Tissue collection and ethical statement
CCRCC tissues were acquired from clinical patients diagnosed with CCRCC in the period of their surgical resection at First Affiliated Hospital of Jiamusi University, then preserved in liquid nitrogen right away. Before this study was started, we have collected 60 CCRCC samples and an equal number of normal para-carcinoma tissues.
All individuals were entirely informed regarding the objective of this research. Tissue collection was performed after the informed consent was signed and in line with the Declaration of Helsinki. Besides, our study has got the authorization of the Ethical Committee of First Affiliated Hospital of Jiamusi University.
Cell culture and transfection
For cell culture, human renal tubular epithelial cell line HK-2 and CCRCC cell lines (786-O, 769-P, CAKI-1 and A498) purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) were cultured using Dulbecco’s modified eagle medium (DMEM; Hyclone, Logan, UT, USA) complemented with 10% fetal bovine serum (FBS; Corning Inc., Corning, NY, USA) and 1% penicillin/streptomycin (Millipore, Billerica, MA, USA) in a humidified incubator. The growing environment was set as the temperature at 37℃ and CO2 of 5%.
For cell transfection, the mixtures of Lipofectamine3000 reagent (Invitrogen, Carlsbad, CA, USA) and vectors or oligonucleotides were pipetted into 786-O and 769-P cells in 96-well plates as per the user’s guideline. The circ_RPL23A overexpression vector (circ_RPL23A), miR-1233 mimic/inhibitor (miR-1233/anti-miR-1233), small interfering RNA (siRNA) targeting ACAT2 (si-ACAT2) and the respective negative controls (Vector, miR-NC, anti-miR-NC and si-NC) were all obtained from RIBOBIO (Guangzhou, China).
Quantitative real-time polymerase chain reaction (qRT-PCR)
Trizol (Sangon, Shanghai, China) was employed for the isolation of total RNA from collected tissues and cells, and the reverse transcription assay (1 μg RNA/sample) was instantly implemented using QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany). Then the qRT-PCR was carried out by QuantiFast SYBR® Green PCR Kit (Qiagen) via the QuantStudio™ 6 Flex Real-Time PCR System (96-well; Applied Biosystems, Carlsbad, CA, USA). And the cycle threshold method (2-∆∆Ct) was exploited to analyze the relative expression levels of related molecules. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) acted as the internal control of circRNA and mRNA, while U6 was used for miRNA normalization. The used primers (Sangon) included circ_RPL23A: Forward (F), 5’-AAGGAACCACTGATGCACCT-3’ and Reverse (R), 5’-TTCCCAATTCGTTCGCTCT-3’; RPL23A: F, 5’-CCCTTTCGTCACAAGATGGCG-3’ and R, 5’-GAATTAGGCAGCTGGACTCA-3’; miR-1233: F, 5’-TGGGAGGCCAGGGCAC-3’; ACAT2: F, 5’-CATGCTGCTGCTCATCTTCT-3’ and R, 5’-ACTGCGGAGACCAGGAACA-3’; GAPDH: F, 5’-ACCCACTCCTCCACCTTTG-3’ and R, 5’-CTGTAGCCAAATTCGTTGTCAT-3’; U6: F, 5’- CTCGCTTCGGCAGCACA-3’ and R, 5’-AACGCTTCACGAATTTGCGT-3’.
Stability analysis
Actinomycin D (Millipore), a transcription inhibitor, was added to the culture medium for 786-O and 769-P cells with a concentration of 2 mg/mL for 0 h, 8 h, 16 h and 24 h. In other hand, 4 μg total RNA was subjected to 12 U Ribonuclease R (RNase R; Epicentre Technologies, Madison, WI, USA) digestion at 37℃ for 1 h. Then circ_RPL23A and RPL23A were determined via qRT-PCR post-treatment.
Cell cycle detection
Propidium iodide (PI) staining was applied for measuring the changes of cell cycle (G0/G1, S and M) through a flow cytometer (BD Biosciences, San Diego, CA, USA) as previously described [20, 21].
Western blot
The standard procedures of western blot contained: protein extraction and quantification by radio-immunoprecipitation assay (RIPA) buffer (Abcam, Cambridge, UK) and a BCA Protein Assay Kit (Abcam); protein separation via the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for 2 h; protein transferring to polyvinylidene fluoride (PVDF) membranes (Abcam) and non-specific binding prevention in Blocking Buffer (Abcam); antibody incubation of primary antibodies (overnight at 4℃) and second antibody (at room temperature for 1 h); protein appearance using a ECL Substrate Kit (Abcam) and analysis by the ImageLab software version 4.1 (Bio-Rad, Hercules, CA, USA). All antibodies were purchased from Abcam and shown as follows: anti-CyclinD1 (ab16663, 1:1000), anti-Cell Cycle Dependent Kinase 4 (anti-CDK4; ab137675, 1:1000), anti-snail (ab180714, 1:1000), anti-E-cadherin (ab15148, 1:1000), anti-ACAT2 (ab131215, 1:1000), anti-GAPDH (ab9485, 1:3000) and goat anti-rabbit IgG H&L (HRP) secondary antibody (ab205718, 1:5000). GAPDH served as an endogenous control for other proteins.
3-(4, 5-dimethylthiazol-2-y1)-2, 5-diphenyl tetrazolium bromide (MTT) assay
MTT assay was administrated for the assessment of cell proliferation. In 96-well plates, inoculated cells (about 1 × 104) for 24 h were transfected with different experimental groups. After 0 d, 1 d, 2 d and 3 d, each well was added with 50 μL 1 × MTT (KeyGen, Nanjing, China) to incubate at 37℃ for 4 h. Then supernatant was sucked out using a micropipette and the optical density (OD) of each well was determined at 490 nm through a microplate reader after the addition of 150 μL dimethylsulfoxide (DMSO; Sangon).
Apoptosis detection
The detection of cell apoptosis was performed using FITC Annexin V Apoptosis Detection Kit I (BD Biosciences) through the flow cytometer (BD Biosciences). 5 μL FITC Annexin V and 5 μL PI were severally pipetted to 100 μL cell suspension in 1 × Bing Buffer (1 × 105 cells). After gentle mixing and lucifugal incubation for 15 min, apoptotic cells were discerned as cells of Annexin V+/PI- and Annexin V+/PI+ in staining, followed by the calculation of apoptosis rate (the apoptotic cells/total cells × 100%).
Transwell assay
The migrated and invaded cells were determined via the 24-well transwell chamber (Corning Inc.), in which cell suspension (produced by serum-free medium) in the upper chamber could be attracted to the lower chamber by 10% FBS+DMEM medium. 24 h later, cell fastening and dyeing were respectively implemented by methanol and crystal violet (Sangon). Whereafter, the migrated and invaded cells were observed and photographed via an inverted microscope (Olympus, Tokyo, Japan). It should be noted that the matrigel (Corning Inc.) needed to envelop the upper chamber in invasion assay before seeding cells, while it was not necessary for migration assay.
Dual-luciferase reporter assay
The gene combination was affirmed via the dual-luciferase reporter assay. For circ_RPL23A and miR-1233, wild-type (wt) or mutant-type (mut) circ_RPL23A sequence that contained the binding sites of miR-1233 or mutated sites was cloned into the pGL3 luciferase basic vector (Promega, Madison, WI, USA) and named as circ_RPL23A-wt and circ_RPL23A-mut. Then they were respectively transfected with miR-1233 or miR-NC into 786-O and 769-P cells, followed by the measurement of the dual-luciferase assay system (Promega) according to the manufacture’s instruction. For ACTA2 and miR-1233, ACAT2-wt and ACAT2-mut plasmids were also constructed as above, and the luciferase activity was assayed. The renilla luciferase intensity was used as the internal reference of firefly luciferase intensity.
Xenograft tumor assay
Six-week-old BALB/c nude mice (n=10) were bought from Animal Center of Chinese Academy of Medical Sciences (Beijing, China) and subcutaneously injected with 2 × 106 786-O cells transfected with circ_RPL23A using Vector as a negative control. After injection for 10 d, tumor volume (length × width2 × 0.5) was measured per 5 days. Tumors were weighed at 30 d after mice were euthanatized by cervical dislocation, then qRT-PCR and western blot were adopted for examining the levels of circ_RPL23A, miR-1233 and ACAT2. All animals were cared in compliance with the National Institutes of Health (USA) guidelines, and this experiment was carried out after the approval of the Institutional and Local Animal Ethics Committee of First Affiliated Hospital of Jiamusi University.
Statistical analysis
SPSS 22.0 was exploited for statistical analysis in our research. All assays were administrated in three duplications and data were shown as mean ± standard deviation (SD). Spearman’s correlation coefficient was used to analyze the linear relation between molecules. Difference analysis was executed using Student’s t-test and one-way analysis of variance (ANOVA) followed by Tukey’s test. P value < 0.05 was deemed as the indication of a significant difference.