The Mechanism And Effect Of RTN4 Protein Over-expression On The Proliferation Of Immortalized Gastric Mucosa Epitheliumgastric GES1 Cells

Objectives: Gastric cancer is a common malignant tumor which is a serious threat to people's health and life in our country.In order to reveal the mechanism of gastric cancer occurrence and development,RTN4 protein was identified from gastric cancer tissues in early study.This study will understand the role and mechanism of gastric cancer occurrence and development,and we can provide experimental data for further elucidate the molecular mechanism of gastric cancer.Methods: 1.The p IRESneo3-RTN4 eukaryotic expression vector was constructed by PCR method.The PIRESneo3-RTN4 eukaryotic expression vector was transfected into immortalized gastric mucosa GES1 cells by liposome transfection.After 500mg/L G418 was screened for 2 weeks,the colony was confluenced and expanded,and then the GES1 cell line of RTN4 over-expression,GES1-RTN4(GES1-p IRESneo3 as control),was established.2.The expression of RTN4 protein in GES1-RTN4(GES1-p IRESneo3)cells was detected by cell immunofluorescence and Western-blot.3.The cell growth curve,plate cloning and soft agar colony forming experiment and flow cytometry were used to observe the effect of RTN4 over-expression on growth and proliferation,cycle and apoptosis in GES1 cells.4.The protein expression of I?B?was detected by Western-blot in GES1-RTN4(GES1-p IRESneo3)cells.Results:1.The results of cell growth curve showed that the growth rate of GES1-RTN4 cells was significantly faster than that of GES1 and GES1-p IRESneo3 cells,and the over-expression of RTN4 promoted the growth and proliferation of GES1 cells(P<0.01).2.The results of plate clone formation experiment showed that the clones numbers of GES1,GES1-p IRESneo3 and GES1-RTN4 cells were 56±8,65±12 and 128±21.The clones number of GES1-RTN4 cells increased significantly than that of GES1 and GES1-p IRESneo3 cells,and the clone size was large(P<0.01).The results showed that the colony formation ability of GES1 cells was increased significantly after over-expression of RTN4.3.The soft agar colony formation assay showed that the colone numbers of GES1,GES1-p IRESneo3 and GES1-RTN4 cells were 64±12,72±16 and 146±28.The colony formation of GES1-RTN4 cells significantly increased(P<0.01),and the clone size was large.The colony forming ability of GES1 cells increased significantly after RTN4 over-expression.4.The flow cytometry showed that the percentage of S phase cells was 45.63±4.62,the percentage of G1 phase cells was 49.57±8.49,and the cell proliferation index was 50.43 in GES1 cells.The percentage of S stage cells was 30.10±6.28,the percentage of G1 phase cells was 48.75±7.86 and the cell proliferation index was 51.25 in GES1-p IRESneo3 cells.The percentage of S stage cells was 55.28±9.15,the percentage of G1 phase cells was 26.88±3.42,and the cell proliferation index was 73.13 in GES1-RTN4 cells.The results showed that the proportion of S phase cells increased significantly,the proportion of G1 phase cells decreased and the cell proliferation index increased after the over expression of RTN4 in GES1 cells(P<0.01).Meanwhile,the apoptotic rate of GES1,GES1-p IRESneo3 and GES1-RTN4 cells were respectively 24.65±4.58,24.43±3.36 and 17.52±1.23.The results showed that the apoptosis rate of the cells decreased after the over-expression of RTN4 in GES1 cells(P<0.01).5.The Western-blot results showed that the expression of I?B? protein in GES1-RTN4 cells decreased compared with that of GES1 and GES1-p IRESneo3 cells(P<0.01).Conclusions: RTN4 promotes the proliferation of GES1 cells by activating the NF-?B signaling pathway to increase the cell proliferation index and inhibit the apoptosis.