| Objective To discuss the role of gastrokine 2(GKN2) on proliferation, migration, invasion, cell cycle and apoptosis in CD44(+)SGC7901 gastric cancer stem cells(GCSCs), and have preliminary illustration on the inhibitory effect of GKN2 on GCSCs,which may provide theoretical basis for the clinical design of effective strategy of gastric cancer.Methods1. GKN2 plasmid vector was constructed transiently transfected into human gastric cancer cell line MKN28, and thenthe transfection efficiency was observed under fluorescence microscope. Western Blot assay was performed to verify whether GKN2 transfection was successful.The secretory protein was collected from culture medium of MKN28 cells transfected by GKN2 plasmid vector, and then purified and concentrated by ultrafiltrationtechnology. BCA proteinquantitative method was employed to quantitate the secretoryprotein. The secretory protein was finally identified to be GKN2 by Coomassie Brilliant Blue Staining and Western Blot.2. CD44(+)cells were separated from SGC7901 cells by Magnetic cell sorting(MACS) technique, and then analyzed the sortingratio using immunofluorescence.3. The role of GKN2 on proliferation, migration, invasion, cell cycle and apoptosis were analyzed by CCK-8, Tumor sphere formation assay,Transwell migration assay, Transwell invasion assay and Flow Cytometry in GCSCs. In addition, we also compared the inhibitory effects of GKN2 with 5-Fu and VP-16 in GCSCs by CCK-8.Results1. The transfection experiment showed that the transfection efficiency of GKN2 plasmid vector was about 91%. Western Blot indicates that the relative expression of GKN2 proteinwas significantly higherin GKN2 transfected group(1.037±0.030) than that in the non-transfected group(0.014±0.003) and that in empty vector group(0.015±0.004)(P < 0.05), which suggested transfection successfully.2. It showed thatthe concentration of the total protein was 27.4g/L and 22.4g/L in transfected GKN2 group and non-transfected GKN2 group respectively after the secretory protein was collected, purified and concentrated. Coomassie Brilliant Blue Staining and Western Blot displayed that the secretoryprotein was GKN2.3. Immunofluorescence results indicated that the CD44(+)cells presented green fluorescence on the cell membrane. The positive rate of CD44 in SGC7901 cells before sorted was only 9%, however, the positive rate in CD44(+)SGC7901 cells was 97%, which suggested that sorting GCSCs from SGC7901 cells was successful.4. CCK-8 test revealed that 10, 20, 40, 80, 160, 320 mg/L of GKN2 protein can inhibit the proliferation of GCSCs after treated for 24 h, 48 h and 72 h, and their inhibitory effect was concentration-time dependence(P<0.05). The IC50 value of GKN2 to GCSCs was 30mg/L.We also found that the inhibitory effect of GKN2 was stronger than that of 5-Fu and VP-16 respectively.5. Tumor sphere formation assay showed that the number of spheres in GKN2 treated group(15.1±0.45) was notably less than that in untreated group(38.6±0.62) and non-GKN2 control group(38.0±0.44)respectively(P < 0.05). Furthormore, The spheres’ diameters and transmittance in GKN2 secretory protein treated groupwere smaller and higher than that in untreated groupand non-GKN2 control grouprespectively(P < 0.05), prompting that GKN2 has a significant inhibitory effect on the sphere formation of GCSCs.6. Transwell migration assay revealed that the amount of migrating cells in GKN2 treated group(69±3)was obviously lower than that inuntreated group(174±6)and non-GKN2 control group(173±7)(P<0.05)respectively. Similarly, transwell invasion assay displayed that the number of cells which transfered from the ring of matrix to the lower chamber in GKN2 treated group(36±6)was significantly lower than that in untreated group(94±12)and non-GKN2 control group(93±9)respectively(P<0.05). All above suggested that GKN2 can remarkably inhibit the migration and invasion ability of GCSCs.7. Flow Cytometry detection results demonstrated that the cell proportion of S phase markedly decreasedin GKN2 treated group,compared with untreated group and non-GKN2 control group(P<0.05);so did the proliferation index.However, the cell proportion of G0/G1 phase increased significantly(P<0.05). The above results indicated that GKN2 can suppress the proliferation and arrest the cells in the G0/G1 phase in GCSCs. Flow Cytometry method also showed that the cell apoptosis rate in GKN2 treated group(15.540±0.530) was obviously higher than that in untreated group(8.083±0.480) and non-GKN2 control group(7.543±0.471)(P< 0.05), which indicated that GKN2 can promote the apoptosis of GCSCs.ConclusionGKN2 can induce proliferation inhibition and apoptosis, G0/G1 phasearrest and suppressthe migration and invasion ability in gastriccancer stem cells. |
