Functions And Associated Mechanisms Of The Immune Homeostatic Molecule Tipe2in The Carcinogenesis Of The Gastric Cells

Objective:To identify the function of a novel immune homeostasis molecule TIPE2in the developmental process from gastritis to gastric cancer, and attempt to interrogate the molecular mechanisms of TIPE2inhibiting the proliferation of gastric cells, thereby to provide a new clue for the study of stomach diseases.Methods:1. At the tissue level, immunohistochemistry (IHC) was applied to detect the TIPE2protein in tissue specimens of gastritis and gastric cancer. Meanwhile, the qRT-PCR was employed to examine the mRNA of TIPE2, as well as some molecules associated with cellular proliferation, such as N-ras, p21CIP1/WAF1,p53, p27, Bcl-2, etc;2. At cellular level, human TIPE2expression plasmid pRK5-tipe2was used to transfect gastric epithelial cells, triggering upregulated expression of the TIPE2. The effects of TIPE2on proliferation and migration were examined in gastric epithelial cell lines as following details:(1) Transfection efficiency estimation:TIPE2expression plasmid pRK5-tipe2and its control plasmid pRK5-mock were transfected into the gastric epithelial cells lines AGS and BGC-823. After48hours and72hours incubation, the TIPE2expressed on mRNA and protein levels were tested via qRT-PCR and western blotting respectively.(2) The colony formation assay, various concentrations of TIPE2expression plasmids and its control plasmids were transfected to AGS and BGC-823cell lines. After48hours, the cells were cultured in the six-well-plates and the cell number was normalized, then the cell clones resolved were counted.(3) Wound healing assay:the adherent AGS and BGC-823cells with TIPE2overexpression were scratches to disrupt the cellular growth and gaps was created with sterile blue pipette tips, medium was changed every24hours and cell growth of the wounded area was observed with a microscope;3. To explore the functions of TIPE2on cell cycle regulation, the AGS and BGC-823cells were tranfected with TIPE2expression plasmid.24hours,48hours and72hours later, cells were collected and flow cytometry analysis (FCS) was performed to investigate the stages of the cell cycle the cells were in.4. Microarray was utilized to screen the possible molecules involved in TIPE2inhibiting cell proliferation; to reveal the molecular pathways related to the proliferation inhibition of gastric epithelial cells:qRT-PCR and western blotting were used to detect the expression changes of cell cycle-related molecules at mRNA and protein levels in TIPE2over-expressed cells.Results:1. Immunohistochemistry results showed that TIPE2protein highly expressed in normal stomach tissues, yet rarely expressed in the stomach chronic inflammatory tissues, even no expression in gastric carcinoma tissues; qRT-PCR data confirmed the immunohistochemistry result, suggesting that the expression of TIPE2really changed in the development of stomach disease;2. After transfected with TIPE2expression plasmid, protein level of TIPE2was proved a significant increment in the gastric epithelial cells. Although cell colony formation ability of the transfected cells was significantly inhibited, yet little difference in its wound healing capability was discovered. These results demonstrated that TIPE2was able to inhibit the proliferation of gastric epithelial cells whereas had no function on their migration.3. FCS results revealed that TIPE2overexpression reduced the ratio of cells in S phase, therefore raised the ration of cells in G1phase, implying that TIPE2is able to inhibit the proliferation of gastric epithelial cells by preventing their transition to S phase.4. The biochips and qRT-PCR results revealed the molecules involved in TIPE2triggered cellular proliferation inhibition, such as N-ras, p21CIPI/WAF1,P27, P53, CDK2, as well as the apoptosis associated gene, Bcl-2.Conclusion:1. TIPE2is down-regulated in the gastritis and gastric cancer tissues and negatively correlates with the development of stomach disease.2. TIPE2reduces the cell proliferation of gastric epithelial cells by repressing their transition to S phase, but has no effect on cell migration.3. TIPE2targets N-ras and its downstream p21CIP1/WAF1/p27/p53molecules to inhibit cellular proliferation, revealing the roles of this new immune homeostasis molecule in gastric disease.