Inhibitory Effect Of Filamin A On Tumor Formation Activity Of SW480 Cells In Nude Mice

Objective: Filamin A(FLNa)were discovered as the first family of non-muscle actin-binding protein, which is mainly distributed in the cytoplasm. FLNa was originally considered to stabilize delicate three -dimensional actin webs. Studies shown that expression of FLNa is lower in colorectal cancer tissue than that in colorectal normal tissue. Expression level of FLNa was positively correlated with the TNM staging,liver metastasis, lymph node metastasis, and the depth of tumor invasion,but not with the patient's age, gender, lesion site, tumor size, the grade of tumor differentiation, gross type, and histological type. In this study, we intend to explore the inhibitory effect of FLNa gene on tumor formation activity of human colon carcinoma cell line SW480 in nude mice.Methods: First, preparation for related reagent to this experiment, then the cell culture. Expressing vector pcDNA3.1/V5-His-TOPO/ FLNa was transfected into SW480 cells by lipofectamine protocols. In the study, we determined the concentration of G418 selection. After G418 selection, the cells SW480/FLNa expressing FLNa stably were obtained. Express- ion of FLNa in tumor cells was confirmed by reverse transcription polymerase chain reaction(RT-PCR) and Western blot. We obtained the SW480/FLNa that stably express FLNa. Cell proliferation in vitro were assessed by MTT assay, the incubation time for the horizontal axis (X axis), the absorbance value for the vertical axis (Y axis), cell growth curve. First, 30 nude mice were divided into 3 groups randomly. Then take the SW480/FLNa, SW480/ pcDNA3.1 and SW480 cells were subcu- taneously injected into nude mice to observe the formation of tumors. After 8 weeks ,all the nude mice were killed, dissection of tumor and weighing, and inhibitory rate of the tumor were measured. The tumor tissue after fixation, dehydration and transparent, wax-embedded baptist and dewa-xing treatment into slices, after HE staining, the morphology of tumor were observed under optical microscope. Finally, application the SPSS 13.0 statistical software for data analysis and processing. And the data using t test, ANOVA, Dunnett-t test was used for correlation test. Set the test levelα= 0.05, P <0.05 was considered statistically significant.Results:1 By using RT-PCR detected the three groups of SW480 cells showed that: SW480/FLNa, SW480/pcDNA3.1 and SW480 cells, mRNA expression levels were 1.27±0.11, 0.12±0.02 and 0.14±0.03. SW480/FLNa group is higher than SW480 group (P = 0.019 <0.05), that is no statistically significant difference between SW480/pcDNA3.1 group and SW480 group(P=0.865> 0.05); The Western blot showed that, SW480 /FLNa, SW480/pcDNA3.1 and SW480 cells, protein expression levels were 0.78±0.03, 0.01±0.00 and 0.01±0.00. SW480/FLNa group is higher than SW 480 group (P = 0.008), That is no statistically signi- ficant difference between SW480/pcDNA3.1 group and SW480 group (P = 0.943). FLNa gene was stably expressed in SW480 cells.2 MTT assay results showed that no statistically significant difference between the three groups cells absorbance in each 24h, the cell growth curve of basic overlap, so the proliferation of different SW480 cells were similar in vitro.3 The experiments showed that tumor growth of SW480/FLNa group is the slowest in three groups. Compared with SW480 group, growth rate and weight of the tumors in SW480/FLNa group were significantly restrained, and the experimental statistical data showed that [(0.67±0.06)g vs (5.90±0.77)g, P= 0.000];the inhibition rate of tumors were increased at 88.7±3.5%(P=0.000).4 Degeneration and necrosis were found in tissues of SW480/FLNa group by microscopical observation.Colusion:1 From the experiments we can recognized that tumor growth of SW480 /FLNa group is the slowest in three groups. 2 In the microscope showed that SW480/pcDNA3.1 and SW480 tumor tissu- es of nude mice showed nests, significantly shaped tumor cells, nuclear chromatin, dark, showed more split-phase,degeneration and necrosis were found in tissues of SW480/FLNa group.In summary, FLNa gene may inhibit the tumor formation activity of SW480 cells in nude mice. It tips that FLNa gene can inhibit of tumor formation. Research for the mechanism, could provide new ideas for tumor therapy.