Mechanism Of ROS Regulating EGFR/MYC Axis By Activating AKT To Promote The Proliferation And Migration Of Esophageal Squamous Cell Carcinoma Cells

BackgroundEsophageal squamous cell carcinoma(ESCC),the most common trpe of esophagus carcinoma(EC)in China,has the poor prognosis though a lot of progress haa been made in diagnosis and treatment.Therefored,it’s essential to understand the mechanism of occurrence and development of ESCC by experiments.Studies have shown that hydrogen peroxide(H2O2)acts an important role in the malignant progression of solid tumors.The underlying mechanism leading to tumor may be to promote heritable mutations in genes that regulate the cell cycle.However,the mechanism of how the Hydrogen peroxide promotes the proliferation and metastasis of ESCC have not been elucidated.As known that epidermal growth factor receptor(EGFR)and MYC,influenced by H2O2,involve in the proliferation and migration of tumor cells.Therefore,the mechanism of H2O2 promoting proliferation and migration of tumor was explored by realizing the relationship among H2O2 and genes in ESCC cells to look for suitable traditional Chinese medicine(TCM).MethodsFirst of all,the effect of hydrogen peroxide on the proliferation and migration of esophageal squamous cell carcinoma was analyzed:(1)CCK-8 cell viability test was used to detect the growth and proliferation of ESCC cells stimulated by H2O2 at low concentration.(2)The migration of ESCC cells stimulated by H2O2 at low concentration was observed by Transwell cell migration assay.(3)The changes of ROS level in ESCC cells stimulated by hydrogen peroxide at low concentration was dected.(4)Westernblot(WB)was used to detect the expression of E-cadherin and Vinmentin in ESCC cells stimulated by low concentration of H2O2.Secondly,the expression and clinical significance of EGFR and MYC in esophageal carcinoma were analyzed:(1)the difference in the expression of EGFR and MYC in esophageal carcinoma(ES)was analyzed by TIMER2.0 database.(2)The relationship between the co-expression of EGFR and MYC and the survival(overall survival,OS;recurrence-free survival,RFS)of patients with ES was analysed by Kaplan-MeierPlotter(PanCancer)database.Thirdly,the relationship among H2O2 and genes(EGFR and MYC)was studied to reveale the mechanism of promoting the proliferation and migration of ESCC:(1)The expression of EGFR and MYC in ESCC cells were detected by WB assay under the stimulating of hydrogen peroxide at low concentration or NAC.(2)The correlation between EGFR and MYC was analyzed by TIMER2.0 and GeneMINIA database,and the correlation between genes was verified by EGFR and MYC inhibitors and immunoprecipitation.(3)The coenrichment pathway of EGFR and MYC was analyzed by STRING database.Related pathway inhibitors were used to block the stimulation of hydrogen peroxide and the expression of EGFR and MYC was dected byWB assay.Finally,the high-frequency therapeutic TCM targeting EGFR and MYC for ESCC were screened by TCMSP,which visualized by the Venn diagram(Venn diagram).Furthermore,the effective components of TCM were analyzed.Results(1)Under the stimulation of low concentration of H2O2 in vitro,the level of ROS in ESCC cells slightly increased,the expression level of E-cadherin decreased(P<0.05),the expression level of Vinmentin increased(P<0.05),and the cell viability increased(P<0.05),thus promoting the proliferation and migration of ESCC cells.(2)The expression levels of EGFR and MYC(P<0.05)were increased in esophageal carcinoma.Survival analysis showed that while the co-expression level of EGFR and MYC increasing,the overall survival time(P=0.0008,HR:8.44,95%CI:1.95-36.6)and recurrence-free survival time(P=0.0398,HR:2.63,95%CI:1.01-6.83)of patients with esophageal cancer were decreased.(3)There was a correlation between EGFR and MYC in ESCC,and the correlation coefficient was 0.463(P<0.05).EGFR and MYC were co-enriched in AKT signal pathway(P<0.05).Low concentration of H2O2 increased the expression of EGFR and MYC in ESCC,and increased the level of p-AKT(P<0.05),but there was no significant change in AKT.After inhibition of AKT pathway,the expression of EGFR and MYC incresing by H2O2 was decreased.(4)The analysis results of traditional Chinese medicine showed that there were 11 kinds of traditional Chinese medicine targeting EGFR and MYC to treat ESCC,including licorice,Hedyotis diffusa,Scutellaria barbata,Panax notoginseng,Salvia miltiorrhiza,Radix Rehmanniae,Honeysuckle,safflower,Huanglian,Solanum nigrum.And most of medicines above contain the active ingredient quercetin.ConclusionAs one of the important components of ROS,H2O2 activates the expression of EGFR and MYC by phosphorylating AKT to promote the proliferation and migration of ESCC cells.