miR-140-3p in exosomes derived from bone-marrow mesenchymal stem cells inhibits pyrotoisosis by targeting PTEN under high-glucose condition

Abstract

BACKGROUND

Diabetes has continuously affected some people for a long time, and among them there are many cases of foot ulcers which appear due to insufficient fibroblast activity. Bone-marrow mesenchymal stem cell (BMSC) secreted exosomes (BMSCs-Exos), although showing good clinical application prospects; however, the function of microRNAs (miRNAs) in exosome is not well understood yet.

AIM

To explore the function of miR-140-3p in BMSC-Exo treatment for high-glucose-induced damage to rat skin fibroblasts (RSFs), as well as its related cell pathways.

METHODS

Exosome materials originated from rat BMSCs and were obtained by precipitating them. Based on the structure, dimensions and external markers (CD9, CD63, CD81), etc., of these cells using transmission electron microscope, nanoparticle tracking analysis and western blotting technologies respectively. Tagging the exosomes with PKH26 to track their internalisation into RSF cells. Using high-throughput miRNA sequencing combined with bioinformatics analysis of samples from BMSCs-Exo cells in normal or hyperglycaemic conditions to find deregulated miRNAs associated with the pyroptotic pathway. After transfecting mimic or inhibitor RNAi-miR-140-3p into rat BMSCs, exosomes were isolated from cell cultures, and their concentrations of miR-140-3p were determined by real-time quantitative polymerase chain reaction (qRT-PCR). The RSFs were divided into three groups: Group Hy-miR-inhibitor-NC-Exo; the Hy-miR-140-3p-inhibitor-Exo group and the Hy-miR-140-3p-inhibitor-Exo-AYC group. Determine whether the cells have increased proliferation or migrated through the CCK-8 assay and the cell-wound-closure experiment, respectively. Determine the levels of pyroptotic-related molecules (NLRP3, interleukin-18, interleukin-1β, caspase-1, and gasdermine-D-N-terminal) using western blotting technology. miR-140-3p’s functional association with its target gene phosphatase and tensin homolog (PTEN) has been verified by qRT-PCR, western blotting and dual-luciferase reporter assay. To examine the interaction of the miR-140-3p/PTEN regulatory loop with fibroblast pyroptosis via western blot. The statistical verification of all results used SPSS 21.0; group differences were compared with a one-way ANOVA.

RESULTS

BMSCs-Exo was successfully isolated and characterised. These vesicles are about 50 nm to 250 nm in diameter, oval or spherical in shape; they strongly express the three surfaces antigens of CD9, CD63 and CD81. PKH26 labelling confirmed that BMSCs-Exo had been absorbed into the RSF cells. miRNA-seq data showed that miR-140-3p was significantly up-regulated in BMSCs-Exo under hyperglycaemia, and it was closely related to the pyroptosis signal transduction pathway. According to qRT-PCR results, compared with the negative control group (1.04 ± 0.03), the level of miR-140-3p in the miR-140-3p mimics group increased by more than 2.5 times; the level in the miR-140-3p inhibitor group decreased by about 46% compared with that in the negative control group (1.07 ± 0.02). Comprehensive comparison showed that there was a statistically significant difference at all levels (F = 1062.839, P < 0.001). Cellular experiments showed that compared with those in the Hy-miR-inhibitor-NC-Exo group, there was a significant decrease in proliferation and migration abilities, as well as an obvious increase in expression levels of pyroptotic protein genes in the Hy-miR-140-3p-inhibitor-Exo group. After adding the pyroptosis pathway inhibitor AYC, the above parameters improved greatly. qRT-PCR, western blotting, and dual-luciferase reporters have demonstrated that PTEN is a specific target of miR-140-3p; it has been effectively repressed by this reduction in PTEN levels. Afterwards’ examination found that PTEN overexpressed promoted fibroblasts to perform pyroptosis; meanwhile, miR-140-3p mimic reversed it.

CONCLUSION

BMSCs-Exo delivered miR-140-3p to target PTEN in cells, suppress NLRP3-mediated pyroptosis, improve fibroblast function damaged by high-glucose conditions, and serve as a new therapeutic strategy for treating diabetics with wounds.

Keywords: Bone marrow mesenchymal stem cell-derived exosomes; Rat fibroblasts; MicroRNA; Phosphatase and tensin homolog; Pyroptosis

Core Tip: Specifically, extracellular vesicles derived from bone marrow mesenchymal stem cells contain miR-140-3p that inhibits pyroptosis in rat dermal fibroblasts caused by hyperglycaemic stimulation through direct inhibition of phosphatase and tensin homolog. By blocking the NLRP3/caspase-1/gasdermine-D-N-terminal-dependent pyroptosis pathway to promote fibroblast proliferation and migration. The miR-140-3p/phosphatase and tensin homolog axis has provided an attractive strategy to treat diabetic foot ulcers.