GEO database was used for the screening of differentially expressed mRNAs and miRNAs between LPS and normal control tissues, where mRNA data were derived from GSE21122 and miRNA data were derived from GSE36982. Screening was performed by GEO2R (Fig. https://www.ncbi.nlm.nih.gov/geo/geo2r/ ). The screening criteria for mRNA were | logFC | > 2, and bonferron corrected P < 0.05, and the screening criteria for miRNA were | logFC | > 1.5, and bonferron corrected P < 0.05. The selected differentially expressed genes were subjected to GO and KEGG functional enrichment analysis by applying STRING, and the screening criteria were taken as P < 0.05 after correction. MiRTarBase(41), miRWalk(42) and TargetScan(43) databases were applied to predict all regulated target genes of differentially expressed miRNAs. Only target genes that were predicted simultaneously in the three databases were included in the analysis. The mRNA-miRNA regulatory network was constructed by overlapping the target genes of differentially expressed miRNAs with the genes of differentially expressed mRNAs. STRING(44) was applied to construct a protein-protein interaction network, and Cytoscape was applied to fuse it with the mRNA-miRNA regulatory network, and the fusion network was topologically analyzed using the CytoHubba plugin to screen the top 10 key nodes. Survival analysis of miR-215-5p expression in the TCGA database with overall survival time for DDL was implemented by LinkedOmics(45). MiR-215-5p was predicted for it regulatory relationship with MDM2 using TargetScan(43).
Cell lines and culture
The human liposarcoma cell line SW-872 was purchased from the American Type Culture Collection (ATCC), and the cell line used in the validation experiment of short tandem repeat (STR) DNA profiling matched exactly with the SW-872 cell line. SW-872 cells were lysed and cultured in DMEM medium containing 10% fetal bovine serum at 37℃ and 5% CO2. SW-872 cells were transferred into a centrifuge tube containing 5ml medium (DMEM + 15% FBS + 1% (Penicillin-Streptomycin Solution)) at a ratio of 1:3, centrifuged to collect cells, suspended with complete medium containing 10% fetal bovine serum, inoculated into a culture dish, gently blown and mixed well, and cultured at 37℃ under 5% CO2 saturated humidity. When the cell density reached 80%, 1 ml 0.25% trypsin was added to digest and collect the cells, which were passaged in a ratio of 1:3, and the culture was expanded at 37 ° C with 5% CO2 saturated humidity.
MiR-215-5p and MDM2 fluorescent real-time quantitative PCR primers were designed by DNAMAN software（table 3） and synthesized by Shanghai Sangon Co. Ltd. 2 ul 5 × PrimeScript Buffer, 0.5 ul PrimeScript RT Enzyme Mix I, 0.5 ul Oligo (dT) 15 ( 15 μM), 0.5 ul Randommers (100 μM), Total RNA 500 ng, RNase Free dH2O were added to 10 ul, respectively, mixed well and briefly centrifuged, heated at 37 ° C for 15 min, 85 ° C for 5 s, and diluted to 35 µl on a PCR instrument to obtain reverse transcribed cDNA. QPCR reaction: Forward primer 10 µ M0.4 ul, 0.4 ul Reverse primer 10 µM, 2XSYBR Select Master Mix (2 ×) 10 ul, RNase-free water 4.8 ul, cDNA template 4 ul, 50 × ROX Reference Dye 20.4 μl, prepared into 20 ul system, mixed well, maintained on the instrument at 95℃, 10 min, 95℃, 15s, 60℃ 60s, 95℃, 15s, 40 cycles, melting curve 60℃ 1 min, 95℃ 15s, 1 cycle, so as to obtain qPCR reaction results, using 2-ΔΔCt method for result quantification calculation.Three biological replicates were performed for each group.
Dual luciferase reporter gene
MiR-215-5p or control was cotransfected with pYr-MirTarget-MDM2 3 ′ UTR into cells according to the groups as indicated using lipofectamine 2000 as transfection reagent, and fluorescence intensity was measured after 48 hours in the presence of firefly luciferase as an internal reference.Three biological replicates were performed for each group.
cell phenotype experiments
The effect of miR-215-5p on cell proliferation activity was explored by MTT colorimetric assay: the above cells were cultured for 24 h, 48 h, and 72 h according to the groups, 10 μL MTT was added to each well and cultured at 37 ° C for 4 h. The culture medium was aspirated and shaken with 150 μL DMSO for 10 min, and the absorbance OD568 of each well was measured by a microplate reader.Three biological replicates were performed for each group.
Flow cytometry was used to investigate the effects of miR-215-5p on cell cycle and apoptosis: cells were taken according to groups, digested with 0.25% trypsin without EDTA, resuspended and washed and fixed in 70% ethanol at 4 ° C for more than 4 h. Ribonuclease (RNase) was added and bathed in water at 37 ° C for 30 min; propidium iodide (PI) working solution was stained in the dark at 4 ° C for 30 min and then detected by flow cytometry. At the same time, the cells were taken according to the groups, digested with 0.25% trypsin without EDTA, resuspended and washed and reacted using the AnnexinV-APC/7-AAD apoptosis detection kit according to the instructions, and detected on the flow cytometer.Three biological replicates were performed for each group.
Cell scratch and invasion assay
Use a marker pen behind the 6-hole plate, use a ruler to make comparison, and evenly draw a horizontal line to pass through the hole at interval of 0.5 cm. The treated cells were divided into groups, inoculated in a six-well plate, and cultured at 37℃ and 5% CO2. When the cell density reached about 90%, a vertical scratch was made, and photographs were taken at 0h and 48h.Three biological replicates were performed for each group.
The treated cells were taken according to groups, washed, resuspended, counted and diluted, inoculated in the prepared Matrigel-transwell reaction system, and cultured in 5% CO2 at 37℃ for 24 hours; the cells were fixed in 70% ice-cold ethanol solution for 1 hour, stained with crystal violet and observed and photographed under a microscope.
Colony formation assay was performed to investigate the effect of miR-215-5p on colony formation: cells were diluted in suspension according to groups and seeded in 6-well plates at a density of 300 cells. Culture for 3 weeks. When clones appeared, they were counted after methanol fixation and stained with crystal violet. Colony formation rate = colony count/inoculated cell count 100%.Three biological replicates were performed for each group.
Detection of MDM2 Expression by Immunofluorescence Single Labeling
In the culture plate, the slides that had climbed the cells were immersed with PBS for 3 times, 3 min each time; the reptiles were fixed with 4% paraformaldehyde for 15 min, and the slides were immersed with PBS for 3 times, 3 min each time; 0.5% TritonX-100 (prepared with PBS) was permeabilized at room temperature for 20 min; the slides were immersed with PBS for 3 times, PBS was blotted with absorbent paper, normal goat serum was added to the slides, and blocked at room temperature for 30 min; the blocking solution was absorbed with absorbent paper, without washing, a sufficient amount of diluted primary antibody (1:100) was added to each slide and placed in a wet box, and incubated overnight at 4 ° C; the reptiles were immersed with PBST for 3 times, 3 min each time, and the excess liquid on the reptiles was blotted with absorbent paper followed by dripping of diluted fluorescent secondary antibody (1:100), and the sections were immersed with PBST for 3 times, 3 min each time. Three biological replicates were performed for each group.
Detection of miR-215-5p expression by FISH
Cell crawls were fixed in 4% paraformaldehyde (DEPC) for 20 min and washed three times by shaking on a destaining shaker in PBS (pH 7.4) for 5 min each. Gene strokes were circled, and proteinase K (20 ug/ml) was added dropwise for digestion for 6 min according to different index characteristics of different tissues. After rinsing with pure water, PBS was washed three times × 5 min. The prehybridization solution was dropped for 1 h in a 37 ° C incubator. The prehybridization solution was poured off, and the hybridization solution (containing probe miR215 at a concentration of 500 nM) was dropped and hybridized overnight at 42 ° C. The hybridization solution was washed off, 2 × SSC, 10 min at 37 ° C, 1 × SSC, 2 × 5 min at 37 ° C, and 10 min at 0.5 × SSC 37 °. If there are more non-specific hybridizations, formamide washing can be added, and the hybridization solution containing secondary standard probes can be dropped and incubated at a dilution ratio of 1:400.42 ° for 3 hours. The latter 2 × SSC, washed at 37 ° C for 10 min, 1 × SSC, washed at 37 ° C for 2 × 5 min, and 0.5 × SSC at 37 ° for 10 min. Drop blocking solution: Drop blocking serum at room temperature for 30 min. Gutta mouse anti-digoxigenin-labeled peroxidase (anti-DIG-HRP): The blocking solution was decanted and anti-DIG-HRP was added. Incubation was performed for 50 min at 37 ° C, followed by three × 5 min PBS washes. Dropping of CY3-TSA: add CY3-TSA reagent and allow to react at room temperature for 5 min, protected from light. After washing with PBS three times × 5 min. The sections were dropped with DAPI staining solution and incubated in the dark for 8 min, rinsed and then dropped with anti-fluorescence quenching mounting medium for mounting. Sections were observed and images were collected under a Nikon upright fluorescence microscope, and DAPI-stained nuclei were blue with Cy3-labeled red light under UV excitation.Three biological replicates were performed for each group.
RG7388 is an MDM2-specific inhibitor. RG7388 was added to SW-872 mimics, and after 48 hours of cell culture, 10 μl cck8 was added to each well and cultured at 37 ° C for 4 hours; the absorbance OD450 of each well was measured by a microplate reader.Three biological replicates were performed for each group.
WB was used to detect the expression changes of caspase-3, MDM2, P53, P21, PCNA, Bax, and Bcl-2
After cell lysis, centrifugation, and BCA quantification in different groups, the protein expression of caspase-3, MDM2, P53, P21, PCNA, Bax, and Bcl-2 was detected. METHODS: 5% stacking gel and 12% separation gel, sample denaturation and sample loading, electrophoresis and membrane transfer were prepared. The membrane was loaded into 5% TBST blocking solution, prepared and shaken at room temperature for 2 h. 5 ml primary antibody diluent was added and kept at 4 ° C overnight. 10 ml of secondary antibody (1:1000 dilution) was placed on a shaker for 2 h. Color was developed and developed by compression exposure in a dark room. Results BandScan was used to analyze the gray value of the film, and the ratio of the intensity of the detected protein band to that of the protein band (%) was calculated, respectively.Three biological replicates were performed for each group.
In this study, SPSS 22.0 software was used for data processing. All data were expressed as mean ± standard deviation (x ± s). One-way analysis of variance was performed for measurement data. LDS method was used for pairwise comparison. The test level α = 0.05, P < 0.05 was considered statistically significant, and P < 0.01 was considered very significant.