2.1 GBM tissues
Fifty-five paired fresh tissue specimens were collected from GBM patients who recruited between March 2013 and September 2017 at Jinan Zhangqiu District Hospital of TCM. All GBM patients have not received any treatment before surgery. The fresh tissues were verified by pathologists and stored at -80°C refrigerator for further use. All patients have provided written consent to allow for research purposes prior to the collection of tissue samples. The histological features of all specimens were confirmed by pathologists according to the WHO criteria. The research was approved by the ethic committee of Jinan Zhangqiu District Hospital of TCM.
2.2 Cell culture
GBM cell lines U251, U87 and A172 were obtained from BeNa Culture Collection (Suzhou, China). Normal human astrocytes (NHA) were purchased from BeNa Culture Collection (Suzhou, China). All the cells were cultured as previously described [23]. Then the cells were maintained in a humidified incubator containing 5% CO2 at 37 °C.
2.3 MiR-1825 mimic and miR-1825 inhibitor
The mimic or inhibitor of miR-1825 was purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China). They were used for increasing or decreasing the expression of miR-1825. A172 cells were the selected cells for further analysis. The transfection was conducted for 48h following the instructions of Lipofectamine 2000 reagent (Invitrogen, Thermo Fisher Scientific).
2.4 Real time PCR (RT-PCR)
The mRNA expression of miR-1825 and CDK14 was measured by RT-PCR. Total RNAs were firstly isolated from GBM tissues and cells using TRIzol reagent (Invitrogen) for both mRNA and miRNA analyses. The miScript Reverse Transcription kit (Beyotime, Haimen, Jiangsu, China) was carried out for producing cDNA. Then, quantitative PCR was conducted by a miScript SYBR-Green PCR kit (Beyotime) at an ABI 7500 Real-time PCR system (Applied Biosystems, Thermo Fisher Scientific). U6 was applied for normalizing miR-1825 relative mRNA expression and GAPDH for normalizing CDK14. 2−ΔΔCq method was used for calculating the gene expression. The primers were shown in supplemental table 1.
2.5 Western blot
The relative proteins level was tested by Western blot. In brief, the total proteins were exacted from GBM cells using RIPA lysis buffer and then conducted the protein concentration by BCA kit (Beyotime). After the equal protein separated by 10% SDS-PAGE, they were transferred to the NC membranes. Then, the membranes were blocked with 5% skimmed milk powder at 37℃ for 1h, incubated with primary antibodies (CDK14, ab167928, 1:1,000; E-cadherin, ab76055, 1:1,000; N-cadherin, ab18203, 1:1,000; Vimentin, ab92547, 1:1,000; β‑catenin, ab32572, 1:1,000; c‑myc, ab32072, 1:1,000; p‑c‑Jun, ab32385, 1:1,000; GAPDH, ab181602, 1:1,000; all from Abcam) at 4℃ overnight, and secondary antibodies at 37℃ for 1h. Finally, an enhanced chemiluminescence (ECL) method and the Image J software were applied for detecting the immune complexes and quantified protein levels, respectively.
2.6 Cell proliferation analysis
Cell proliferation was evaluated by 3‑ (4, 5‑Dimethylthiazolyl‑2)‑ 2,5‑diphenyltetrazolium bromide (MTT) assay. Briefly, A172 cells were placed in 96-well plates at a density of 2,000 cells / well. When the cells were cultured for 1, 2, 3, 4 days, MTT solution (20 μl) was added to each well and incubated for another 4 h at 37˚C. Then, the medium was removed and dimethyl sulfoxide (DMSO) was added to dissolve formazan crystals by swirling gently. Finally, the optical density was detected at a wavelength of 490 nm using a microplate reader.
2.7 Cell migration and invasion analysis
The invasiveness and metastasis of U87 cells were measured by Transwell assay. Detection of invasion and migration was similar, except for the upper chamber coated with Matrigel. Briefly, the cells were added to the upper chamber and DMEM medium containing 20% FBS was added to the lower chamber. After incubation for 24h, the cells traversed the membrane were fixed, stained and counted. These traversed cells were used to evaluate cell invasion and migration.
2.8 Immunohistochemistry analysis
Immunohistochemistry was applied for detecting CDK14 protein density. Firstly, the paraffin sections were obtained from GBM tissues. Then they were incubated with 3% H2O2 for 15min. After blocking with goat serum for 2h at room temperature, the sections were incubated with primary antibodies at 4℃ overnight, and secondary antibodies at 37℃ for 1h. Next, the sections were performed staining using DAB solution, followed by alcohol dehydration, xylene decolorization and neutral resin sealing. Finally, the results were observed and photographed by a microscopic.
2.9 Tumor growth analysis
Xenograft tumor formation assays were applied for measuring the tumor growth in vivo. The Animal Research Committee of Jinan Zhangqiu District Hospital of TCM approved these animal experiments. The operation meets the standards for laboratory animal care and use in Jinan Zhangqiu District Hospital of TCM. A172 cells (1×105) treated with miR-1825 mimic or NC were injected into right flank of nude mice subcutaneously, which were purchased from Shanghai Laboratory Animal Center (Shanghai, China). Then, the tumor size and weight was measured every four days for 28 days by a vernier caliper and electronic scale, respectively. All mouse experiments were carried out in accordance with institutional guidelines and regulations of the government.
2.10 Luciferase activity analysis
Bioinformatic analysis algorithm TargetScan (http://www. targetscan.org/vert_72/) was used to predict the targets of miR-1825. The binding site of miR-1825 in the 3’-untranslated region (3’-UTR) of CDK14 (wild-type or mutant) was cloned into
pGL3-reporter vector (Promega, Madison, WI, USA). Then, A172 cells were con-transfected with the NC vector and miR-1825 mimic using Lipofectamine 2000 (Invitrogen). After transfection for 48h, the luciferase activity was tested by the Dual-Luciferase Reporter Assay System (Promega), and Renilla luciferase activity was used to normalize the data.
2.11 Statistics analysis
The values were represented as mean ± SD. All experimental conditions were repeated in duplicate independently. Data was analyzed by SPSS 22.0 statistical software (SPSS, Inc.) and the statistics was performed by GraphPad Prism 6. Student’s t-test was applied for comparing the difference between two groups and Tukey’s post hoc test of one‑way analysis of variance (one‑way ANOVA) was carried out for comparing the differences between more than two groups. Pearson test was applied for determining the relationship between miR-1825 and CDK14. Log-rank test was applied for analyzing the survival rate. P<0.05 was considered statistically significant.