The Effect And Mechanism Of MiR-29a On The Biological Activity Of Infantile Hemangioma Endothelial Cells

Infantile hemangioma is a common benign tumor of endothelial cells in infancy.The clinical features of the tumor are the proliferating phase,the involuting phase and the involuted phase.The onset of the disease occurred in 3-6 months after birth and rapidly entered the proliferating phase.In the proliferating phase,because of the rapid increase of blood flow in the hemangioma and the rapid proliferation of the tumor,the tumor volume can be increased rapidly,resulting in oppression of the surrounding tissues and organs symptoms.In involuting phase,the tumor body ceases to grow and spontaneously degenerates.Most of the children can remain unobvious skin changes.However,some infantile hemangiomas can cause serious complications.At present,the treatment of infantile hemangioma has been gradually unified.This is mainly because its pathogenesis remains unclear.It is generally accepted that apoptosis of endothelial cells is an important factor in the regression of infantile hemangioma in involuting phase.Micro RNA is a kind of small molecule RNA in nature.This kind of RNA has important significance for maintaining the stability of the internal environment and regulating the differentiation of cells and tissues.This kind of RNA does not encode proteins,but it can inhibit the formation and the expression of m RNA,which can inhibit the formation and function of target proteins.At present,there are many literatures that miRNA has an important relationship with apoptosis.In prostate cancer[1],miRNA-34 can down-regulate the expression of Bcl-2 protein,inhibit cell proliferation and promote cell apoptosis.In malignant glioma[2],miRNA-153 can down-regulate Bcl-2 protein and promote cell apoptosis.In rectal cancer,miRNA-195[3] manifest low expression.overexpression of miRNA-195 can induce apoptosis of rectal cancer cells.through micro RNA chip,We previously found mi R-29a-3p is obviously different between the proliferation and regression stage.in many literatures,mi R-29 family members also have been found to inhibit the development of tumor through the action of apoptosis related factors.In malignant hepatocellular carcinoma cell lines[4],mi R-29 a can regulate the expression of anti-apoptotic protein Mcl-1 and Bcl-2,which leads to apoptosis of hepatocell?lar carcinoma cells.The expression of mi R-29 a was down-regulated in chronic lymphocytic leukemia,lung cancer and breast cancer.Up-regulation of mi R-29 a can activate the expression of P53,which inhibit the proliferation of CDC42,Tcl-1 and other related genes,to promote cell apoptosis.In this study,we investigated the effect of mi R-29 a on the biological activity of infantile hemangioma and its role in the regression of infantile hemangiomas.In order to understand the mechanism of spontaneous regression of infantile hemangioma.Part one:Culture and identification of infantile hemangioma endothelial cells.Method Specimens of fresh proliferative infantile hemangiomas were collected under aseptic conditions and transferred to the laboratory within 6 hours.Cells were cultured by tissue explants adherent method.After several weeks of culture,sufficient amount of cells were collected,CD31 positive cells were recollected by CD31 Macs.The cells were examined by flow cytometry of CD31-FITC to ensure the positive cell rate was above 90%.The growth curve was drawn and Tube forming experiment were made.by CD31,v WF,Glut-1,VEGFA cell surface markers immunofluorescence detection,we identified the cells for the infant hemangioma endothelial cells.Result Through the culture of proliferating infantile hemangioma tissue block,cells collection and CD31 immunomagnetic separation,we obtained a sufficient amount of cells.By flow cytometry after the CD31-FITC test,the positive rate of the cells was almost 96%.by CD31,v WF,Glut-1,VEGFAcell surface markers immunofluorescence detection,the antigen above were all strong positive.Conclusion We collected specimens of proliferating infantile hemangiomas,and cultured in vitro.The cells were confirmed by morphology and immunofluorescence antibody test.then we could make sure those cells to be the endothelial cells of infantile hemangioma we required.Part Two:The effect of mi R-29 a on the biological activity of hemangioma endothelial cells.Method To construct lentiviral vector Lv-hsa-mi R-29 a and Ubi-MCS-SV40-EGFP-IRESpuromycin in vitro.Lv-hsa-mi R-29 a and Ubi-MCS-SV40-EGFP-IRES-puromycin were transfected into endothelial cells.The expression of mi R-29 a was observed by fluorescence microscopy and q RT-PCR.After overexpression of mi R-29 a,MTT assay was used to detect the proliferation of endothelial cells.Flow cytometry was used to detect the cell cycle and apoptosis,cell invasion ability was detected by Transwell.Result Under the fluorescence microscope,the expression of FITC was about 95%.q RT-PCR results showed that mi R-29 a gene expression abundance was 2.643 times higher than the negative control group.After transfection of 72 h,the apoptosis rate of infantile hemangioma endothelial cells was over 10.82%,which was significantly higher than the other groups.The expressions of cell cycle?invasion and proliferation of mi R-29 a were not statistically significant.Conclusion The stable and high expression of mi R-29 a cell model could be constructed by the transfection of Lv-hsa-mi R-29 a.After the high expression of mi R-29 a,the early apoptosis of endothelial cells in infantile hemangioma was significantly changed,but there was no significant effect on cell cycle?invasion and proliferation.Part Three:Study on the mechanism of mi R-29 a in infantile hemangioma.Method By miRNA gene microarray and Target Scan database for comparison,we selected some factors as the target gene to examine.The dual luciferase assay showed that Mcl-1 could bind to mi R-29 a directly.Through the Western-blot detection method,we further verified that Mcl-1,VEGFA and Bcl-2 protein levels were significantly changed in mi R-29 a cells of infantile hemangioma.,we choosed the Stat3 and Akt3 to further study whether mi R-29 a could regulate the expression of Mcl-1 by regulating Mcl-1 upstream molecules.Result Through the miRNA chip screening and database analysis,we selected some factors to examine.Mcl-1 was identified as a possible downstream target protein by dual luciferase assay.The Mcl-1,VEGFA and Bcl-2 protein levels were confirmed by Western-blot assay.At the same time,the Stat3 and Akt3 protein levels were not significantly changed by Western-blot assay.Conclusion The expression of Mcl-1,VEGFA and Bcl-2 could be inhibited by up-regulating mi R-29 a in the endothelial cells of infantile hemangioma.Dual luciferase assay revealed that Mcl-1 was a direct interaction of mi R-29 a.Western-blot detection showed that the expression of Stat3 and Ark3 was not correlated with Mcl-1,and could not inhibit or activate the expression of Stat3 and Ark3.