The Effect Of Bisphenol S On The Biological Characteristics Of Malignant Hemangioma Cells By Regulating Basic Fibroblast Growth Factor

Research BackgroundHemangioma is a common benign disease,which is caused by overgrowth of mesoderm vascular tissue.Hemangioma will bring heavy physical and mental burden to patients,especially for patients with skin lesions.Drug therapy and surgery are the main methods of hemangioma treatment.However,25%of hemangioma patients still have symptoms after resection.It is reported that about 75%-80%of hemangioma patients are women.The detailed reasons for women’s superiority are not clear.Estradiol levels in healthy children are reported to be lower than in patients with hemangioma.The level of serum estradiol in proliferative hemangioma was higher than that in involute phase.Laboratory studies have shown that estrogen can promote the proliferation of hemangioma cells in vitro,which may depend on some growth factors and be inhibited by tamoxifen.In addition,estrogen and VEGF have synergistic effect on the proliferation of hemangioma cells.All these data indicate that estrogen signaling has a positive effect on the progression of hemangioma.Vascular endothelial growth factor（VEGF）is a family of six ligands.VEGFA is the main growth factor related to endothelial proliferation,migration and survival.Fibroblast growth factor（FGFs）is composed of 22 molecules,which is related to the structure.According to the new nomenclature,FGF is continuously numbered and basic fibroblast growth factor（bFGF）is called FGF-2.VEGF and bFGF seem to be important for angiogenesis and angiogenesis.However,the morphological differences induced by FGF and VEGF demonstrated the different roles of these growth factors in angiogenesis.As we all know,VEGF is a powerful inducer of vascular permeability,which can cause edema and lead to the formation of high concentration hemangioma.However,these adverse effects have not been reported.FGF induced newly formed blood vessels are usually characterized by functional maturity and increased investment in parietal cells.Bisphenol s can induce epithelial mesenchymal transition（EMT）in hemangioma cells through the induction of snail.Objective:（1）To explore the mechanism of bisphenol s inducing malignant change of hemangioma cells by regulating basic fibroblast growth factor.（2）To explore the mechanism of BPS increasing the proliferation and migration of HA cells by increasing the expression of basic fibroblast growth factor（bFGF）in HA cells.（3）To explore the molecular mechanism of BPS increasing bFGF expression by activating and increasing HIF-1α.（4）To explore the mechanism of BPS triggering the malignant development of HA cells by increasing the expression of bFGF by up-regulating HIF-1αand down-regulating miR-195.Methods:（1）The primary ha derived endothelial cells（HDEC）and CRL‐2586 cells were cultured in DMEM medium containing 10%FBS,and the medium containing the same concentration（less than 0.5%）DMSO（non-toxic effect on cells）was used as the control.Cell proliferation test,cell community formation test,cell cycle analysis,real-time RT-PCR analysis,Western blot analysis,enzyme-linked immunosorbent assay,promoter activity analysis.（2）Hemangio-derived endothelial cells（HDEC）were used in the study,and the cells were stored in 10%endothelial basal medium（EBM-2）.HDEC was inoculated into RPMI1640,supplemented with10%heat-inactivated fetal bovine serum,streptomycin and penicillin（100 U/mL）,and cultured at 37℃in a humidified atmosphere with 5%CO₂.According to the experimental requirements,the cells were divided into the following groups:control group（normally cultured cell line as the experimental control）,BPS treatment group（using BPS treated cells at a concentration of 80μM）,miR-424 overexpression group（100 pmol miR-424 mimics were added to HDEC for transfection）,inhibitor group（BPS-induced HDEC was treated with BAY 11-7082）.Real-time analysis of bFGF,VEGF,FGFR1,and p65 mRNA by reverse transcription quantitative PCR（RT-qPCR）;analysis of HDEC proliferation activity by MTT assay;analysis of apoptosis by flow cytometry;detection of cell migration by Transwell analysis;detection of miR by western blot analysis Effect of-424 on bFGF/FGFR1 pathway protein expression.（3）The endothelial cell line was derived from hemangioma tissue.RPMI-1640medium（Invitrogen）supplemented with 10 ng/ml epidermal growth factor and 15%fetal bovine serum plus 100 U/ml penicillin and 100μg/ml streptomycin.,Carlsbad,California,USA)cell line cultured in a humid environment containing 5%CO₂ at37℃.According to the experiment,the cultured cells were divided into the following groups:control group（normally cultured cell line as the experimental control）,BPS treatment group（the cells were treated with 100μMBPS and incubated for 24 hours）,and HIF-1αinhibition group（used before BPS treatment Concentration of HIF-1αinhibitor NS398 was incubated with cells）,siRNA knockdown HIF-1αgroup（transfected with antibiotic-free human endothelial serum-free medium using X-tremeGene siRNA transfection reagent,each sample was used 1μg siRNA）,the culture conditions of the cells in each group are the same except for the experimental treatment.Western blot analysis of HIF-1αand bFGF protein expression in cells;MTT assay of cell proliferation;TUNEL analysis of cell apoptosis;reverse transcription quantitative PCR（RT-qPCR）real-time analysis of mRNA expression of STAT3 and Bcl-2.（4）As a substitute for Bisphenol A（BPA）,Bisphenol S（BPS）has been applied to consumer products in our daily lives.With a similar chemical structure to BPA,BPS has also been proven to be an exogenous endocrine disrupting chemical.HA-derived endothelial cells（HDEC）cell line was purchased from Beijing Anbiqi Biotechnology Co.,Ltd.HDEC cell line was supplemented with Dulbecco’s modified Eagle’s medium with 10%heat-inactivated fetal calf serum,100 U/ml penicillin and 100μg/mL streptomycin,and placed in a humid atmosphere at 37°C with 5%CO₂.Lv-HIF-1αoverexpression vector and miR-195 mimic or inhibitor were transfected into HAs cells,and further functional experiments were performed.The protein content of miR-195,bFGF and VEGF after BPS induction was detected by Western blot.Cell proliferation activity was evaluated by MTT assay.Apoptosis index was analyzed by flow cytometry.MiRWalk 2.0 was used for biological information analysis to screen the target genes of miR-195.A pmirGLO reporter vector carrying HIF-1αwild type（WT）3’-UTR or a mutated 3’-UTR was co-transfected with miR-195 mimic or inhibitor into HDEC cells.48 hours after transfection,the luciferase activity was checked using a dual luciferase reporting system.The expressions of miR-195,HIF-1αand bFGF were detected by qRT-PCR.Results:（1）Cell community formation analysis showed that 10nm BPS could also significantly trigger the colonization of HDEC and crl-2586 cells.Cell cycle analysis showed that the number of cells in G0/G1 phase decreased in BPS group.BPS can trigger the proliferation of HA cells by inducing cell cycle transition.BFGF expression was increased in BPS ha cells.BFGF is involved in the proliferation of HA cells induced by bps.BFGF can improve the transcription and mRNA stability of bFGF.NF-κB is involved in BPS induced bFGF expression in Ha cells.Mir-155-5p is involved in the increased bFGF mRNA stability of BPS.（2）The mRNA expression of bFGF,VEGF,and FGFR1 was increased in the BPS-treated group compared with the control group（P<0.05）.This indicates that BPS promotes the transcription and expression of bFGF,VEGF and FGFR1 in hemangio cells.At 24 hours and 36 hours,the BPS treatment group had a higher cell proliferation rate than the control group（P<0.05）,and at 72 hours,the BPS treatment group had a higher cell proliferation rate than the control group（P<0.01）.Compared with the control group,the apoptosis rate of the BPS-treated group was lower（P<0.05）,and the cell viability of the BPS-treated group was higher than that of the control group（P<0.05）.The number of cell migration increased in the BPS-treated group compared with the control group（P<0.05）,and the number of cell invasion increased in the BPS-treated group compared with the control group（P<0.05）,indicating that BPS can promote cell migration and invasion.The p65mRNA expression of the inhibitor group was higher than that of the BPS-treated group（P<0.05）.There was no difference in p65mRNA expression of the BPS-treated group compared to the control group（P>0.05）.The bFGFmRNA expression of the inhibitor group was lower than that of the BPS-treated group and the control group（P<0.05）,The expression of bFGFmRNA was increased in the BPS-treated group compared with the control group（P<0.05）.The overexpression group of miR-424 had lower bFGF/FGFR1 and ERK1/2 protein expression than the BPS treatment group and control group（P<0.05）,and the BPS treatment group had higher bFGF/FGFR1 and ERK1/2 protein expression（P<0.05）.This shows that miR-424 overexpression blocks the bFGF/FGFR1 pathway,and miR-424 overexpression significantly inhibits ERK1/2 phosphorylation.（3）BPS（0μM）compared with BPS（50μM）HIF-1αand bFGF protein expression（P<0.05）,BPS（50μM）compared with BPS（200μM）HIF-1αand bFGF protein expression（P<0.05）.In a dose-dependent manner,HIF-1αand bFGF protein expression was promoted.The expression of HIF-1αand bFGF protein was higher at 72 hours than at48 hours and 24 hours（P<0.05）,and the expression of HIF-1αand bFGF protein at 48hours was higher than that at 24 hours（P<0.05）,indicating that HIF The expression of-1αand bFGF increased at 24 h and continued to increase until 72 h.Cell proliferation was measured by MTT.Cell proliferation increased in the BPS-treated group at 24hours and 48 hours compared with the control group（P<0.05）,and cell proliferation in the control group decreased at 72 hours compared with the BPS-treated group（P<0.01）.Compared with the control group,the expression of HIF-1αand bFGF protein was increased in the BPS treatment group（P<0.05）,and the expression of HIF-1αand bFGF protein was decreased in the HIF-1αinhibition group compared with the BPS treatment group（P<0.05）.The results indicate that the effect of BPS on cells is to increase bFGF protein expression through HIF-1αactivation.Compared with the BPS-treated group,the expression of STAT3 and Bcl-2 mRNA was lower in the control group（P<0.05）,and the expression of STAT3 and Bcl-2 mRNA was decreased in the siRNA knockdown HIF-1αgroup than in the BPS-treated group（P<0.05）.siRNA knockdown of HIF-1αreduces mRNA expression of STAT3 and Bcl-2.These results indicate that HIF-1αplays an important role in the promotion of STAT3 signaling by BPS-mediated hemangio cells.（4）Compared with the control group,the miR-195 protein content and bFGF and VEGF protein were increased in the HA group.The miR-195 protein content and bFGF and VEGF protein content were further increased in the BPS-induced group compared with the HA group（P<0.05）.Compared with the blank control group（3.46±0.52,2.17±0.24）,the miR-195 mimic transfection group（2.53±0.37,8.53±0.85）had a lower cell proliferation activity and an increased apoptosis rate（P<0.05）.Compared with the blank plasmid transfection group（2.37±0.26,5.64±0.16）,the miR-195 inhibitor transfection group（3.89±0.22,2.11±0.23）had a higher cell proliferation activity and apoptotic rate（P<0.05）.miR-195 mimics reduce the luciferase activity of HIF-1αWT 3’-UTR,while miR-195 inhibitors enhance this effect,but they all have an effect on HIF-1αmutant 3’-UTR luciferase Activity has no effect.miR-195 mimics increase the expression of miR-195 and decrease the expression of HIF-1αand bFGF,while miR-195 inhibitors decrease the expression of miR-195 and increase the expression of HIF-1αand bFGF.Conclusion:（1）BPS can induce cell proliferation and cell cycle transformation to induce malignant transformation of HA cells.This process is realized by BPS activating p65 to cause down-regulation of mir-155-5p and increase the expression of bFGF.（2）BPS can induce cell proliferation and trigger the occurrence of tumors in HA cells.This process is completed by activating ERK1/2 and the bFGF/FGFR1 signaling pathway,upregulating the expression of bFGF,and promoting the proliferation and migration of HA cells.（3）BPS promotes the expression of bFGF in hemangio cells by activating and increasing hypoxia-inducible factor（HIF）-1α,and induces the proliferation and development of hemangio cells.（4）miR-195 targets HIF-1α,regulates the expression of bFGF and VEGF,promotes the proliferation of BPS-induced HA cells and inhibits their apoptosis,and inhibits the development of HA in the skin.And miR-195 up-regulation and HIF-1αdown-regulation provide potential therapeutic targets for HAs.As the prenatal period is sensitive to BPS exposure,we recommend that pregnant women and babies take special care to avoid exposure to BPS（for example,reduce the use of plastic products,PCP,canned food,and skin contact with thermal paper）.