| Chapter ?:the effect of propranolol on the biological behavior of HUVECs and the expression of VEGF,VEGF-A,FLT-1,FLT-2 and FOXF-1PrefaceWith a prevalence of 4-5%,infantile hemangioma are the most common benign soft tissue tumors of infancy.The main pathological features of hemangioma are excessive formation of blood vessels and subsequent spontaneous degeneration of blood vessels.It usually arises in the first few weeks of life,then into a fast-growing period of about 8 to 12 months,followed by a slow spontaneous involution phase.Most infantile hemangiomas do not require treatment and regress spontaneously.However,about 10-15%of high-risk hemangiomas require treatment,especially at specific sites.Failure to intervene actively can lead to serious complications,such as obstruction,ulcers,disfigurement,destruction of the anatomical structure or physiological function of important organs,etc.For these high-risk infantile hemangiomas,it should be actively intervened to prevent and reduce the occurrence of complications.Infantile haemangioma is the result of dysregulation of both vasculogenesis and angiogenesis.There are many hypotheses about the pathogenesis of hemangioma,but the triggers that initiate development of infantile haemangioma are still a matter of debate.A large number of studies have shown that the occurrence of hemangioma is related to the overexpression of vascular endothelial growth factor(VEGF).In order to explore the effective and safe treatment for infantile hemangioma,researchers and clinicians at home and abroad have been making great efforts.Corticosteroids have been considered as the first choice for the treatment of infantile hemangioma.In 2008,doctors L e aute-Labr e ze and his colleague of Bordeaux Childrenundefineds Hospital in France found that oral propranolol could lighten the color and reduce the volume of hemangioma in infants.Subsequently,a large number of cases about propranolol treatment of infantile hemangioma have been published at home and abroad.Propranolol has been proved to be effective in the treatment of hemangioma in a wide range of clinical cases and has replaced corticosteroids as the first-line drug in the treatment of infantile hemangioma.Propranolol is non-selective ?-adrenoceptor blocker,which mainly treats cardiovascular diseases such as hypertension,arrhythmias,angina pectoris and so on.However,how propranolol can effectively inhibit the proliferation of hemangioma has not been widely accepted so far,and the specific mechanism of propranolol needs to be further studied.In previous studies,our team found that FOXF1 was specifically expressed in hemangioma tissues of infants and young children,suggesting that FOXF1 may play an important role in the regulation of pathological neovascularization.In addition,it has been reported that FOXF1 is an indispensable transcription factor in the formation of embryonic blood vessels by VEGF signaling pathwayIt is suggested that the mechanism of propranolol in the effective treatment of hemangioma may be related to the signal pathway related to the inhibition of angiogenesis,and one of the most important signal pathways related to the formation of hemangioma is the VEGF signal pathway.With the deepening of the understanding of related molecules,it is understood that FOXF1 may play an important role in the regulation of angiogenesis,and it has even been reported that FOXF1 is a necessary transcription factor of the VEGF signaling pathway in the process of angiogenesis.Based on the above findings,we speculate whether propranolol has an effect on FOXF1.Does it affect the expression of FOXF1 and then the expression of VEGFsignal-related molecules so as to achieve the effect of treatment of infantile hemangioma?There is still no research on the correlation between FOXF1 and VEGF signal pathway in hemangioma,so it is necessary to explore it experimentally.Objective:To observe the effects of propranolol on proliferation,apoptosis and migration of human umbilical vein endothelial cells(HUVECs),and to investigate the effects of propranolol on the expression of VEGF,VEGF-A,FLT-1,FLT-2 and FOXF-1 in HUVECs in order to explore the underlying mechanism of propranolol in IH in vitro.Methods:HUVECs were treated with propranolol(0,0.15,1.5 and 15 ? M/L)at different concentrations.Cell viability,migration,and apoptosis were examined using Cell Counting Kit-8,transwell assay,and flow cytometry analysis,respectively.In addition,the expressions and concentrations of VEGF,VEGF-A,FLT1,FLT2,and FOXF1 were assessed using real-time polymerase chain reaction,Western blot assay,and enzyme-linked immunosorbent assay.(Elisa).Results:Cell viability was reduced at 0.15,1.5,and 15? M/L concentration groups compared with that at the control group(P<0.05,P<0.01,or P<0.001,).The greatest decrease in HUVEC viability was observed at 15? M/L group.Therefore,we chose 15,M/L of propranolol for the following experiments.Subsequently,flow cytometry analysis showed that HUVECs apoptosis was gradually increased with cell incubation in the propranolol group,and its largest addition on the 6th day with nearly 26%(P<0.05,P<0.01,or P<0.001).Conversely,cell migration was decreased by propranolol treatment(P<0.05 or P<0.01).Hence,we found that propranolol could increase cell apoptosis and reduce viability,migration in HUVECs.VEGF and VEGF-A expressions and concentrations were significantly decreased on the 2,3,and 6 day group compared with the control group(P<0.05 orP<0.01).Similarly,the messenger RNA(mRNA)and protein levels of FLT1,FLT2,and FOXF1 were clearly downregulated after day 3(P<0.05 orP<0.01).Conclusion:Propranolol can inhibit the proliferation of human umbilical vein endothelial cells,promote apoptosis and decrease the migration ability of HUVECs.Propranolol inhibited the expression of VEGF,VEGF-A,FLT-1,FLT-2 and FOXF-1 in a concentration-dependent manner,suggesting that the inhibitory effect of propranolol on hemangioma was related to the VEGF signaling pathway mediated by FOXF-1.Chapter ?:Propranolol regulates the biological behavior of HUVECs and the expression of related molecules by inhibiting the expression of miR-4295.PrefaceMicroRNA is a revolutionary discovery in the field of biology.It introduces miRNAs as a key factor in regulating gene expression and controlling many physiological processes,such as cell development,proliferation,differentiation and apoptosis.Its research will have a profound impact on the development of post-transcriptional gene regulation.The significance of miRNAs in cancer was first confirmed by observing the down-regulation of mir-15a and miR-16-1 in most patients with chronic lymphoblastic leukemia.Since then,there has been a great deal of evidence to support the involvement of miRNAs in different stages of tumor formation and development,suggesting that miRNAs is a promising biomarker for tumor diagnosis and prognosis and a potential therapeutic target.Moreover,miRNAs has been shown to affect angiogenesis,suggesting that miRNAs is important in cancer and other angiogenic diseases.Recent studies have found that miRNA-4295 is a new neoplastic miRNA,but its expression and biological function in infantile hemangioma have not been reported.In view of this,we hypothesized that miRNA-4295 was involved in the treatment of IH with propranolol,and further observed the effect of propranolol on HUVEC in vitro under the action of miRNA-4295.It provides a new theoretical basis for the treatment mechanism of infantile hemangioma.Objective:To compare and analyze the expression of miR-4295 mRNA in infantile hemangioma and observe the effect of up-regulated miR-4295 expression on the biological behavior of HUVEC induced by propranolol by miR-4295 mimic.In order to further explore the underlying mechanism of propranolol in the treatment of hemangioma,provide a new target for the treatment of hemangioma and predict the prognosis of indicators.Methods:Quantitative reverse transcription PCR was used to detect the expression of miR-4295 mRNA in 24 cases of hemangioma tissues and its matched normal tissues.At the same time,the expression of miR-4295 mRNA in HUVECs treated with propranolol was detected also.MiR-4295 mimic was used to up-regulate the expression of miR-4295 mRNA.Cck-8,flow cytometry and Transwell assay were used to observe the effects of propranolol on cell proliferation,apoptosis and cell migration induced by miR-4295.The expression and concentration of VEGF,VEGF-A,FLT-1,FLT-2 and FOXF-1 were detected by quantitative reverse transcription PCR,Westernblot and Elisa.Results:The results showed that miR-4295 expression was significantly increased in IH tissues compared with that in the correspondingly adjacent tissues(P<0.01).MiR-4295 expression was downregulated on thel,2,3,and 6-day group compared with the control group(P<0.05 or P<0.01).The expression of HUVECs,miR-4295 gene was up-regulated by miR-4295 mimic transfection(P<0.01).Compared with propranolol + NC group,the proliferation rate,migration ability and apoptosis rate of propranolol + miR-4295 mimic group increased significantly(P<0.001).On the 3rd and 6th day of culture,the relative expression levels and concentrations of VEGF and VEGF-A mRNA in propranolol and propranolol +NC groups were significantly lower than those in the blank control group(control group).The relative expression level and concentration of VEGF and VEGF-A mRNA in propranolol + miR-4295 mimic group were significantly lower than those in blank control group,the difference was statistically significant,but the relative expression level of VEGF mRNA in propranolol group and propranolol NC group was significantly higher than that in propranolol group and propranolol NC group.The difference is statistically significant.In addition,the expression trend of FLT-1,FLT-2 and FOXF-1 was consistent with that of VEGF.Conclusion:The expression of miR-4295 in IH tissue is significantly increased,showing the activity of tumorigenic miR-4295.Up-regulation of miR-4295 expression could reduce the effect of propranolol on the biological behavior and related molecules of HUVECs,suggesting that propranolol inhibits the growth of hemangioma through miR-4295-mediated inhibition of VEGF signaling pathway-related molecules. |
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