Expression And Gene Mutation Analysis Of TTF-1 On The Lung Adenocarcinoma Cells And Influence On Biological Behavior Of GLC-82 Cells After TTF-1 Gene Transfection

Background and ObjectivesLung cancer is a common malignant tumor in the worldwide.The morbidity and mortality of lung cancer rank first in commonly seen malignant tumors.Meanwhile it is the main cause resulting in malignant pleural effusion.About 10%lung cancer patients have pleural effussion as their first clinical symptom and 30%-40%lung cancer patients have pleural effussion in their disease progression.Lung cancer patients with pleural effussion have bad prognosis and survial.In order to take effective measures for patients with pleural effussion accurate and immediate diagnosis are necessary.So deep research into the carcinogenesis,development and metastasis of lung cancer to establish scientifically and effectively diagnostic methods is of great significance to the early diagnosis,improvement of survial and cure rate,judgement of prognosis of lung cancer.Thyroid transcription factor-1(TTF-1),with the gene name TITF-1/NKX2.1,is a homeodomain-containing nuclear transcription protein of Nkx2 gene family that is expressed in thyroid,lung and brain.It is closely correlated with the histogenesis,functional maintenance of these organs,tumorigenesis and development of other diseases.TTF-1 is localized to 14q 13 and has 371-amino acid which is 98%identical to the rat sequence.By genomic sequence analysis the TTF-1 gene spans approximately 3.3 kb and contains 2 exons and 1 intron.There is a conserved homeodomain region of 60 amino acids between region 160 and 220.In normal lung tissue,TTF-1 is expressed in mature typeâ…¡alveolar epithelial cells and epithelial cells of the developing airway but not expressed in typeâ… alveolar epithelial cells.TTF-1 mediates the expression of surfactant protein A,B,C and Clara cell secretory protein(CCSP).By now,it is the only transcription factor that mediates these four lung special genes.Willemsen et al.found that a de novo heterozygous insertion mutation 859-860insC in the TTF-1 gene of a 23-year-old man exhibited unexplained combinations of chorea,mental retardation,primary hypothyroidism,and chronic lung disease.Introduction of a name for the disorder,e.g.Brain-Thyroid-Lung syndrome. The man died from large cell lung carcinoma at the age of 23.Kang Y et al.found that the decreasded expressin of TTF-1 was associated with mouse lung cancer but the mechanism was not clear.Whether the TTF-1 gene mutation predisposed to the development of lung cancer remains speculative and needs further investigation.Studies show that TTF-1 is expressed in different kinds of lung carcinomas with different intensities.TTF-1 is a marker of adenocarcinoma and small cell carcinoma of the lung.TTF-1 is little expressed in other primary or metastatic adenocarcinoma exclude lung adenocarcinoma and thyroid adenocarcinoma.So it is a useful and reliable marker for metastatic pulmonary adenocarcinoma in diagnosis of malignant pleural.But there are no studies on quantitative analysis of TTF-1 expression in malignant pleural.It is not clear whether its gene mutation in malignant pleural is as the same as its primary site.Whether the TTF-1 gene mutation predisposed to the tumorigenesis,development and metastasis of pulmonary adenocarcinoma remains speculative.Wether it can be used as a tumor marker for pulmonary adenocarcinoma diagnosis in supernatant of malignant pleural effusion is still unknown.So,our study aims to detect the expression of TTF-1 protein and mRNA in metastatic adenocarcinoma cells in malignant pleural to reveal the characteristics of TTF-1 expression in adenocarcinoma cells from different origins in malignant pleural. Quantitative analysis of the intensities of TTF-1 protein and mRNA were performed to reveal the charicterristics of TTF-1 for metastatic pulmonary adenocarcinoma in diagnosis of malignant pleural.The.Polymerase chain reaction-single strand conformation polymorphism(PCR-SSCP) and DNA sequencing were used to analyse TTF-1 gene mutation and its relationship with the carcinogenesis of lung cancer.To construct eukaryotic fluotrscent expressin vector pEGFP-N1-TTF-1,induce the vector express in human adenocarcinoma cell line GLC-82 and observe the behavior changes of GLC-82 after TTF-1 transfection.Methods1.A series of pleural specimens were available from the Pathology Department of Nanfang Hospital of Guangzhou in China from 2006,Jun to 2007.Mar.The primary sites of origin were confirmed clinically or histologically in all patients.There were 37 adenocarcinomas from lung,4 adenocarcinomas from stomach,3 adenocarcinomas from breast,2 adenocarcinomas from ovary,1 endometrial carcinoma and 1 metastatic colon carcinoma.Pap,Giemsa and PAS stained smears and cell block were prepared for each case.2.Immunohistochemical staining and in situ hybridisation were used to detect TTF-1 protein and TTF-1mRNA expression on the cell block sections.Leica Q500 MC image analysis system was used to detect the intensity of TTF-1 protein and TTF-1mRNA expression.To explore the clinical usefulness of TTF-1 as a marker for metastatic pulmonary adenocarcinoma cells in diagnosis of malignant pleural. 3.Polymerase Chain Reaction-Single Strand Conformation Polymorphism(PCR-SSCP) and DNA sequencing analysis were used to analyze TTF-1 gene mutation.Three specific primers for exon 1 and exon 2 of TTF-1 gene were synthesized and used for PCR amplification.PCR products were detected using PCR-SSCP.DNA sequencing analysis was used to analyze fragments with anomalous migrating bands by PCR-SSCP detection to identify the type of mutation and analyze the changes of gene function.4.Immunocytochemistry,RT-PCR and western blot were used to detect TTF-1 protein and TTF-1 mRNA expression in human adenocarcinoma cell line-GLC-82.5.Effect of TTF-1 expression on the biological behaviors of human adenocarcinoma cell line-GLC-82.TTF-1 cDNA was transfected into GLC-82,which had no endogenous TTF-1 expression.The TTF-1 expression in transfectant was determined by RT-PCR, immunohistochemistry and Western blot.The biological behaviors of tranfectant were investigated by MTT assay,Plate colony formation,wound-healing assay and flow cytometry in vitro.Moreover,tumorigenesis of tumor cells after transfection was observed in nude mice models in vivo by whole-body visualizing methods.Results1.Pap,Giemsa and PAS stain were used to observed the morphologic features of metastatic adenocarcinoma cells from different origins in malignant pleural.These tumor cells had the samilar morphologic features for example,forming cell clusters, marked variation in size and shape,marked atipical nuclei,mucus in plasma and so on. Sometimes adenocarcinoma cells can form the glandular structure in HE smears.But these morphologic features can not determine the primary site of metastatic adenocarcinoma cells in serous pleural. 2.Immunohistochemical staining was used to detect TTF-1 protein expression on the cell block sections.TTF-1 was expressed in 28 lung adenocarcinomas while none of the 11 extrapulmonary adenocarcinomas expressed TTF-1.The sensitivity and specificity of the TTF-1 for the adenocarcinomas of lung origin were 75.7%and 100%, respectively and there is a significant difference between them(x²=19.978,p=0.000). TTF-1 positive unit(PU) is bigger in adenocarcinomas from lung than in those of extrapulmonary adenocarcinomas and there is a significant difference between them (t=13.208,p=0.000).3.In situ hybridisation was used to detect TTF-1mRNA expression on the cell block sections.TTF-1 mRNA was expressed in 21 lung adenocarcinomas while none of the 11 extrapulmonary adenocarcinomas expressed TTF-1 mRNA.The sensitivity and specificity for the adenocarcinomas of lung origin were 56.75%and 100%, respectively and there is a significant difference between them(x²=11.099,p=0.001). TTF-1 mRNA positive unit(PU) is bigger in adenocarcinomas from lung than in those of extrapulmonary adenocarcinomas and there is a significant difference between them (t=9.901,p=0.000).4.PCR-SSCP technique was used to detect TTF-1 gene mutation in 37 cases of metastatic adenocarcinomas from lung origin and 20 cases of normal lung tissues. After 3 times of silver staining,no abnormal migrating bands were observed in fragment a amplified from primer 1A,1S of exon 1 of TTF-1 gene in 37 cases of metastatic adenocarcinomas from lung origin and 20 cases of normal lung tissues. Abnormal migrating bands were observed in fragment b amplified from primer 2A,2S of exon 2 of TTF-1 gene in 4 cases of metastatic lung adenocarcinomas.Heterozygous single nucleotide absence and substitutions were conformed by DNA sequence.The mutation rate is 10.8%.5.The TTF-1 expression in human adenocarcinoma cell line GLC-82 was determined by RT-PCR,immunohistochemistry and Western blot.The result shows that there no TTF-1 expression in GLC-82 cell.6.Effect of TTF-1 expression on the biological behaviors of human adenocarcinoma cell line-GLC-82.(1) The pEGFP-N1 plasmid and pEGFP-N1-TTF-1 plasmid were transfected respectively into GLC-82,which had no endogenous TTF-1 expression by cationic liposomes-Lipofectamine ^TM2000.Stable EGFP and TTF-1 expression cells(GLC-82-EGFP and GLC-82-TTF-1) were acqired through G418 selective culture.The TTF-1 expression was detected by RT-PCR and western blot.(2) There was no significant effects of extra TTF-1 expression on cell proliferation which were assessed by MTT(F=0.388,P=0.863),plate colony formation assay( x²=1.029,P=0.598),wound-healing assay(F=0.650,P=0.690) and flow cytometry(F=0.960,P=0.435;F=0.542,P=0.608) in vitro.The result indicated that there was no significant inhibitory of cell proliferation in GLC-82-TTF-1 cell as compared with GLC-82 and GLC-82- EGFP cells.The effect of TTF-1 expression in vivo tumor growth was detected by subcutaneous growth of GLC-82,GLC-82-EGFP and GLC-82-TTF-1 cells in nude mice models for thirty days.Extra expression of TTF-1 had no significant effects on tumor growth and cell proliferation in vivo(F=0.099,P=0.986).Conclusions1.TTF-1 protein and TTF-1 PU detection can be used as a tissue special marker in distiguishing pulmonary adenocarcinoma cells from extrapulmonary adenocarcinoma cells in pleural effusion.The TTF-1 PU betwwen pulmonary adenocarcinoma cells and extrapulmonary adenocarcinoma cells in pleural effusion has no overlop.So the TTF-1...