2.1 Clinical samples of RCC
A total of 20 pairs of RCC and adjacent non-tumoral tissues were collected from January 2010 to August 2014 in the Department of Urology, the Third Affiliated Hospital of Soochow University. TNM staging of RCC was defined based on the Fuhrman histologic grading system. Each subject recruited was followed up till December 2018. This study got approval of the Institutional Research Ethics Committee of the Third Affiliated Hospital of Soochow University.
2.2 QRT-PCR
Cells were lysed using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) for isolating RNAs. Qualified RNAs were reversely transcribed into cDNAs using Primescript RT Reagent Kit (Takara, Otsu, Japan), followed by qRT-PCR using SYBR®Premix Ex Taq™ (Takara, Japan). GAPDH was the internal reference. Each sample was performed in triplicate, and relative level was calculated by 2-ΔΔCt. Plasmids were constructed using Primer 5.0 as follows. APOC1: 5’-GAAGGAGTTTGGAAACACACTG-3’ (forward) and 5’-CATCTTGGCAGAAAGTTCACTC-3’ (reverse); GAPDH: 5’-GAGAGACCCTCACTGCTG-3’ (forward) and 5’-GATGGTACATGACAAGGTGC-3’ (reverse).
2.3 Western blot
Cells or tissues were lysed in RIPA on ice for 15 min, and the mixture was centrifuged for isolating protein samples. The concentration of protein was determined by BCA method. Protein samples with the adjusted same concentration were separated by SDS-PAGE, and loaded on PVDF membrane. The membrane was cut into small pieces according to the molecular size and blocked in 5% skim milk for 2 h. They were incubated with primary (Cell Signaling Technology) and secondary antibodies, followed by band exposure using ECL and grey value analyses.
2.4 Cell culture and transfection
RCC cell lines (7860, 769P, ACHN, CAKI-1, OS-RC-2) and the normal human epithelial cells of renal tubules (HK-2) were provided by ATCC. Cells were cultured in a humidified environment with 5% CO2 at 37℃. Except for CAKI-1 cells cultivated in McCoy’s 5A, the remaining were in RPMI-1640 (GIBCO, Carlsbad, USA). 10% fetal bovine serum (FBS, GIBCO) and 1% penicillin/streptomycin (Invitrogen) were supplemented in the medium.
Transfection lentiviruses (phU6-EGFP-shRNA-APOC1, sh-NC, oe-APOC1 and oe-NC) were synthesized by GeneChem, Shanghai, China. Cell transfection was routinely conducted.
2.5 CCK-8 assay
Cells were inoculated in a 96-well plate with 3×103 cells/well. At day 1, 2, 3 and 4, optical density at 450 nm of each sample was recorded using the CCK-8 kit (Dojindo Laboratories, Kumamoto, Japan) for plotting the viability curves.
2.6 Colony formation assay
Cells were inoculated in the 6-well plate with 1×103 cells/well. Medium was replaced once a week in the first week, and twice in the second week. Visible colonies were washed by PBS, fixed in methanol for 20 min and dyed with 0.1% crystal violet (Sigma-Aldrich) for 20 min. Colonies were captured for counting.
2.7 Flow cytometry
Cells were fixed in 70% cold ethanol for 2 h. Later, they were incubated in 100 μL of RNase at 37°C for 30 min, followed by induction of 5 μL of AnnexinV-FITC and 5 μL of PI at room temperature in the dark. Cell cycle progression was determined using flow cytometry (FACScan, BD Biosciences) in triplicate.
2.8 Transwell assay
200 μl of serum-free suspension containing 2×104 cells and 500 μl of medium containing 10% FBS was respectively added on the top and bottom of a Transwell insert (8 μm diameter), and cultured for 48 h. Migratory cells on the bottom were induced with methanol for 15 min, and crystal violet for 20 min. Five random fields per sample were selected for capturing using a microscope and cell counting. Invasive cells were examined using Transwell inserts pre-coated with Matrigel (Invitrogen).
2.9 Tumorigenicity assay
Tumorigenesis assay in nude mice was approved by the Committee on Animal Ethics and Use of Soochow University. Ten female nude mice with 5 weeks old were administrated with 7×106 CAKI-1 cells transfected with sh-NC (n=5) or sh-APOC1 (n=5) diluted in 150 µl of PBS in the posterior flank. Tumor width and length were recorded with an interval of one week. Mice were sacrificed at the 6th week for collecting tumor tissues. Tumor volume was calculated using the formula: Tumor width2 × tumor length / 2. Positive expression of APOC1 was detected by immunoprecipitant staining.
2.10 Statistical processing
SPSS 22.0 was used for statistical analyses and data were expressed as mean ± standard deviation. Differences between groups were compared by the t-test. Kaplan-Meier survival curves were depicted to analyze overall survival in RCC patients. The potential influence of APOC1 on clinical features of RCC was analyzed by Chi-square analysis. Each experiment was repeated in triplicate. A significant difference was considered at the level of p < 0.05.