The kidney cancer or the normal tissues were obtained from The Affiliated Hospital of Qingdao University Hospital (The Affiliated Hospital of Qingdao University, China). None therapies were administrated on the patients prior to the surgery. We obtained informed consents from all patients, and the present study was with the permission of the Medical Ethics Committee of the The Affiliated Hospital of Qingdao University Hospital.
The kidney cancer cells ACHN and CAKI-1 (ATCC, Manassas, Virginia, USA) were considered as the main study materials. The cells were cultured with RPMI-1640 (Gibco BRL, Grand Island, New York, USA) plus 10% fetal bovine serum (FBS, Gibco, Waltham, Massachusetts, USA), at 37°C with the humidity of 95% air and 5% CO2, in line with the suggestions from ATCC.
Small-interfering RNA (si)-circNRIP1 (si-circNRIP1) and miR-505 inhibitor as well as their corresponding negative controls (NC) (Life Technologies Corporation, Carlsbad, California, USA) were synthesized to alter the productions of the circNRIP1 and the miR-505 in ACHN and CAKI-1 cell lines. They were transfected into the cells via lipofectamine 3000 reagent (Invitrogen, Carlsbad, California, USA) for further experiment. Three days after transfection was selected as the optimum harvest time owing the highest transfection efficiency.
Cell viability assessing
The CCK-8 was introduced to detect cells viability in the present study. Firstly, different group cells were seeded into the 96-well plate with 5 × 103 cells/well, and cultivated in a CO2 incubator, for 24 h, at 37°C for further assays. Besides, referring to the instruction, the CCK-8 solution was injected and co-incubated with the cells for 1 h, after the 24 h conventional culture. Finally, the microplate reader (Bio-Rad, Hercules, California, USA) was introduced for absorbance measurement under 450 nm.
Colony formation assay
The 6-well plates, which contained 500 cells/well, were put into the carbon dioxide incubator and the cells were cultured for 2 weeks. After that, cells were fixed with paraformaldehyde (Sigma-Aldrich, St. Louis, Missouri, USA) and stained by crystal violet (Sigma-Aldrich). The colony formation ability was measured with a microscope (Olympus IX81, Tokyo, Japan).
Cell migration assay
A modified two-chamber migration assay (Millipore, Bedford, Massachusetts, USA) was conducted to test cells migration. The cells suspension were prepared with 200 μL serum-free medium (Gibco BRL) with a concentration of 5 × 104 cells/ml. Followed by a two days conventional cultivation, the suspension was then injected in the upper chamber while 600 μL of serum-containing medium (Gibco BRL) was complemented into the lower chamber. After incubation, cells were rinsed 1-2 times with phosphate buffer saline (PBS), and the non-traversed cells were removed with a wet cotton swab. Moreover, the methanol (Beyotime Biotechnology, Shanghai, China) (20 min) and the crystal violet (Sigma-Aldrich) (10-20 min) were then individually used for cells handling. Finally, five fields were randomly selected for cell counting and photographing under the microscope (Canon, Tokyo, Japan).
The cells apoptotic rate was measured by Annexin V-FITC/PI detection kit (Beijing Biosea Biotechnology, China) and the flow cytometer (Beckman Coulter, California, USA). Totally, these treated cells, which were adjusted and prepared in the 6-well plate with 1 × 105 cells/well density and cultured at 37°C for 24 h, were washed and re-suspended with PBS. Annexin V-FITC and PI were mixed under 1:1, and co-incubated with the cell suspension in the dark for 15 min to constitute the sample for flow cytometer detecting.
After cells collection, 1 × PBS buffer and 4°C RIPA buffer (Beyotime Biotechnology) containing protease inhibitors (Roche, Basel, Switzerland) was respectively used for total proteins extraction. The BCA™ Protein Assay Kit (Pierce, Appleton, Wisconsin, USA) was applied for protein quantification and the Bio-Rad Bis-Tris Gel system (Bio-Rad, Hercules, California, USA) was helped to form the western blot system. The primary antibodies were diluted with 5% blocking buffer, followed by the soak of polyvinylidene difluoride (PVDF) membrane (Millipore), which carried proteins blots at 4°C overnight. The primary antibodies were including anti-Bcl-2 (ab32124), anti-Bax (ab53154), anti-cleaved-caspase-3 (ab2302), anti-matrix metalloproteinase (MMP)-2 (ab92536), anti-MMP-9 (ab38898), anti-AMPK (ab131512), anti-p-AMPK (ab240058), anti-PI3K (ab40776), anti-p-PI3K (ab191606), anti-p-AKT (ab192623), anti-AKT (ab106693), anti-mTOR (ab32028), anti-p-mTOR (ab109268) and anti-β-actin (ab8227). Following 1 h attachment of horseradish peroxidase (HRP) signed secondary antibody, goat anti-rabbit (HRP) (ab7090) at 25°C, the PVDF membrane was put into the Bio-Rad ChemiDoc™ XRS system (Bio-Rad) with the addition of the Immobilon Western HRP Substrate (200 μL) (Millipore). Image Lab™ Software (Bio-Rad) was adopted to capture and quantify the protein bands.
Quantitative reverse transcription polymerase chain reaction (qRT-PCR)
Trizol reagent (Invitrogen, Carlsbad, California, USA) and the DNaseI (Promega, Madison, wisconsin, USA) were used for RNA extracting. The MultiscribeRTkit (Applied Biosystems) and random hexamers or oligo (dT) was consumed for the miRNA reverse transcription and qRT-PCR assessing. Experiment records were managed through 2–△△Ct method and normalized with β-actin or U6.
The SPSS statistical software version 19.0 was the main tool for statistical analysis. The experimental data were showed as the mean + standard deviation (SD). Analysis of variance (ANOVA) or t-test was responsible for P values. P < 0.05 was considered to be statistically significant. All experiments in this study were repeated 3 times at least.