TCGA databases and clinical specimens
The mRNA expression chip data and accompanying clinical information were downloaded from the TCGA Research Network. The data was analyzed using GraphPad Prism. Paraffin-embedded glioma tissues (WHO II-IV) was obtained from patients undergoing neurosurgery in Qilu Hospital of Shandong University (n=30). Normal brain tissue samples were collected from 5 patients with severe traumatic brain injury who had partially resected normal brain.
Immunohistochemistry(IHC)
Sections were obtained from paraffin-embedded tissues of normal brains and of different grades of human gliomas. The sections were dewaxed and deparaffinized in xylene and rehydrated in graded alcohol solutions. Sections were heated in tris-EDTA buffer for 30 mins to extract antigens. The slides were blocked with 10% normal goat serum (#5425; Cell Signaling Technology; USA) and incubated with primary antibody (rabbit anti-RPS27L monoclonal antibody, 1:100 dilution, #AP33216PU-N; Acris Antibodies GmbH; GERMANY) (mouse anti-Ki67 antibody, 1:800 dilution, #9449; Cell Signaling Technology; USA) (rabbit anti-Bcl-2 antibody, 1:150 dilution, #40994; Cell Signaling Technology; USA) (rabbit anti-MMP2 antibody, 1:150 dilution, #40994; Cell Signaling Technology; USA) at 4 °C overnight. The images were visualized by following standard protocols using horseradish-peroxidase-conjugated secondary antibody and 3, 3′-diaminobenzidine (DAB) as the substrate. Sections were avoided light with hematoxylin, then dehydrated and secured. Images were caught with Leica DM 2500 microscope.
Cell culture
GBM cell lines U87MG and A172 human were purchased from the Chinese Academy of Sciences (Shanghai) culture bank. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Thermo Fisher Scientific; Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, USA). Cells were maintained at 37 °C in a humidified chamber containing 5% CO2.
RPS27L silencing
siRNA targeting RPS27L was synthesized (GenePharma; Shanghai, China). LipofectamineTM 3000 reagent (Thermo Fisher Scientific; USA) was used to transfect siRNAs according to manufacturer's protocol. Stable knockdown of RPS27L in cells were produced by sh-RPS27L lentivirus (GenePharma; Shanghai, China). Knockdown efficiency was evaluated by qRT-PCR and Western blotting. siRNA sequences (n = 2) are the following: si-RPS27L#1: 5′-GCCUUUGGCUAGAGAUUUATT-3′; and si-RPS27L#2: 5′-GAAGGGUGUUCAUUUAGAATT-3′. The first sequence of siRNA was used for stable knockdown and for the functional assays in vitro.
Quantitative real-time PCR (qRT-PCR)
RNA isolation, reverse transcription and quantitative expression were carried out according to the instructions. Total RNA was isolated from tissue or cultured cells using Trizol reagent (Invitrogen, USA) and DNA was generated using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA). The cDNA was subjected to real-time PCR using the quantitative PCR System Mx-3000P (Stratagene). The RPS27L primers were 5-GTCTGGTAGGGCTGAGCTTG-3 and 5-GGTGATCTTGTAGCAACCTGGA-3. The primers for GAPDH were 5-GCACCGTCAAGGCTGAGAAC-3 and 5-TGGTGAAGACGCCAGTGGA-3.
Western blotting
Harvested cells are thermally denatured in the RIPA cell lysis buffer. The protein lysate was run on SDS-PAGE and the protein was transferred to the PVDF membrane. Blots are cultured with primary antibodies against RPS27L (1:500 dilution; #AP33216PU-N; Acris Antibodies GmbH; GERMANY); p21, CDK4, Cyclin D1, Cyclin E1, MMP2, MMP9, Bcl-2, Bax, cleaved Caspase-3, GAPDH (Cell Signaling Technology; Danvers, MA, USA). Specific proteins were detected with enhanced chemiluminescence (ECL, Millipore, Bedford, MA, USA). GAPDH was used as the loading control.
5-ethynyl-2'-deoxyuridine (EdU) cell proliferation assay
An EdU cell proliferation assay kit (RiboBio, #C10310-1; Guangzhou, China) was used to measure the glioma cell proliferation according to the manufacturer's protocol. The glioma cells were incubated in 250 µl EdU solution for 2 h. Cells were fixed in 4% paraformaldehyde for 15min, 0.4% Triton X-100 penetrability for 10min, and incubated with 250 μl Apollo® reagent for 30min. Then the cells were stained with Hoechst 33342 for 30min. The ratio of EDU positive cells (red) to the total number of Hoechst 33342 positive cells (blue) was used as cell proliferation rate.
Cell counting kit (CCK)-8 assay
Cell viability was assessed using CCK-8 assay according to the manufacturer's instructions (Dojindo, Kumamoto, Japan). Cells (2×103 /well) were incubated in 96 well plates for 24, 48, and 72 h. CCK-8 solutions (10 μL) were added to each well, the plates were incubated for 1 h at 37°C, and the absorbance of OD450 was measured with a Microplate Reader (Bio-RAD).
Flow cytometry
In the cell cycle analysis, U87MG and A172 glioma cells were collected, re-suspended and stained with propidium iodide (PI) (BD Biosciences; USA) for 20 min in the presence of RNase A. In the apoptosis assay, U87MG and A172 cells were stained with Annexin V-FITC (BD Biosciences; USA) and PI for 20 min. Cells were analyzed using flow cytometry (BD Biosciences; USA) according to manufacturer's instructions.
3D tumor spheroid invasion assay
Glioma cells were seeded into a 3D culture qualified 96-well spheroid-formation plate (5×103 cells/well) and incubated in the spheroid-formation matrix (Trevigen, Gaithersburg, MD, USA) and DMEM containing 10% FBS for 72 h. When the spheroids grew to a diameter of >200 mm, the invasion matrix (Trevigen, Gaithersburg, MD, USA) was infused. The spheroids at 24 h were regarded as a reference point for measuring the area invaded by the sprouting cells. Spheroids were imaged every 24 h using a Leica microscope.
Transwell invasion and migration assays
To further assess the invasiveness of glioma cells, we coated the filter with Matrigel. The glioma cells were added to the superior lumen in serum-free medium. The bottom chambers were filled with DMEM (10% FBS). After incubation for 24 h, the superior lumen cells were taken out with a cotton swab, fixed with 4% paraformaldehyde for 15 min, and stained with crystal violet for 15 min. The adherent cells in each well were randomly imaged in 5 fields. To measure migration, the filter was not precoated with a substrate.
Orthotopic glioma xenografts
To evaluate the tumorigenesis in vivo, U87MG luciferase cells (1 × 106) were infected with Lentivirus sh-RPS27L (the sequence was same as si-RPS27L#1) or Lentivirus control. Then we implanted them stereotactically into the brain of nude mice (SLAC laboratory animal Center; Shanghai, China). We detected the intracranial tumor growth by Bioluminescence imaging on days 7, 14, 21 and 28. Kaplan-Meier survival curves were plotted to show survival time. The brain tissues with tumor were collected, formalin-fixed and paraffin-embedded, sectioned (4 μm) and incubated with antibodies against RPS27L, Ki-67, MMP2 and Bcl2.
Statistical analysis
We used oneway ANOVA test or Student’s t test to data comparisons with the help of GraphPad Prism 6 software. The data was presented as the mean ± standard error of three independent experiments. Kaplan-Meier survival curves were also analyzed by log-rank tests using GraphPad Prism software. All tests were two-sided, and P-values <0.05 were considered to be statistically significant.