The Expression And Regulation Of Ras Signaling Pathway In Hemangioma Endothelial Cells

Objective:To observe the expression of key signaling molecules of Ras signaling pathway in childhood hemangioma tissues at different stages,regulate the expression of Ras signaling pathway in human hemangioma endothelial cells cultured in vitro,and explore the effect of Ras signaling pathway on hemangioma vascular endothelial cells and the mechanism.Methods:1.Tissue samples from the department of pediatric surgery of the Affiliated Hospital of Zunyi Medical University that were surgically removed and pathologically diagnosed as capillary hemangioma between 2005 and 2017 were collected,and reclassified and divided according to Mulliken classification and ki-67 expression,then 30proliferative hemangioma and 30 degenerative hemangioma samples were extracted,respectively.The expression of Ras,Raf-1,TSC2,mTOR,p70s6K and 4E-BP1 in proliferative and degenerative hemangiomas was detected by immunohistochemistry and compared.2.Human hemangioma endothelial cells were cultured in vitro?provided by BeNa pioneering biotechnology co.,LTD.,product name:HemECs,article number:BNCC340867?and subcultured to the third generation.Logarithmic growth phase cells were taken and treated with serum starvation for 24 hours.Grouping and treatment:4mL medium containing 50?mol/L of propranolol was added to the propranolol group,4mL medium containing 0.1ng/L of estradiol was added to the estradiol group,and 4mL complete medium was added to the control group.Cells were further incubated for 24 hours to collect cells.Western blot was used to detect the expressions of Ras,Raf-1,TSC2,mTOR,p70s6K,and 4E-BP1 in hemangioma vascular endothelial cells,and RT-PCR was used to detect the expressions of Ras,Raf-1,TSC2,mTOR,p70s6K and 4E-BP1 mRNA.Meanwhile,the cell cycle and apoptosis rate of vascular endothelial cells in hemangioma were detected by flow cytometry.Finally,the relationship between the expression levels of Ras,Raf-1,TSC2,mTOR,p70s6K and 4E-BP1 in each group and the vascular endothelial cell cycle distribution and apoptosis was analyzed.Results:1.In proliferative hemangioma?n=30?and degenerative hemangioma?n=30?,the mean optical density?Mean OD?of TSC2,4E-BP1,p70s6K,Ras,Raf-1 and mTOR are0.176±0.024/0.2373±0.030,0.181±0.027/0.218±0.030,0.159±0.031/0.228±0.032,0.369±0.034/0.337±0.032,0.362±0.041/0.327±0.038,0.531±0.079/0.508±0.055respectively.Mean OD of Ras and Raf-1 in proliferative hemangioma was significantly higher than that in degenerative hemangioma?t=3.665,3.478,P<0.01?,and Mean OD of mTOR was higher than that in degenerative hemangioma?t=2.252,P<0.05?.Mean OD of TSC2,p70s6K and 4E-BP1 in proliferative hemangiomas were significantly lower than that in regressive hemangiomas?t=8.636,8.365,4.709,P<0.01?.2.The total apoptosis rate?%?of hemangioma endothelial cells in the propranolol group,the estradiol group and the control group were15.41±0.75,3.41±0.40 and 4.84±0.45,respectively.Compared with the control group,the apoptosis rate in the propranolol group was significantly increased,while that in the estradiol group was decreased?F=419.30,P<0.01?.3.The cell cycle?%?of hemangioma endothelial cells in the propanolol group,estradiol groupandcontrolgroupwasG₀/G₁?74.53±1.66/50.47±1.05/60.21±1.14?,S?22.35±0.92/42.60±0.74/32.93±1.23?,G₂/M?3.14±1.10/6.92±1.04/6.84±0.36?,Three comparisons,the difference was significant??²=13.77,P<0.01?.Compared with the control group,the cells growth of propranolol group was stagnated in G₀/G₁ phase,and the number of cells in S phase was decreased.In estradiol group,G₀/G₁ phase cells were decreased while S phase cells were significantly increased.4.Compared with the control group,protein expression multiples of TSC2,4E-BP1 and p70s6K in the propranolol group?n=3?were 1.568±0.192,1.534±0.140 and1.690±0.123respectively,which were significantly higher than those in the control group?F=44.42,136.41 and 66.75,P<0.01?.Ras,Raf-1 and mTOR,were 0.585±0.030?0.527±0.040 and 0.430±0.045,respectively,significantly lower than the control group?F=109.96,43.41and726.30,P<0.01?.The protein expression levels of TSC2,4E-BP1 and p70s6K in the estradiol group?n=3?are 0.673±0.067,0.662±0.092 and 0.563±0.077respectively,which were significantly lower than those in the control group?F=44.42,66.75 and 136.41 P<0.01?.Ras,Raf-1 and mTOR,were1.530±0.131,1.534±0.225 and1.548±0.042,respectively,significantly higher than the control group?F=109.96,43.41and 726.30,P<0.01?.5.Compared with the control group,the mRNA expression multiples of TSC2,4E-BP1 and p70s6K in the propranolol group?n=3?were 2.138±0.243,3.139±0.331 and 2.297±0.099and respectively,which were significantly higher than those in the control group?F=246.34,74.92 and 38.10,P<0.01?.The mRNA of Ras,Raf-1 and mTOR,were 0.555±0.076,0.339±0.068 and 0.403±0.103,respectively,significantly lower than the control group?F=47.42,96.34 and 36.03,P<0.01?.The mRNA expression multiples of TSC2,4E-BP1and p70s6K in the estradiol group?n=3?were 0.439±0.102,0.481±0.086 and 0.614±0.060respectively,which were significantly lower than those in the control group?F=246.34,74.92,and 38.10,P<0.01?.The mRNA of Ras,Raf-1 and mTOR are 1.849±0.214,1.604±0.094 and 1.434±0.127 respectively,significantly higher than the control group?F=47.42,96.34 and 36.03,P<0.01?.Conclusions:1.The expression of key signaling molecules of Ras signaling pathway was found in vascular endothelial cells of hemangioma in children.The expression of Ras,Raf-1 and mTOR in the proliferative phase of hemangioma was higher than that in the degenerative phase,while the expression of TSC2,p70s6K and 4E-BP1 was lower than that in the degenerative phase.2.Propranolol negatively regulates Ras signaling pathway,up-regulates TSC2,inhibits mTOR/p70s6k signaling pathway,and stasis the cell cycle of cultured human hemangioma endothelial cells in G₀/G₁ phase,and induces cell apoptosis.Estrogen was the opposite.