Culture of OSC
MG63, HOS, U2OS, Saos2 cells were resuscitated and cultured in MEM medium containing 10% fetal bovine serum (100 U/ mL penicillin, 100 μg/ mL streptomycin) in a cell incubator at 37℃ and 5% CO2. After 80% of the cells converged, the medium was removed and PBS was added for washing once or twice. After removing PBS, 1 mL trypsin was added for digestion for 1-3 min, and 3 mL of the complete medium was added to neutralize trypsin and stop digestion. The digested cells were transferred to a 15 mL centrifuge tube and centrifuged at 1000 RPM for 5 min. After the supernatant was dumped, the cells were added to the 3 mL medium to resuspend, and the cells were subcultured in a petri dish at 1:3.
Culture of cisplatin resistant OSC
When the OSC grew to the logarithmic stage, 0.1 mg/L cisplatin (CDDP) was added to the culture medium. After 24 h, the culture medium without CDDP was replaced. After the stable growth of cells, the passage was carried out. The above method was used for continuous treatment for 5 times, and the CDDP concentration was changed to 0.2 mg/L for 5 times, and the CDDP concentration was changed to 0.5 mg/L for 3 times. After a total of 190 days of induction, OSC could grow stably and pass normally under the condition of 0.5 mg/L CDDP, indicating that the cell line could tolerate 0.5 mg/L CDDP, and the drug-resistant strain was named OSC/CDDP.
Reverse transcription-polymerase chain reaction (RT-PCR)
Screening of OS cell lines and detection of miR-331 expression
By measuring the expression of miR-331 in OS cell lines through RT-PCR, OS cell lines to be studied were selected.
Direct Zol RNA microPrep Intracellular RNA was extracted in the toolkit and exosome RNA was extracted through the miRCUTTE RNA isolation toolkit. The cDNA was synthesized by SuperScript III and TaqMan through 200ng isolated RNA The ABI Prism 7500 sequence detection system was amplified.2− ΔΔ CT method was used to determine the value mRNA expression of the corresponding miRNA or gene (miRNA should take U6 as internal reference)
24h before OSC transfection, the cells were digested with trypsin, and the concentration was adjusted to 2×105 cells/ mL, and the cells were inoculated in 6-well culture plates. After the cells were bonded, they were cultured in 2 mL DMEM medium containing serum and without antibiotics. During transfection, the mimics (miR-199a mimics) were diluted with 200 μL Opti-MEM medium, mixed gently, and stood for 5 min at room temperature. At the same time, 5μL Lipofectamine 2000 was diluted with 200 μL Opti-Mem I medium, mixed gently, and stood for 5 min at room temperature. The diluted mimics (miR-199a mimics) and Lipofectamine 2000 were mixed and let stand for 25 min at room temperature, then added into the cell culture well and shaken evenly. Cells were placed in an incubator at 37℃ with 5% CO2 for 30 h, and then transferred to 3 culture plates with a diameter of 10 cm. After transfection, the next experiment was carried out, that is, the expression level of miR-199a in cells was detected by qRT-PCR 48 h after culture.
When OSC grew to 70-75% convergence, the original culture medium was discarded and washed with PBS buffer for 3 times. Serum-free medium was added for further culture for 36 ~ 48 h. The culture medium was collected and centrifuged at 4 ° C for 300×g for 10 min to remove the remaining cells, followed by centrifugation at 4 ° C for 20 min at 2000×g to remove the cell fragments, and centrifugation at 4 ° C for 45 min at 11,000 ×g to further remove the impurities and retain the supernatant. The supernatant was centrifuged at 110,000×g at 4℃ for 90 min, then the supernatant was discarded, 1 mL of 1×PBS was added to each centrifuge tube to resuspend the precipitation, and the suspension in each tube was collected in a centrifuge tube, and then centrifuged at 110,000×g at 4℃ for 70 min. Discard the supernatant and resuspend the precipitation with 100 μL 1×PBS.
PKH26 labeled exosomes
100 μL of 100 μg/ mL of exogenous body weight was suspended in 1 mL of PBS, then 4 μL of PKH26 fluorescent dye solution was added and incubated at 37 ℃ for 20 min. Centrifuge at 100,000 ×g for 70 min, discard the supernatant, suspend the exogenous body weight in 10 mL PBS, and centrifuge at 100,000 ×g at 4 ℃ for 70 min. The excess dye was removed, supernatant was discarded, and the exogenous body weight was suspended in 100 μL PBS for reserve. The labeled PKH26 and OSC were cultured in MEM medium containing 10% fetal bovine serum (100 U/mL penicillin, 100 ug/mL streptomycin) in a cell incubator at 37℃ and 5% CO2 for 12 h. The medium was removed, the cells were washed twice with PBS, fixed with 4% paraformaldehyde and stained with DAPI. The cells were observed with red PKH26 under confocal fluorescence microscope.
Cy3-miR-199a mimic was transfected into OSC/CDDP cells. After that, OSC /CDDP (1x106 / well) and OSCwere co-cultured at a 1:1 ratio on trans-well plate for 12h. The upper compartment was OSC/CDDP, the lower compartment was OSC. In the control group, only CY3 was transfected without miR-199a. After rinsing the MG63 twice with PBS, the fluorescence of Cy3 in the OSC was observed under confocal microscope.
Verification of exosome function
Exosomes extracted from OSC/CDDP and OSC were divided into three groups: drug-resistant exosome group (Exo/CDDP), OSC exosome group (Exo/S), and blank control group PBS (PBS).The exosomes (2 μg) were added into OSC (1x106 / well) to establish the experimental group. After co-culture for 12 hours, 1-60ng/ mL CDDP (4 gradients of 1, 10, 30 and 60) was added to the three groups, respectively, for 24 hours, and then the cell activity was detected by MTT. The migration ability was detected by scratch test, which was divided into four groups: MG63, MG63+ ExO /S, MG63+ ExO /CDDP, and MG63/CDDP. The exosomes (2 μg) were co-cultured with cells (1x106/ well) for 12 hours, and then the wound healing assay was performed.
After CDDP treatment, 10 μL MTT was added to each well for 3 h, α-MEM was removed from the complete medium, 150 μL dimethyl sulfoxide was added to each well and shaken evenly. The absorbance was measured at 570 nm by enzyme linked immunoassay.
Wound Healing assay
The OSCs were scraped with a pipet of 200 μL. Photos were taken at 0h and 24 h. The distance from the damaged area to the disappearance at 0h was used as the quantification and relative invasion rate of remodeling.
Verification of miRNA carrying function in exosomes
The cells were divided into three groups: OSC/CDDP, OSC+Exo/CDDP, OSC+ Exo/CDDP+GW4869 (10 μm, cultured for 24 h), the expression of miR-199a was detected by RT-PCR. Identifying the key factors for exosomes to transport miRNAs from OSC/CDDP. Then, by knocking out the Drosha protein of MG63/CDDP, the activity of exosomal-miRNA was inhibited. The expression of miR-331 and miR-199a in exosomes was detected by RT-PCR to determine whether miRNA was inhibited by knocking out the Drosha protein. Then, the exosomes of OSC/CDDP were extracted and co-cultured with OSC. The exosomes were divided into three groups: OSC+Exo/CDDP (treated with Drosha), OSC+Exo/CDDP (not treated with Drosha) and OSC+PBS. After 12 hours of culture,2 μm CDDP was added to each well for MTT detection.
The MDC staining
Cells of each group were mixed and cultured with MDC at 37℃ and room temperature. The IOD value was then determined under a confocal fluorescence microscope using anti-fluorescence quenching slides and DAPI staining.
Ice lysis of cells or exosomes in RIPA lysates containing protease inhibitors. Equal amounts of total proteins between different groups were separated in SDS-PAGE gel (100V,1.5h) and transferred to 0.22 μm polyethylenedifluoride (PVDF) membrane at (280mA,1.5h).Then it was treated with 5% skim milk powder washing buffer at room temperature for 1 hour and added with primary antibody at 4℃ overnight.On the second day, the membrane was cleaned with TBST and incubated with secondary antibody, and each band was detected using an enhanced chemiluminescence kit.
Inoculation of tumor cells
OSCs were cultured in MEM medium containing 20% fetal bovine serum, 100 U/ mL penicillin, 100 μg/ mL streptomycin, and cultured in an incubator containing 5% CO2 at 37℃. 80% ~ 90% of the cells growing in the culture dish were digested with trypsin, centrifuged at 1000 RPM, supernatant was removed, the cells were washed by adding PBS, and then resuspend by adding PBS again. Cells were counted and the number was adjusted to 2×106 cells/mL. According to the number of nude mice, a sufficient amount of cell suspension was prepared for use when inoculating nude mice. The prepared cell suspension was stored in a refrigerator at 4℃ and inoculated into nude mice as soon as possible.The injection site was uniformly 0.3 cm from the right forearm armpit of the nude mice on the back. After iodine volt cotton ball disinfection, a 1 mL syringe was used to extract 0.2 mL of the mixed cell suspension (density: 2×106/ mL). After the needle of the syringe penetrated the skin of the nude mice, the needle was slightly picked up, moved to the injection site, and the cell suspension was injected slowly. After the injection, the sterile cotton ball is pressed into the needle, and the experimental instruments and waste are disposed of innocently. The newly modeled nude mice were put into cages with new padding, and the mental state, activity and feeding condition of the nude mice, and whether there was redness and swelling at the injection site were observed the next day.
Grouping and drug intervention
When nodules appeared in nude mice (about 7 days after inoculation), the NS group was intraperitoneally injected with 100 μL of normal saline twice a week for 5 weeks. CDDP group was intraperitoneally injected with CDDP (2 mg/kg/ time) twice a week for 5 weeks. Specific groups are as follows:MG63 group (n=6), MG63 +Exo group (n=6), MG63+Exo/CDDP group (n=6) ,MG63+Exo/CDDP+miR-331-3p inhibitor group (n=6), MG63/CDDP group (n=6).
Animal observation: the body weight, mental state, excitement, biting and activity of the nude mice were recorded. Tumor measurement: After tumor formation was visible to the naked eye, the transplanted tumor was measured with a ververier caliper to determine the short diameter (W) and long diameter (L). The tumor volume was calculated according to the formula V (cm3) =L×W2/2, and the tumor size was measured twice a week.The final sampling: 6 weeks after tumor grafting in nude mice, tumor tissue was collected, the volume and weight of tumor were calculated, fixation with 4% paraformaldehyde.
The obtained tissues were embedded in paraffin sections, sealed with endogenous peroxidase, antigen repair, exposed to antigen determinants, and incubated with primary and secondary antibodies. DAB staining was followed by redyeing, dehydration, transparency, sealing and observation under light microscope.
Each experiment was performed three times and the results are shown as the mean ± standard deviation. SPSS 23.0 software (SPSS, Inc.) was used for statistical analysis. Differences among three or more groups were compared by one‑way analysis of variance (ANOVA); Tukey's test was used form comparisons of data between two groups. P<0.05 was considered, to indicate a statistically significant difference.