGA data[18]. We analyzed the relationship between C1QBP mRNA expression and overall survival of PCa patients using UCSC Xena.
Patients and tissue samples and Ethical Statement
We collected paraffin embedded tissues of 100 PCa and 50 BPH patients who were surgically treatment at the Second Hospital of Tianjin Medical University between 2016 and 2020. Clinical information on age, serum PSA, gleason score and metastasis were collected to analyze the association of C1QBP with clinicopathology.
IHC staining
The formalin fixed paraffin embedded tissues were cut into 5μm, dewaxed with xylene, hydrated with gradient ethanol, and then antigen repair was completed in citrate buffer (Solarbio, China). Endogenous peroxidase activity was blocked using a peroxide blocker (Solarbio, China), 5% bovine serum albumin (Solarbio, China) was used to block the non-specific antigen. Finally, tissues were incubated with C1QBP antibody (Abcam, USA, ab101267, 1:1,500) at 4°C overnight. After rinsing with PBS, the sections were incubated with secondary antibody (ZSGB-BIO, China) for 1 h at room temperature and detected with HRP DAB detection kit (ZSGB-BIO, China). All sections were scored for percentage and intensity of staining by two independent investigators. The percentage of cells in grade 1-4 was graded as follows: 1, 0-10% of tumor cells were positive staining; 2, 11-50% of tumor cells were positive staining; 3, 51-75% of tumor cells were positive staining; 4, more than 75% of tumor cells were positive. The final score was calculated by combining the percentage and intensity scores.
Cell culture and establishment of stable cell lines
The human PCa cell lines PC-3, DU145, 22RV1, LNcap and the human embryonic kidney HEK-293T cells were acquired from American Type Culture Collection (ATCC, USA). The cells were grown in DMEM and RPMI 1640 (Biological Industries, Israel) supplemented with 10% fetal bovine serum(BSA)(Excell Bio, New Zealand) and 1×penicillin/streptomycin (Biological Industries, Israel). Cells were maintained at 37℃ with 5%CO2. To generate C1QBP knockdown stable cell lines, 293T cells were transfected with lentiviral(pLKO.1-SCR, pLKO.1-shC1QBP)together with lentivirus packaging plasmids(psPAX2, pMD2.G) using Lipofectamine 2000(Invitrogen, USA). The lentivirus supernatant was collected and infected PCa cells. The three primer sequences of pLKO.1-shC1QBP were listed in Supplemental Table1.
RNA extraction and quantitative Real-time PCR
Total RNA was extracted with Trizol reagent (Ambion, USA) and cDNA was obtained by reverse transcription of RNA with FastQuant RT Kit (Tiangen, China). The primer sequences were shown in Supplemental Table 2. The expressions of target genes were normalized to GAPDH.
Western blot
Cells were lysed in SDS lysis buffer containing 1× protease inhibitor cocktail (Roche Applied Science, Germany)and the protein sample was separated by SDS-PAGE electrophoresis and transferred to polyvinylidene fluoride membrane (Millipore, USA). After blocked with 5% BSA, the membranes were incubated with β-actin (Affinity Biosciences, USA, T0022; 1:4,000) and C1QBP (Abcam, USA, ab101267, 1:3,000) overnight at 4℃. Finally, the membranes were incubated with HRP-conjugated secondary antibody for 1hour and visualized with an ECL system (Millipore, USA).
Liquid chromatography tandem Mass spectrometry (LC–MS/MS)
Cells were disrupted using 8 M urea solution in 50 mM triethylammonium bicarbonate, and then the cell debris was removed by centrifugation at 20,000 g for 30 minutes. The proteins were aided sample preparation (FASP) with 10 Kd filter (Millipore, USA)and digested by incubating with trypsin at a protease: protein ratio of 1:40 at 37℃ overnight. Then the peptides were dried with Savant SpeedVac (ThermoFisher Scientific, USA) and resuspended in 0.1% formic acid (FA). Peptides from each fraction were analyzed by nano-LC-MS/MS using an Easy-nLC 1,000 system (ThermoFisher Scientific, USA) coupled to an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific, USA) with a nanoelectrospray ion source (Thermo Fisher Scientific). Protein quantitation was performed in R (3.6.1) by using the unique peptide intensities exported from the Maxquant. The data matrix was normalized by the quantile method, log2 transformed, and imputed by the “imputeLCMD” package. Student’s t-test was performed for statistical analysis, and proteins with p<0.05 and fold change>1.5 were considered to be significantly changed.
CCK-8 proliferation Assay
After starvation with serum-free RPMI 1640 medium, 1,000 PC-3 cells per well were grown in 96 well plates. After the cells were plastered, 10 μl of cell counting kit-8 (Biosharp, China) was added to each 100 μl of medium. Cells were incubated at 37℃ for 2 hours and the absorbance was measured at 450 nm. All plates were assayed at 0, 24, 48, 72 and 96 hour time points to observe cell proliferation.
Cell migration Assay
Migration assay was performed using transwell chambers (Millipore, USA) containing polycarbonate membranes with 8 μm pores. PC-3 cell was starved in serum-free RPMI 1640 medium for 4 hours and then added to the upper chamber, the RPMI 1640 medium containing 20% FBS was added to the lower chamber. After incubation for 12 hours at 37℃, cells in the upper layer of the chambers were wiped off, while cells attached to the submembrane surface were fixed and stained, and then counted in five randomly selected areas. Each group of cells was repeated in three chambers.
Triglyceride determination Assay
Cells were digested by trypsin and washed 1-2 times with 10 × PBS, then centrifuged at 1,000 rpm for 10 minutes. Supernatant was discarded and cells resuspended in 10×PBS were sonicated and fragmented. The appropriate amount of cell lysate and working solution (NJJC, China) were mixed, and then the absorbance was measured at 510 nm to reflect the triglyceride content.
Oil red O staining
Cells were fixed by Oil Red O fixative, isopropyl alcohol was added to soak the cells for 5 minutes. Then, freshly prepared Oil Red O dye was added to stain the lipid droplets, while the nuclei were stained with hematoxylin. Cells were observed under a microscope, and five fields of view per well were randomly selected for photography. Cells that were not stained with hematoxylin could be eluted with isopropanol and the absorbance was measured at 510 nm to indirectly reflect the lipid droplets content.
Statistical Analysis
All statistical tests were calculated using SPSS version 21.0 and GraphPad Prism 9.0. Analysis of t-test, Mann-Whitney test and two-way ANOVA were used for statistical analyses. Differences of p<0.05 were considered statistically significant.