| Background and Object Esophageal cancer is one of the most common malignant tumors of human digestive system,according to the data from International Agency for Research on Cancer(IARC)in 2012,there are about 455800 new cases of esophageal cancer around the world every year,The incidence of esophageal cancer is about 5.90/one hundred thousand,The incidence ranks eighth in all the malignant tumors;About 400200 patients die from esophageal cancer every year,The standardized mortality rate is about 5.00/one hundred thousand,ranks sixth in all the malignant tumors,So esophageal cancer becomes a serious threat to the lives and the health of human beings.Esophageal cancer has the characteristic of regional distribution,The incidence and mortality rates of esophageal cancer in china are both the highest in the world,according to the national cancer center statistics in 2012 the incidence of esophageal cancer is about 21.17/one hundred thousand,ranks the fifth in all kinds of malignant tumors,and the mortality rate is about 15.58/one hundred thousand,ranks the fourth in all kinds of malignant tumors.The most common pathological type of esophageal cancer in European couctries and America is esophageal adenocarcinoma,While in China esophageal squamous cell carcinoma is the main pathological pattern,accounts for more than 90% of all esophageal cancer patients.The causes of esophageal cancer were not yet completely clear,the treatment strategy is surgical treatment combined with chemotherapy and radiotherapy at present.But due to early symptoms of esophageal cancer is not obvious and the lack of the early diagnosis of simple,reliable technique,70% of esophageal cancer patients have been in the middle or late course when they come to the hosipital;because of the lack of specific,sensitive tumor markers,local recurrence and distant metastasis seriously influence the quality of life and prognosis of the patients;Sensitive targets and corresponding drugs for the treatment of esophageal cancer have not been found so far.So the treatment of esophageal cancer is far from satisfactory,5 years survival rate of esophageal cancer is only 30-40% on average.So we sho?ld do theoretical and basic researches based on the characteristics of esophageal cancer in China.Looking for tumor markers of high sensitivity and specificity,therapeutic targets and corresponding drugs is of great significance.Mitogen induced gene 6(Mig-6)is a newly discovered tumor suppressor gene,Absent gene expression or decreased protein expression of Mig-6 was found in breast cancer,endometrial carcinoma,hepatocellular carcinoma,non-small cell lung cancer,pancreatic cancer,ovarian cancer,skin cancer and other human malignant tumors;Mig-6 knockout mice develop spontaneous tumors in various organs and tissues such as lung,gallbladder,bile duct,uterus,gastrointestinal tract and so on.Vitro studies have found that Mig-6 could inhibit tumor cell proliferation,invasion,metastasis and induce apoptosis.But the effect of Mig-6 in esophageal has not been reported,whether Mig-6 play a suppressor role in esophageal cancer deserves further studies.In this study we detect the Mig-6 expression in human esophageal squamous carcinoma tissues and normal esophageal epithelium tissues,and explore its relationgship with the patients clinical and pathological characteristics,we examined the m RNA expression levels of Mig-6 in human esophageal squamous carcinoma cell lines TE13 EC109 and KYSE510.The Mig-6 m RNA expression level of TE13 was lower,and TE13 was chosen for the next studies.Mig-6 was upregulated in TE13 by transfection.The changes of proliferation and apoptosis in TE13 esophageal squamous cancer cells were observed.Further we investigate the expression profile after Mig-6 affected in esophagealsquamous cell carcinoma line TEl3,To explore the possible mechanism of Mig-6 in esophageal squamous cell carcinoma.Methods: 1.Realtime polymerase chain reaction(Real-time PCR)was used to detect the expression level of Mig-6 m RNA in human esophageal squamous carcinoma tissues and corresponding normal esophageal epithelial tissues(?5cm from the edge of tumor);Immunohistochemical technique was used to detect the expression of Mig-6 protein in esophageal squamous carcinoma tissues and normal esophageal epithelial tissues,if the expression level of Mig-6 was lower in esophageal squamous cell carcinoma tissues than normal esophageal epithelial tissues.Analyze its relationgships with the gender,age,tumor location,TNM stages,tumor differentiation and other clinical pathological parameters.2.Human esophageal squamous cancer cell lines TE13,EC109 and KYSE510 were cultivated conventionally;Real-time PCR was used to choose the cell line whose Mig-6 m RNA expression level was lower.TE13 whose Mig-6 m RNA expression level was lower was chosen for the next studies.Mig-6 expression was up-reg?lated by transfection in human esophageal squamous cancer cell line TE13.Real-time PCR and Western Blotting were used to verify the transfection efficiency.3.Human esophageal squamous cancer cells TE13 whose Mig-6 gene m RNA expression level was already verified lower were divided into two groups: The experimental group was transfected with eukaryotic expression vectors pc DNA3.1(+)/Mig-6 using the lipofectamin transfection reagent.The control group was transfected with pc DNA3.1(+).24,48 and 72 hours after transfection,MTT test was used to detect the cell growth of esophageal squamous cancer cells.The growth curve was drawed and growth inhibition rate was recorded;The apoptosis of the cells was observed 48 hours after transfection by fluid cytology test.4.Mig-6 was up-reg?lated by transfection using the lipofectamin transfection reagent.Agilent whole human genome oligo microarray(4×44K)was used to screening the the differences of genes(fold change ? 2 or ? 0.5).The differences of genes and the signaling pathways were analysed.Results: 1.Mig-6 gene m RNA expression level in esophageal squamous carcinoma tissues was relatively lower than that in normal esophageal epithelium tissues(1.00VS1.67±0.16,t=22.627,P=0.000).Immunohistochemical analysis showed that the positive rate of Mig-6 protein expression was lower in esophageal squamous carcinoma tissues compared to that in normal esophageal epithelium tissues(x~2=21.679,P=0.000).The low expression of Mig-6 was related with TNM stages(x~2=4.21,P=0.04)and tumor differentiation(x~2=8.438,P=0.004)and there were no relations between the expression of Mig-6 and the patients' gender (x~2=0.859,P=0.354),age(x~2=0.892,P=0.741)and tumor location(x~2=0.647,P=0.732).2.The result of Real-time PCR showed that the Mig-6 gene m RNA expression level was lower in human esophageal squamous cancer cell lines TE13 compared to that in EC109 and KYSE510.TE13 was chosen for the next studies(1.00VS6.770±1.17VS133.59±5.44,F=3262.019,P=0.000).3.Mig-6 was up-regulated in TE13 by transfection using the lipofectamin transfection reagent.The transfection efficiency was verified by Real-time PCR and Western Blotting.MTT assay results showed that the cell growth was restrained,the fluid cytology results show that the cell apoptosis rate increased.4.Agilent whole human genome oligo microarray results: There are 2212 different genes totally.1141 genes up-regulated and 1071 genes down-regulated.Mainly involved in signaling pathways as follws: MAPK signaling pathway,cytokinescytokines interaction and cell adhesion molecules(CAMs).Conclusion: 1.Mig-6 gene m RNA and protein expression levels were lower in esophageal squamous cell carcinoma tissues.The low expression of Mig-6 was associated with TNM stages and tumor differentiation,and Mig-6 might be an important prognostic factor in human esophageal squamous cell carcinoma.2.With the up-regulated of Mig-6 in esophageal squamous cell carcinoma.The cell growth was restrained and the the rate of cell apoptosis increased.Mig-6 might play an important role in the development and progression of cancer.3.With the up-regulation of Mig-6 in TE13 by transfection,genes expression changed widespread.The potential upstream or downstream genes of Mig-6 maily involved in the signaling pathways such as MAPK signaling pathway,cytokinescytokines interaction and cell adhesion molecules(CAMs). |
PDF Full Text Request