2.1 Bioinformatics analysis
Expression data of APOC1 in RCC and paracancerous tissues, as well as corresponding clinicopathologic profiles were downloaded from The Cancer Genome Atlas (TCGA) database (https://cancergenome.nih.gov/). Expression differences between cancer and normal tissues were analyzed by edger function, and prognosis was analyzed by survival function. After data processing, expression levels of APOC1 and survival curves of RCC patients were determined.
2.2 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. Recruited subjects were followed up through telephone and outpatient review till December 2018, including physical conditions, cancer recurrence, death, etc. No patient received chemotherapy or radiation before surgery and the collected tissues were preserved in liquid nitrogen. All participants read and signed an informed consent, which conformed to standards for the use of human subjects. This study got approval of the Institutional Research Ethics Committee of the Third Affiliated Hospital of Soochow University.
2.3 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 purchased from the ATCC. Cells were cultivated in a humidified environment with 5% CO2 at 37℃. Except for CAKI-1 cells that were 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.
For the overexpression of APOC1 and Wnt3a, the full cDNA sequences of APOC1 and Wnt3a were synthesized and inserted into pLVX-puro vector to generate pLVX-APOC1, pLVX-Wnt3a, and empty vector was used as negative control. The pLKO vectors containing APOC1 or Wnt3a shRNA sequences were constructed by GeneChem (Shanghai, China). The lentiviruses containing (phU6-EGFP-shRNA-APOC1, sh-Wnt3a, sh-NC, oe-APOC1, oe-Wnt3a and oe-NC) vectors were packaged and provided by GeneChem (Shanghai, China). Lentiviral infection was performed in CAKI-1 and 769P cell lines. Pools of stable transductants were generated by puromycin (4μg/ml) selection for 2 weeks.
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, and normalized to that of GAPDH. Oligo primers 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).
2.5 Western blot
Cells or RCC 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. After adjusting protein samples to the same concentration, they were denaturated, separated by SDS-PAGE, and loaded on PVDF membrane. The membrane was cut into small pieces according to the molecular size and blocked in TBST containing 5% skim milk for 2 h. They were incubated with primary (1:1000) and secondary antibodies (1:3000), followed by band exposure using ECL and grey value analyses using ImageJ software. Antibodies were purchased from Abcam (APOC1, Wnt3a, β-Catenin, TCF7, CCND1, Vimentin) and Cell Signaling Technology (GAPDH, MMP2, MMP9, anti-rabbit and anti-mouse secondary antibodies).
2.6 CCK-8 assay
CAKI-1 and 769P 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.
2.7 Colony formation assay
CAKI-1 and 769P 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 following week. Two weeks later, visible colonies were washed by PBS, fixed in methanol for 20 min and dyed with 0.1% crystal violet (Sigma-Aldrich) for 20 min. After washing in PBS, stained colonies were captured for counting.
2.8 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, and 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.
2.9 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 (pore size = 8 μm; Costar, Corning, NY, USA), 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), following the same procedures as abovementioned.
2.10 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), respectively. Briefly, 7×106 transfected CAKI-1 cells were diluted in 150 µl of PBS, which was subcutaneously injected into a single side of the posterior flank of each mouse. 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 (mm3) was calculated using the formula: Tumor width2 × tumor length / 2. Immunohistochemistry was performed to evaluate positive expressions of Ki-67, Vimentin and N-cadherin in xenograft tissues.
2.11 Immunohistochemistry (IHC)
Positive expression of APOC1 in RCC tissues was evaluated using the tissue microarray by IHC. Tissue microarray was incubated with the primary antibody at 4℃ overnight and HRP-conjugated secondary antibody on the other day, followed by diaminobenzidine dyeing. IHC staining was independently assessed by two experienced pathologists. RCC tissues were divided into low- and high-staining groups based on IHC staining results for further analysis.
2.12 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.