Acquisition, isolation and culture of hBMSCs.
HBMSCs were collected from a patient (male, 32 years old) who underwent femoral shaft fracture surgery in the Tianjin Medical University General Hospital. The experiment was examined and approved by the Ethics Committee of Tianjin Medical University General Hospital, and the relevant ethical agreement was signed. The consent of the patient and their families was obtained in advance when the bone marrow tissue was collected, and the purpose of the experiment was informed.
HBMSCs were isolated by density gradient centrifugation (Ficoll-PaqueTM Plus, GE Healthcare), according to the manufacturer’s instructions, and hBMSCs were cultured in low glucose Dulbecco's Modified Eagle's Medium (DMEM-LG; Thermo Fisher Scientific, Inc.), supplemented with 10% MSC-qualified fetal bovine serum (Thermo Fisher Scientific, Inc.), and 1% penicillin/streptomycin (Thermo Fisher Scientific, Inc.). Human glioma LN229, U87-MG and U251-MG cell lines were long-term subcultured cell lines in our laboratory. The LN229, U87-MG and U251-MG cell lines were subjected to a short tandem repeat (STR) test. The STR result demonstrated that the U87-MG cell line used in the present study matched the U87-MG cell line from ATCC. However, the U87-MG cell line from ATCC may not be the original GBM cell line from the University of Uppsala established in 1968. It has been reported that the U87-MG cell line from ATCC is of CNS origin and is likely to be derived from another patient with glioma, although its source is unknown. Therefore, the aforementioned misidentifications of U87-MG ATCC did not affect the outcomes of the present study. LN229, U87-MG and U251-MG cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Inc.) and were cultured in a humidified 5% CO2 incubator maintained at 37˚C (Thermo Scientific 3131).
Oil Red O, Alizarin Red and Von Kossa staining.
Oil Red O, Alizarin Red and Von Kossa staining were used for lipogenic and osteoblastic staining, respectively. The well-grown third-generation hBMSCs were digested at a regulated concentration of 5×104/ml and inoculated on a 6-well culture plate with cover glass. The cells were completely adherent to the glass after 24 h.
Lipogenic inducers (3µg/ml insulin, 0.45 mM isobutyl methyl xanthine, 1 µM Rosiglitazone, 100nM dexamethasone) (Sigma, US) were used for induction for 3 days(21). The solution was changed twice a week for another 15 days. The cells were then fixed with 4% paraformaldehyde and observed by oil red O staining.
The reagents used for osteogenic induction were 10 mM β-glycerophosphate (Sigma, US), 50 µg/ml vitamin C and 10 nM dexamethasone (Sigma, US) (21). After 2–3 days, the culture medium was changed into osteogenic induction medium, and the culture was continued for 3–4 weeks. The changes in cell morphology were visualized on an Olympus upright BX53 microscope. Alizarin Red and Von Kossa staining were used 4 weeks after osteoblast-induced differentiation to observe the calcium deposition of osteoblast differentiation.
Lentiviral vectors expressing miR-451 and enhanced green fluorescent protein (EGFP) were constructed from Shanghai GenePharma Co., Ltd (China). The coding sequences were miRNA-451, 5’-AAACCGUUACCAUUACUGAGUU-3’ and miRNA-NC, 5’-UUCUCCGAAC
GUGUCACGUTT-3’. The well-grown hBMSCs were routinely passed on and incubated until they reached 80% fusion. One day prior to infection, 2×105 hBMSCs were placed on 24-well plates, followed by infection with lentivirus expressing miR-451(MOI = 20). The cells were incubated for 24 h and replaced with fresh medium. After 72 h, the fresh medium was changed and the supernatant was collected, filtered, and placed in the refrigerator at -80˚C for the subsequent assays.
RNA extraction from cells, extracellular and tissues and qRT-PCR.
The total RNA was extracted with Trizol reagent from cells, culture supernatants and tissues (Thermo Fisher Scientific, Inc.). Tumor tissue was ground into powder under liquid nitrogen and then transferred to 1.5 ml centrifuge tube and added 1 ml Trizol to centrifuge tube. The subsequent operation was consistent with RNA extraction of cells. As for extracellular RNA extraction, 25 fmol of C. elegans cel-mir-39 standard RNA (Ribobio, Guangzhou, China) was added to each sample as a spike-in control [25 27]. Before isopropanol precipitation, Dr.GenTLE Precipitation Carrier (TAKARA#9094, RR820A, Takara, Japan) was added as a co-precipitant to promote the yield of extracellular RNA. Then, the RNA (2 µg) was reverse-transcribed with GoScript reverse transcription system (Promega Corporation) to synthesize cDNA. The GoTaq®qPCR Master Mix (Promega Corporation) was used to detect mRNA expression status on ABI QuantStudio 3, and GAPDH expression was used as an internal control. The miR-451 qPCR Quantitation Kit and U6 snRNA qPCR Kit were synthesized by Shanghai GenePharma Co., Ltd. The PCR process followed the steps identified by the Hairpin-it™ miRNAs quantitative PCR kit. The U6 snRNA quantitative PCR calibration kit was used to detect the PCR primers of U6 snRNA in each group to correct the errors. The oligonucleotide primers used for quantitative PCR were as follows: miRNA-451, 5’-GCGGCGCAAAGAATTCTCCT-3’ forward and 5’-GTGCAGGGTCCGAGGT-3’ reverse; U6, 5’-ATTGGAACGATACA GAGAAGATT-3’ forward and 5’-GGAACGCTTCACGAATTTG-3’ reverse (Shanghai GenePharma Co., Ltd.). The following PCR program was used: 95˚C for 3 min, 40 cycles of amplification (12 sec at 95˚C, 40–60 sec at 62˚C and 1 min at 72˚C) and a final extension step (72˚C for 2 min). Other primers: CAB39, 5’-GAGCATGGCTGTTCTGGAAAAGC-3’ forward and 5’- GCTACTGCTTCTG TCTGAGGCT-3’ reverse; LKB1, 5’-GGCACCCT CAAAATCTCCGA-3’ forward and 5’-CTTGAAGCCGGAGAAGGTGT-3’ reverse; AMPK, 5’-CCTGTGA CAAGCACTTACTCC-3’ forward and 5’-CTCTGTG GAGTAGCAGTCCC-3’ reverse; HIF-1α, 5’-CATCAGCTATTTGCGTGTGAGGA-3’ forward and 5’-AGCAA TTCATCTGTGCTTTCATGTC-3’ reverse; VEGF, 5’-CCTGGTGGACATCTTCCAGGAGTACC − 3’ forward and 5’-GAAGCTCATC TCTCCTATGTGCTGGC-3’ reverse; and GAPDH, 5’-TGTGGGCATCAATG GATTTGG-3’ forward and 5’-ACACCATGTATTCCGGGTCAAT-3’ reverse. All PCR experiments were performed in triplicate.
The glioma cells treated with supernatant as described above were routinely digested and inoculated in a 96-well plate with 2×103 cells (200 µl)/ well. 20 µl MTT solution (5 mg/ml; KeyGEN) was added to each well at 1, 2, 3, 4, 5 and 6 days after 48 h of culture. The supernatant was discarded and 200 µl DMSO was added to each well. Following incubation for 20 min at room temperature, a microplate reader (BioTek, China) was used to detect the light absorption value of each well at a wavelength of 570 nm. The tumor cell proliferation rate was then calculated.
Plate clonal formation experiment.
Trypsin was used to digest the cells at the logarithmic growth stage, single-cell suspension was prepared and counted, and the cell density was adjusted to 1×103/ml. Six-well plates were taken, 1 ml cell suspension was added to each well, and the cells were incubated for 12 h in a 5% CO2 and 37˚C saturated humidity environment. Next, 2 ml medium was added to each well, and the liquid was changed once every 3 days, and terminated at ~ 14 days. When obvious clones were formed in the petri dish, the culture medium was removed, washed with phosphate-buffered saline (PBS), fixed with 4% formaldehyde (Merck KGaA) for 15 min, discarded with formaldehyde, and dried in air. Next, 2.5% crystal violet (Merck KGaA) was added for staining for 30 min and eluted with PBS. The number of clones was counted under the microscope (Olympus, BX53) and the experiment was repeated 3 times.
Identification of hBMSC surface markers, cell cycle analysis and cell apoptosis analysis were performed by flow cytometry. When the cells were cultured to the third generation in vitro, the cells were washed twice with PBS and digested by 0.25% trypsin when they reached ~ 80% confluence. Cell suspensions were collected and centrifuged at 157 ×g for 5 min. After the supernatant was discarded, cell precipitation was resuspended with PBS. The cell concentration was adjusted to ~ 1×106/ml, and 100 µl cell suspension was taken respectively. FITC labeled-CD29 (SAB4700397; Merck KGaA), CD34 (SAB4700703; Merck KGaA), CD44 (SAB4700182; Merck KGaA), CD45 (SAB4700480; Merck KGaA), CD71 (SAB4700518; Merck KGaA) and HLA-DR (SAB4700658; Merck KGaA) antibodies were diluted to the reaction concentration, and staining was performed at room temperature for 30 min in the dark. Following washing once with PBS, cells were resuspended and detected by flow cytometry.
Cell cycle assay and apoptosis assay.
After the tumor cells were incubated with the supernatant for 48 h, cells were digested to prepare the cell suspension, which was fully suspended by PBS (0.01 M, pH 7.2). The cell suspension was fixed with 75% ethanol overnight at 4˚C. Next, propidium iodide staining solution was used for staining and detection. Anexin V-FITC Apoptosis Detecion kit (APOAF; Merck KGaA) was used for apoptosis analysis and flow cytometry was performed in accordance with the manufacturer's instructions.
Transwell and wound-healing assays.
Matrigel was solidified by adding 60 µl diluted Matrigel on the polycarbonate film (diameter, 6.5 mm) in a Transwell chamber (BD Biosciences) and left to stand at 37˚C for 30 min. Single-cell suspension was prepared by serum-free medium, and each group was made into three wells. A total of 1×105 cells/well were accurately added in the upper chamber (48 h after culture with the BMSCs supernatant). The cell suspension liquid was accumulated to 100–200 µl and placed in an incubator containing 5% CO2 for 24 h at 37˚C. After 24 h of culture, the upper chamber was removed and the surface of the cells was wiped with a wet cotton swab. Cells were then dyed with hematoxylin, thoroughly rinsed with Clearmont seal slice and dried room temperature. Finally, they were observed under an inverted microscope (Olympus, BX53). Three visual fields were randomly selected in and around the center of each membrane to count the number of cells passing through micropores. Each cell line was divided three groups: control, hBMSCs and Lv-miR-451-hBMSCs groups with 3 wells in each group. The glioma cells used in the wound-healing experiment were prepared into a single-cell suspension with an adjusted density of 1×105/ml. Next, 2 ml/well was inoculated into the 6-well plates and incubated for 24 h. When the cell density reached 90% of the tiled culture plate, every 3 scratch positions in the 6-well plates were selected and marked on the bottom surface with a 20-µl pipette nozzle to make a straight-line scratch in the hole, and then the cells were scraped off with PBS and washed, and then incubated in a constant temperature incubator. Different time points were selected to mark the scratch position of 6-well plates in advance for microscopic observation and measurements, and then the mobility was calculated.
Chemotactic capacity of BMSCs via the Transwell migration assay in vitro.
To monitor the dynamic migration of hBMSCs towards chemoattractants, a Boyden Chamber-based assay was performed. Serum-free medium was placed in the top chamber to hydrate the membrane. Lv-miR-451-hBMSC and hBMSCs (2×104 cells) were added in the upper chamber (Corning Costar, USA) and the cells were allowed to settle to the bottom. Conditioned media harvested from U87-MG, was added to the lower chambers. After 24 h of incubation, the non-migrated cells were scraped from the upper surface of the membrane with cotton swabs. Then, the membrane was stained with crystal violet (Solarbio, Beijing, China). Each sample was tested in triplicate, and a representative field in each well was counted to determine the number of migrated cells. To collect conditioned media, normal culture media was removed from U87 cells and replaced with Alpha-MEM (without FBS) for 48 hours after which the conditioned media was collected for use.
Protein extraction and western blotting.
The cultured glioma cell lines were further cultured with hBMSC supernatant in incubator at 37˚C. After 48 h, the cultured cells were rinsed with PBS, drained, cultured with 200 µl RIPA cell lysis buffer (Thermo Fisher Scientific, Inc.), transferred to an Eppendorf tube and chilled in a bath for 30 min, and the protein concentration was measured using the bicinchoninic acid (Merck KGaA) method. Dithiothreitol (40 µl) and loading buffer (160 µl) were added into each tube, boiled for 15–20 min, cooled and centrifuged at 22000 ×g for 15 min, and then the protein supernatant was separated for use.
The same amount of protein was isolated from glioma cells in each group, separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes (EMD Millipore) using a Mini Trans-Blot apparatus (Bio-Rad Laboratories, Inc.). Finally, the PVDF membrane was incubated with PVDF membrane sealing solution (Tiangen Biotech Co.) at 37˚C for 1 h and then incubated with mouse antibodies against GAPDH (2118; Cell Signaling Technology, Inc.), CAB39 (2716; Cell Signaling Technology, US), LKB1 (3050; Cell Signaling Technology, Inc.), AMPK (5832; Cell Signaling Technology, Inc.), HIF-1α (14179; Cell Signaling Technology, Inc.), VEGF (Santa Cruz Biotechnology, Inc.). On the next day, the PVDF membrane was washed with a large amount of PBST 3 times for 5 min each, and the membrane was then incubated with HRP-conjugated goat anti-rabbit or anti-mouse secondary antibodies (7074/7076; Cell Signaling Technology, Inc.) at 37˚C for 1 h using shaker. The membrane was washed again with PBST 3 times for 5 min each. Finally, the membrane was placed face up in a petri dish and performed the chemiluminescent analysis using the ECL Protein Detection Kit (Pierce). Protein bands were analyzed using the gel imaging system (Syngene Europe).
All mouse experiments were conducted according to protocols approved by the Tianjin Medical University Animal Care and Use Committee and the reporting in the manuscript followed the recommendations in the ARRIVE guidelines. This study was carried out following all the relevant guidelines and regulations (for animals and humans). Specific pathogen-free BALB/C-NU female nude mice aged 4–6 weeks and weighing 15–18 g were purchased from the Institute of Medical Experimental Animals, Chinese Academy of Medical Sciences.
Next, the intracranial glioma model was established. Briefly, U87-MG cells were infected with the luciferase lentivirus (Shanghai GenePharma Co., Ltd.), and the U87-MG cells were suspended in serum-free DMEM medium to make the cell suspension. To generate orthotopic tumors, 5×104 U87-MG cells were stereotactically injected into the brains of nude mice in a total volume of 3 µl using the previously described stereotactic instrument (Stoelting Co., USA). After 7 days of intracranial tumor cell inoculation, mice were randomly divided into 3 groups: The first group was mainly administered Lv-miR-451-hBMSCs (1×106) (n = 9) by tail vein, the second group was administered uninfected hBMSCs (n = 9) and the third group was just administered PBS (n = 9).
In order to obtain tumor growth status in live animals of different treatment groups, the mice were anesthetized and injected intraperitoneally with D-luciferin (150 mg/kg, beetle luciferin, potassium salt; E1605; Promega Corporation) 15 min prior to imaging with the IVIS imaging system (PerkinElmer, Inc.) for 10–120 sec. Imaging was performed once a week and stopped after 6 weeks. The diet, mental state and physical activity of the nude mice were observed every day, and the weight change of the three groups was monitored once every 3 days. The weight change curve was plotted at the end of the experiment. Six weeks after implantation, three animals from each group were sacrificed, and the brains were collected for H&E, TUNEL staining and immunohistochemistry (IHC). The remaining six mice in each group were used for survival analysis.
Lv-miR-451-hBMSC (1×106) were injected into the tail veins of the intracranial glioma -bearing mice (n = 3). The mice were sacrificed after 24 hours post-injection, and the tumors were removed and snap-frozen for immunofluorescence staining. The slides from frozen tumor tissues were incubated with DAPI. The slides were rinsed several times to remove all free DAPI, and then viewed under a fluorescent confocal microscope (FV1200, Olympus).
H&E staining and IHC.
In order to conduct histological analysis, tumor tissues were fixed in 10% neutral buffered formalin for H&E staining and IHC. The paraffin-embedded brain sections were treated with xylene and then hydrated with ethanol and distilled water. Next, they were washed with PBS for 3 times, 5 min each time, then dyed with hematoxylin for 5 min, and washed with distilled water. The slices were stained with eosin for 2 min, and rinsed with distilled water. Following normal dehydration and neutral glue sealing, the slices were observed and photographed under an Olympus upright BX53 microscope.
For IHC, paraffin sections were dewaxed in xylene I and II each 15 min, anhydrous ethanol I and II, 95% ethanol, and 80% ethanol and distilled water every 15 min. Next, antigen retrieval was performed using sodium citrate (0.01 M, pH = 6) buffer at 97°C for 20 min. Slides were then washed in PBS prior to incubation in 3% H2O2 for 5 min to block endogenous peroxidases. After washing in PBS, slides were blocked with blocking serum for 30 min at room temperature. Next, the slides were incubated at 4°C overnight in a 1:100 dilution with primary antibodies against CAB39 (2716; Cell Signaling Technology), LKB1 (3050; Cell Signaling Technology), AMPK (5832; Cell Signaling Technology), HIF-1α (14179; Cell Signaling Technology), VEGF (MA5-13182; Thermo Fisher Scientific) before incubated with a biotin-labeled secondary antibody (1:100 dilution) for 1 h at 37°C, followed by incubation with ABC-peroxidase and diaminobenzidine (DAB). The slides were then counterstained with hematoxylin and mounted.
The TUNEL apoptosis kit (Roche Diagnostics) was used to detect the apoptosis of tumor cells in each group in situ. The paraffin sections were routinely hydrated, and the endogenous peroxidase was inactivated by H2O2 at room temperature for 30 min. Then, the paraffin sections were washed with PBS (0.01 M, pH 7.2–7.4) 3 times for 5 min. The cover glass was soaked in permeable solution at room temperature for 30 min (0.1% Triton X-100, 0.1% sodium citrate, freshly prepared). The labeled solution was added with 20–25 µl in a wet box at 4˚C overnight, and then washed with PBS (0.01 M, pH 7.2–7.4) 3 times for 5 min. DAPI was diluted with 0.01 M PBS (pH 7.2–7.4) at 1:500 and the nuclei were restrained for 5 min. And the paraffin sections were washed with PBS (0.01 M, pH 7.2 ~ 7.4) 3 times for 5 min, and covered with 0.5 M Na2CO3-50% glycerol. IX81 fluorescence microscope was used for observation and photography.
SPSS 19.0 statistical software (IBM Corp.), χ2 test and one-way ANOVA were used for statistical analysis, and P < 0.05 was considered to indicate a statistically significant difference. One-way ANOVA was used to analyze the difference in the photon number in the in vivo images of the intracranial glioma model, as well as the weight change of mice. Kaplan-Meier survival analysis was used for survival, and the log-rank test was performed.