Cell culture
Human embryonic kidney 293T cells and four RCC cell lines, ACHN, A498, Caki-1 and 786-O, were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, Grand Island, NY, USA), 100 µl/ml penicillin and 100 mg/ml streptomycin. All the cells were incubated at 37 °C in a 5% CO2 atmosphere.
Clinical sample collection
A total of 20 pairs of ccRCC and adjacent normal tissues were collected from Affiliated Sanming First Hospital of Fujian Medical University (Sanming, China) between May 2016 and May 2018. The study was approved by the Institutional Ethics Committee of Affiliated Sanming First Hospital of Fujian Medical University, and written informed consent was obtained from all patients. The tumor samples were histopathologically diagnosed as ccRCC and were stored in liquid nitrogen immediately after surgical resection until further use.
Cell Transfection
ACHN or 786-O cells were seeded into 6-well plates at a density of 60%. Synthesized miR-125b mimic or inhibitor was transiently transfected into cells with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). MiR-125b inhibitors (sequence: 5’-UCACAAGUUAGGGUCUCAGGGA-3’), NC inhibitor (sequence: 5’-UCUACUCUUUCUAGGAGGUUGUGA-3’), miR-125b mimics (sequence: 5’-UCCCUGAGACCCUAACUUGUGA-3’), and NC mimic (sequence: 5’-UCACAACCUCCUAGAAAGAGUAGA-3’) were purchased from GenePharma (Shanghai, China). The efficiency of miR-125b expression regulation was confirmed using qRT-PCR.
RNA Isolation and qRT-PCR
Total RNA was extracted from the tissues and cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. For miR-125b detection, Taqman assays were employed, and U6 was used as an internal control. For mRNA analysis, total RNAs were reverse transcribed into cDNA using the RT MasterMix Kit (Abm). Then, qRT-PCR was performed using SYBR Green-I as the fluorogenic dye. The relative expression fold changes were calculated by the 2-∆∆CT method.
Cell proliferation assay
Cell proliferation was measured using the CCK-8 (Dojindo, Kumamoto, Japan) assay. Cells (786-O and ACHN) transfected with miR-125b mimic and inhibitor were seeded into 96-well culture plates at a density of 1 x 104 cells/well. The proliferation assay was carried out for 4 days, and cell growth was measured every 24 hours. Briefly, 10 μl of CCK-8 solution was added to each well, and the plate was incubated at 37 °C for 24 hours. Then, the absorbance of each sample was measured at 450 nm using a microplate reader. All experiments were performed in triplicate.
Cell apoptosis assay
Cell apoptosis analysis was performed by flow cytometry using a fluorescein isothiocyanate (FITC) and propidium iodide (PI) kit (Vazyme, Nanjing, China). Cells were harvested after treatment with miR-125b mimic or inhibitor and washed with precooled PBS. The cells were mixed with 500 μL of binding buffer and 5 μL of FITC and PI and incubated for 10 minutes at room temperature (20 to 25 °C). Apoptosis rates were then measured by flow cytometry (FACS, Partec AG, Arlesheim, Switzerland), and the percentage of apoptotic cells was calculated. Each experiment contained three replicates for each condition.
Cell migration and invasion assays
Cell migration was measured using a Transwell chamber plate (Corning, Bedford, MA, USA). Briefly, 1×105 cells were seeded onto the Transwell insert 48 hours after transient transfection. Twenty percent fetal bovine serum was used as a chemoattractant. After 48 hours of incubation at 37 °C, cells that did not migrate through the pores of the Transwell insert were manually removed with a cotton swab. The cells present at the bottom of the membrane were fixed in 4% polymethanol for 15 minutes and then visualized by incubation with 0.1% crystal violet for 20 minutes. Independent experiments were repeated three times. The invasion assay was performed in a similar manner to the migration assay with the only difference being that the upper layer of the Transwell chamber membrane uniformly covered the Matrigel. Two independent experiments were performed in triplicate for each experiment.
miRNA target prediction and luciferase reporter assay
TargetScan was used to predict miRNAs that could potentially target VDR and identify possible binding regions. The VDR 3’-untranslated region (UTR) contained miR-125b binding sites. The dual luciferase reporter system (Beyotime, Shanghai, China) was then used to verify luciferase activity. The VDR 3'-UTR cDNA sequence, including the mutant or wild-type miR-125b binding region, was amplified and cloned into the pGL3 luciferase vector (Promega, Madison, WI, USA). Next, 786-O cells were transfected with luciferase reporter plasmids and ether miR-125b mimics or NC using Lipofectamine 2000, according to the manuscript protocol. Then, the activity of luciferase was determined using a luminometer (Promega, Madison, WI, USA) and measured based on that of the empty pGL3 vector.
Western blotting
VDR expression was assessed by Western blotting using standard protocols. Briefly, cells were harvested 48 hours after transfection, and lysates were prepared in radioimmunoprecipitation assay buffer. Equal amounts of protein, as determined by the Bradford Protein Assay (Beyotime, Shanghai, China), were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Beyotime, Shanghai, China). Five percent skim milk powder was added for 2 hours at room temperature, and after incubation with a peroxidase-conjugated secondary antibody (Beyotime, Shanghai, China), the blot was developed using an enhanced chemiluminescence reagent and exposed to X-ray film to detect the labeled protein. β-actin served as a reference standard for protein expression.
Statistical analysis
All statistical analyses were performed using GraphPad Prism 7 and SPSS 22, and all the data are presented as the mean ± SD. P<0.05 was considered to indicate a significant difference.