Stable Transfection Of The DMBT1 Plasmid Into Gallbladder Carcinoma Cell Line GBC-SD And Effects On Its Biological Behavior

The DMBT1(deleted in malignant brain tumors 1) gene is located at chromosome 10q 25.3—26.1,a possible tumor suppressor locus indicated by the refinement of the losses of heterozygosity in various cancers[5].The repetitive structure of the gene has been proposed to increase its susceptibility to genomic instability,which leads to the homozygous/hemizygous deletion observed in a significant number of medulloblastomas and glioblastoma multiforme.Abnormalities of its expression in brain,lung and gastrointestinal tumors have also been reported.Based on these findings,DMBT1 was considered as a candidate tumor suppressorgene.However, studies on its functions and its relationship with tumorigenesis are still at an initial stage.Whether the gene belongs to classical anti-oncogenes is still a controversy. Gallbladder cancer is the fifth most common gastrointestinal malignancy,and ranks first among all the biliary malignant tumors with an increasing incidence in recent years[6-8].The prognosis for the carcinoma is still very poor for its early distant metastasis.It frequently occurs with a lack of specific symptoms,which may constitute the fundamental cause of all above[9,10].As a consequence,early diagnosis and treatment as well as effective control over its infiltration and metastasis is the sticking point provided that better clinical outcome is desired.Nonetheless,the animal tumor models and cell lines with complete biological characteristics are indispensable for the research.Shimura H established ten types of gallbladder cell lines and the only gallbladder cell line GBC-SD in China was established by Liubo[11-14].Although part of their biological characteristics have been investigated and research have been made on some tumor markers,scanty reports have toughed on the mechanism for gallbladder carcinoma infiltration and metastasis as well as relevant oncogenes and anti-oncogenes.In the present study,we aimed at transfecting the cDNA of DMBT1 into the gallbladder cell line GBC-SD and exploring the influence of DMBT1 up-regulation on the biological behavior of gallbladder carcinoma cells in vitro.Immunohistochemistry was primarily applied to investigate the expression of DMBT1 in gallbladder carcinoma and adjacent tissues;Western blot and RT-PCR were in turn employed to detect the mRNA and protein expression level of DMBT1 after transfection of DMBT1 cDNA into the cell line GBC-SD with Lipofectamine and confirmed the success of stable transfection;MTT showed a slackened pace of cell growth and immunofluorescence demonstrated a much higher percentage of apoptotic cells after stable transfection;wound-healing and invasion assays found a descendent ability of cell migration;finally,western blot was used again to probe into the mechanisms of the changes above and found increased level of E-cadherin expression but yet decreased level of CD15 expression;In addition,cell adhesion and aggregation assay manifested an elevated ability of cell aggregating. Taken together,our results indicate that over -expression of DMBT1 in the cell line GBC-SD can significantly inhibit cell growth,induce cell apoptosis in serum-free medium and suspend invasive process,including cell adhesion and migration,which further upholds the previous findings that DMBT1 possesses some properties that classical anti-oncogenes have.