All experimental procedures involving animals were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and Guide for the Care and Use of Laboratory Animals in China. The present study complied with the ethical standards and was approved by the Medical Ethics Committee of Taizhou University Medical School (approval no. 2019‑215; Taizhou, China).
TCGA data analysis
RCC tissue samples and their corresponding clinicopathological parameters were downloaded from TCGA database. We selected 525 cases of RCC and 72 cases of solid normal who had RCC. For TCGA data, the edgeR package was used for screening differentially expressed genes (DEGs), using the criteria of adjusted P-value <0.01 and log2|fold change| >2 as the cut-off. To analyze the potential prognostic value of KIR2DL4, the cut-off point was set using the median value of KIR2DL4 expression in TCGA cohort to categorize into high and low expression subgroups.
Cell culture and reagents
Human RCC cell lines, Caki-1 and 769-P, and the 293T cell line were obtained from The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences and maintained in the appropriate medium as suggested by the supplier. All cells were cultured at 37˚C in a humidified, mycoplasma-free atmosphere with 5% CO2. Polybrene and puromycin were purchased from MedChemeExpress (Shanghai, China).
The pLenti-EF1a-EGFP-P2A-Puro-CMV-KIR2DL4-3Flag plasmid was obtained from OBiO Technology (Shanghai) Corp., Ltd. The insert sequence of KIR2DL4 (accession no. NM_002255) was subcloned into the lentiviral vector.
For the creation of the short hairpin RNA (shRNA) plasmid, the 5’-GGTCTATATGAGAAACCTT-3’, 5’-CCCAGCATCAATGGAACAT-3’ or 5’-CCTTCTAGTAGTTGGCCTT-3’ sequences were subcloned into the pLKD-CMV-G&PR-U6 vector separately to establish a KIR2DL4-shRNA vector. The negative control sequence used was TTCTCCGAACGTGTCACGT.
pMD2.G was a gift from Didier Trono (Addgene plasmid no. 12259; Addgene, Inc.) whereas psPAX2 was a gift from Didier Trono (Addgene plasmid no. 12260; Addgene, Inc.). Both of these aforementioned plasmids were used for lentivirus assembly.
RCC samples were obtained from Taizhou University Hospital. The patients provided written informed consent prior to participation in the study, which was ethically approved by the Medical Ethics Committee of Taizhou University Hospital (approval no. TZZXYY2019‑027; Taizhou, China). The tissues were formalin-fixed and paraffin-embedded, and then cut into 4-μm sections. The slices were then processed for immunohistochemistry by following the standard methods as previously described. Antibodies against KIR2DL4 were purchased from Abcam.
Immunoblotting assay were conducted by following standard procedures  using antibodies against KIR2DL4 (Abcam), AKT, phosphorylated AKT (pAKT; Cell Signaling Technology, Inc.) and GAPDH (Santa Cruz Biotechnology, Inc.).
Reverse transcription-quantitative PCR
Total RNA was extracted with TRIzol® (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturers’ protocol and was reverse transcribed using the PrimeScript™ RT reagent kit (Takara Biotechnology Co., Ltd.). The resultant cDNA was amplified by qPCR using a KIR2DL4-specific primer pair. The primer sequences for KIR2DL4 were as follows: 5’-GTTGGATCACTGCATTTTCACAC-3’ (forward) and 5’-GGCTCAGCATTTGGAAGTT CTATAC-3’ (reverse). 5’-GCACCGTCAAGGCTGAGAAC-3’ (forward) and 5’-GCCTTCTC CATGGTGGTGAA-3’ (reverse) were the primer pairs used for GAPDH. Thermocycling was programmed according to instructions as follows: Initial denaturation at 95˚C for 30 sec, followed by 40 cycles of 95˚C for 5 sec, 60˚C for 30 sec and then 95˚C for 15 sec, 60˚C for 60 sec and 95˚C for 15 sec. Gene expression was assessed using the 2-ΔΔCq method and mRNA levels of KIR2DL4 were normalized to those of GAPDH in the identical sample.
Cell viability was assessed using a MTT assay. Briefly, cells (1x103 cells per well) were placed in a 96-well plate in complete culture medium. After 4, 24, 48, 72 and 96 h, the media was carefully aspirated before 50 µl serum-free media and 50 µl MTT solution was added into each well. The plates were then incubated at 37˚C for another 3 h. After incubation, 150 µl MTT solvent was added into each well before the plates were wrapped in foil and shaken on an orbital shaker for 15 min, then absorbance at 490 nm was read in each well.
Flow cytometry analysis of DNA content
DNA content was measured using flow cytometry (CytoFLEX S; Beckman Coulter, Inc.) after fixation with 70% ethanol and propidium iodide staining. Kaluza Analysis software (Beckman Coulter, Inc.) was used for subsequent analysis.
Colony formation assay
Initially, a 4% low melting-point agarose stock was made with ddH2O. The 24-well plates were then coated with 0.8% low melting-point agarose in RPMI-1640 medium supplemented with 10% FBS, which was used as the base agarose. Cells were suspended in RPMI-1640 media containing 0.4% agarose and seeded at a density of 1,000 cells per well in a 24-well plate as the top layer and supplemented with 100 μl 10% FBS medium every 3 days. After incubating for 14 days at 37˚C under 5% CO2, cell colony-forming units were counted.
In vivo xenograft model and tumorigenicity
Male BALB/C‑nu/nu mice aged 4‑5 weeks were obtained from Shanghai SIPPR-Bk Lab Animal Co., Ltd. (Shanghai, China). The mice were housed in sterile cages under laminar airflow hoods at 20˚C, in a specific pathogen‑free environment under a 12‑h light/dark cycle and provided with autoclaved chow and water ad libitum. The present study was ethically approved by the Medical Ethics Committee of Taizhou College of Medicine (approval, no. TZYXY2019‑215; Taizhou, China). In total, 1x107 parallel-controlled or KIR2DL4-overexpressed Caki-1 cells were transplanted subcutaneously into the flank of six mice 6 mice in each group. Tumor volumes were measured with calipers twice per week and calculated using the formula volume = (width)2 x length/2. After 12 weeks, animals were sacrificed by cervical dislocation before the solid tumors were removed and weighed. The largest diameter of tumors isolated from all mice examined was 1.106 cm, with no multiple tumors observed.
Total RNA was extracted using the miRNeasy Mini kit (Cat. no. 217004; Qiagen GmbH) and inspected for quality by electrophoresis using an Agilent Bioanalyzer 2100 (Agilent technologies, Inc.). mRNA was concentrated by Oligo (dT) magnetic beads with 3-5 μg total RNA used.
Next, mRNA was fragmented in a Mg2+ solution, which was then reverse transcribed using random primers to synthesize the first strand and the second strand cDNA, which became a double-stranded cDNA. After the 3’ ends were adenylated and the 5’ ends were repaired, “Y” adapters were ligated to both ends of the double-stranded cDNA. After fragment selection, PCR amplification and purification, the product was inspected for quality by electrophoresis with an Agilent Bioanalyzer 2100 (Agilent technologies, Inc.). In this manner, a standard sequencing library was completely constructed, which was sequenced on a HiSeq sequencer.
Statistical analysis was performed with GraphPad Prism version 8 (GraphPad Software, Inc.). Data are presented as the Mean ± SD. Statistical differences were determined using a Student’s t-test or ANOVA. P<0.05 was considered to indicate a statistically significant difference.