PRL-3 Promotes The Peritoneal Metastasis Of Gastric Carcinoma Through PI3K/Akt Signaling Pathway By Regulating PTEN

PURPOSE:To investigate whether PRL-3 regulates the expression of PTEN through its phosphatase activity,and further by regulating the PI3K/Akt signaling pathways that leads to the peritoneal metastasis of gastric carcinoma.METHODS:(1)Detect the protein expression of PRL-3,PTEN and p-PTEN by Western blot in 21 specimens of normal gastric mucosa,49 specimens of gastric carcinoma without peritoneal metastasis,and 23 specimens of gastric carcinoma with peritoneal metastasis.We also detect the enzyme activity of PRL-3 in these tissues.(2)Detect the protein expression of PRL-3,PTEN and p-PTEN by Western blot,the enzyme activity of PRL-3 in a human gastric mucosa cell lines GES-1 and five human gastric adenocarcinoma cancer cell lines SGC7901,MKN28,MGC803,AGS and MKN45.(3)Construct a wild type plasmid EGFP-PRL-3 and a mutant type plasmid EGFP-PRL-3(D72A/C104S),which was transfected into SGC7901 cells.Furthermore,we detected the enzyme activity of PRL-3 and the protein expression of PRL-3,PTEN and p-PTEN by Western blot,(4)Construct the PRL-3 high expression plasmid of EGFP-PRL-3 and PRL-3 interference plasmid of PRL-3-RNAi,which were transfected into SGC7901 cells.Furthermore,we detected the enzyme activity of PRL-3 and the protein expression of PRL-3,PTEN,p-PTEN,Akt,p-Akt,MMP-2 and MMP-9 by Western blot.(5)Construct a PTEN-knockdown plasmid PTEN siRNA and transfect it into the PRL-3 low expression cell lines.Furthermore,we detected the enzyme activity of PRL-3 and the protein expression of PRL-3,PTEN,p-PTEN,Akt,p-Akt,MMP-2 and MMP-9 by Western blot.RESULTS:(1)In the gastric normal gastric mucosa and gastric carcinoma tissues,the level of PRL-3 protein expression was as follows: the gastric carcinoma with peritoneal metastasis>the gastric carcinoma without peritoneal metastasis> the normal gastric mucosa.The level of PRL-3 enzyme activity was as follows: the gastric carcinoma with peritoneal metastasis> the gastric carcinoma without peritoneal metastasis> the normal gastric mucosa.The level of PTEN protein expression was as follows: the normal gastric mucosa> the gastric carcinoma without peritoneal metastasis> the gastric carcinoma with peritoneal metastasis.The ratio of p-PTEN/PTEN was as follows: the gastric carcinoma with peritoneal metastasis> the gastric carcinoma without peritoneal metastasis> the normal gastric mucosa.(2)Changes in the expression level of protein in the cell lines was as following,PRL-3 expressed higher in gastric cancer cell line than that of normal gastric mucosa cell lines,SGC7901 cells was the highest.PRL-3 phosphatase activity was higher in gastric cancer cell line than that of normal gastric mucosa cell lines.PTEN expression level in the gastric cancer cell lines was below than the normal gastric mucosa cell lines.The ratio of p-PTEN/PTEN in the gastric cancer cell lines was higher than the normal gastric mucosa cell lines.(3)The enzyme activity of PRL-3 was that the wild type was higher than the mutant type.The protein expression level of PRL-3 was that both of the wild type and the mutant type were higher than the control group.PTEN expression level was that the wild type was lower than the mutant type.The ratio of p-PTEN/PTEN was that the wild type was higher than the mutant type.The cell lines invasion and metastasis ability was that the wild type was higher than the mutant type.(4)PRL-3 phosphatase activity in PRL-3 high expression cell lines was higher than PRL-3 low expression cell lines.In the PRL-3 high expression cell lines,the Akt protein expression was increased and the ratio of p-Akt/Akt was also higher,at the same time,the expression of MMP-2 and MMP-9 was also higher in the PRL-3 high expression cell lines than in the PRL-3 low expression cell lines.When we added MMP-2/MMP-9 inhibitor I into the PRL-3 higher expression cell lines,the cell invasion and metastasis ability was decreased.(5)After adding the PTEN siRNA plasmid into the PRL-3 low expression cell lines,the expression of PTEN was decreased,the ratio of p-Akt/Akt and the expression of MMP-2 and MMP-9 was partly increased,the cell invasion and metastasis ability were also partly recovered.CONCLUSION:PRL-3 down-regulates the expression of PTEN,further to activate PI3K/Akt signaling pathway,raise the expression of MMP-2 and MMP–9,leading to promote the peritoneal metastasis of gastric carcinoma.