The Mechanism Of Proliferation Of Hemangioma Cells Induced By MEHP Via Hippo YAP Signal

Research BackgroundHemangioma is a kind of tumor formed by the proliferation of vascular endothelial cells.75%~80%of hemangioma patients are female.It is considered to be the most common benign tumor,in addition,hemangioma patients do not need any treatment because they come from hair regression for many years.However,10%of hemangiomas will grow rapidly or even become life-threatening.One stage of rapid proliferation is the main feature of hemangioma.However,the cause of rapid proliferation is not well explained.Therefore,there is an urgent need to study more risk factors related to the progression of hemangioma.Phthalates are used in various food packaging,household products,medical devices,pharmaceutical formulations and industrial plastics.About 6 million tons of phthalates are produced every year in the world.Humans are exposed to phthalates by skin,inhalation and absorption.Dioctyl phthalate(DEHP)is one of the most widely used phthalates,which can make medical devices more flexible.People may be exposed to high levels of DEHP through plastic medical devices.DEHP can be easily leached from plastics as a highly hydrophobic material after contact with serum,blood and other lipophilic fluids.Once DEHP enters the human body,it is rapidly metabolized into its active metabolite,mono(2-ethylhexyl)phthalate(MEHP),which is then preferentially absorbed.Recently,studies have shown that DEHP and MEHP can promote tumor progression by triggering cell proliferation,migration and invasion.Di(2-ethylhexyl)phthalate[di(2-ethylhexyl)phalate,DEHP]is a widely used plasticizer,which can be metabolized into mono(2-ethylhexyl)phthalate,MEHP].Inhalation is an important exposure pathway for two phthalates.Their effects on lung include inflammation,enlarged airspace and changes in cell differentiation.It has been shown that alveolar epithelial cells are targets of phthalates.However,the potential role and mechanism of MEHP in Ha development have not been well explained.DEHP is widely used in the production of PVC medical materials.It can be metabolized into MEHP,which is considered to be the most toxic metabolite of MEHP to human body.Recently,studies have shown that DEHP and MEHP can promote tumor progression by triggering cell proliferation,migration and invasion.MEHP is a kind of chemical substance that destroys endocrine,and has been widely used in the production of plastic containers for food and beverage,adhesives,paints and dental sealants.More and more evidences show that MEHP has negative effects on human health.MEHP has excellent stability.So far,MEHP has been widely used in industrial products all over the world,including detergent,phenolic resin,electroplating solvent and thermal paper.Epidemiological studies have shown that MEHP can be widely detected in human urine and blood.For example,81%of urine samples from the United States and Asia contain detectable MEHP,indicating that the biosafety of MEHP is an urgent public health issue.More and more evidences show that MEHP has strong estrogen reaction in vivo and in vitro,which leads to the destruction of endocrine function.New reports show that MEHP can trigger the proliferation,migration and invasion of breast cancer cells.However,the potential effect of MEHP on the progress of HA has not been well explained,so it has become my current research focus.1、Mechanism of in vitro proliferation and migration of HA cells by mono(2-ethylhexyl)phthalate(MEHP)Objective:To investigate the mechanism of proliferation and migration of HA cells in vitro by mono(2-ethylhexyl)phthalate(MEHP).Methods:Hemangioendothelial cells were used in our experiments and stored in modified Eagle’s medium containing15%fetal calf serum and penicillin/streptomycin(100μg/ml).The medium was changed daily during cell growth,and when the cells reached 80-90%confluence,the cell suspension was subcultured at a ratio of 1:3.The cultured cells were divided into two groups according to the experimental requirements:control group(normal cultured cell line as experimental control),MEHP induction group(cell 5 x 10~4cells/well was inoculated into 6-well plate.After 8 hours of incubation The cells were treated with 10 nM MEHP and cultured for 24 hours).Real-time analysis of mRNA expression of YAP and IRF-1 by reverse transcription quantitative PCR(RT-qPCR);Western blot analysis of related protein expression in cells;Scratch invasion assay to detect cell migration;Detection of cells by apoptosis detection kit Apoptosis and relative viability;MTT assay compared cell proliferation.Results:The expression of YAP and IRF-1 mRNA in MEHP-induced group was higher than that in control group(P<0.05).MEHP can increase the transcription of YAP by IRF-1.The expression of E-cadherin was higher in the control group than in the MEHP-induced group(P<0.05).The expression of MMP9 protein in the MEHP-induced group was higher than that in the control group(P<0.05).It is indicated that MEHP promotes the process of cell epithelial-mesenchymal transition.The number of cell migration in the control group was lower than that in the MEHP-induced group(P<0.05),and the number of cells in the MEHP-induced group was higher than that in the control group(P<0.05).This indicates that MEHP can induce cell migration and invasion.The cells were tested for apoptosis at 24 hours and 72 hours according to the manufacturer’s protocol using an apoptosis assay kit.The apoptosis rate of the control group was higher than that of the MEHP-induced group at 24 hours(P<0.05),and the 72 hours of MEHP.The apoptosis rate of the induction group was lower than that of the control group(P<0.01).At the10th hour after different cultures,there was no difference in cell proliferation between the control group and the MEHP-induced group(P>0.05).At 24 hours and 36 hours,the number of cells in the MEHP-induced group was higher than that in the control group(P<0.05).On the third day of the test,the control group was found to have a lower cell proliferation than the MEHP-induced group(P<0.01).Proliferative capacity is enhanced in the presence of cell exposure and MEHP.Conclusion:MEHP can promote the proliferation of HA cells by up-regulating YAP expression and promote the expression of factors E-cadherin and MMP9 related to epithelial-mesenchymal transition.2、Blocking MEHP-triggered HA cell proliferation by inhibiting YAPObjective:To investigate the mechanism of blocking the proliferation of HA cells triggered by MEHP by inhibiting YAP.Methods:HemEC was isolated from 7infantile hemangioma patients admitted to our hospital.The cells were subcultured no more than five times before application to the experiment.HemEC was cultured under standard conditions in alpha-MEM supplemented with 10%heat-inactivated fetal bovine serum,0.25%fungal zone and 1%gentamicin.According to the experimental requirements,the cells cultured in the Yuan Dynasty were cultured and treated differently:the control group(the normal primary cultured HemEC cells were used for culture counting and observation),and the MEHP treatment group(the control cells were given at the beginning of the culture).The concentration of MEHP induced treatment,and then continued to culture),YAP silencing group(transfected cells with siRNA against YAP,inhibited YAP expression).The effect of MEHP intervention and YAP expression restriction on cells in different time periods was determined by MTT cell proliferation assay;apoptosis assay kit and 2-methoxy-4-nitro-5-sulfophenyl group were used to detect each group of cells.Apoptosis and viability;cell migration activity was measured by cell scratching plane migration assay.Western blotting,RNA extraction and semi-quantitative RNA analysis,enzyme-linked immunosorbent assay(ELISA)were used to detect the expression of YAP,CTGF and ANKRD1 in each group.Results:At the 12th hour,the proliferation of MEHP intervention group was higher than that of YAP silence group(P<0.05),there was no difference between the control group and the MEHP intervention group(P>0.05).At 24 hours,the proliferation of the control group and the MEHP intervention group was higher than that of the YAP-suppressed group(P<0.05).The cell proliferation of the MEHP intervention group was higher than that of the control group and the YAP-suppressed group(P<0.05).The proliferation of the YAP-suppressed group was lower than that of the control group(P<0.05).The cells were induced to proliferate after MEHP treatment,and the proliferation of the cells was inhibited after YAP expression was inhibited.The apoptosis of MEHP intervention group was lower than that of control group and YAP silence group(P<0.05).The MEHP intervention group was compared with YAP silence group(P<0.01),indicating that MEHP inhibited apoptosis or decreased YAP expression.Die.The cell viability of the control group and the YAP-suppressed group was lower than that of the MEHP-treated group(P<0.05),and the cell viability of the MEHP-treated group was higher than that of the YAP-suppressed group(P<0.01).The migration of MEHP intervention group was higher than that of control group and YAP silence group(P<0.05),and the number of cell migration in YAP-suppressed group was lower than that of control group(P<0.05).The number of cell migration in the YAP-suppressed group was lower than that in the MEHP-treated group(P<0.01).The expressions of YAP,CTGF and ANKRD1 in MEHP intervention group were significantly higher than those in control group(P<0.05).The expression of YAP,CTGF and ANKRD1 protein in YAP-suppressed group was lower than that in control group(P<0.05).The expressions of YAP,CTGF and ANKRD1 mRNA in the MEHP intervention group were higher than those in the control group and the YAP-suppressed group(P<0.05).The expressions of YAP,CTGF and ANKRD1 mRNA in the control group were higher than those in the YAP-suppressed group(P<0.05).This indicates that the expression of target gene CTGF and ANKRD1 is also inhibited after YAP expression is inhibited.The levels of YAP,CTGF and ANKRD1in the MEHP intervention group were higher than those in the control group and the YAP-suppressed group(P<0.05).The levels of YAP,CTGF and ANKRD1 in the control group were higher than those in the YAP-suppressed group(P<0.05).Conclusion:MEHP can promote the proliferation and migration of HA cells by up-regulating the expression of YAP.YAP can be used as a potential target for the treatment of hemangioma.3、MEHP regulates HA cell activation and function by reducing the phosphorylation level of LATS1(Ser909)in HA cellsObjective:The aim of this study was to investigate the mechanism by which MEHP regulates the activation and function of HA cells by reducing the phosphorylation level of LATS1(Ser909)in HA cells.Methods:From January 2017to May 2019,the Department of Oncology of the Third People’s Hospital of the City was diagnosed as a tissue sample of HA and normal skin tissue.Endothelial cells(HemEC)isolated from proliferative hemangiomas of 3 infants were used in this experiment.According to the experimental requirements,HA patients’tumor samples and normal healthy skin tissue samples were divided into HA group and healthy group for related index analysis.The cultured hemangio cells were divided into a control group(conventional cultured tumor cell line,which was not treated)and a MEHP-treated group(control group was treated with a certain concentration of MEHP for 12 hours on the basis of tumor cell culture).The verteporfin+MEHP treatment group(the MEHP-treated group was based on the YAP-TEAD inhibitor verteporfin)was used to investigate the effect of MEHP induction on HemECs proliferation.RT-qPCR was used to analyze the mRNA expression of LATS1(Ser909)and LATS2(Ser872)in two groups.The proliferation of HemECs in different periods was determined by MTT.The expression of PCNA in the two groups was detected by Western blot.The ROS was detected by flow cytometry.Results:RT-qPCR was used to analyze the mRNA expression of LATS1(Ser909)and LATS2(Ser872)in the two groups.The mRNA expression of LATS1(Ser909)and LATS2(Ser872)in the MEHP-treated group was lower than that in the control group(P<0.05).,indicating that MEHP treatment can reduce the phosphorylation of LATS1/2(Ser909/Ser872).The proliferation of the two groups was determined by MTT at different times.There was no difference between the control group and the MEHP-treated group at the first hour(P>0.05).The proliferation of the MEHP-treated group was higher than that of the control group at the 12th and 36th hours.P<0.05),the proliferation of MEHP group was higher than that of the control group at 72 hours(P<0.01).YAP is the most important transposon of Hippo pathway,which can regulate organ size and tumorigenesis.The expression of YAP in healthy and tumor groups was detected by Western blot.The protein expression of YAP in HA group was higher than that in healthy group(P<0.05)..Western blot analysis showed the expression of PCNA in the two groups.The expression of PCNA in the MEHP-treated group was higher than that in the control group(P<0.05).The ROS content was detected by flow cytometry.The ROS content in the control group was higher than that in the MEHP-treated group,indicating that MEHP treatment can reduce ROS and resist H2O2-induced apoptosis of HemECs(P<0.05).Conclusion:MEHP can increase the protein stability of YAP by reducing the phosphorylation of tumor suppressor kinase LATS1(Ser909),thereby increasing the proliferation and viability of HA cells.