Serum Specific MicroRNAs In Infantile Hemangioma:A Preliminary Study

Objective:This study is to investigate the specific microRNAs in the serum of patients with infantile hemangioma and identify the effects of the microRNAs on cell proliferation and apoptosis.Method : 1.Selecting the same stage infantiles without hemangioma as control samples,significant different miRNAs including: hsa-miR-518a-3p,hsa-miR-518 e,hsa-miR-519 a,hsa-miR-512-3p,hsa-miR-216 b,hsa-miR-544 were identified and verified by qRT-PCR with Taqman probe.2.After the specimen of hemangioma was collected from the surgery,it was digested by the collagenase and cells were collected and purified by anti-CD133–coated magnetic beads to separate the CD133 positive stem cells from the specimen.To testify the cells separated,cell membrane antibody measurement including CD29,CD31,CD34,CD44,CD45,CD90,CD105 and CD144 were analyzed by flow cytometry,the differentiation of the cells was carried out with adipogenic media and osteogenic media to study the multilineage differentiation,tube formation experiments of stem cells was done on Matrigel and in vivo subcutaneous murine model of IH was established.3.HemSC were transfected with miR-518a-3p mimics、miR-518a-3p inhibitor、miR-518a-3p mimics NC and miR-518a-3p inhibitor NC.After transfection,cells were observed under inverted fluorescence microscope to analyze the transfection efficiency.With the utilization of RT-PCR,the content of miR-518a-3p was evaluated by SYGR Green.CCK-8 kit was used to identify the cell proliferation ability of HemSC.With the fluorescence staining reagent of Annexin V/7-AAD,the effect of miR-518a-3p on cell cycle and apoptosis on HemSC were measured by flow cytometry.Results: 1.Serum miR-518a-3p and miR-518 e with fold change of 276.740(P<0.001)and 1889.889(P<0.001)each has a remarkable difference in the infantile hemangioma patients and is specific compared to normal infantiles.Serum miR-518a-3p has a higher expression in the patients with infantile hemangioma.2.Flow cytometric analyses for endothelial and mesenchymal cell-surface markers showed that HemSC expressed CD29(99.5%),CD44(97.9%),CD90(87.6%)and CD105(98.5%),barely expressed CD31(0.2%),CD34(0.1%),CD45(0.1%)and CD144(0.1%).HemSC display tube formation and clonogenic and multilineage differentiation ability in vitro.HemSC form hemangioma-like lesions in immunodeficient mice.3.The fluorescence analysis post-transfection showed that miR-518a-3p mimics can slightly enhance the proliferation ability and miR-518a-3p inhibitors can inhibit the stem cells to proliferate.Cell cycle analysis showed that the percentage of S phase was increased,accompanied with decrease in G2/M phase after the treatment of miR-518a-3p mimics,although the difference wasn’t remarkable.Cell apoptosis results showed that miR-518a-3p mimics was able to induce a lower percentage of apoptosis to HemSC and opposite effect of miR-518a-3p inhibitors.Conclusion: Serum miR-518a-3p and miR-518 e has the potential for the early differential diagnosis in hemangioma.The expression level of miR-518a-3p of HemSC was significantly increased by the administration of miR-518a-3p mimics,resulting in the enhancement of cell proliferation,cell cycle percentage in S phase and inhibition of apoptosis of the stem cells.