2.1 Clinical specimen collection and processing
Forty-five cancerous and adjacent non-cancer tissues of patients with glioma who underwent resection in the Affiliated Hospital of Hebei University from March 2014 to March 2015 were selected. None of the patients received adjuvant treatment such as chemotherapy and radiotherapy before surgery. The control group specimens were obtained from the normal tissues of the same patient (at least 1 cm from the surgical margin), and no cancer cells were found through pathological examination. The glioma was diagnosed pathologically according to World Health Organization (WHO) criteria. Immediately after removal, all specimens were stored in -196℃ liquid nitrogen until used for RNA extraction. The Ethics Committee of Affiliated Hospital of Hebei University approved our study and all of the involved patients signed informed consent.
2.2 Cell culture
Human normal glial cell HEB and glioma cell lines (U87, TJ861, TJ905, U251, H4 and A172) were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). The cells were incubated with RPMI1640 (Thermo Fisher Scientific, MA, USA) containing 10% fetal bovine serum (FBS) (Thermo Fisher Scientific,MA, USA) and 1% penicillin/streptomycin (Invitrogen, CA, USA)) at 37℃ and 5% CO2. Cells during the logarithmic growth phase were treated with 0.25% trypsin (Thermo Fisher, HyClone, Utah, USA) for trypsinization and passage. It was found that CircPIP5K1A was the lowest expressed in U87 cells and the highest expressed in A172. Therefore, U87 and A172 cells were chosen as the research object in subsequent studies.
2.3 Cell transfection
CircPIP5K1A overexpression plasmids (pcDNA3.1-CircRNA PIP5K1A) and its small inference RNA (Si-CircPIP5K1A), miR-515-5p mimics, TCF12 overexpression plasmids (pcDNA3.1-TCF12) and its small inference RNA (Si-TCF12) and the corresponding negative controls were obtained from GenePharma (Shanghai, China). U87 and A172 cells were taken and seeded in 24-well culture plates at 3 × 105 cells/well, then incubated at 37 ℃ with 5% CO2 for 24 hours before transfection using Lipofectamine® 3000 (Invitrogen; ThermoFisherScientific, Inc.) according to the supplier's instructions. RT-PCR was used to determine the transfection efficiency, and the cells were incubated at 37˚C with 5% CO2 for 24 hours for further analysis.
Firstly, total RNA from tissues or cells was extracted using TRIzol reagent (Invitrogen, Waltham, MA, USA). Then, Nanodrop-spectrophotometer was employed to measure RNA concentration and purity. Subsequently, we used a PrimeScript-RT Kit (Madison, WI, USA) to reverse-transcribe 1 µg of total RNA to synthesize its complementary DNA (cDNA), and then adopted SYBR® Premix-Ex-Taq ™ (Takara, TX, USA) and ABI7300 system for real-time polymerase chain reaction (RT-PCR) of cDNA according to the manufacturer's protocol. The total volume of the PCR system was 30 µl, and each sample contained 300 µg of cDNA. The amplification was initially performed at 95℃ for 10 minutes for 45 cycles. Namely, 95 ℃ for 10 s, 60℃ for 30 s, and 85℃ for 20 s. We converted all fluorescence data to relative quantification, β-actin was the internal reference of CircRNA PIP5K1A and TCF12, while U6 was the internal reference of miR-515-5p. The RT-PCR experiment was repeated three times. The primers were designed and synthesized by Guangzhou Ruibo Company. CircPIP5K1A: forward primer 5'-AGATTCCCTAACCTCAACCAGA-3’, reverse primer 5'-CGAATGTTCTTGCCACCTGC-3' ; TCF12: forward primer 5'-TCTGCCCCTAGATGAGACCT-3’, reverse primer 5'-GGCAATCATTCGGTCCTGTC-3'; miR-515-5p, forward primer: 5'-TTCTCCAAAAGAAAGCACTTTCTG-3, reverse primer 5'-CTCGCTTCGGCAGCACA-3'; GAPDH: forward primer 5'-TGATCTTCATGGTCGACGGT-3, reverse primer 5'-CCACGAGACCACCACCTACAACT-3'; U6, forward primer 5'-CTCGCTTCGGCAGCACA-3 ', reverse primer 5'-AACGCTTCACGAATTTGCGT-3'.
2.5 CCK 8 assay
CCK 8 assay was used to detect cell viability using a CCK8 kit (Beyotime, Shanghai, China). Firstly, 100 µL cell suspension at 2 × 105/ml of glioma cells U87 and A172 were inoculated into 96-well plates per well. On the next day, the original culture medium was removed and cell culture medium was supplemented. Subsequently, the medium was taken out after cultured in an incubator with 5% CO2 for 3 hours, 6 hours, 12 hours, and 24 hours, respectively. Finally, CCK8 reagent at 10 µL/well was added and incubated for 2 hours, and the OD value at 450 nm wavelength was obtained by microplate.
2.6 BrdU assay
Firstly, cells at the logarithmic phase were added with 10 µmol/L BrdU (Sigma, Shanghai, China). After DNA denaturation, the cells were incubated with BrdU primary antibody (Abcam, ab6326,1:1000, CA, USA) at room temperature for 2 hours. Then, fluorescent secondary antibodies were added and incubated for 2 hours at room temperature. Later on, the nucleus was labeled by 10 µmol/L Hoechst33342. Finally, fluorescence inverted microscope was used for imaging and statistical analysis.
2.7 Transwell assay
Transwell chambers (Corting, NY, USA) were coated with 200 mg/mL Matrigel (BD, SanJose, USA) and incubated overnight. Then, U87 and A172 cells (5 × 105/ml) was suspended by serum-free RPMI1640 medium and 200 µL cell suspension was added into the upper chambers. Subsequently, RPMI1640 (500 µL) containing 10% FBS was placed in the lower chambers as a chemotactic agent. After incubation for 24 hours, all uninvaded cells were removed. Then, matrigel membranes were fixed with paraformaldehyde and then dyed with crystal violet buffer. At last, the number of invaded cells was counted by a phase contrast microscope (Olympus, Tokyo, Japan). The experiment was repeated three times.
2.8 RIP experiment
Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Merck Millipore, USA) was used to conduct RIP assay. 2 × 107 U87 cells transfected with miR-505-5p or its negative control were collected and added with 200 µL of RIP Lysis Buffer, then they were cleaved on the ice for 5 min and centrifuged at 1500 rpm for 15 min to obtain the supernatant. Then, the extract was incubated with anti-Ago2 or anti-IgG (Sigma) overnight. Subsequently, the supernatant was discarded after magnetic beads were washed with Wash Buffer for 5 times, and then protease K lysate was added to the magnetic beads for lysis at 55℃ for 30 min. Finally, the supernatant was placed in a new centrifuge tube, total RNA was extracted by phenol-chloroform-isoamyl alcohol extraction and purified by isopropanol centrifugation. The level of CircPIP5K1A and TCF12 were tested by RT-PCR.
2.9 Dual luciferase reporter assay
All luciferase reporter vectors (CircPIP5K1A-WT, CircPIP5K1A-MUT, TCF12-WT, TCT12-MUT) were obtained from Promega (Promega, Madison, WI, USA). U87 cells (4.5 × 104) were seeded in 48-well plates to 70% confluence. Then, the U87 cells were co-transfected with miR-515-5p or negative control with CircPIP5K1A-WT, CircPIP5K1A-MUT, TCF12-WT and TCF12-MUT using liposome 2000. Forty-eight hours after transfection, luciferase activity was determined according to manufacturer's guidelines. All experiments were made in triplicate and repeated three times.
2.10 Western blot
The cells were collected and then washed with cold PBS for 3 times, and 100 ~ 200 µL RIPA lysate (Beyotime Biotcchnology, Shanghai,China) was added to lyse the cells on ice. Then, the protein in the lysates were isolated by centrifugation and the protein concentration was determined by Bradford method. Equal protein in each group was isolated on 10% SDS-PAGE, and then the proteins in the gel were transferred to PVDF membranes (Millipore, Bedford, MA, USA). Subsequently, the membranes were blocked at 4℃ for 1 hour by 5% BSA, and incubated with primary antibodies anti-TCF12 antibody (ab70746, 1:1000, abcam, MA, USA), anti-PI3K antibody (ab191606,1:1000, abcam, MA, USA), anti-PI3k (phosphoY607) antibody (ab182651, 1:1000, abcam, MA, USA), anti-pan-AKT antibody (ab18785, 1:1000, abcam, MA, USA), anti-AKT (phospho T308) antibody (ab38449, 1:1000, abcam,MA,USA), anti-Bax antibody (ab32503,1: 1000, Abcam,MA,USA), Anti-Bcl-2 antibody (ab59348,1:1000, abcam,MA,USA), Anti-Caspase 3 antibody (ab13847, 1:1000, abcam, MA, USA), Anti-E-cadherin antibody (ab16505, 1:1000, abcam, MA, USA), Anti-Vimentin-antibody (ab92547, 1:1000, abcam, MA, USA) and Anti-N-cadherin antibody (ab18203,1:1000,abcam,MA,USA) at 4℃ overnight. After being washed with TBST for 2 times, the membranes were incubated with HRP labeled goat-anti-rabbit secondary antibody (ab205718, 1:2500, abcam) at room temperature for 1 hour. Finally, the membranes were washed for 3 times, exposed with ECL color reagent (Millipore,Bedford,MA,USA), and imaged with a scanner.
2.11 Flow cytometry
After being treated with different factors, the cells were trypsinized, and then collected by centrifugation (1500r/min, 3 min). The harvested cells were handled according to cell apoptosis detection kit(Shanghai Aladdin Bio-Chem Technology Co., LTD): after the cells were washed with PBS twice, 400 µL precooling PBS was added, then 10 µL of AnnexinV-FITC and 5 µL of PI were supplemented respectively and incubated at 4℃ in the darkness for 30 min. Immediately after that, flow cytometry instrument was adopted to measure cell apoptosis, and the percentage of apoptotic cells was calculated after computer processing.
2.12 Tumor formation assay in nude mice
Firstly, 4-6-week-old BALB/c-nu nude mice were selected to construct tumor formation model. U87 and A172 cells in the logarithmic phase were chosen, and the cell concentration was adjusted to 2 × 108/ml. Subsequently, 0.1 ml cell suspension was injected subcutaneously into the armpit of the left forelimb of each nude mouse. Each group had a total of 10 mice. The survival rate, weight and status of the mice were monitored, and the size and weight of the tumor in the newly dead mice were measured within 25 days after the injection.
2.13 Statistical Methods
SPSS22.0 software (SPSS Inc., Chicago, IL, USA) was used for statistical analysis, and the results were expressed as mean ± SD (`x ± s). The measurement data between the two groups were compared by t test, and P < 0.05 was considered statistically significant.