Cell isolation and culture
Bone marrow was extracted from the posterior superior iliac spine of healthy volunteers under sterile conditions. Then, bone marrow-derived mesenchymal stem cells (BMMSCs) were isolated and purified by density gradient centrifugation as previously reported . BMMSCs at passage two were used in experiments and cultured in medium composed of 90% Dulbecco’s modified Eagle’s medium (DMEM, Gibco, New York,
USA) and 10% fetal bovine serum (FBS, TIANHANG, Zhejiang, China) at 37 °C in a 5% CO2 atmosphere. The culture medium was replaced every third day. Peripheral blood mononuclear cells (PBMCs) were isolated via density gradient centrifugation. CD14+ monocytes were isolated from PBMCs with CD14 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany).
Monocyte migration assay
Polycarbonate Membrane Transwell® Inserts (5-µm pores, Corning, New York, USA) were used to perform monocyte migration experiments. MSCs (2 × 104) or cell-free MSC culture supernatants were placed in the lower chambers, and CD14+ monocytes (1 × 106) were seeded in the upper chambers. Both sets of cells were cultured in RPMI 1640 medium supplemented with 10% FBS at 37 ℃ in a 5% CO2 atmosphere. Twelve hours later, the culture supernatants in the lower chambers were collected, and the number of CD14+ monocytes was determined via flow cytometry.
Macrophage polarization assay
CD14+ monocytes were cocultured with MSCs using Polycarbonate Membrane Transwell® Inserts (0.4-µm pores). CD14+ monocytes (2 × 105) were seeded in the lower chambers, and MSCs (2 × 104) were seeded in the upper chambers. Recombinant human M-CSF (25 ng/ml, PeproTech, New Jersey, USA) was added to activate monocytes. Five days later, lipopolysaccharide (LPS; 50 ng/ml, SigmaAldrich, Darmstadt, Germany) and recombinant human interferon-γ (IFN-γ; 20 ng/ml, PeproTech) were added to induce macrophage polarization to the M1 phenotype, and recombinant human IL-4 (20 ng/ml, PeproTech) and IL-10 (20 ng/ml, PeproTech) were added to induce macrophage polarization to the M2 phenotype. Twenty-four hours later, the phenotype of the macrophages was determined by flow cytometry. Macrophages were digested, incubated with anti-HLA-DR-PE antibody or anti-CD206-BV421 antibody (BD Biosciences, California, USA) and then incubated with fixation medium (Invitrogen, Massachusetts, USA) for 15 min. After three washes with PBS, the cells were incubated with permeabilization medium plus an anti-CD68-FITC antibody (BD Biosciences) for 30 min.
Rapid amplification of cDNA ends (RACE)
A SMARTer RACE amplification kit (Clontech, California, USA) was used to perform RACE. In brief, total RNA was extracted from MSCs and used to synthesize first-strand cDNA for 3′- and 5′-RACE. Then, 5′-RACE and 3′-RACE PCRs were performed using specific primers (Additional file 1: Table S1), and 3′- and 5′ nested PCRs were performed using products from the aforementioned PCRs as templates. DNA gel electrophoresis was used to identify the target fragments. These fragments were collected and cloned into a plasmid, and the full-length cDNAs were subsequently evaluated via sequencing.
siRNAs specific for MRF, IRF1, HNRNPD and a negative control siRNA were generated by IGE (Guangzhou, China). MSCs were transfected with siRNAs using Opti-MEM (GIBCO) and Lipofectamine RNAiMAX (Thermo Fisher, Massachusetts, USA) according to the manufacturer’s directions. Knockdown efficiency was analyzed via qRT–PCR or western blotting after 72 h.
A full-length MRF-overexpression lentivirus and a negative control were generated by OBiO (Shanghai, China). MSCs were infected with the MRF-overexpression lentivirus or vector (multiplicity of infection: 50) using 5 µg/ml polybrene. The medium containing the lentivirus was replaced with fresh medium 24 h later. Overexpression efficiency was analyzed via qRT–PCR after 96 h.
Dual-luciferase reporter assay
The promoter sequence of MRF from − 2000 to + 100 bp relative to the transcription start site and the antisense sequence were synthesized and separately cloned into a pGL4.10 vector. 293 T cells were transfected with the above vectors followed by transfection with siRNAs to silence IRF1 using Lipofectamine 3000 (Invitrogen). All experimental group cells were transfected with pRL-TK plasmids as an internal control. Luciferase activities were detected with a Dual-Luciferase Reporter Assay System (Promega, E1910, Massachusetts, USA). The relative luciferase intensity is shown as the numerical value of firefly luciferase activity divided by Renilla luciferase activity.
RNA pull-down and mass spectrometry
In vitro transcription of MRF and the antisense sequence was performed with a TranscriptAid T7 High Yield Transcription Kit (Thermo, K0441). The RNA products were purified and subsequently biotinylated using a Pierce RNA 3′ End Desthiobiotinylation Kit (Thermo, 20163). MSC protein lysates were prepared using standard IP lysis buffer with protease inhibitor. RNA pull-down was performed using a Pierce Magnetic RNA‒Protein Pull-Down Kit (Thermo, 20164) according to the manufacturer’s instructions. Briefly, the biotinylated RNA was captured with streptavidin magnetic beads, and the labeled RNA-magnetic bead complexes were then incubated with MSC protein lysates for 60 min. The protein-biotinylated RNA-magnetic bead complexes were collected with a magnetic stand, and RNA-binding proteins were washed with buffer and finally eluted for subsequent analysis. Silver staining was used to detect distinct protein bands, and further identification was performed using mass spectrometry analysis with an LC–MS/MS system (Thermo Scientific Q Exactive).
Cell cytoplasmic and nuclear fractionation
A PARIS kit (Thermo Fisher) was used to separate the cell cytoplasmic and nuclear fractions. The RNA in each fraction was extracted and reverse transcribed into cDNA. The distribution of target mRNA in each fraction was analyzed via qRT–PCR. U6 and MALAT1 were used as positive controls for the nuclear fraction, while GAPDH and ACTIN were used as positive controls for the cytoplasmic fraction.
RNA extraction and quantitative real-time PCR
To extract total RNA from MSCs, first, MSCs were washed 3 times with phosphate-buffered saline, TRIzol (TaKaRa, Dalian, China) was added to lyse the cells, and extraction was performed according to the manufacturer’s instructions. Specifically, to extract total RNA from MSCs cocultured with monocytes to assess the RNA level of MCP1 and eliminate interference from MCP1 derived from monocytes, a transwell system was used to perform coculture. MSCs (2 × 104) were seeded in the lower chambers, and CD14+ monocytes (1 × 106) were cultured in the upper chambers. Then, the upper chambers containing monocytes and supernatants were removed, MSCs in the lower chambers were washed 3 times with phosphate-buffered saline, and TRIzol (TaKaRa) was added to lyse the cells. Total RNA was reverse transcribed into cDNA with a PrimeScript PT Reagent Kit (TaKaRa). Quantitative real-time PCR was performed using a SYBR Premix Ex Taq Kit (TaKaRa). The PCR procedure was set as follows: 95 °C for 1 min, 40 cycles at 95 °C for 30 s, 58 °C for 20 s, and 72 °C for 30 s, and finally 72 °C for 5 min for full elongation of the products. The results were analyzed using the 2−ΔΔCt method, and GAPDH was considered the reference gene to calculate relative gene expression. The forward and reverse primers are listed in Additional file 1: Table S1.
Western blotting analysis
To collect proteins, cell lysates were extracted with RIPA lysis buffer containing 1% protease and phosphatase inhibitors. Specifically, to detect the MCP1 protein level in MSCs cocultured with monocytes and eliminate interference from MCP1 derived from monocytes, coculture was performed as described above, MSCs in the lower chambers were washed 3 times with phosphate-buffered saline, and RIPA buffer was added to extract cell lysates. Then, the proteins were separated via sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to polyvinylidene fluoride membranes. The membranes were blocked and incubated with primary antibodies, followed by incubation with HRP-conjugated secondary antibodies. Protein levels were detected with a chemiluminescent HRP substrate (Millipore, Vermont, USA) and quantified using ImageJ. The following antibodies were used in this study: anti-MCP1 (Abcam, ab9669, Cambridge, UK), anti-IRF1 (Cell Signaling Technology, 8478S, Massachusetts, USA), anti-HNRNPD (Cell Signaling Technology, 12382S), anti-GAPDH (Cell Signaling Technology, 5174S) and anti-β-tubulin (Cell Signaling Technology, 2128S).
To detect MCP1 secreted from MSCs cocultured with monocytes, coculture was performed as described above. After removal of the upper chambers and culture supernatants, fresh medium was added to the lower chambers, and MSCs were cultured for an additional 24 h to eliminate the interference from MCP1 produced by monocytes. Then, cell culture supernatants were collected and analyzed with a human MCP1 ELISA kit (R&D, DCP00, Minnesota, USA) according to the manufacturer’s instructions. Briefly, the supernatants and standard were added to a microplate and incubated for 2 h at room temperature. Then, the samples were aspirated and washed 3 times with washing buffer, and human MCP-1 conjugate was added to the microplate and incubated for 1 h at room temperature. After three washes, substrate and stop solutions were successively added to the wells. Optical density (O.D.) was measured with a microplate reader, and a standard curve was created using standard samples. Accurate MCP1 concentrations were calculated based on the standard curve.
LncRNA and mRNA high-throughput sequencing was performed as previously described . Briefly, MSCs (n = 5) cocultured with or without CD14+ monocytes were separately treated with TRIzol (TaKaRa). RNA was extracted according to the manufacturer’s protocol, and RNA integrity was evaluated with an Agilent 2200 TapeStation (Agilent Technologies, USA). Then, RNA was fragmented into average sizes of approximately 200 nt and reverse transcribed into single-stranded cDNAs. Double-stranded cDNAs were synthesized, purified and treated with terminal repair and ligation primers using the NEBNext® Ultra™ RNA Library Prep Kit for Illumina (NEB, USA). After PCR amplification and purification, libraries were paired-end sequenced (PE150) on an Illumina HiSeq 3000 platform at Guangzhou RiboBio Co., Ltd. (Guangzhou, China). Differential expression was analyzed using DESeq2 with |log2Fold Change|> 1 and Q value < 0.05.
A heatmap was drawn with pheatmap (v1.0.12) based on differentially expressed genes. An advanced volcano plot was generated using the OmicStudio tools (https://www.omicstudio.cn/tool) based on OmicStudioClassic (v1.3.14) and OmicStudioKit (v1.9.0). A coexpression-based GO enrichment analysis of lncRNAs was performed using the AnnoLnc2 database (http://annolnc.gao-lab.org/) , and significantly enriched GO terms with a Q value < 0.05 were reported as putative functional annotations of the lncRNAs. The GO enrichment analysis of differentially expressed genes was performed with the DAVID database (https://david.ncifcrf.gov/), and a P value < 0.05 was considered to indicate significant enrichment in the annotation categories. GO terms related to the immune response were selected to perform a GOChord analysis using the R package GOplot (v1.0.2). Potential transcription factors binding to the MRF promotor were predicted via the JASPAR database (https://jaspar.genereg.net/), and the top 30 predicted transcription factors were considered for subsequent screening.
Monocyte recruitment and macrophage polarization in vivo
NOD/SCID mice were subcutaneously injected with 0.5 mg/kg recombinant human M-CSF (PeproTech, 300-25) before adoptive transfer [20, 21]. For monocyte recruitment, human MSCs pretreated with MRF siRNA or the negative control were intraperitoneally injected into the mice. Twelve hours later, 1 × 107 human CD14+ monocytes labeled with CFSE were intravenously injected into each mouse. After 24 h, the adoptively transferred mice were sacrificed, and peritoneal lavage fluid and spleen cells were collected for flow cytometry analysis. For macrophage polarization, human MSCs were pretreated with MRF siRNA or negative control, human macrophages were activated by human M-CSF and then stimulated with LPS and recombinant human IFN-γ to induce M1 polarization or with recombinant human IL-4 and IL-10 to induce M2 polarization, similar to the in vitro induction of macrophage polarization. Then, 1 × 105 human MSCs and 1 × 106 macrophages labeled with CFSE were simultaneously injected into the abdominal cavity of NOD/SCID mice. Two days later, transplanted mice were sacrificed, and peritoneal lavage fluid was collected to detect the MFI of human HLA-DR and human CD206 in CFSE-labeled cells via flow cytometry.
The data in this study were analyzed using SPSS 26.0. The results are presented as the mean ± standard deviation (SD). Independent-sample t tests were used to compare two experimental groups. One-way ANOVA with Bonferroni’s test was used to analyze differences among three or more groups. P < 0.05 was considered to indicate a significant difference.