Cell culture and establishment of stable cell lines
Human RCC cell lines 786-O and ACHN and HEK-293T were obtained from American Type Culture Collection (ATCC, USA). The cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) and Eagle’s Minimum Essential Medium (MEM) (HAKATA, China) supplemented 10% fetal bovine serum (HAKATA, China) and 1 × penicillin/streptomycin (Biological Industries, Israel) in a humidified 37 ℃ incubator in the presence of 5% CO2.
To construct C1QBP overexpression and knocked-down stable cell lines, HEK-293T cells were co-transfected with lentiviral vectors (Promega, USA), including pLKO.1-Scr, pCDH-C1QBP, pCDH or pLKO.1-shC1QBP and with packaging plasmid system consists of pSEV-REV, pMD 2.G and pRRE for 48 h using lipofectamine 2000 (Invitrogen, USA). The lentivirus supernatant was collected, centrifuged and filtered with 0.45 μm filter. Then 786-O and ACHN cells were infected with the lentivirus supernatant respectively for 48 h and selected for 10 days with 2 μg/ml puromycin (Sangon Biotech, China) or 100 mg/ml G418 (MDBio Inc, China), subsequently maintained with 0.5 μg/ml puromycin or 25 mg/ml G418. The expression of C1QBP in the stable cell lines were determined using qRT-PCR and western blot.
RNA extraction, reverse transcription and quantitative real-time PCR
Total RNA extraction, reverse transcription and qRT‐PCR analysis were performed as previously described [9]. The primers for qRT‐PCR analysis were synthesized by HuaDa Gene. The primer sequences were showed in Table 1 (page 32). The expressions of target genes were normalized to GAPDH and the data were analyzed by the 2−ΔΔCt method.
Western blot
Proteins were extracted with SDS lysis buffer and 1 × protease inhibitor cocktail (Roche, Germany) and the concentrations were measured by Enhanced BCA Protein assay kit (Thermo Fisher Scientific, USA). Proteins were separated by SDS-PAGE, then transferred onto a PVDF membrane (Millipore, USA), followed by blocking with 5% milk (BD, USA) in TBST (Tris‐buffered saline with Tween‐20) solution for 1.5 h at room temperature. Primary antibodies were incubated at 4 ℃ overnight. with corresponding primary antibodies to C1QBP (Abcam, USA, ab101267; 1:2000), XDH (Affinity, DF8111; 1:1000), caspase-3 (Abcam, ab184787; 1:2000), bcl2 (Abcam, ab182858; 1:2000), bax (Abcam, ab32503; 1:2000) and β-actin (Affinity Biosciences, T0022; 1:3000). In the next morning, after washing three times, the membranes were incubated with HRP‐conjugated goat anti‐rabbit IgG (Affinity, S0001; 1:5000) or goat anti‐mouse IgG (Affinity, S0002; 1:4000) secondary antibodies for 1h at room temperature, and developed by ECL Western Blotting Detection Reagents (Millipore, USA). The expressions of target proteins were normalized with β-actin.
Metabolomics Relative-quantitative Analysis
Well-growth and logarithmic 786-O C1QBP overexpression and control cells(1 × 107)were washed twice with ice-cold PBS, and then washed cells with ice-cold saline (0.9 % sodium chloride solution) quickly, discarding the supernatant completely after each wash. Finally, cells were collected in a 1.5 ml centrifuge tube with 1 ml of methanol: acetonitrile: water (2:2:1, v/v) and stored at -80 ℃ after quick freezing with liquid nitrogen.
The samples were thawed and mixed with 1ml cold methanol/acetonitrile/H2O(2:2:1,v/v/v)and sonicated at low temperature (30 min/once, twice) . Followed by centrifuged for 20 min (14000 g, 4 ℃) and the supernatant was dried in a vacuum centrifuge. For LC-MS analysis, the samples were re-dissolved in 100 μl acetonitrile/water (1:1, v/v) and adequately vortexed, and then centrifuged (14000 g, 4 ℃, 15 min). The supernatants were collected for LC-MS/MS analysis using an UHPLC (1290 Infinity LC, Agilent Technologies) coupled to triple quadrupole mass spectrometer.
MRM Analyzer software was used to extract chromatographic peak area and retention time. Detected metabolites in pooled samples with coefficient of variation (CV) less than 30 % were denoted as reproducible measurements. In order to find the different expressed metabolites, statistical analyses between two sample groups were performed by calculating the fold changes and p values of metabolites using Student t test, p < 0.05, fold changes > 1.5, were marked as the significantly changed metabolites between sample groups.
Xanthine/hypoxanthine detection
The level of xanthine/hypoxanthine was detected by using Xanthine/Hypoxanthine Colorimetric/Fluorometric Assay Kit (Bio Vision, USA,) in RCC cells. According to the manufacturer’s protocol, cells (1 × 106) were lysed with 100 μl ice cold Xanthine Assay Buffer for 10 min on ice, centrifuged at 12000 rpm for 5 min followed by collecting the supernatant. The mixture of samples and reaction buffer were incubated for 30 min at room temperature, protected from light. Measure color at λ = 570 nm using Microplate reader. Actually, calculating xanthine/hypoxanthine concentration (noml/ml) in the sample according to the standard curve.
Human patients
Human patient cohorts were used to explore the clinical importance of C1QBP and XDH expression in RCC. We collected 57 pairs of primary RCC tissues and corresponding para-carcinoma tissues. All RCC tissues were surgically removed and paraffin-embedded in the Second Hospital of Tianjin Medical University between January 2013 and December 2018 with the patients’ consent and the approval from the ethical committee. Criteria for inclusion were that patients with pathological diagnosis of RCC and patients had undergone radical nephrectomy with no preoperative and postoperative adjuvant therapy. In addition, patient clinical pathological parameters were collected including gender, age, tumor size, tumor stage and Fuhrman grade.
IHC staining
Tissues were formalin-fixed and embedded in paraffin blocks and then cut into 5 µm thickness sections. Tissue sections on glass slides were deparaffinized in xylene, and dehydrated in gradient ethanol, followed by blocking of endogenous peroxidase. Repaired antigens with boiling citrate buffer and blocked non-specific background staining with 5% BSA (Solarbio, China). The tissue sections were incubated with primary antibody C1QBP (Santa Cruz Biotechnology, USA) or XDH overnight at 4 ℃. The second day, after washing with PBS, the slides were immunoblotted using Universal kit (mouse/rabbit polymer detection system) (ZSGB.BIO, China). Sections were developed by peroxidase substrate DAB Detection Kit (ZSGB.BIO, China) and then counterstained by hematoxylin. Finally, covered slides with neutral resin.
All immunostaining slides were scored by two independent researchers for the percentage and intensity of cells showing specific immunostaining signals. The score of percentage of positively stained cells was evaluated from 0 to 5: 0, (0–1) % cells positive; 1, (1–5) % cells positive; 2, (6–10) % cells positive; 3, (11–20) % cells positive; 4, (21–50) % cells positive; and 5, (51–100) % of cells positive. The score of immunostaining intensity as follows: 0, no staining; 1, yellow-brown staining; 2, brown staining. The final score was calculated by adding the percentage and intensity scores and recorded as negative (0–3) and positive (4–7).
RNA stability
RCC cells were treated with 5 μg/ml actinomycin D (Med Chem Express, USA) at indicated time point 0 min, 15 min, 30 min, 45 min, 60 min. The RNA was extracted using Trizol (Ambion, USA) and detected XDH mRNA levels by qRT-PCR.
Reactive Oxygen Species (ROS) Assay
The cells were detected with ROS Assay Kit (Beyotime Biotechnology, China) according to the manufacture’s instruction. RCC cells were seeded at a density of 3 × 105 cells/well in 6-well plate and cultured overnight. The cells were treated with 10 μM DCFH-DA and incubated at 37 ℃ for 20 min. Subsequently, washing cells three times to fully remove the DCFH-DA from liquid. And in positive group, cells were treated with 50 mg/ml Rosup for 20 min. Afterwards, cells were collected and the levels of ROS were detected by fluorescent microplate reader at λ = 488 nm/525 nm or detected via flow cytometry.
Annexin V/PI apoptosis assay
Renal cancer cells were detected with Annexin V-FITC apoptosis assay kit (Absin bioscience, China) according to the manufacture’s instruction. The cells were harvested and counted and 5 × 105 cells were resuspended with 300 μl 1 × binding buffer. Subsequently, cells were stained with Annexin V-FITC and PI for 15 min in the dark, and detected by flow cytometer (BD FACS Verse, USA). The results were analyzed by Flow Jo vX.0.7 software (Flow Jo, USA).
Small interfering RNA
To investigate the role of XDH in RCC cells, XDH was knocked down by transient transfection of small interfering RNA (siRNA). Three independent XDH siRNA oligonucleotides and non-targeting sequences (negative controls) (Ribo Bio, China) were transfected into ACHN and 786-O cells using Lipofectamine 2000 and 50 nmol/L of each siRNA according to the manufacture’s protocol. The medium containing siRNAs were replaced 4-6 h after transfection.
Total RNA was extracted 24 h after transfection and protein was extracted 48 h after transfection. The knockdown of gene expression was assessed by western blot and qRT‐PCR. The sequences of XDH siRNA were listed in Table 2 (page 32).
Xenograft tumor growth and metastasis
Luciferase-labeled C1QBP overexpression and control of ACHN cells were stably constructed through lentivirus-mediated luciferase plasmids (ACHN-pCDH-luc and ACHN-pCDH-C1QBP-luc) and efficiencies of transduction including luminescence intensity and protein expression were verified using microplate reader and western blot, respectively. BALB/c Nude mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) (n = 6 each group, 6–8 weeks old, male: female = 1:1) were anesthetized and surgery was performed to expose the left kidney on the back of the mouse. Subsequently, tumor cells (1 × 106 cells, mixed with Matrigel, 1:1) were injected under the renal capsule and incisions were sutured. Eight weeks later, mice were intraperitoneally injected with luciferin (30 µg per mouse), and detected bioluminescence signals of primary tumors and metastasis in liver and lung by the live IVIS imaging system (Perkin Elmer, USA). Mice were sacrificed, and tumors of the left kidney, liver and lungs were removed. The weight of primary tumor of mice were calculated by using minus the weight of corresponding right kidney. Primary RCC tumors, livers, and lungs were fixed in formalin, embedded in paraffin and then cut into sections. The proteins expressions in sections of primary RCC tumor were observed by IHC staining with antibodies of C1QBP, XDH, caspase-3, bax and bcl2, and metastases of lungs and livers of mice were examined with HE staining. Actually, the difference between two groups were analyzed using t test.
Xenograft tumor growth and metastasis
Liver and lung tissues of BALB/c mice were formalin-fixed and embedded in paraffin and then cut into 5 µm thickness sections. Tissue sections on glass slides were deparaffinized in xylene, and hydrated in gradient ethanol from high to low concentrations and soaked in distilled water, followed by staining with hematoxylin and eosin. Next, the sections were dehydrated in gradient ethanol from low to high concentrations ethanol and hyalinized in xylene then covered glasses with neutral resin. Sections were observed the metastases under the microscope and took pictures.
Statistical analysis
Statistical analysis was performed by GraphPad Prism 7.0 and SPSS 21.0 software. All data were represented as the mean ± SD. Differences between two groups were used t- test. Correlation analysis between C1QBP and XDH expressions was analyzed with Spearman test. χ2 test was used for analysis of the correlations between protein expression and clinicopathologic features. P < 0.05 was considered as statistically significant.