1.1 Bioinformatics analysis
TCGA is currently the largest cancer genetic information database. ccRCC miRNA-seq (normal: 71, tumor: 536) and mRNA-seq (normal: 72, tumor: 530) expression data were downloaded from TCGA database (https://portal.gdc.cancer.gov/). “edgeR” package was employed to obtain the differentially expressed miRNAs and mRNAs (DEmiRNAs, DEmRNAs) with threshold set as |logFC|>2 and FDR<0.05, while “survival” package was used for survival analysis to identify the target DEmiRNA. miRDIP (http://ophid.utoronto.ca/mirDIP/index.jsp#r) and miRDB (http://mirdb.org/) databases were applied to conduct target prediction for the miRNA. The mRNA with binding sites of the miRNA was identified from the intersection of DEmRNAs in TCGA database and predicted genes.
1.2 Cell culture
Human renal tubular cell line HK-2 (BNCC339833), human renal carcinoma cell line A498 (BNCC100609), and human ccRCC cell lines 786-0 (BNCC338472), 769-P (BNCC100976) and Caki-1 (BNCC100682) were all purchased from BeNa Culture Collection (BNCC; China). All cell lines were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; BNCC351841, BNCC, China) containing 10% fetal bovine serum (FBS), and maintained in an incubator with 5% CO2 at 37 ℃.
1.3 Cell transfection
miR-129-5p mimics (miR-mimics), mimics NC (miR-NC), pcDNA3.1-SPN (oe-SPN) and pcDNA3.1 (oe-NC) accessed from Ribobio (China) were transfected into ccRCC cell line 786-0 by Lipofectamine 2000 kit (Invitrogen, Carlsbad, USA) in accordance with instructions. After 24 h of transfection, transfected cells were used for subsequent experiments.
1.4 Real-Time fluorescence quantitative PCR
Total RNA was isolated from cells using TRIzol kit (Life Technologies, USA), and its concentration was measured by NanoDrop 2000 system (Thermo Fisher Scientific, Inc., USA). miRNA was reversely transcribed into cDNA by miScript II RT kit (Qiagen, USA), while mRNA was transcribed into cDNA by PrimeScript RT Master Mix (Takara, P.R. China). miRNA expression and mRNA expression were detected by miScript SYBR Green PCR Kit (Qiagen, Germany) and SYBR ® Premix Ex Taq TM II (Takara Bio Inc., Japan), respectively. qRT-PCR was performed to assess miR-129-5p and SPN mRNA expression through Applied Biosystems® 7500 Real‐Time PCR Systems (Thermo Fisher Scientific, MA) with U6 and GAPDH taken as internal reference respectively. Primer sequences used in qRT-PCR were listed in Table 1. The relative expression of miR-129-5p and SPN mRNA was presented by 2-ΔΔCt method. The experiment was repeated three times.
1.5 Western blot
Total proteins were harvested after cells were lysed by RIPA lysis buffer, and protein concentration was assayed by BCA kit (Beyotime, China). After being denatured at a high temperature, proteins were isolated by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride membranes (PVDF; Millipore), which were then blocked with 5% skim milk for 2 h. Then the membranes were incubated with primary antibodies overnight at 4 ℃. Primary antibodies including mouse anti-SPN (ab9088, 1:100) and mouse anti-β-Actin (ab20272, 1:5000) were both purchased from Abcam (Shanghai, China). Subsequently, the membranes were incubated with secondary antibody goat anti-mouse IgG (ab205719; abcam, Shanghai, China). After culture for 2 h at room temperature, all protein bands were visualized using an enhanced chemiluminescence kit (GE Healthcare, USA). The experiment was conducted in triplicate.
1.6 Cell proliferation assay
CCK-8 assay was applied for the detection of cell proliferative ability. Cells were seeded into 96-well plates at a density of 2×104 cells/well and incubated with 5% CO2 at 37 ℃. 10 μl CCK-8 solution (CK04; Dojindo Laboratories, Japan) was added to each well at 0 h, 24 h, 48 h, 72 h and 96 h, followed by incubation for 2 h with 5% CO2 at 37 ℃. The absorbance at 450 nm was measured by Microplate reader (Multiskan MK3, Thermo Fisher Scientific Inc., MA). The experiment was repeated three times.
1.7 Colony formation assay
Colony formation assay was employed for the determination of colony forming ability. Transfected cells were planted into 6-well plates with a density of 1×103 cells/well, and each procedure was run in triplicate. Transfected cells were then incubated in complete mediums for 14 days until the colonies were visualized to naked eyes. Cell colonies were fixed in 4% paraformaldehyde for 15 min at room temperature and then stained by 0.05% crystal violet (Thermo Fisher, USA) for 20 min. Sterile water was used to wash away the crystal violet in each well. Colonies (over 50 cells) were calculated in each well. The experiment was performed three times.
1.8 Transwell invasion assay
24-well Transwell chambers (8 μm in aperture, BD Biosciences) were used for Transwell invasion assay. Approximately 2×104 cells were placed into the upper chambers coated with Matrigel matrix, while the lower chambers were filled with DMEM supplemented with 10% FBS. After being cultured for 48 h at 37 ℃, the non-invaded cells were removed through a wet swab cotton, while the invaded cells were stained with 0.1% crystal violet. The images of cells were captured under the inverted microscope (DSX510i, Olympus, Japan), and five fields were randomly selected to count the invaded cells. The experiment was conducted three times.
1.9 Wound healing assay
Wound healing assay was carried out to detect cell migratory ability. Cells were placed into 6-well plates. When cells grew to 80% confluence, cell monolayers were wounded using a 200 μl pipette tip. Mediums were used to wash the cells twice to remove the isolated cells. Then, the cells remained were cultured in fresh medium for another 24 h. Cell migration was observed and images at 0 h and 24 h were caught. The experiment was repeated three times.
1.10 Dual-luciferase assay
To determine the binding relationship between miR-129-5p and SPN 3’-UTR, luciferase vectors pmirGLO (Promega, USA) fused with wild type (WT) SPN 3’-UTR or mutant (MUT) SPN 3’-UTR were established. ccRCC cells 786-0 were seeded into 96-well plates (3×105 cells/well), and 100 nM miR-mimics/miR-NC and -WT/-MUT were co-transfected into cells. After culture for 48 h, luciferase activity was measured by dual-luciferase reporter assay system (Promega, USA). The experiment was performed in triplicate.
1.11 Statistical analysis
All data were analyzed by GraphPad Prism 6.0 (La Jolla,CA), and the results were expressed in mean ± standard deviation. The comparison between two groups was analyzed by t test. * means p<0.05, and p<0.05 was considered statistically significant.