| Background and AimNRG1,the most characteristic gene in NRGs family,locates on the 8th chromosome and encodes six kinds of proteins.NRG1 is considered to be an excellent ligand for HER family and can activate the HER signaling axis through combining with HER3/HER4 in an autocrine and paracrine manner,thus causes tumorigenesis.This study aims to investigate the function of NRG1 in esophageal squamous cell carcinoma(ESCC)cells and evaluate the target therapeutic efficiency of ESCC with high NRG1 expression,as well as explore the possible molecular mechanism.MethodsAnalysis of NRG1 expression in ESCC1.The expression of NRG1 in esophageal carcinoma(EC)was analyzed using GEPIA database.2.The expression of NRG1 in normal esophageal cells HEEC and ESCC cells TE1,ECa109,KYSE150,EC9706 as well as KYSE70 cells was detected by Western blot.Effects of NRG1 on ESCC cells and molecular mechanism1.The NRG1 secretion in culture medium of ESCC cells with high expression of NRG1 was detected by ELISA assay.2.Gene enrichment analysis and protein-protein interaction network analysis were performed in NRG1 signaling pathway using Metascape and String database.3.According to above results,the conditioned medium(CM)was prepared using the medium cultured KYSE150 cells with high NRG1 expression and used to treat ECa 109 cells with low expression of NRG1,cell proliferation and migration was subsequently investigated by Colony formation assay,MTT assay,Wounding healing assay and Transwell assay.ECa109 cells cultured with normal medium,medium supplemented with exogenous NRG1 protein(PNRG1)or medium cultured KYSE150 cells interfered by NRG1 siRNA(si-1)(CMsi-1)were used as controls.The expression of EMT process,apoptosis,HER2,HER3,p-HER2(Y1248),p-HER3(Y1289),proteins in MAPK and PI3K signaling pathway-related proteins was detected by Western blot.4.After KYSE150 cells were treated with PNRG1,cell proliferation and migration were investigated by Colony formation assay,MTT assay,Wounding healing assay and Transwell assay,and expression of proteins above was detected by Western blot.5.A certain number of ECa109 cells mixed with PNRG1 or a certain number of KYSE150 cells were inoculated subcutaneously in nude mice,the tumor-forming ability of the cells was evaluated.Effects of targeting NRG1 and its receptor on ESCC cells and molecular mechanism1.KYSE150 cells were treated with Afatinib,or ECa109 cells were treated with PNRG1/CM and PNRG1/CM+Afatinib.Cell proliferation,migration and apoptosis were detected by MTT assay,Colony formation assay,Wounding healing assay,Transwell assay and Flow cytometry.2.After KYSE150 cells were treated with si-1,si-1+Afatinib,si-1+PNRG1,respectively,proliferation,migration and apoptosis of cells above were detected by Colony formation assay,MTT assay,Wounding healing assay,Transwell assay and Flow cytometry.3.After KYSE150 cells were treated with Afatinib and si-1,respectively,expression of proteins above was detected by Western blot.Targeting NRG1 and its downstream targets on the growth of ESCC1.KYSE150 cells were subcutaneously inoculated into nude mice and the effects of Afatinib,si-1 and si-1+Afatinib on tumor growth were detected.The potential toxicity was preliminary evaluated according to body weight,relative organ weights,H&E staining of liver and kidney tissues and blood routine of nude mice.ResultsHigh expression of NRG1 in ESCC1.The analysed result using GEPIA database showed that expression of NRG1 in EC was significantly higher than that in normal tissues.2.Expression of NRG1 in KYSE150 and KYSE70 cells was significantly higher than that in normal esophageal epithelial cells,especailly in KYSE150 cells.NRG1 promotes ESCC through the activation of HER3-HER2/PI3K/MAPK pathway1.ELISA results showed that NRG1 protein was detected in culture medium of KYSE150 cells.2.Bioinformatics analysis showed that NRG1 signaling pathway might be involved in apoptosis and Epithelial-mesenchymal transformation,and NRG1 could be interacted with HER3,PIK3CA and AKT1.3.NRG1 activates the HER3-HER2/PI3K/MAPK signaling pathway to promote proliferation and migration of ESCC cells and inhibit cell apoptosis1)PNRG1 and CM significantly promoted the proliferation and migration of ECa109 cell,increased expression of N-Cadherin,Vimentin,Slug,Bcl2,p-HER2(Y1248),p-HER3(Y1289),p-AKT(Ser473),p-ERK(Thr202/Tyr204)and p-MEK1/2(Ser217/221),and inhibited expression of E-Cadherin,Cleaved-PARP and Bax in ECa109 cell,while CMsi-1 had no effect on the proliferation and migration ability of ECa109 cell.2)PNRG1 significantly promoted the proliferation and migration of KYSE150 cells,and had similar effects on the expression of proteins in KYSE150 cells with that in ECa109 cells.4.NRG1 promotes tumor formation of ESCC cells.Tumorigenesis ability and tumorigenesis rate of ECa109 cells mixed with PNRG1 or KYSE150 cells were significantly enhanced and tumorigenesis time was significantly shortened,in nude mice compared to ECa109 cells alone.Targeting NRG1 or its downstream targets inhibited ESCC by affecting the NRG1/HER3-HER2/PI3K/MAPK signaling pathway.1.Afatinib can inhibit the proliferation and migration of KYSE150 cells with high expression of NRG1 and promote cell apoptosis,and can inhibit the promotion of PNRG1/CM on the proliferation and migration of ECa109 cells.2.Afatinib and si-1 significantly inhibited the proliferation,migration and promoted apoptosis of KYSE150 cells,and the combination of Afatinib and si-1 was more effective,while PNRG1 weakened the effects of si-1.3.Both Afatinib and si-1 inhibited the expression of N-Cadherin,Vimentin,Slug,Bcl2,p-HER2(Y1248),p-HER3(Y1289),p-AKT(Ser473),p-ERK(Thr202/Tyr204)and p-MEKl/2(Ser217/221)in KYSE150 cells,and promoted expression of E-Cadherin,Cleaved-PARP and Bax.Targeting NRG1 or its downstream inhibited the growth of ESCC xenografts1.Both si-1 and Afatinib alone or combined significantly inhibited the growth of xenograft from KYSE150 cells,especially the combination group.The mortality,body weights,relative organ weights and blood routine of nude mice had no significant difference in every group.Conclusions1.NRG1 promotes ESCC by activating HER3-HER2/MAPK/PI3K signaling axis.2.Afatinib or NRG1 siRNA could inhibit ESCC by inhibitng NRG1/HER3-HER2/MAPK/PI3K signaling axis. |
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