The Effect And Mechanism Of MiR-206 On Cell Proliferation Of Infantile Hemangioma Endothelial Cells

As one of the most common benign tumors of infancy, infantile hemangioma(IH) affects 4-10% of infants, with a predilection for female, Caucasian, low birth weight, and prema-ture infants, multiple birth, and elder pregnant woman. According to the development and pathology, IH can be separated into three different phases, including proliferating phase, involuting phase, and involuted phase. The proliferating phase mostly starts from birth and ends within the first five months, with the characteristic of rapid proliferation of endothelial cells. Almost 10% IH can complete involuting in the first year, and the rest needs 9-10 years, or even longer time to achieve this. IH has been defined as benign tumor with the tendency of involuting, however, the mechanism of involuting in IH still remains unknown.MicroRNA (miRNA) belongs to small molecular single-stranded RNA, are main regulatory sequences of gene expression in eukaryotic cells. We analyzed the specimens of different phase of IH, and preliminary results showed that among all the phases, the expression level of miR-206 varies. miR-206 was proven to increase with the development of IH, especially in the involuted phased,24 fold higher than that in the involuting phase, suggesting the miR-206 as an post-transcriptional regulating factor in IH.The signaling pathway of Transforming growth factor (TGF?) is playing an important role in the regulation of tumor incidence and vascular development, as well as the whole phases of IH, which is a benign tumor of endothelial cells. It has been demonstrated that TGF? is lowly expressed in early proliferating phase and late involuting phase, but highly expressed in middle and late proliferating phase and early involuting phase, indicating that TGF? may be involved in the down-regulation of endothelial cell proliferation, to promote the involuting of IH. However, the mechanism to regulate TGF? remains unknown.Based on the above points, we focus on miR-206 and TGF?, investigate their relationship, as well as their effect on the infantile hemangioma endothelial cells (HemEC), to address the mechanism of involuting of IH.Part 1 The isolation, culture and identification of Human umbilical vein endothelial cell and Infantile hemangioma endothelial cellsObjectiveThe isolation, purification, culture and identification of Human umbilical vein endothelial cells (HUVEC) and Infantile hemangioma endothelial cells (HemEC).MethodThe specimen of human umbilical cord (1cm) was collected, followed by the adherence of human umbilical vein to flask, and culture for 10 days. The morphology of cells were observed under the microscope, and the new cells, crawled from the original tissue and in the shape of paver stone, were picked out and seeded into a new flask followed by independent culture for 3-4 days. The new cells were proliferated, sub-cultured, and saved.The specimen of hemangioma was collected after the surgery, followed by primary culture of the tissue of hemangioma for 6-7 days. The new crawled cells were then collected, cultured and sub-cultured. When cell number was enough, cells were purified by CD31 immunomagnetic beads to separate the CD31 positive cells from infantile hemangioma.Morphology of two cell lines were compared, and the cell growth curve was measured, Cell obtained from tissue culture were identified by the following experiments, including tube formation experiments of Vascular endothelial cells on Matrigel, of Acetyl low density lipoprotein intake analysis of endothelial cells. Additionally, cell immunofluorescence and the membrane antibody measurement were analyzed by flow cytometry.ResultsAfter the tissue culture of human umbilical vein and purification by the morphology under the microscope, flow cytometry results showed that the positive percentage of CD31 of HUVEC reached to 97%, whereas the positive percentage of CD31 of HemEC reached to 93% after the tissue culture of infant hemangioma and purification by immunomagnetic beads. Both cell lines showed positive results in intake experiments, but cell growth curve indicated that HemEC had a stronger cell proliferation ability than HUVEC. Moreover, HemEC had better ability in tube formation than HUVEC, leading to earlier in tube formation and cell accumulation. Cell immunofluorescence results demonstrated both cell lines were positive in CD31, CD105, VEGF, GLUT1 and vWF.ConclusionAfter collecting the human umbilical cord (1cm), tissue culture, and selection by cell morphology, HUVEC were obtained and identified. By tissue culture of infant hemangioma and purification by immunomagnetic beads, HemEC were collected and identified. These methods were easy and efficient, and the identified cells were highly purified with good condition and enough amount. HemEC showed higher cell proliferation ability and faster tube formation ability than HUVEC.Part 2 The influence of miR-206 on cell proliferation of Infantile hemangioma endothelial cellsObjectiveIdentify the effect of miR-206 on cell proliferation and apoptosis in HemEC.MethodHUVEC and HemEC were transfected with miR-206 mimics, miR-206 mimics NC, miR-206 inhibitor or miR-206 inhibitor NC.8 hours after transfection, cells were observed under inverted fluorescence microscope to analyze the transfection efficiency. With the utilization of RT-PCR 24 hours later, the content of miRNA-206 was evulated by Taqman probe and SYGR Green. CCK-8 kit was used to identify the cell proliferation ability of HemEC. With the fluorescence staining reagent of PI and AnnexinV-FITC/PI, the effect of miR-206 on cell cycle and apoptosis on HemEC were measured by flow cytometry.ResultsThe fluorescence analysis post-transfection showed that miR-206 mimics was successfully transfected into HemEC with high transfection efficiency.TaqMan probes results showed that miR-206 mimics can significantly up-regulate the level of miR-206 in HUVEC and HemEC, as the content of miR-206 in miR-206 mimics group was 50000 fold and 7500 fold higher than that in miR-206 NC group respectively(P<0.001).SYBR Green results also showed that miR-206 mimics can significantly up-regulate the level of miR-206 in HUVEC and HemEC, as the content of miR-206 in miR-206 mimics group was 70000 fold and 100000 fold higher than that in miR-206 NC group(P<0.001).CCK-8 kit results showed that the cell proliferation ability of HemEC was significantly reduced post-transfection of miR-206(P<0.05), and the inhibitory ability of miR-206 reached to the peak 24 hours after transfection(P<0.01).Cell cycle analyais showed that the percentage of S phase was reduced, accompanied increase in G2 phase after the treatment of miR-206.Cell apoptosis results showed that miR-206 was not able to induce apoptosis to HemEC.ConclusionThe expression level of miR-206 of HemEC was significantly increased by the administration of miR-206 mimics, resulting in the inhibition of cell proliferation and cell cycle arrest in G2, rather than apoptosis.Part 3 The mechanism of miR-206 to inhibit the cell growth of Infantile hemangioma endothelial cellsObjectiveInvestigate the mechanism of miR-206 to inhibit the cell growth of infantile hemangioma endothelial cells.MethodsWith the utilization of KEGG data, the potential pathways regulated by miR-206 were investigated. The content of TGF?1, TGF?2, TGF?3, BMP2, BMP4 and BMP7 in the medium of miR-206 transfected HemEC were measured by Elisa. Moreover, the mRNA level of smad2, smad4 and smad7 in miR-206 transfected HemEC were analyzed by RT-PCR with the fluorescence staining reagent of SYBR Green. The expression of smad2, pp-smad2, smad4 and smad7 in miR-206 transfected HemEC were measured by Western Blot. The effect of TGF?1 on cell proliferation of HemEC was measured by CCK-8 kit. Flow cytometry was utilized by evaluate the effect on cell cycle and apoptosis by TGF?1 in HemEC.ResultsAccording to the signal pathway analysis results, we chose TGF-beta signaling pathway as the most potential pathway that miR-206 can target and regulate. Elisa results indicated that the secreted TGF?1 in miR-206 mimics transfected HemEC was significantly higher than that cells in miR-206 NC group (P<0.001), and the secretion of TGF?1 was significantly reduced by miR-206 inhibitor (P<0.001).RT-PCT results demonstrated that the mRNA of smad7 was significantly increased by miR-206 mimics (P<0.01), but reduced by miR-206 inhibitor (P<0.05).Western Blot results showed that smad7 protein was highly expressed in miR-206 mimics transfected HemEC, but lowly expressed in miR-206 inhibitor transfected HemEC.CCK-8 analysis results showed that TGF?1 can inhibit proliferation of HemEC in a dose-and time-dependent manner (P<0.01, P<0.05).Cell cycle results showed that TGF?1 can lead to the increase in S phase of HemEC.Cell apoptosis results demonstrated that TGF?1 was not able to induce apoptosis in various concentrations.ConclusionTGF?1 was increased by miR-206 in HemEC, followed by up-regulation of smad7. TGF?1 can inhibit the proliferation of HemEC, and the inhibitory ability was gradually increased with the increase of concentration. Cell cycle of HemEC was arrested in S phase after the treatment of TGF?1 (5ng/mL), without significant influence on cell apoptosis.Based on the above, we can draw the conclusion that miR-206 is an important regulatory factor in the development of IH, since it can up-regulate TGF?1 and smad7 in the down streaming of TGF? pathway, therefore result in the inhibition of cell proliferation of HemEC.