Patients and Clinical tissue samples
Fifty-eight patients who underwent surgery at the Department of Neurosurgery in the First Affiliated of Chengdu Medical College from July 2015 to September 2016 were included in the study, and classified according to the WHO guidelines for the classification of tumours of the central nervous system . This study mainly collected the information from the patients’ medical records, and the detail information was showed in Table 1. The survival rate was calculated from the data of surgical resection to Dec 31, 2020. The human glioma tissue of the patients or the control group was immediately immersed in liquid nitrogen until RNA extraction.
The written informed consents were collected from all patients, and this study was approved by the Ethics Committee of the First Affiliated of Chengdu Medical College (IRB number: 20140332).
Clinicopathological characterisitcs of RMRP expression in glioma patients.
| || |
| || |
| || |
| || |
| || |
Recurrence time in months [mean (sd)]&
| || |
Survival time in months [mean (sd)]&
| || |
|*p < 0.05 was considered statistically significant.|
|# Low/high expression was determined by the sample mean. Pearson chi-square test was utilized to analyze the clinical data.|
|&p-value from t test.|
The human glioma cell lines A172, SHG44, U251, LN229 as well as the normal human astrocytes (NHAs) were obtained from the American Type Culture Collection (Manassas, VA, USA). All cell lines were maintained in DMEM medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) containing 10% FBS (Gibco) and cultured in a 5% CO2 incubator at 37°C.
RNA extraction and quantitative RT-PCR analysis
The total RNA was isolated from human glioma tissues and cell lines using TRIzol reagent (Invitrogen). For quantitative RT-PCR, the total RNA was reverse-transcribed into complementary DNA using the reverse transcription kit (Invitrogen), according to the manufacturer’s instruction. Further, the SYBR green PCR kit (Toyobo, Nipro, Osaka, Japan) was used to conduct real-time PCR with the 7500 Fast Real-time PCR system (Applied Biosystems, Waltham, MA, USA). The 2−ΔΔCt method was used for calculating the target gene expression levels, and GAPDH or U6, as endogenous control, was used to normalize the target gene expression levels, respectively. The primer sequences were as follows: RMRP, F-5’-CACTGCCTATGTGCACGACT-3’, R-5’AGAGTCCGGCAAGAAGAACA-3’; miR-466, F-5’-GGGATTCCCCTGGGTGTCAACACTGGC-3’, R-5’-TGCGCAGCACAGTTCCACGGGTC-3’; CXCR4, F-5’-ACCCCTTCATTGACCTCAACTA-3’, R-5’-TCTCGCTCCTGGAAGATGGTGA-3’; GAPDH: F-5’-AATGGGCAGCCGTTAGGAAA-3’, R-5’TGAAGGGGTCATTGATGGCA-3’; and U6: F-5’-CTCGCTTCGGCAGCACATATA-3’, R-5’ACGCTTCACGAATTTGAGTGTC-3’. All assays were performed three times.
The small short hairpin RNA (shRNA) targeting RMRP, control shRNA, pcDNA-RMRP, and the control plasmid pcDNA3.1 were provided by Novogene (Beijing, China). The detailed sequences of all targeted genes were used: shRMRP: (F) 5’GGCCAAACCCUCAAUGAAUTT-3’ and (R) 5’AUUCAUUGAGGGUUUGGCCTG-3’; shCtrl: (F) 5’UUCUCCGAACGUGUCACGUTT-3’ and (R) 5’ACGUGACACGUUCGGAGAATT-3’; pcDNA-RMRP: (F) 5’CGATCTTAATTAAGGGGTACCAAAGTCCACTCTG-3’ and (R) 5’TCAGTGGCGCGCCTTTTTCGTGAGTACACAATAGTCATC-3’. The miR-466 mimic (5’-ACCUGGCAUACAAUGUAGAUUU-3’), miR-negative control (NC) mimic (5’-UUCUCCGAACGUGUCACGUTT-3’), miR-466 inhibitor (5’-UGAGGUAGUAGGUUGUAUAGUU-3) and miR-NC inhibitor (5’-CAGUACUUUUGUGUAGUACAA-3’) were provided by Shanghai Sangon Biotech Corporation. The corresponding plasmid or miRNAs (50 nM) were transfected into A172 and U251 cell lines with a density of 105/well in 6-well plates by Lipofectamine 2000 reagent (GE Healthcare Life Sciences, Lafayette, CO, USA), and the cells were collected for analysis at 48 h after transfection.
A172 and U251 cell lines were added to a 96-well dish at a density of 103 cells per well and then transfected with pcDNA-RMRP, sh-RMRP, miRNA, pcDNA-CXCR4, or shCXCR4. Subsequently, at 0, 24, 48, 72, and 96 h, 10 µl MTT solution was added to each well. After 4 h of culture, the supernatant was discarded, and then 100 µl of DMSO was added. The absorbance at 570 nm was determined using an enzyme-labeling instrument (EL808 BioTek Instruments, Winooski, VT, USA).
BrdU proliferation assay
A cell proliferation assay was performed using a cell proliferation ELISA-BrdU (colorimetric) Kit (Roche Diagnostics, CA, USA), according to the manufacturer’s protocol. Briefly, the A172 and U251 cell lines were cultured into a 96-well plate at a density of 103 cells per well. Overnight, the cells were transfected with the corresponding pcDNA-RMRP, sh-RMRP, miRNA, pcDNA-CXCR4, or shCXCR4 at 37°C. After 24 h of incubation, cell proliferation was analyzed.
The A172 and U251 cells were seeded into 6-well plates at a density of 105 cells per well, and the pcDNA-RMRP, sh-RMRP, miRNA, pcDNA-CXCR4, or shCXCR4 were transfected into cells. After that, a cell apoptosis assay was conducted via flow cytometry using the Annexin V-FITC Apoptosis Detection Kit (Beyotime, China). Briefly, cells were collected with annexin‐V/FITC (5 µl) and propidium iodide (10 µl), and were incubated for 15 min at room temperature in the dark. The cell apoptosis rate was analyzed within 1 h using a FACS caliber cytometer (BD Biosciences, Franklin Lakes, NJ, USA).
Cell cycle analysis
The A172 and U251 cells were transfected with pcDNA-RMRP, sh-RMRP, miRNA, pcDNA-CXCR4, or shCXCR4 for 24 h. After that, the cells were collected by trypsinization and washed with PBS that was precooled on ice. The cells were fixed in 70% methanol and incubated at 4 ℃ for 1 h. The cells were then centrifuged and added with RNase at 37 ℃ and incubated for 30 min. The cells were stained by propidium iodide for 1 h. The cell cycle of pretreated cells was analyzed using FACScan flow cytometer, and the data were presented by CellQuest software.
The Western blot assay used in this study was according to the following previously described methods. Total proteins were extracted from A172 cells using a protein isolation kit (Tiangen, Beijing, China), and the protein concentrations were quantified through the Pierce BCA assay (Thermo Scientific, CA, USA). Equal amounts of protein samples were separated by SDS-PAGE and then electrophoretically transferred to a polyvinylidene fluoride membrane (Milipore, CA, USA). The antibodies against CDK4, Cyclin-D1, CDK2, Cyclin-E, β-actin, and CXCR4 (Santa Cruz Biotechnology, CA, USA) as the primary antibody (all obtained from Cell signaling Technology, Beverly, MA, USA) were used in the Western blot assay, and peroxidase-conjugated anti-IgG (Abcam) was also used as the secondary antibody. Monoclonal β-actin or GAPDH antibody (Sigma) was used as the control. The blots were detected using an enhanced chemiluminescent kit (Pierce, Rockford, IL, USA). The data of Western blot bands were quantified by Image J software.
The nude mice were used to determine the tumorigenesis as described previously. Briefly, 4-week-old female BALB/c nude mice were subcutaneously injected with U373MG cells (105 cell in 200 mL/mouse) transfected with shRMRP, shCtrl, miR-466 mimic or mimic control at a single site under their right front leg. After 2 weeks, the tumor sizes were measured every 3 days. After 5 tests (14, 17, 20, 23and 26 days), the tumor volumes were calculated. The average weight of the excised tumor was measured at the time of sacrifice at 5 weeks after the initial injection.
All the procedures were approved by the Animal Management rule of the First Affiliated of Chengdu Medical College.
All experiments were carried out in triplicate, and the GraphPad Prism 5 software (GraphPad Software Inc., San Diego, CA, USA) was used for all statistical analyses. Data for each group of each experiment were presented as mean ± standard deviation (SD). The overall survival rate was calculated using the Kaplan–Meier method. Two-tailed Student’s t-test or analysis of variance followed by Turkey’s post hoc test was applied to analyze significance, and the differences were considered statistically significant at P < 0.01.