| ObjectiveThe present study was to demonstrate the expression level of integrin ?1 subunit in gastric epithelial cells GES-1 and cell appoptosis and proliferation rates of GES-1 after H.pylori infection.The expression level of integrin ?1 subunit in gastric cancer cell MGC803 and cell adhesion rates,migration rates and cell skeleton morpholory of MGC803 were also indicated in the present study.These results were together to provide more theoretical basis for understanding the pathogenic mechanism of H.pylori.MethodsThe expression level of integrin ?1 in GES-1 cells was detected by flow cytometry,and cell apoptosis and proliferation of GES-1 cells were detected by Flow cytometry and CCK-8 methods respectively after H.pylori infection.The siRNA of integrin ?1 was used to decrease the expression level of integrin ?1 in GES-1 cells.The mRNA expression of integrin ?1 in MGC803 cells was detected by RT-qPCR methods,and the protein expression levels of integrin ?1 in MG3C80 cells were identified by Flow cytometry(FCM)and Western blot.Subsequently,the siRNA of integrin ?1 was used to decrease the expression level of integrin ?1 in MGC803 cells.And MTT was used to test cell adhesion rates,transwell champer was applied to detect cell migration rates and phalloidine was utilized to confirm the skeletal rearrangement.Finally,the signal pathway related protein Paxillin,FAK,PI3K,AKT,mTOR,Grb2 and MAPK were detected by Western Blot.Results1.GES-1 were co-cultured with H.pylori for 12,24 and 48 h.Cell apoptosis rates of the control group were(10.80±0.71)%,(12.23±0.06)%and(12.30±2.12)%at 12,24 and 48 h,respectively.And cell apoptosis rates in GES-1 cells after H.pylori infection were(24.20±0.14)%,(30.05±2.47)%and(26.40±1.91)%at 12,24 and 48 h,respectively.The differences are statistically significant(t=-26.28,-7.701 and-12.855,P<0.05);2.Cell proliferation of GES-1 cells was inhibited at 6,24 and 48 h after bacterial infection,and cell proliferation inhibition rates were(35.00±3.22)%,(40.96±2.45)%and(8.00±3.33)%,respectively.3.GES-1 were co-cultured with H.pylori for 24 and 48 h.The mean fluorescence intensity(MFI)of integrin ?1 were(1616.33±24.70)and(1834.67±17.01)at 24 and 48 h respectively in the control group.The mean fluorescence intensity(MFI)were(1484±60.89)and(1376±14.11)at 24 and 48 h in the GES-1 cells after H.pylori infection.The differences between control and H.pylori infection group were statistically significant(t=3.488 and 35.95,P<0.05)at 24 and 48 h.4.The siRNA of integrin ?1 was used to reduce the expression of integrin ?1 in GES-1 cells.Cell apoptosis rateswere(18.75±3.59)%and(39.75±3.68)%in the control group,and(26.25±4.11)%and(55.30±5.57)%in the siRNA-integrin?1 group at 24 and 48 h,respectively.The difference was statistically significant(t=-2.746 and-4.656,P<0.05).5.The cell proliferation of siRNA-integrin?1 group was inhibited and the inhibition rates were(23.77±2.98)%,(18.42±1.57)%,(16.64±4.57)%and(15.96±5.30)%at 24,36,48 and 72 h respectively.6.MGC803 cells were co-cultured with H.pylori for 24 and 48h,subsequently the expression of integrin ?1 was detected by flow cytometry.The mean fluorescence intensity(MFI)of control group were(4072.33±42.48)and(3779.33±211.50)at 24 and 48 h,respectively.The H.pylori-infection group were(3485.67±109.09)and(2103.00±389.77)at 24 and 48 h,respectively.The differences were statistically significant(t=8.680 and 6.547,P<0.05)between control and H.pylori-infection group.The relative quantity(RQ)of integrin ?1 was 0.0651±0.0293 at 24 h in MGC803 cells treated with siRNA of ?1,and the differences was statistically significant(t=20.609,P<0.05).The relative quantity(RQ)of integrin ?1 was 2.77±1.90515 at 48 h in MGC803 cells treated with siRNA of ?1 and there is no significant differences(t=-1.606,P>0.05);the expression of integrin ?1 in protein levels was detected and the blot in H.pylori-infection group was lighter compared to the control group by Western Blot.7.There are three groups of integrin ?1 siRNA used to decrease the integrin ?1 expression levels.The mRNA expression levels were reduced to 0.04±0.02,0.02±0.01,0.01±0.00 in the three integrin ?1 siRNA groups,which was integrin ?1 siRNA in the control group.The differences among integrin ?1 siRNA groups and control group were statistically significant(t=9.029,9.181 and 9.219 respectively,P<0.05).The third group of integrin ?1 siRNA was selected to further study.The protein expression levels of integrin ?1 siRNA group were significantly decreased in MSC803 cells by flow cytometry and Western Blot.8.Transwell champer was applied to detect cell migration rates of MGC803.The captured photos were imported into the software Image-Pro Plus,by which cell numbers were counted.The numbers that penetrate the membrane were 941.1±2.00,616.72±54.62,848.50±33.91,563.33±54.62,631.60127.27 and 527.67136.83 in the control group,H.pylori-infection group,control-siRNA group,integrin ?1 siRNA group,control-siRNA+H.pylori group and integrin ?1 siRNA+H.pylori group respectively.The results showed that cell numbers penetrated the membrane were significant affected by H.pylori infection and integrin?1 siRNA treatement(t=9.705,P<0.05and t=13.123,P<0.05,respectively).Further,the cell numbers penetrated the membrane in the integrin ?1 siRNA+H.pylori group were significantly different compared to other groups(t= 17.242,2.341,16.717,and 4.757,7.310,P<0.05).9.MTT was used to examine MGC803 cell adhesion rates.Cells were incubated with MTT for several hours and dissolved by DMSO,absorbance was gathered at the wavelength of 570 nm.The OD were 0.56±0.01,0.29±0.01,0.46±0.01,0.36±0.04,0.26±0.01 and 0.18±0.03 in the control group,H.pylori-infection group,control-siRNA group,integrin ?1 siRNA group,control-siRNA+H.pylori group and integrin(31 siRNA+H.pylori group respectively.The results showed that the OD were significant affected by H.pylori infection and integrin ?1 siRNA treatement(t=9.705 and 13.123,P<0.05).Further,the OD in the integrin ?1 siRNA+H.pylori group were significantly lower compared to other groups(t=20.178,5.516,21.701,14.497 and 4.605,P<0.05).10.MGC803 cell cytoskeleton was stained by phalloidin.Compared with control group,H.pylori-infection and integrin ?1 siRNA cells both became small,cell shrinkage and cell edge pseudopodia reduced.The cells could not stretch normally,which spread in the cell culture plate.The results suggested that the cytoskeleton rearrangement might occur in MGC803 cells after H.pylori infection or integrin(31 siRNA treatment.11.The signal moleculars p-Paxillin,p-FAK,FAK,PI3K,AKT,mTOR,Grb2,Ras and ?-actin were detected tby Western Blot.Compared with control group,the p-Paxillin p-FAK,FAK,PI3K/AKT/mTOR,Grb2,Raswere decreased in MGC803 cells after H.pylori infection or integrin ?1 siRNA treatmentConclusions1.The integrin ?1 in GES-1 cells might modulate cell apoptosis and cell proliferation after H.pylori infection.2.The integrin ?1 in MGC803 cells might regulate cell adhesion,cell migration and rearrangement of cytoskeleton after H.pylori infection.3.The signal pathways of FAK-Paxillin,PI3K/AKT/mTOR and Ras/raf/MEK/MAPK might be involved in cell adhesion,cell migration and cytockeleton rearrangement. |
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