| Objective:Gastric cancer is one of the most harmfulest malignant tumor to human health,and China is one of the highest gastric carcinoma incidence rate areas in the world.Patients after radical gastrectomy,even with adjuvant chemotherapy,the 5-year survival rate was only nearly 40%. Therefore, the etiology and pathogenesis of gastric cancer need further study, it's significance to seek some new therapy targets and intervention strategies to improve the curative of gastric cancer.In recent years, the research of gastric cancer about cell metastasis and cell cycle regulation in the field of prevention and treatment shows enormous potentiality.The researches of control protein in cell level is expected to further elaborate the pathogenesis of gastric cancer,and provide some new targets for the treatment of gastric cancer.We separated 70 differente expression proteins in gastric cancer and adjacent normal mucosa tissues via two-dimensional electrophoresis and mass spectrometry identification in the early study. Then preliminary screening by Western blot and real-time PCR, we selected the protein ERp29 as our research target because it expression decrease obviously and haven't in-depth study in gastric cancer.At first,we test the expression of ERp29 in gastric cancer and adjacent normal mucosa tissues through the tissue microarray and immunohistochemical technique.the results show that the expression of ERp29 in gastric cancer is strongly correlation with clinical pathological parameters such as gender?lymphatic metastasis and TNM stages(P<0.05).Thoes research results show that the ERp29 may play a role in gastric cancer cell proliferation?invasion and metastasis.Methods: At first we identify the expressin of ERp29 in differnt gastric cancer cell lines by western blot,and choose two strains of gastric cancer cell lines for constructe ERp29 overexpression stable cell lines by the method of four plasmid lentivirus infection.Find the correlation between ERp29 and cell proliferation, migration, invasion and other biological behavior gastric cancer cell by CCK-8 assay?clony formation?transwell migration assay?matrigel invasion assay and wound healing. Then constructe silence stable cell lines and the same cell behavior assay to prove the reliability of results.Then prove influence between ERp29 and gastric cancer cells through the in vivo expression. Validated ERp29 alter the abilities of cancer cells proliferation and metastasis through what kind of mechanism by Western-blot. And use the Gene set enrichment analysis to search the mechanism that ERp29 may affect. Verify the mechanism through Western blot.At last use inhibitors to reverse proof ERp29 correlation with the signal path.Results: 1. Validate the expression of ERp29 in different gastric cancer cells through Western-blot,choose the poor differentiation gastric cancer cell MGC803, and the middle differentiation gastric cancer cell SGC7901 for constructe stable gastric cancer cell lines.;2.the result of cancer cell biology behavior experiments in ERp29 overexpression stable cell lines MGC803?SGC7901 show that ERp29 inhibite the gastric cancer cells abilities of migration and invasion.but don't associated with proliferation;3.In contrast,the ERp29 knokdown cell line MGC803 achieve opposite experimental results compare with the overexpression cell line. the sh-ERp29 MGC803 enhancethe cell abilities of migration and invasion,and don't associated with proliferation,too; 4.the results of nude mouse tail vein inject pulmonary metastasis models show ERp29 overexpression group have less and small lung carcinoma nodule in tissue slices; 5.Through real-time PCR we prove ERp29 expression may be associated with RNA level changes of MET markers;And further testify that ERp29 is associated with MET by immunofluorescence and Western-blot.;6.The results of GESA plot show that ERp29 is associated with PI3K/Akt signal pathway.we find that the expression of pAkt(Ser473)?pAkt(Thr308) are decreased in ERp29 high expression gastric cancer cell lines,and GSK3?(Ser9) expression increase obviously; the expression of p-mTOR decrease when ERp29 expression increased,but the expression of ?-catenin has no change through Western-blot;7.Use PI3 K inhibitor LY294002 for sh-ERp29MGC803,the abilities of migration and invasion of the cell line is obviously suppressed, further proof ERp29 inhibite gastric cancer cell lines abilities of migration through PI3K- AKT signal pathways.Conclusion:ERp29 can suppress the abilitise of gastric cancer of the migration and invasion, but hve no influence on the gastric cancer cell proliferation.The mechanism that ERp29 can inhibite the abilitise of gastric cancer of the migration and invasion is through promote MET.The further mechanism is able to through the inhibition of PI3K/Akt signaling pathways leading to MET phenomenon. |
PDF Full Text Request