Patient tissue samples
This study was approved by the Ethics Committee of the Second Affiliated Hospital of Medical School, Xi’an Jiaotong University (Xi’an, China), which was conducted according to the Helsinki Declaration. The RCC tissue samples were collected from 42 RCC patients (45.0 ± 15.3 years) who experienced surgical resection but did not receive chemotherapy or radiation therapy. The corresponding adjacent normal tissues were also taken from the edge of the RCC tissues by 3 ~ 4 cm which were no obvious tumor cells. All tissues were frozen in liquid nitrogen and stored at -80 oC for subsequent testing.
Cell lines and cell culture
The human renal epithelial cell lines (HK-2 and 293T) and RCC cell lines (Caki-1, ACHN, 786-P and 786-O) utilized in the present study were purchased from the Institute of Cell Research, Chinese Academy of Sciences (Shanghai, China). The authenticity of cell lines has been verified by short tandem repeat analysis. All cells were cultured in Roswell Park Memorial Institute 1640 medium (RPMI-1640, Gibco, BRL, Carlsbad, CA, USA) and supplemented with of 10 % fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA), then incubated in a 37 oC incubator with a humidified atmosphere of 5% CO2.
Reverse transcription polymerase chain reaction (RT-qPCR)
Total RNA was extracted from cells and tissues according to the instruction of TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and purified with the RNeasy Maxi kit (Qiagen, Dusseldorf, Germany) according to the manufacturer's protocols. MiScript Reverse Transcription kit (Qiagen, Dusseldorf, Germany) was used to reverse transcription with 1 µg total RNA of each sample and miScript SYBR® Green PCR kit (Qiagen, Dusseldorf, Germany) was used to detect the expression level of miR-6838-5p following the manufacturer's protocols. We used U6 or GADPH as endogenous control, respectively. PCR program in real-time PCR system (ABI7500, Applied Biosystems, Waltham, MA, USA), the detailed process was as follows: 1 minute at 95 oC followed by 35 cycles of 20 seconds at 95 oC, then 10 seconds at 56 oC and 15 s at 72 oC. Finally, the relative expression levels of miR-6838-5p were calculated using the 2-ΔΔCt method.
The NC mimic, miR-6838-5p mimic, NC inhibitor, anti-miR-6838-5p, Scramble and DMTF1 siRNA, Vector were purchased from Invitrogen (Carlsbad, CA, USA). DMTF1 overexpressed plasmid (pcDNA3.1-DMTF1) was synthesized by Thermo Fisher Scientific (Waltham, MA, USA). Transfection and co-transfection of these oligonucleotides were carried out with Lipofectamine Plus reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.
We used RIPA lysis buffer (CW Biotech, Beijing, China) to lyse the cells to extract total protein and measure protein concentration by BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). To separate protein samples in equal amounts, 10% SDS-PAGE must be used. Next, we transferred and blocked the complex onto a nitrocellulose membrane (General Electric Co, USA). After blocked with 5% skim milk, the blots were probed with primary antibodies. Following washed with PBS, the horseradish peroxidase-conjugated Goat Anti-Rabbit IgG secondary antibodies (ab205718, 1：2, 000) were added. Next, we incubated the membrane for 1 hour at room temperature. The primary antibodies used in this study including Rabbit Anti-DMTF1 antibody (ab246945, 1:500), Rabbit Anti-PTEN antibody (ab32199, 1:500) , Rabbit Anti-p53 antibody (ab32389, 1:100), Rabbit Anti-MDM2 antibody (ab38618, 1:400) and Rabbit Anti-ARF antibody (ab77581, 1:300) (Cell Signaling Technology, Inc. Danvers, MD, USA). We then uniformly added the chemiluminescence reagent to the film and developed the image with a developer. We used Tubulin as an endogenous control to normalize protein expression. The fold change versus control group was the demonstration of target proteins’ relative expression levels.
Cell viability assay
The CCK-8 proliferation assay kit was used to determine the proliferation of cells. First, cells were incubated for 24 hours, then 3 × 103 cells were seeded into 96-well plates and cells were transfected with the indicated RNA duplexes. Then, the cells were treated with 10 μL of the cell counting solution (WST-8, Dojindo Laboratories, Tokyo) at 0, 24 and 72 hours, respectively, and incubated for an additional 2 hours. The absorbance (OD) value was measured at 450 nm using a microplate reader.
Transwell invasion assay
The invasive ability of ACHN and 786-O cells was determined using an 8.0 µm 24-well Boyden chamber. For the intrusion test, first, we pre-coated Matrigel in the chamber, then inoculated 2 × 105 cells in the serum-free medium into the upper layer of the chamber, and then added the intact medium as a chemotactic agent to the lower chamber. After 24 hours, we fixed the membrane with methanol and stained with 0.1% crystal violet, then selected five randomly selected fields of view (× 100) to count the invading cells.
Colony formation assays
For colony formation analysis, cells were seeded into 6-well plates at a concentration of 500 or 2000 cells/well, ensuring that the medium was changed every 5 days. After the cells were incubated for 7-14 days, cells were washed with PBS, fixed with 4% paraformaldehyde, and then stained with 0.5% crystal violet. Finally, the number of colony formations was calculated.
Cells were washed with cold PBS and fixed in cold 75% ethanol for 30 minutes at 4 oC. The fixed cells were washed three times and treated with ribonuclease. The resulting cells were stained with propidium iodide (50 μg/mL) overnight at 4oC (Abeam, Cambridge, UK) and then subjected to flow cytometry analysis at 488 nm challenge (BD Biosciences, Franklin Lakes, NJ, USA).
Vector construction and luciferase assay
The TargetScan Human database (http://www.targetscan.org/) was used to predict potential targets of miR-6838-5p. The 3'UTR of the DMTF1 sequence was amplified and cloned into a pGL4.10-report vector (Promega Corporation, Madison, WI, USA). Equal quantities of the pGL4.10-3'UTR-DMTF1 and Renilla expression vector pRL-TK (Promega Corporation) were co-transfected into the HEK293 cells. The luciferase activity was measured 48 h post-transfection using the Dual-Glo Luciferase Assay system (Promega Corporation).
Statistical analyses were performed on SPSS 19.0 (IBM SPSS, Armonk, NY, USA). All quantitative results were presented as the mean ± standard deviation (SEM) of three independent experiments. The significance of the differences between two groups, unless for paired comparison which was noted specially, was conducted with a two-tailed Student’s t-test. Analysis of variance (ANOVA) was used to compare the significance between multiple groups. The correlations between miR-6838-5p and clinical characteristics were analyzed by Chi-square test. P-values < 0.05 were considered to be statistically significant.