Effects Of P130CAS On The Proliferation And Apoptosis Of Gastric Cancer Cells

Aim: To detect the expression of p130CAS gene in gastric cancer cells，clarifythe effects of overexpression or knock down of p130CAS on proliferation andapoptosis of gastric cancer cells，explore effects of p130CAS on the malignantbiological phenotype and possible molecular mechanism of gastric cancer，clarify thepotential value of p130CAS in the treatment of gastric cancer.Methods:1.Lentivirus expressing p130CAS-targeting siRNA and p130CASwere produced in AGS cells by using the corresponding plasmid and the packagingplasmids. AGS cells were infected with LV-siRNA-Control,LV-p130CAS-siRNA,LV-Control or LV-p130CAS, and stable transfectants were collected. Western-blotwere used to confirm the validation of lentivirus.2.The phosphorylation levels of p130CAS were detection by Westernblot onAGS/p130CAS expression, AGS/p130CAS block and the corresponding control celllines.3.Study effects of cells growth of AGS cells infected with recombinant lentiviralvector harboring RNAi sequence and overexpression sequence. The absorbancevalues of each cell groups was performed by MTT analysis. Comparison theproliferation of each cell groups after lentivirus transfection.4.Study effects of apoptosis of AGS cells infected with recombinant lentiviralvector harboring RNAi sequence and overexpression sequence. Using AnnexinV-PIflow cytometry experiment to detect the apoptosis rate of each cell group, andcompare between these groups.Results:1.The recombinant lentiviral vectors carrying human p130CAS-targetingsiRNA and p130CAS were constructed successfully. The recombinant lentiviralvector can efficiently infect AGS cells.The expression levels of p130CAS mRNA andprotein of AGS cells infected with recombinant lentiviral vector carrying human p130CAS enhanced, compared with either negative control group or uninfected cellsgroup. And，the expression levels of p130CAS mRNA and protein of AGS cellsinfected with recombinant lentiviral vector carrying human p130CAS-targetingsiRNA reduced, compared with either negative control group or uninfected cellsgroup.2. Phosphorylation of p130CAS can be detected in all gastric cancer cells westudied.The phosphorylation levels of AGS/p130CAS blocking cell lines aresignificantly decreased, while obviously increasing of the phosphorylation level hasbeen found in the AGS/p130CAS high expression cell lines.3.Cells were harvested3d post infection, absorbance values of cells wasmeasuered by MTT. Viability of cells infected with recombinant lentiviral vectorcarrying human p130CAS-targeting siRNA was lower than the cells infected withnegative control group and uninfected AGS cells (P<0.05).However，AGS cellsinfected with recombinant lentiviral vector carrying human p130CAS were notobvious on the effect of cell growth.4. FACS analysis was used to assess apoptosis. The apoptotic increasedstatistical significantly compared with uninfected AGS cells and negative controlgroup. However，compared with uninfected AGS cells and negative control group，theapoptotic decreased in AGS cells infected with recombinant lentiviral vector carryinghuman p130CAS.Conclusion:1.Lentiviral vector can efficienctly infect the gastric cancer cell linesAGS cells.2. Phosphorylation of p130CAS can be detected in all gastric cancer cellswe studied.3.Downregulation of p130CAS gene expression levels can inhibit theproliferation and promote apoptosis of gastric cancer cell lines AGScell.4.Upregulation of p130CAS gene expression can inhibit the apoptosis of gastriccancer cell lines AGS cells，but has no obvious influence on the proliferation of AGScells.