Four human RCC cell lines (786-O, ACHN, Caki-1, and 769-P) and human tubular epithelial cells (HK-2) were purchased from ATCC (Rockville, MD, USA). The four RCC cell lines were cultured in DMEM (Gibco, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. HK-2 cells were cultured in keratinocyte serum-free medium (K-SFM) supplemented with 5% FBS.
Small interfering RNAs (siRNAs) against SNHG17 (siSNHG17#1, siSNHG17#2) or H2AX (siH2AX), as well as siRNA scramble control (siSCR), were generated from Genepharma (Shanghai, China). The cDNA of human SNHG17 was amplified and cloned into the vector pcDNA3.1 and termed pcDNA3.1/SNHG17. The sequence was confirmed by DNA sequencing. The microRNA inhibitor, mimics, and negative control (NC inhibitor and NC mimic) were generated from Ribobio (Guangzhou, China).
Transfection of plasmids was conducted using Lipofectamine 3000 Reagent (Invitrogen, Carlsbad, CA, USA). After transfection for 24 h, RCC cells were collected for subsequent analyses. The knockdown and overexpression efficiencies were evaluated by quantitative RT–PCR (qRT-PCR).
Short hairpin RNA (shRNA) targeting SNHG17 (shSNHG17) was produced by GenePharma (Shanghai, China). ACHN cells (5 × 106) stably expressing shSNHG17, SNHG17, or control vectors, generated from Genepharma (Shanghai, China), were implanted subcutaneously into the dorsal flank regions of five-week-old male nude mice (Shanghai lingchang Laboratory Animals, China; n = 5 in each group). All mice experiments had been approved by the Committee of the Ethics of Animal Experiments of Zhengzhou University. The mice's tumor growth was detected every five days and calculated according to the formula: width2 × length/2. One month after the inoculation, the mice were executed, and each tumor sample was harvested for further experiments.
Eighty-four RCC tumor tissue (TT) samples and matched adjacent nontumor tissue (ANT) samples were collected from the Department of Urology, the First Affiliated Hospital of Zhengzhou University. Tissue samples were immediately snap-frozen in liquid nitrogen for long term preservation until RNA extraction. Based on the 2010 American Joint Committee on Cancer (AJCC) tumor-node-metastasis (TNM) classification, all patients were staged.  Detailed information was shown in Supplementary Table s1. The study was executed following the ethics committee of this hospital. All the patients provided written informed consent.
Trizol reagent (Invitrogen) was used for RNA isolation. The Reverse Transcription Kit (Takara, Tokyo, Japan) was used for reverse transcription of extracted RNA. The expression of indicated genes was normalized to endogenous reference controls, GAPDH and U6, and was valued using 2−ΔΔCt method.  Primer sequences were displayed in Supplementary Table s2. Primers for miR-328-3p and other candidate miRNAs were produced from RiboBio (Guangzhou).
RIPA lysis buffer (Thermo Scientific) containing 0.5% PMSF was used to separate the cells' proteins. Equivalent proteins (30 µg) were isolated on 10% SDS-PAGE gel and then transferred to PVDF membranes (Millipore, Billerica, MA, USA). After incubation in blocking solution for 1 h, proteins were immunoblotted with primary antibodies: H2AX (1:1000; Abcam, Cambridge, MA, USA) or β-actin (1:2000; Abcam, Cambridge, MA, USA) overnight at 4 °C. Following incubation with the secondary antibody, the blots were finally detected using enhanced chemiluminescence (Pierce, Rockford, IL, USA). Protein expression levels were normalized to β-actin and quantitated with ImageJ (https://imagej.nih.gov/ij/).
Subcellular fractionation and fluorescence in situ hybridization (FISH)
A PARIS Kit (Thermo Fisher Scientific, Waltham, Mass., USA) was used to isolate nuclear and cytoplasmic RNA, which were then detected by quantitative RT-PCR. GAPDH and U6 were served as endogenous reference controls in the cytoplasm and nucleus, respectively. The FISH assay was used to determine the subcellular location of SNHG17 in ACHN cells. The cell suspension was diluted to 100 cells/µL and seeded in the autoclaved glass slides. After cells were grown for 24 hours, cells were fixed in 4% paraformaldehyde at room temperature for 10 min. ACHN cells were then hybridized with fluorescein-labeled antisense RNA probes specific for SNHG17 (GenePharma) overnight at 37 °C. Slides were rinsed, washed, and then counterstained with 4’-6’diamidino-2-phenylindole (DAPI). Images were acquired with a fluorescence microscope (IX70, Olympus, Japan).
We used CCK8 (Dojindo, Japan) and CellTiter-Glo luminescent cell viability kit (Promega Corporation, Madison, WI, USA) to examine cell proliferation. For CCK-8 assay, RCC cell lines (1.0 × 103 cells per well) were cultured in a 96-well plate for 24 h after transfection. An enzyme-linked immunosorbent assay (ELISA) plate reader was then used to detect the absorbance of 575 nm at 0, 24, 48, and 72 h. For CellTiter-Glo luminescent cell viability assay, the transfected RCC cells were seeded in 96-well plates at a density of 1 × 104 cells/well in culture medium and cultured overnight at 37 ˚C. Following this, 100 µl CellTiter-Glo solution (Promega Corporation, Madison, WI, USA), mixed with the culture medium, was incubated for 20 min at room temperature, and the intensity of luminescence was detected.
RCC cell lines (786-O and ACHN), seeded in 6-well plates, were cultured at 37 °C for 24 h. After the cells were collected and lysed in ice-cold cell lysis buffer (50 µl), caspase-3, -8, and − 9 colorimetric assay kits (Abcam, Cambridge, MA, USA) were used to examine the activity of caspases. A microtiter plate reader (Benchmark, Bio-Rad, USA) at OD405 nm was used to read samples.
Scratch wound-healing assay
The scratch assay was conducted in 24-well plates with a serum-free medium until they reached 100% confluence, and then a wound on monolayer cells across each well's center was generated using a pipette tip. Then, cell motility was tested according to gap breadth at the indicated time points by a phase‐contrast microscope (Nikon, Tochigi, Japan).
In a 24-well plate Transwell system (8-µm pore size, BD Biosciences, USA), RCC cell lines (4 × 104) in serum‐free medium were loaded to the upper chamber (500 µL for each chamber) with Matrigel‐uncoated (for migration) or -coated membrane (for invasion). Culture medium supplemented with 20% FBS was loaded to the lower chamber. Following incubation for 24 hours, cells on the lower surface were immobilized in 4% paraformaldehyde solution and stained with 0.1% crystal violet solution. A light microscope (Olympus, Tokyo, Japan) was employed to count the cells and take pictures with three independent fields.
Bioinformatics analysis, luciferase assay and RNA immunoprecipitation (RIP)
Data mining and bioinformatics analyses were performed following StarBase v3.0 (http://starbase.sysu.edu.cn/), the Lnclocator, LNCipedia (https://lncipedia.org), miRDB (www.mircode.org), and Gepia (http://ualcan.path.uab.edu/index.html). The predicted binding sites of miR-328-3p with SNHG17 were obtained from online software StarBase v3.0. Mutant-type (mut) vectors (SNHG17 mut and H2AX mut) were produced by a Mutagenesis Kit (QIAGEN, California, USA). These vectors were cloned into pGL3-Basic luciferase plasmids (Promega, Madison, WI, USA). These vectors were then co-transfected into RCC cells using Lipofectamine 3000 Reagent (Invitrogen, Carlsbad, CA, USA). Following transfection for 36 hours, luciferase activities were measured in RCC cells using a luciferase assay system (Promega).
To perform RIP assay, we utilized an EZ-Magna RIP™ RNA‐Binding Protein Immunoprecipitation Kit (Millipore). After RCC cell lines (786-O and ACHN) were lysed in RIP buffer, precipitated RNAs were acquired from the cell lysate supernatant using magnetic beads preincubated with antibodies (Ago2, Abcam or control IgG, Millipore). The purified RNAs were analyzed by qRT‐PCR.
Biotinylated RNA pull-down assay
RCC cells (786-O and ACHN, 1 × 106) were transfected with biotinylated SNHG17 or control (50 nmol/L) using Lipofectamine 3000. Following culture for 48 h, cells were pelleted at 1000 rpm, and then cell pellets were resuspended in 0.8 ml lysis buffer. Cell lysates were acquired by centrifugation at 10,000 g. Simultaneously, streptavidin- coupled magnetic beads (Invitrogen) were coated with yeast tRNA (Invitrogen) and incubated at 4 °C for 2.5 hours. Then the beads were washed with splitting buffer and resuspended with lysis buffer. Sample lysates were mixed with precoated beads and incubated overnight at 4 °C. The binding RNAs were washed and eluted, and then the expression levels of RNAs was analyzed using qRT-PCR assay.
Experiments were executed in triplicate, and the data were shown as mean ± SD. Statistical analyses and graphs were performed using Statistical Program for Social Sciences (SPSS) 19.0 (SPSS Inc., Chicago, IL, USA) and GraphPad Prism version 8.0 (GraphPad Inc., La Jolla, CA, USA). When appropriate, the chi-squared test, student's t test, variance analysis, and Spearman correlation test were used. Survival analyses, including overall survival (OS) and relapse-free survival (RFS), were conducted using the Kaplan–Meier curves and cox’s proportional hazards regression model. P < 0.05 was considered statistically significant.