Research On Expression And Function Of Rassfia In Human Gallbladder Carcinoma Tissues And Cells

BackgroundGallbladder carcinoma is the most frequent malignant tumor of the biliary system. The clinical patients were mostly in advanced stage due to its hard diagnose early, and its five years survival rate is very low. It is a hot research area to find some new antineoplastic drugs in gallbladder carcinoma comprehensive treatments.Tumor’s generation, development is a process of multi-factor, multi-step and multi-stage, which concerned with the activation of many oncogenes and the inactivation of tumor suppressor genes (TSG). As a tumor suppressor gene, RASSF1encodes more than three isoformes including RASSF1A, RASSF1B and RASSF1C. However, many studies shown that the expression of RASSF1A was found missing in a variety of tumors, such as gallbladder cancer, gastric cancer, bladder cancer, lung cancer, breast cancer, nasopharynx cancer and prostate cancer, etc. Now, aberrant hypermethylation of promoter CpG island is recognized as an important mechanism for gene inactivation as an alternative to gene mutation or deletion in tumorigenesis. Research shown that the promoter of RASSF1A gene was hypermethylation in gallbladder cancer, but its mechanism of action remains unclear in tumorigenesis.ObjectiveOur purpose was to investigate the expression of RASSF1A and the relations between it and the clinical in gallbladder cancer tissues and cell lines. By establishing a gallbladder cancer cell line GBC-SD expressing RASSF1A stably, we studied that the impact of RASSF1A over expressing on the biological behavior of GBC-SD. And it is well worthy to explore the pathogenesis of gallbladder cancer and gene therapy. Methods1.RNA was extracted from frozen tissues of20gallbladder cancer and adjacent tissues, RASSF1A mRNA expression was detected by semi-quantitative RT-PCR and qRT-PCR, and the relation between the expression of RASSF1A and clinical pathologic in gallbladder cancer was analyzed. Besides, the expression of RASSF1A was detected in four gallbladder cancer cell lines by RT-PCR.2.Aberrant promoter methylation of RASSF1A was detected by MS-PCR in gallbladder cancer and adjacent tissues.3.Lentivirus vector was used for establishing the stable gallbladder cancer cell line GBC-SD expressing RASSF1A.4.The impact of RASSF1A over expressing on the biological behavior of GBC-SD was analyzed. The proliferation of transfected cells was examined by MTT assay and Colony forming experiment, and the cell apoptosis and cell cycle were detected by flow cytometry. The migration and invasive ability of transfected cells was measured separately via scratch test and Trans well assay. The nude mouse transplantation tumor experiment was performed in order to observe the impact of RASSF1A over expressing on the tumorigenesis of GBC-SD in vivo. The RASSF1A mRNA and protein expression were detected by immunocytochemical staining of Ki67and Western blotting.Results1. The loss frequency of RASSF1A expression was35.0%in gallbladder cancer. The level of RASSF1A expression and the clinical stages of gallbladder cancer had a close relationship (P<0.05), and it showed low expression in clinical mid-late stage cases (Ⅳ-Ⅴ). However, the level of RASSF1A expression was not related to sex, age, tumor location, tumor size and histological grade. The deletion of RASSF1A expression was detected in the four gallbladder cancer cell lines GBC-SD, NOZ, TGBCITKB and OCUG1, but there was higher expression in normal gallbladder tissues.2. The hypermethylation of RASSF1A in gallbladder cancer is higher than that in adjacent tissues. 3. We successfully built up a GBC-SD-RASSF1A cell line with transfection rate of over90%, which can stably express high level of GFP after two months. The expression of RASSF1A was very low or loss in controls, but it was increased by nearly eight times in GBC-SD-RASSF1A cells.4. The RASSF1A-over-expression significantly inhibited the proliferation and colony formation of GBC-SD in vitro, and induced apoptosis and cell cycle arrest at G1phase, which only had a small impact on cell invasion and migration ability.5. The over-expression of RASSF1A significantly inhibited the growth of tumor. Compared with control groups, the growth of tumor in RASSF1A over expressing group slowed down, its tumor volume and weight all decreased significantly. The positive stain of Ki67was reduced in tumor tissues over expressing RASSF1A. After the tumorigenesis of GBC-SD-RASSF1A cells, the RASSF1A stablely expressed in tumor tissues.Conclusions1.The RASSF1A gene was downregulated or loss in human gallbladder cancer tissues and cell lines, and this downregulation was closely related to the hypermethylation of promoter CpG island.2. GBC-SD-RASSF1A cell line was successfully built up, and the expression of RASSF1A was increased by nearly eight times in the cell line.3.The RASSF1A-over-expression significantly inhibited the proliferation of GBC-SD in vitro, and induced apoptosis and cell cycle arrest at G1phase, which only had a small impact on cell invasion and migration ability.4.The RASSF1A overexpressing can efficiently inhibit the growth of tumor in gallbladder cancer.