| Background:Non-small cell lung cancer(NSCLC)is the leading cause of cancer-related death worldwide.At present,with the introduction of molecular targeted drugs and immunosuppressants,the treatment strategy of NSCLC has changed greatly,but the overall effect is still not satisfactory.Members of the ErbB family are often overexpressed,amplified or mutated in a variety of cancers,making it an important therapeutic target for malignant tumors.So far,most studies on the role of ErbB receptors in the pathogenesis of cancer have focused on EGFR or ErbB2,and drug development is parallel to this.There are few studies on the function of ErbB3 and its role in human diseases.At prcsent,studics have found that ErbB3 is a factor that promotes tumor occurrence and development,and is considered to be a potential target for cancer therapy.Ebpl is the main binding protein of ErbB3 and the human homologous protein of mouse protein P2AG4,which is regulated by cell cycle.It has been reported that Ebpl is highly expressed in human brain gliomas,resulting in down-regulation of p53 protein expression,promoting the proliferation of tumor cells in vitro and in vivo,resulting in poor clinical treatment.It has also been reported that the high expression of Ebpl in breast cancer is closely related to clinical stage,pathological grade and poor prognosis.Whether Ebpl can regulate the carcinogenic activity of ErbB3 or has the independent activity of ErbB3 in promoting cancer progression remains to be fully elucidated.Up to now,the expression,biological function and molecular mechanism of Ebpl in lung cancer are not clear,so exploring the role and mechanism of Ebpl in NSCLC is of great significance to explore new molecular targets of NSCLC therapy and improve the prognosis of NSCLC patients.Objective:To investigate the expression level of Ebpl in NSCLC tissues,analyze the correlation between Ebp1 expression and clinicopathological parameters,and explore the effect and mechanism of Ebpl on NSCLC proliferation,invasion and metastasis.It will provide a new target for clinical treatment of NSCLC.Methods:1.Database and tissues specimens analysis:(1)The expression of EbplmRNA in NSCLC tissues and normal tissues were retrieved through TCGA and Oncomine database.Then the relations between Ebpl and survival of NSCLC patients were also analyzed by TCGA and Oncomine database;(2)The expression levels of Ebpl protein in NSCLC and paracancerous tissues were detected by immunohistochemistry;(3)The expression levels of Ebpl protein in NSCLC were compared with paracancerous tissues,and analyzed the correlations between Ebp1,Ki-67 and the clinicopathological features;(4)The Kaplan-Meier survival analysis was performed to identify the correlations Ebpl expression and the prognosis of NSCLC.2.In vitro experiments:(1)The expression of Ebpl protein in bronchial epithelial cell(16HBE)and NSCLC cells(A549,H522,PC9,H650)were detected by Western blotting,Then A549 and PC9 cells with relatively high expression of Ebp1 were selected for subsequent experiments;(2)Ebp1 was silenced b y short hairpin RNA(shRNA)techniquegene in A549 and PC9 cells.Then the expression of Ebpl in thetransfected cells was detected by Western blotting;(3)Ebpl protein localization was detected in A549 and PC9 cells and verified its silencing effect by immunofluorescence assay;(4)The effects of Ebp1 on the proliferation of A549 and PC9 cells were detected by colony formation,soft AGAR cloning and CCK-8 assay;(5)The effects of differentially expressed Ebpl inA549 and PC9 cell cycle was detected by flow cytometry;(6)The effects of differentially expressed Ebpl on the migration and invasion of A549 and PC9 cells was detected by the scratch,transwell assay;(7)The effect of Ebpl silenced was used to detected on EMT-related marker protein expression in A549 and PC9 cells by western blotting assay;(8)EMT-related markers Vimentin and N-cadherinin A549 and PC9 cells after silenced of Ebpl was used to detected the expression and localization by immunofluorescence assay.3.In vivo experiments:(1)The transfected PC9 cells were cultured and inoculated subcutaneously into the right shoulder of nude mousenude mice to establish a transplantation tumor model of NSCLC;(2)The effect of Ebpl silenced on the formation and growth of xenografts in nude mice was observed.After mice were sacrificed,the tumors were separated and calculated volume;(3)Theexpression of Ebp1、Ki-67、CyclinDl and EMT-related marker MMP-2、Slug、Snail of transplanted tumors were detected by immunohistochemistry;Results:1.Database and tissues specimens analysis:(1)TCGA and Oncomine database analysis showed that EbplmRNA in NSCLCwere highly expression in NSCLCwhich were associated with poor prognosis of NSCLC patients;(2)The expressionof Ebpl protein in NSCLC tissue was significantly higher than in paracancer tissues(*P<0.05);(3)The expression of Ebpl protein was significantly correlated with the pathological grade,clinical stage and lymph node metastasis in NSCLC tissue(*P<0.05),but not correlation with age,gender,tumor location and size(P>0.05);(4)Ebpl protein in NSCLC tissue was positively correlated with the expression of ki-67 proliferation index(*P<0.05);(5)The Kaplan-Meier analysis showed the NSCLC patients with high Ebpl expression had significantly lower overall survival than patients with low Ebpl expression,showing a negative correlation(**P<0.01);2.In vitro experiments:(1)Western blotting showed silencing of Ebpl inhibited the expression of Ebp1 protein in NSCLC cells(***P<0.001);(2)Immunofluorescence showed that Ebp 1 protein was located in cytoplasm and nucleus,and was mainly expressed in cytoplasm;(3)CCK-8、colony formation and soft AGAR cloning experience showed silenced with Ebpl inhibited the proliferation and colony formation ability of A549 and PC9 cells(***P<0.001);(4)Cell cycle experiment showed the low expression of Ebpl protein,A549 and PC9cell cycle arrest occurred in the G0/G1 phase to inhibit the proliferation ability;(5)Scratch and transwell experiment showed that knockdown of Ebp1 expression significantly inhibited the migration and invasion ability ofA549 and PC9 cells(***P<0.001);(6)Western blotting showed that knockdown of Ebp1 expression up-regulate the expression of epithelial cell marker(E-cadherin)(**P<0.001)and down-regulate the expression of interstitial cell markers(N-cadherin.Vimentin,)and transcription Snai,Slug)and matrix metalloproteinase(MMP-2)of NSCLC cells(***P<0.001):(7)Immunofluorescence showed that the Vimentin and N-cadherinn were located in the cytoplasm of A549 and PC9 cells.and knockdown of Ebpl expression the levels of Vimentin and n-cadherinn were significantly decreased(***P<0.001)3.In vivo experiments:(1)The mouse xenograft model was successfully established.Its showed that Ebp1 silencing significantly inhibited tumor volume(**P<0.01);(2)Immunohistochemical showed that after silencingEbpl,transplantation tumor tissues Ebpl,Ki-67,Cyclin D1 expression were significantly decreased(***P<0.001),the expression of interstitial cell markers(Slug,Snail)decreased obviously(***P<0.001),matrix metalloproteinase(MMP-2)was slightly decreased(**P<0.01).Conclusion:1.Ebpl was highly expressed in NSCLC tissues,which were closely related to the poor prognosis of NSCLCpatients.2.Ebpl has the ability to promote the proliferation,invasion and metastasis of NSCLC cells.3.Ebpl participates the invasion and metastasis of NSCLC cells via regulation EMT progression. |
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