The Primary Research Of The Effect And Mechanism Of Pokemon On The Expresion Of Smad4 In Gastric Cancer Cells

ObjectiveTo explore the effect and mechanism of Pokemon on the expression of Smad4 in gastric cancer cells.MethodsThe gastric cancer cell SGC7901 and MGC803 in vitro culture was the research object. 1. Q-PCR technology and Western blot assay were performed to detect the expressions of Smad4 level in SGC7901 and MGC803 cells. 2. Different lengths of the Smad4 promoter were amplified by PCR and cloned into the plasmid p GL4.10-Basic. Then the constructed recombinent plasmids(named Smad4PP1/ Smad4PP2/ Smad4PP3) were identified by PCR, enzyme digestion and DNA sequenceing.and then three plasmids(Pokemon/p GL4.10-Basic?PTK?Smad4PP1?Smad4PP2?Smad4PP3) co-transfected into SGC7901 and MGC803 cells. The dual- luciferase Reporter Assay System was used to quantitate the reporter vector luciferase activity.3. The relationship between overexpression of Pokemon and EPCAM+/CD44+ gastric cancer stem cells was detected by the flow cytometry.Results1.Pokemon could inhibits Smad4 express in human gastric cancer SGC7901 and MGC803 cells Western blot assay suggested that Pokemon functions act as a negative regulator in Smad4.2. Pokemon could inhibits Smad4 express through the inhibition of Smad4 promoter activity in human gastric cancer SGC7901 and MGC803 cells.1)A Smad4 promoter- luciferase reporter vector was constructed.The three kinds of promoter fragment of Smad4 gene were successfully constructed and cloned into the p GL4.10-Basic reporter vector,and named Smad4PP1?Smad4PP2?Smad4PP3. 2) Analysis of p GL4.10-Smad4-promoter luciferase activity. The luciferase activity of the promoter construct was significantly higher than that of the p GL4.10-Basic promoter in SGC7901 and MGC803 cells,the difference was statistically significant between them(P<0.05). 3) Pokemon could inhibited Smad4 promoter express in human gastric cancer SGC7901 and MGC803 cells.3. Pokemon could upregulated EPCAM+/CD44+ the radiation of gastric cancer stem cells SGC7901 and MGC 803 cells.Conclusion1. Pokemon could inhibited Smad4 express through the inhibition of Smad4 promoter activity in human gastric cancer SGC7901 and MGC803 cells.2. Pokemon could upregulated EPCAM+/CD44+ the radiation of gastric cancer stem cells SGC7901 and MGC 803 cells.