Mechanism Research On Regulation Of Midkine Gene On Antitumoral Effect Of Cisplatin On Gastric Cancer

Part ? Expression of MK in gastric cancer tissues and cancer cellsObjective:To investigate the different expression of MK between GC tissue and matched adjacent non-tumor tissues,between GC cell lines and normal gastric mucosa epithelial cell lines.Methods:Collected 17 pairs of surgical removal GC specimens.Detect the expression of MK in GC tissue and matched adjacent non-tumor tissues by western blot,H&E-stained and immunohistochemical analysis.Results:1 MK was significantly up-regulated in GC tissues,compared with that detected in matched adjacent non-tumor tissues(p<0.01).2 MK was up-regulated in GC cell lines(803,AGS,MKN45,SGC-7901),as compared with normal gastric mucosa epithelial cell lines(GES-1).The expression of MK in four gastric cancer cell lines were AGS>MKN-45>SGC-7901>803.Conclusion:MK is up-regulated in GC,maybe it plays important roles in the process of GC development.Part ? RhMK attenuates the cytotoxic effect of cisplatin on AGS cellsObjective:To investigate the effects of rhMK on proliferation and apoptosis in AGS cells undercisplatin treatment.Methods:Constructed MK overexpression model by rhMK treatment in vitro,and verify the treatment effect by western blot;CCK-8 assay was used to assess the effect of MK on AGS cell proliferation;the measurement of flow cytometry for Annexin V/PI was used to assess the induction of apoptosis.Results:1 Choose AGS cells as model in vitro,slight increase were observed in AGS treated with rhMK compared to NC control(p>0.05),.but not obviously.2 CCK-8 assay shown that Cisplatin can damage AGS cells,but rhMK can partly reduce the damage by Cisplatin(p<0.05).3 For flow cytometry:the result showed that as comparing to the NS group,the effect of intervene by rhMK on AGS is small.Cisplatin can induce the necrosis and apoptosis of AGS cells obviously,early apoptosis(28.73.±0.97)%,late apoptosis(18.87±0.97)%,necrosis(10.82±1.38)%;and rhMK can reverse the cell damage by Cisplation,early apoptosis(26.55±7.37)%,late apoptosis(14.39±2.53)%,necrosis(4.93±3.82)%?Conclusion:RhMK may attenuate the cytotoxic effect of cisplatin on AGS cells.Part ? The cytotoxic effect of cisplatin on AGS cells is promoted by suppressing MK expressionin vitroandvivoObjective:To investigate the effects of downstream target MK on proliferation and apoptosis in AGS cells and in vivo under cisplatin treatment.Methods:Inhibited the expression of MK by MK-siRNAs in GC cell AGS,and verify the silence efficiency by western blot;CCK-8 assay was used to assess the effect of cisplatin on AGS cells promoted by suppressing MK expression on AGS cell proliferation;the measurement of flow cytometry for Annexin V/PI was used to assess the induction of apoptosis.Immunofluorescence was used to detect the expression of Delta-likel and Jagged 1.Constructednude mice GC tumor model.Inhibited the expression of MK by MK-siRNAs in vivo.Measured the tumor volume and tumor weigh.Immunofluorescence was used to detect the expression of Delta-like 1 and PCNA.Western blot was to assess the effect of cisplatin and MK siRNA on the expression of Notch signaling pathway related proteins in AGS in vitro and vivo.Results:1 Western blot show that significantly decreased MK expression were observed in AGS transfected with MK siRNA compared to NC control(p<0.01).2 CCK-8 assay shown that reduced cell proliferation was observed in si-MK+cisplatin group compared to NC control(p<0.01).3 For Annexin V/PI assay:the result showed that as comparing to the NC control(61.8%),the percentage of apoptosis cells in AGS transfected with si-MK(74%)was distinctly increased.4 For western blot and immunofluorescence assay,comparing to the NC control,the expression of the Notch signaling pathway proteins decreased.5 Significantly decreased the tumor volume and tumor weight were observed in vivo transfected with MK siRNA compared to NC control(p<0.01).6 For western blot and immunofluorescence assay,comparing to the NC control,the expression of the Notch signaling pathway protein decreased in vivo(p<0.01).Conclusion:The cytotoxic effect of cisplatin on AGS cells may be promoted by suppressing MK expression in vitro and vivo and according to regulate Notch signal pathway.