The Effect Of CD44v3 On The Invasion Of Lung Adenocarcinoma Cell Line A549

Part one The effect on the invasion of lung adenocarcinoma cell line A549 by interferering with CD44v3Objective To study the effect of CD44v3 on the invasion of lung adenocarcinoma cell line A549.Methods The plasmid expression vector was constructed by pRNAT-H1.1/Neo(6.1kb) which can transcribe the short hairpin RNA (shRNA) stably and can interfere with the expression of CD44v3. After the vector was transfected to the cell line A549 the CD44v3 level was examined by the fluorenscent immunohistochemical approach and Western Blot. A in vitro invasion model of cancer cells was made by the Boyden chamber and the artificial matrix membrane. The number of the cells through the artificial matrix membrane in every group was counted to compare the change of the invasion capability with each other.Result It was detected first that the lung adenocarcinoma cell line A549 can express CD44v3, the molecular weight of which is 130 kd. The plasmid expression vector we made can knock down CD44v3 level expressed from the cell line A549 effectively. When the transfection efficiency was 48.1% the CD44v3 level was cut down 46%. The invasion capability of cancer cells of the interference group and the empty plasmid group was 4.0±1.22 and 13.20±4.92 respectively from the in vitro invasion model of cancer cells. The invasion capability of cancer cells of the former reduced 70% when compared with the latter. The difference between them is significant and P<0.01. As the same as the interference group, the monoclonal antibody of CD44v3 (positive control group) can reduce the invasion capability of the cell line A549 also. When compared the positive control group with the interference group the difference between two groups was not significant and P>0.05.Conclusion Lung adenocarcinoma cell line A549 can express CD44v3. The technique of RNAi can knock down the expression. The invasion capability of cell line A549 can be repressed in vitro when the expression of CD44v3 is knock-down.Part two CD44v3 expressed on the lung adenocarcinoma cell line A549 and its invading behavior in vivoObjective It is the purpose to study the relationship between CD44v3 expressed on lung adenocarcinoma cell line A549 and its invading behavior in vivo.Methods We have constructed previously a CD44v3 interfering RNA plasmid expressing vector and transfected it successfully into the A549 cell. According to the different A549 cells in experiment we sorted the experiment into three groups. Wild group is the A549 cells untreated and empty vehicle group is the A549 cell transfected with empty plasmid and interference group is the A549 cells transfected with the CD44v3 interfering RNA plasmid expressing vector. Suspended cell was made for three groups and these three group cells were inoculated into athymic nude BALB/c mice subcutaneously. The growth of the implant was observed and the tumor with ambient tissue including skin was resected for sampling in the seventh week when the nude mice were sacrificed and the the skin of the tumor ulcerated. Pathological section and immunohistochemistry were examined under microscope which were stained with hematoxylin and eosin (HE) and diaminobezidine (DAB) respectively. The transplanted success rate, growth incubation period, tumor size, the extent of the tumor invading ambient tissue and photometry on image analysis among three groups were compared.Result Although the transplanted success rate is different among three groups there is no significant difference on statistics. The growth incubation period of the interference group is longer than that of wild group and empty vehicle group whereas there is no difference between the two latters. The tumor sizse of is smaller than that of wild group and empty vehicle group whereas there is no difference between the two latters. It can be observed in the wild group and the empty vehicle group clearly under microscope by HE staining that the tumor invade ambient tissue in some extent which is not present in the interference group. The immunohistochemistry staining demonstrate the color of the wild group and the empty vehicle group is as the same as the positive controll of gastric carcinoma and there is no difference on the photometry of image analysis among them. The color of interference group is as the same as the negative controll and there is no difference on the photometry of image analysis between them. There are some cancer cells invading into the tissue around the tumor in the wild group and the empty vehicle group which is not present in the interference group.Conclusion The invading behavior of lung adenocarcinoma cell line A549 is associated in vivo with its expression of CD44v3 and can be restrained by knock-down the expression of CD44v3.