Abstract
Background Hepatoblastoma (HB) is the most common malignant tumor of the liver. MMP9 plays an essential role in HB. The purpose of our study was to screen for differentially expressed lncRNAs and miRNAs that targeted MMP9. Based on this, the role of lncRNA NEAT1/miR-132/MMP9 in HB and the mechanisms involved were discussed. Methods Bioinformatics analysis was used to screen the differentially expressed lncRNAs and miRNAs targeting MMP9. Exosomes were extracted from HB cells and normal liver cells for characterization and identification. Exosome uptake assay was conducted to determine whether exosomes were absorbed by bone marrow stromal cells (BMSCs). α-SMA, fibronectin, and s-100 expressions in tissues and cells were detected by IHC and ICC. lncRNA XIST, lncRNA NEAT1, miR-132, and MMP9 expressions were characterized by qRT-PCR. Western blot was performed to measure MMP9, α-SMA, and s-100 expressions. Flow cytometry was used to stain α-SMA, s-100. Bioinformatics and dual-luciferase reporter assay were applied to verify the interaction between lncRNA NEAT1 and miR-132, and miR-132 and MMP9. The effect of lncRNA NEAT1 on the development of HB in nude mice was studied. Results Differentially expressed lncRNA NEAT1/miR-132/MMP9 was obtained through bioinformatics analysis and cell verification. HB-derived exosomal lncRNA NEAT1 regulated miR-132 and MMP9 expression in BMSCs. In addition, HB-derived exosomal lncRNA NEAT1 promoted BMSCs differentiation toward invasive myofibroblast via miR-132/MMP9 axis. LncRNA NEAT1 regulated MMP9 through miR-132. Tumor formation experiments in nude mice showed that HB-derived exosomal lncRNA NEAT1 could affect the development of HB. Conclusion HB-derived exosomal lncRNA NEAT1 induced BMSCs differentiation into tumor-supporting myofibroblasts via modulating miR-132/MMP9 axis, which provided a new target for HB treatment.
Highlights
Hepatoblastoma (HB) is a rare malignant tumor derived from pluripotent stem cells, most common in children and mainly occurs in the first two years of life [1, 2]
Total proteins were extracted from tissues and cells using RIPA lysis buffer according to the instructions, and the protein was quantitated according to the BCA protein assay kit. e loading buffer of SDS-PAGE was mixed, and the mixture was heated in a boiling water bath at 100°C for 5 min. e proteins were adsorbed on the PVDF membrane through gel electrophoresis. e membranes were sealed with 5% skim milk solution at room temperature for 2 h, and incubated with primary antibodies MMP9 (AB38898, 1 : 1000, Abcam), α-SMA (14395-1-AP, 1 : 2000, Proteintech), fibronectin (66042-1-IG, 1 : 100, Proteintech), s-100 (16105-1-AP, 1 : 1000, Proteintech), and β-actin (66009-1-Ig, 1 : 5000, Proteintech) at 4°C overnight
ICC results showed that compared with the bone marrow stromal cells (BMSCs) + liver cell exosome group, α-SMA, fibronectin, and s-100 were positive in the BMSC + HB cell exosome group (Figure 4(b)). ese results indicated that HB-derived exosomal lncRNA NEAT1 regulated miR-132 and MMP9 expression in BMSCs
Summary
Hepatoblastoma (HB) is the most common malignant tumor of the liver. MMP9 plays an essential role in HB. e purpose of our study was to screen for differentially expressed lncRNAs and miRNAs that targeted MMP9. E purpose of our study was to screen for differentially expressed lncRNAs and miRNAs that targeted MMP9. The role of lncRNA NEAT1/miR-132/MMP9 in HB and the mechanisms involved were discussed. Bioinformatics analysis was used to screen the differentially expressed lncRNAs and miRNAs targeting MMP9. LncRNA XIST, lncRNA NEAT1, miR-132, and MMP9 expressions were characterized by qRT-PCR. Expressed lncRNA NEAT1/miR-132/MMP9 was obtained through bioinformatics analysis and cell verification. HB-derived exosomal lncRNA NEAT1 regulated miR-132 and MMP9 expression in BMSCs. In addition, HB-derived exosomal lncRNA NEAT1 promoted BMSCs differentiation toward invasive myofibroblast via miR-132/MMP9 axis. Tumor formation experiments in nude mice showed that HB-derived exosomal lncRNA NEAT1 could affect the development of HB. HB-derived exosomal lncRNA NEAT1 induced BMSCs differentiation into tumorsupporting myofibroblasts via modulating miR-132/MMP9 axis, which provided a new target for HB treatment
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