Expression And Clinical Significance Of Nrf2 And FAT4 In Esophageal Carcinoma

BackgroundEsophageal carcinoma(EC)is the eighth most common cancer worldwide,due to limited clinical methods for early diagnosis,it often has a very poor prognosis,5-year survival rate ranges from 10%to 25%.The invasion and metastasis of esophageal cancer is involved by multiple factors,which are regulated by various signaling pathways.Therefore Looking for the important molecules involved in the development of esophageal cancer and explore its molecular mechanism of esophageal carcinoma,it has important significance to improve the early diagnosis of esophageal cancer,search for new treatment targets and improve the survival rate of patients.In recent years,some studys discovered that Nrf2 is essential to maintain cellular redox homeostasis,play a protective role by regulating cell adaptation in the oxidation and exogenous stress,and inhibit the development and metastasis of cancer,meanwhile the activation of Nrf2 contributes to progression and radiochemotherapy resistance of carcinoma.FAT protein is one of the largest molecules in the cadherin superfamily,which is involved in the regulation of morphogenesis,growth control,and polarity of planar cells.FAT1 and FAT4 are closely related to the occurrence and development of tumors.At present,there is little report on the relationship between FAT4 and superficial esophageal carcinoma and esophageal carcinoma,and the relationship between Nrf2 and the development of superficial esophageal cancer has not been reported yet.ObjectivesThe aim of this study was to investigate the expression of Nrf2 and FAT4 in superficial esophageal carcinoma and esophageal cancer tissue,expression of Nrf2 and FAT4 mRNA in peripheral blood mononuclear cell,further analysis the relationship among the clinical pathological parameters.Methods1.Case collectionWe collected 43 tissues and peripheral bloods from endoscopic submucosal dissection for superficial esophageal squamous neoplasms and surgical treatment for esophageal carcinoma between May 2016 and November 2016 at The second hospital of Shandong University,with peripheral blood of 15 cases of non-gastrointestinal cancer and other chronic diseases as control.Exclusion criteria:1)patients receiving chemotherapy or radiotherapy before surgery,2)patients with two or more types of tumors at the same time.According to the 7th AJCC cancer staging system,Out of the 43 patients,11 cases were stage I,8 cases were stage Ⅱ,12 cases were stage III/IV,Patients with stage IV were found to be unable to undergo radical resection and performed palliative treatment.In the case of T staging,there were fourteen T1/T2 lesions,seventeen T3/T4 lesions.Non lymph node metastasis was found in 17 patients,and lymph node metastasis was found in 14 patients.Two lesions were located in cervical and upper esophagus,eighteen lesions were located in the middle esophagus,twenty-three lesions were located in the lower esophagus.Out of all 43 lesions,32 lesions were less than 5cm,11 lesions were more than 5cm.2.Immunohistochemistry staining of Nrf2 and FAT4The paraffin sections were dewaxed in water,and antigen retrieval was performed in EDTA for 15 min.PBS washed three times,3%H202 blocking endogenous peroxides,goat serum blocking 30min,The sections were incubated with primary antibody overnight at 4℃.Sections were washed and then incubated with appropriate biotinylated secondary antibodies(30 minites),and visualized with DAB.The sections were counterstained with haematoxylin,then washed and dehydrated.Known positive reaction was used as positive control,PBS was used as a negative control3.Quantitative RT-PCR of Nrf2 and FAT4 mRNAPeripheral blood mononuclear cells were extracted from human peripheral blood,through Human lymphocyte separation solution,and trizol splited cells,mRNA was extracted by chloroform isopropanol alcohol precipitation method,and measured total concentration.Take 1μl total mRNA for reverse transcription,PCR amplification:qPCR was performed by 25μl reaction system and referred to the specific operation instructions of SYBR(?)Premix Ex Taq TM(RR420A)4.Statistical analysisComparison of enumeration data was preformed using Pearson chi-square test or Fisher’s exact test,measurement data comparison was preformed using t-test.All statistical analyses were carried out using SPSS 21.0 software,P<0.05 was considered statistically significant.Results1.Expression of Nrf2 in esophageal squamous cell carcinoma tissues and adjacent tissuesNrf2 scattered light yellow or brown particles,mainly located in nucleus,scattered in the cytoplasm.The positive expression rate of nrf2 protein in esophageal squamous cell carcinoma tissues and normal esophageal tissues were 62.79%(27/43),13.95%(6/43),it had statistically significant differences between the two groups(x2=21.684,P<0.05).The positive expression rate of Nrf2 in superficial esophageal carcinoma and advanced esophageal carcinoma had statistical significance(χ2= 18.496,P<0.001),The positive expression rates of Nrf2 in normal esophageal squamous epithelium and squamous cell carcinoma(P<0.001),squamous cell carcinoma and intraepithelial neoplasia(P<0.001)had statistical significance;There was no significant difference between normal squamous cell carcinoma and intraepithelial neoplasia(P>0.05).The clinical pathological factor analysis showed that the expression of Nrf2 protein was closely related to length of the lesion,depth of invasion,T staging(P<0.05),but it had not significant correlation with lymph node metastasis,the lesion location and TNM staging(P>0.05).2.Expression of FAT4 in esophageal squamous cell carcinoma tissues and adjacent tissuesImmunohistochemistry presented:The positive expression of FAT4 mainly presented yellow or brown in cytoplasm or nucleus.The positive expression rate of FAT4 protein in esophageal squamous cell carcinoma tissues and normal esophageal tissues were 32.56%(14/43)、65.12%(28/43),it had statistically significant differences between the two groups(χ2=9.118,P=0.005<0.05).The positive expression rate of FAT4 in superficial esophageal carcinoma and advanced esophageal carcinoma had statistical significance(x2=9.918,P=0.003<0.05),The positive expression rates of FAT4 in normal esophageal squamous epithelium and intraepithelial neoplasia(P=0.188>0.05),intraepithelial neoplasia and squamous cell carcinoma(P=0.482>0.05)hadn’t statistical significance;There was significant difference between esophageal squamous epithelium and intraepithelial neoplasia(P=0.004<0.05).The clinical pathological factor analysis showed that the expression of FAT4 protein was closely related to depth of invasion,T staging and TNM staging(P<0.05),but it has not significant correlation with the lesion location,length of the lesion,and lymph node metastasis(P>0.05).4.The expression of Nrf2 mRNA and FAT4 mRNA in peripheral blood mononuclear cellsThe results of qPCR showed that there was no significant difference in the expression level of Nrf2 mRNA and FAT4 mRNA in mononuclear cells in 43 patients and15 controls(P=0.068>0.05 and P=0.263>0.05,respectively).ConclusionThrough detecting the expression of Nrf2 and FAT4 protein,we can understand the malignant degree of esophageal cancer from the molecular biology level,and play a guiding role in the clinical treatment.It may be helpful to judge the prognosis.