FOXA3 On The Growth,Migration And Invasion Of Esophageal Squamous Cell Carcinoma

Background and objectiveEsophageal carcinoma is one of the most common malignant tumors in the world,and its morbidity and mortality rank 7th and 6th among all malignant tumors worldwide.Esophageal carcinoma can be divided into two common histological types: esophageal squamous cell carcinoma(ESCC)and esophageal adenocarcinoma(EAC).And China is a country with a high incidence of esophageal carcinoma which its morbidity and mortality surpass the world average.Due to the absence of serous layer in the esophagus and the close connection between the esophagus and the mediastinal pleura,lymph node metastasis and surrounding organ invasion might occur in the early stage of esophageal cancer.In addition,because there are no obvious symptoms in the early stage of esophageal cancer,many patients have progressed to advanced stages before being diagnosed and lack effective treatment by then.Although with improvements in healthcare these years and the great progress achieved in diagnosis and treatment of esophageal cancer,according to official statistics,the 5-year survival rate of esophageal cancer in China has increased from about 20% to about 30% in last decade,but the overall prognosis is still not satisfying.According to official data,86.3% of esophageal cancer cases in China are ESCC,and ESCC and esophageal adenocarcinoma EAC are actually two different diseases in terms of biological characteristics.But their treatment so far has been roughly the same,usually depending on which stage the tumour is in.In recent years,Due to the the blowout of the researches on molecular targeted therapy and immunotherapy for malignant tumors and promising results obtained from their rapid development,it is more necessary to distinguish them from the molecular biological characteristics so as to implement more precise and targeted treatment of esophageal carcinoma.FOX family(forkhead box),characterized by a segment of 110 amino acids that is highly conserved and highly homologous to the corresponding region of the Drosophila forkhead gene,is further divided into 19 subfamilies from FOXA to FOXS according to the sequence of the forkhead box.The FOXA subfamily,which is considered necessary for endoderm formation and the normal development of related organs such as liver,pancreas,lung and prostate,is also involved in the development of many diseases by regulating multiple target genes in liver,pancreas and adipose tissue,and also closely associated with malignant cancers.The FOXA subfamily can be specifically divided into three members: FOXA1,FOXA2 and FOXA3.Until now,most of studies on cancers are about FOXA1 and FOXA2,but FOXA3’s fewer.Most of researches about FOXA3 still focus on the field of cell differentiation,organ formation,energy metabolism,liver regeneration and hepatic cell reprogramming.Recent studies have found that FOXA3 expression was reduced in Hepatocellular carcinoma(HCC)tissues and further decreased in HCC metastatic lesions and recurrent lesions,and the low expression of FOXA3 was associated with the malignant phenotype of HCC and the poor prognosis of patients.FOXA3 can promote the differentiation of HCC cells as well as HCC stem cells,inhibit the proliferation of HCC cells and promote their response to sorafenib.Another research have found that FOXA3 is highly expressed in lung cancer tissues and cells and is considered to have a poor prognosis.Although the expression and role of FOXA3 in ESCC is unclear,based on the findings of FOXA3 itself in the above malignant cancers and the roles of FOXAl and FOXA2 taking part in the occurrence and development of ESCC,EAC and Barrett’s esophagus,we speculate that FOXA3 may also play an important role in ESCC.To evaluate its feasibility as a prognostic marker for ESCC,and to explore the role of FOXA3 in the proliferation,invasion and migration of ESCC,this study is mainly divided into two parts:Part I: The correlation between FOXA3 expression and clinical characteristic and prognosis of patients in ESCCObjective: To study the correlation between FOXA3 expression and clinical characteristic and prognosis in ESCCMethods: Collect 11 cases of ESCC tissues to detect the expression of FOXA3 m RNA by real-time PCR and collect 93 paraffin specimens of ESCC to detect the expression of FOXA3 by immunohistochemical staining.Analyze the correlation between FOXA3 and clinical characteristic.The relationship between FOXA3 and overall survival rate of patients was analyzed by Kaplan-Meier analysis,and Cox regression analysis was used to analyze independent prognostic risk factors.Results: The expression of FOXA3 in ESCC was significantly reduced,and the low expression of FOXA3 in ESCC was significantly correlated with the presence of lymph node metastasis.(P =0.002).There was no significantly correlation between FOXA3 expression and age,sex,tumor size,tumor differentiation,and tumor t-stage.Through the follow-up of patients after operation,we found that the overall survival rate of patients with low FOXA3 expression was significantly lower than that of patients with high expression(P =0.001).COX regression analysis of low expression of FOXA3 was an independent risk factor for prognosis of patients with ESCC(P =0.032).Conclusion: FOXA3 is poorly expressed in ESCC tissues and the low expression of FOXA3 in ESCC is closely related to lymph node metastasis.The low expression of FOXA3 is an independent risk factor for poor prognosis in patients with ESCC.Part Ⅱ: Effect of FOXA3 on proliferation,migration and invasion of ESCCObjective: To investigate the effect of FOXA3 on proliferation,migration and invasion of ESCCMethods: 1.ESCC cell lines Te-1 and Eca-109 were transfected with lentivirus to up-regulate FOXA3 expression,Real-time PCR and Western-blot were used to detect FOXA3 expression.to ensure that the interference was effective.2.CCK-8 assay,Ed U assay and colony forming assay were performed on the TE-1 and Eca-109 cell lines of the Ad-FOXA3 and Ad-Con groups in vitro.Wound healing assay and Transwell migration and invasion assay were used to detect changes in the migration and invasive ability of the two groups of cells.3.In vivo experiment,two groups of Eca-109 cells were injected subcutaneously at nude mice.Mice were killed after 6 weeks,and the tumors were removed to compare the tumour weights of two groups.Results: 1.Ad-FOXA3 group showed significantly higher expression of FOXA3 than control group after lentivirus transfection.2.In vitro,CCK-8 assay,Ed U assay and colony forming assay showed that the proliferation ability of ESCC cells decreased significantly after up-regulating FOXA3 expression,meanwhile the ability of invasion and migration of ESCC cells was significantly decreased after up-regulating FOXA3 expression.And also after up-regulation of FOXA3,the weight of tumor was significantly lighter than that of the control group.Conclusion: After up-regulating FOXA3 expression in ESCC cell line,the proliferation ability and invasion and migration ability of ESCC decreased obviously.SummaryThe expression of FOXA3 was significantly decreased in ESCC tissues,and the low expression of FOXA3 in patients with ESCC was closely related to the lymph node metastasis,and was also an independent risk factor for the poor prognosis of patients with ESCC.In vitro and in vivo experiments we showed that the proliferation ability and invasion and migration ability of ESCC cells were obviously decreased after up-regulating FOXA3 expression in ESCC cell lines.Based on the above studies,we can draw a preliminary conclusion: FOXA3 in ESCC may be a tumor suppressor gene involved in the proliferation,invasion and metastasis of ESCC.In the next step,animal models of tumor metastasis need to be constructed to further clarify the role of FOXA3 in ESCC.FOXA3 is expected to be a promising molecular marker for early diagnosis of ESCC,an effective prognostic indicator and a key target for molecular targeted therapy.