Study Of TIPE2 Regulates Gastric Cell Proliferation Through IRF4-induced Signaling

Objective:To identify the function of the negative regulator of TLR signaling TIPE2 in the development process from gastritis to gastric cancer,and study the molecular mechanisms of TIPE2 inhibiting the proliferation of gastric cancer through regulating IRF4.Methods:l.at the tissue level,immunohistochemistry(IHC) was applied to detect the TIPE2 protein in tissue specimens of gastritis and gastric cancer.Meanwhile,the qRT-PCR was employed to examine the mRNA of TIPE2. Extract RNA from the Liver,kidney, intestine,heart,stomach tissue of the TIPE2 knockout mice. Detect the levels of mRNA and protein of IRF4 through Western blotting and qRT-PCR.2.At cellular level,human TIPE2 expression plasmid pRK5-tipe2 was used to transfect gastric epithelial cells,triggering upregulated expression of the TIPE2.The effects of TIPE2 on proliferation was examined in gastric epithelial cell lines as the following details:(1)Transfection efficiency estimation:TIPE2 expression plasmid pRK5-tipe2 and its control plasmid pRK5-mock were transfected into the gastric epithelial cells line AGS.After 48 hours and 72 hours incubation,the TIPE2 expressed on mRNA and protein levels were tested via Western blotting and qRT-PCR respectively.(2)The colony formation assay:various concentrations as 2ug,4ug.8ug of T1PE2 expression plasmids and its control plasmids were transfected to AGS cell line.After 48 hours,the cells were cultured in the six-well-plates and the cell number was normalized,then the cell clones resolved were counted.3.To explore the function of TIPE2 on cell cycle regulation,the AGS cells were transfected with TIPE2 expression plasmid.24 hours,48 hoursand 72 hours later,cells were collected and flow cytometry analysis was performed to investigate the stages of the cell cycle the cells were in.4.Microrray was utilized to screen the possible molecules involved in TIPE2.Inhibiting cell proliferation;To reveal the molecular pathways related to the proliferation inhibition of gastric epithelial cells:Western blotting and qRT-PCR were used to detect the expression changes of cell cycle-related molecules at mRNA and protein levels in TIPE2 over-expressed cells.Results:1.immunohistochemistry results showed that TIPE2 protein highly expressed in normal stomach chronic inflammatory tissues,yet rarely expressed in the stomach inflammatory tissues,even no expression in gastric carcinoma tissues;qRT-PCR data confirmed the immunohistochemistry result,suggesting that the expression of TIPE2 really changed in the development of stomach disease.2.After transfected with TIPE2 expression plasmid,protein level of TIPE2 was proved a significant increment in the gastric epithelial cells.These results demonstrated that TIPE2 was able to inhibit the proliferation of gastric epithelial cells.3.flow cytometry analysis results revealed that TIPE2 overexpression reduced the ratio of cells in S phase..therefore raised the ration of cells in G1 phase, implying that TIPE2 is able to inhibit the proliferation of gastric epithelial cells by preventing their transition to S phase.4.The biochips and the results of Western blotting revealed the expression of IRF4 positively correlates with TIPE2;and its expression decreased significantly in various organs of the TIPE2 knockout mice.5.It is further demonstrated that TIPE2 inhibit the proliferation of gastric epithelial cells by regulating IRF4 through transfecting Si-IRF4-RNA to TIPE2 transfeced cells. 6.Pathway inhibitor results show that TIPE2 regulate IRF4 by NF-κB signaling pathwayConclusion1.TIPE2 is down-regulated in the gastritis and gastric cancer tissues and negatively correlates with the development of stomach disease. 2.TIPE2 reduces the cell proliferation of gastric epithelial cells by repressing their transition to S phase.3.TIPE2 inhibit the proliferation of gastric epithelial cells by upregulating the expression of IRF4 through the NF-κB signaling pathway.