Ethics statement
The study was performed with the approval of the Ethics Committee of the University of South China. All participants signed written informed consent before our investigation. The experimental animals were treated according to the Guide for the Care and Use of Laboratory Animals.
Bioinformatics analysis
RCC-related dataset GSE100666 was obtained through the GEO database, which contained 3 normal tissue samples and 3 RCC cancer tissue samples. From this dataset, differentially expressed genes were selected using the “limma” package in R language with the thresholds of |logFC|> 1 and p < 0.05. The differential expression of HDAC3 in pan-cancer was predicted using a bioinformatics website StarBase, and the downstream regulatory miRNAs of HDAC3 were obtained using TransmiR v2.0 and RNAInter, followed by intersection using the jvenn tool. Additionally, RCC-related miRNAs were retrieved through the GeneCards database and screened based on the correlation score, and the screened ones were intersected with the aforementioned downstream regulatory miRNAs of HDAC3. Next, the miRNA target genes were predicted using miRDB, StarBase, TargetScan, and mirDIP tools jointly and the acquired genes were intersected using the jvenn tool. The differentially expressed genes from GSE100666 dataset were then intersected with the candidate miRNA target genes using the jvenn tool.
Clinical sample collection
Paired RCC tissues and adjacent non-cancerous tissues (at a distance > 5 cm from tumor tissues) were resected from 58 RCC patients (40 males, 18 females, mean age: 47.03 ± 8.71 years ranging between 35 and 70 years). The enrolled patients were not treated by chemotherapy or radiotherapy prior to surgical resection. Patients with infectious diseases, autoimmune diseases, or multiple primary carcinomas were excluded.
Isolation and identification of CD3 + T cells
The serum was removed through centrifugation supplemented with heparin anticoagulant. A triple volume of red cell lysis buffer was added to lyse the blood cells for 10 min. The supernatant was removed following 5-min centrifugation. The cells were then rinsed by 5 mL sterile phosphate-buffered saline (PBS) followed by removal of the supernatant through centrifugation. Afterward, cells were re-suspended in sterile PBS before cell counting. Flow cytometric analysis was followed to sort out PE-CD3+ T cells (130-113-129, 1: 50, Miltenyi Biotec Technology & Trading, Bergisch Gladbach, Germany).
Co-culture of RCC cells and T cells
The CD3+ T cells (105 cells/96-well plate) tagged by 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) were co-cultured with the transfected RCC cells in the medium replenished with recombinant human interleukin-2 (rhIL-2) (20 IU/mL), anti-CD3 (2 μg /mL), and anti-CD28 (1 μg/mL). 48 h later, flow cytometric analysis was conducted.
Cell culture
The cells applied for cell experiments included RCC cell lines, Caki-1 (cultured with McCoy’s 5a Medium Modified + 10% fetal bovine serum [FBS]), A498 (cultured with Dulbecco’s Modified Eagle’s medium [DMEM] + 10% FBS), 786-O (cultured with Roswell Park Memorial Institute [RPMI]-1640 + 10% FBS), and 769-P (cultured with RPMI-1640 + 10% FBS), as well as normal renal cell line HK-2 (cultured with keratinocyte serum-free medium + 0.05 mg/mL bovine pituitary extract + 5 ng/mL epidermal growth factor) from American Type Culture Collection (Rockefeller, Maryland, USA). Cell culture was performed in an incubator at 37 °C with 5% CO2.
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
Tissue and cellular total RNA extraction was conducted utilizing the miRNeasy Mini Kit (QIAGEN, GmbH, Hilden, Germany) and TRIzol reagent (15,596,026, Invitrogen, Carlsbad, CA, USA), respectively. NanoDrop ND-1000 Spectrophotometry (NanoDrop Products, Wilmington, DE, USA) was introduced to measure the RNA quality and concentration. For mRNA detection, complementary RNA (cDNA) was obtained by reverse transcription using reverse transcription kits (RR047A, Takara, Japan). For miRNA detection, the cDNA of miRNA containing polyA tails was obtained employing polyA-tailing detection kits (b532451, Sangon, Shanghai, China) which contained miRNA universal PCR reverse primer and U6 universal PCR reverse primer. Primer sequences were synthesized by Sigma-Aldrich (St Louis, MO, USA), which are presented in Additional file 3: Table S1. U6 was selected as an internal reference for miR-195-5p, while glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was applied as an internal reference for SGK1 and HDAC3. Their relative transcription levels were calculated employing a relative quantitative method (the 2−△△Ct method) [41].
Western blot analysis
Total protein isolated from cells and tissues was lysed in 100 µL of lysis buffer at 4 °C for 30 min, followed by 20 min of centrifugation at 12,000 r/min, 4 °C to collect the supernatant. The protein concentration measurement was realized with a bicinchoninic acid kit (20201ES76, Yeasen Biotech Co., Ltd., Shanghai, China). Following sodium dodecyl sulfate–polyacrylamide gel electrophoresis, the isolated proteins were loaded onto a nitrocellulose filter membrane. The protein-carried membrane was blocked with 5% skimmed milk powder at 4 °C overnight and further probed with specific primary antibodies at 4 °C overnight, including rabbit anti-mouse antibodies to PDCD5 (ab75430, 1: 1000, Abcam Inc., Cambridge, UK), HDAC3 (ab32369, 1: 1000, Abcam), SGK1 (ab32374, 1: 500, Abcam), and CD3 (ab16669, 1: 25, Abcam). The membrane was re-probed with diluted horseradish peroxidase-marked goat anti-rabbit immunoglobulin G (IgG) antibody (ab6721, 1: 5000, Abcam) at ambient temperature for 1 h. After visualization, development, and photographic fixing, gray values of protein bands were analyzed by Quantity One software followed by quantitative protein analysis with GAPDH (ab181602, 1: 5000, Abcam) as the internal reference.
Coimmunoprecipitation (co-IP) assay
The A498 cells were lysed in NP-40 buffer (consisting of 25 mm Tris–HCl (pH 7.4), 150 mM NaCl, 1 mm EDTA, 5% glycerol, and 1% NP-40) which contained a complete EDTA-free protease inhibitor mixture (Roche Life Science). Next, the cell extract was cultured overnight at 4 °C with allogeneic rabbit anti-IgG (ab172730, Abcam) or PDCD5 antibody (ab75430, Abcam) or HDAC3 (ab32369, Abcam)-coupled protein G beads (10004D, Roche Life Science). Finally, immune complexes were harvested for Western blot analysis.
Chromatin immunoprecipitation (ChIP) assay
A498 cells were non-treated or manipulated with siRNA for 48 h, cross-linked with formaldehyde, and then subjected to cell lysis and ultrasonic treatment. Chromatin complexes were immunoprecipitated with the use of anti-HDAC3, K27-acetylated histone H3 (Ab4729, Abcam), or homotyped matched anti-IgG (as NC). Following incubation with Dynabeads protein G beads (10004D, Life Technologies), the complexes were harvested. The cross-linking of DNA protein was reversed by heating. After DNA purification, semi-qPCR amplification was performed using the primers covering the HDAC3-binding site within the miR-195-5p promoter. Genomic regions lacking HDAC3-binding sites upstream of GAPDH were used as controls. The qPCR amplification was conducted for purified DNA analysis on LightCycler 480 (Roche Life Science). The input DNA was utilized for normalization of antibody-immunoprecipitated DNA. The primer sequences were: F: CTGGAGCAGCACAGCCAATA, R: AGCTTCCCTGGCTCTAGCA.
5-Ethynyl-2′-deoxyuridine (EdU)
The cells were fixed with 4% paraformaldehyde for 15 min and permeabilized in 1% Triton X-100 for 20 min, followed by incubation with 10 μmol/L EdU (Life Technologies) for 1.5 h. Cells were incubated with the culture medium supplemented with click-it reaction mixture at room temperature for 30 min, stained with Hoechst 33,342 for 30 min, and observed under a fluorescence microscope (Olympus). The cell proliferation rate was obtained based on the proportion of EdU-positive cells.
Cell transfection
Cells were seeded into a 6-well plate for 24 h. After achieving 70% of confluence, 20 μL Lipofectamine 2000 (11,668,019, Thermo Fisher Scientific) was diluted in 500 μL serum-free medium and allowed to stand for 5 min. Meanwhile, the plasmids and liposomes were gently mixed and allowed to incubate together for 20 min. The cells were washed by serum-free medium for 3 times. Every 2 mL serum-free medium was supplemented into each well, and a liposome mixture was added for another incubation for 5–24 h. Following 3 times of washing by serum-free medium, cells were cultured in 20% DMEM without antibiotics for 48 h. The medium was renewed 6 h post transfection. The cell culture lasted for 48 h before their use for subsequent experiments.
Dual-luciferase reporter gene assay
The 3′-untranslated region (3′-UTR) of SGK1 wild type (WT) and mutant (MUT) was artificially synthesized. After restriction enzyme digestion on pmiR-RB-REPORT™, gene fragments WT and MUT were, respectively, inserted into the pmiR-RB-REPORT™ vector (which was constructed by Guangzhou RiboBio Co., Ltd., Guangzhou, Guangdong, China). WT and MUT reporter plasmids together with NC mimic or miR-195-5p mimic were introduced into HEK293T cells, while blank vectors served as controls. 48 h following treatment, the supernatant of cell lysate was harvested through centrifugation for 3–5 min. With the application of the Dual-Luciferase Reporter Assay Kit (YDJ2714, Shanghai Yuduo Biotech Co., Ltd., Shanghai, China), luciferase activity was analyzed in the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA). The relative luciferase activity = relative light unit (RLU) of firefly luciferase/RLU of Renilla luciferase.
Cell surface antigen detection
Following dispersion into a single cell suspension, the collected cells were re-suspended in binding buffer (BD Biosciences). Before use, pacific blue-interferon-γ (IFN-γ) (BioLegend, #505,817, Rat, 1: 50) was subjected to fixation and permeabilization. The pacific blue-IFN-γ or PerCP-CD3 (BioLegend, #100,326, Armenian Hamster, 1: 100) was added into T cells, followed by detection on a BD FACS Canto II flow cytometer (BD Immunocytometry Systems, San Jose, CA, USA) as well as analysis using Flow Jo software.
CD3 + T cell proliferation assessment
The 96-well plate was enveloped with a tetramer antibody of anti-CD3/anti-CD28 (STEMCELL Technologies, Vancouver, Canada). CD3+ T cells were stimulated with IL-2 (20 IU/mL) and then tagged with CFSE (S1076, Solarbio Co., Ltd., Beijing, China) before co-incubation with RCC cells in RPMI-1640 medium at 37 °C with 5% CO2. The cells with low CFSE signals shown by flow cytometric analysis were regarded as proliferated cells.
Enzyme-linked immunosorbent assay (ELISA)
The content of IFN-γ, released from T cells, was measured in strict accordance with the manual of ELISA kits (JLC5013, Jingkang Bioengineering Co., Ltd., Shanghai, China).
Xenograft tumor in nude mice
A498 cells (106) were re-suspended in 100 µL normal saline and subcutaneously delivered into the armpit of nude mice with immunodeficiency (n = 8). The tumor volume measurement was implemented every 2 days. The tumors were resected and imaged before RT-qPCR and cell surface antigen detection.
Statistical analysis
All experimental data were analyzed using the SPSS 21.0 software (IBM Corp. Armonk, NY, USA). Measurement data were summarized as mean ± standard deviation. Following normal distribution and homogeneity of variance, data between two groups in paired design were compared by paired t test and those in unpaired design were analyzed by unpaired t test. Multigroup comparisons were performed by one-way analysis of variance (ANOVA) with assistance of Tukey’s post hoc test. Data among multiple groups at various time points were analyzed employing Bonferroni-corrected repeated measures ANOVA. A value of p < 0.05 was deemed as statistically significant.