| Purpose Research about cell signal pathway is one of important methods in an attempt to reveal the tumorigenesis.Win/?-cantenin signaling pathway is conservative and mainly regulates cell growth,development and differentiation.If Win/?-cantenin abnormally activate or disorder,cell will inevitably be leaded to abnormalities in its differentiation,growth and biological behaviour.COX-2 is the rate-limiting enzyme when arachidonic acid transform into prostaglandins and oncogene correlative inflammation.COX-2 often causes tumor occurrence and development through promoting cell proliferation and suppressing cell apoptosis.A large number of researches have revealed that COX-2 over-expression is closely related to the occurrence and development of gastric cancer.Meanwhile,many studies have showed Wnt/p-catenin signaling pathway is abnormally activated in gastric cancer.The relationships between COX-2 over-expression and abnormal activation of Wnt/?-catenin signaling pathway in gastric cancer is unclear.Therefore,the purpose of this study to reveal whether COX-2/PGE2 regulates expressing activity of Wnt/?-catenin signaling pathway or not by means of investigating the expression changes of?-catenin,C-myc and CyclinD1 which are key molecule of Wnt/?-catenin signaling pathway when COX-2/PGE2 expression is varied in human gastric cell line.This research may provide experimental evidence and theoretical foundation for COX-2 gene therapy to target tumor suppressed.Methods 1.Reverse transcription-polymerase chain reaction(RT-PCR)and Western Blotting were used,respectively,to detect the expression changes of ?-catenin,C-myc and CyclinD1,which are key molecule of Wnt/?-catenin signaling pathway,when the expression of prostaglandin E2(PGE2)was increased in human gastric cancer lines MKN-45 and SGC-7901.Cell counting kit-8(CCK-8)assay was employed to investigate cell proliferation after human gastric cancer cell lines MKN-45 and SGC-7901 were cultivated in moderately going up PGE2 medium density.2.COX-2 SealthTM siRNA,a small interfering RNA,which targeted to COX-2 gene was synthesized by chemical way according to the design principles.Then,COX-2 SealthTM siRNA was transfected into human gastric cancer cell lines MKN-45 and SGC-7901 with Lipofectamine2000 and COX-2 expression level both mRNA and protein were assayed to assure COX-2 gene knock-down.3.Under the condition of COX-2 gene down-regulation,the expression changes of ?-catenin,C-myc and CyclinD1 in human gastric cancer cell lines MKN-45 and SGC-7901were detected by RT-PCR and Western Blotting.In above condition,the cell viability of human gastric cancer cell lines MKN-45 and SGC-7901 was evaluated by CCK-8.4.The changes of cell cycle and apoptosis were assessed by applying with flow cytometry when COX-2 gene is kept knock-down.Result:There is no significant difference of their proliferative activity between 0.01umol/l PGE2 and control group after human gastric cancer cell lines MKN-45 and SGC-7901 incubated with after 24h,48h and 72h.Compared with control,the proliferative activity of 0.1-1.0umol/l PGE2 group have significantly difference(p<0.01)and showed a dose-dependent increasing manner,but in 10umol/l PGE2 group revealed no difference after 24h,48h and 72h.10umol/l PGE2 displayed cytotoxicity and inhibited cell proliferation.Compared with control,the expression of ?-cantenin,CyclinD1 and C-myc,both in mRNA and protein levels,showed a significant difference with uptrend(P<0.05)in 0.1umol/l,1.0umol/l PGE2 group respectively.Compared with control,however,their expression was no significant difference in 0.01umol/l and in 10umol/l PGE2 group.The results also showed protein level of intracellular phospho-GSK-3? was significantly difference with down regulation(p<0.05)in 0.1umol/l and in 1.Oumol/l PGE2 group.Screened for Optimization transfection among three of COX-2 StealthTM siRNA,HSS-183840(numbered COX-2 StealthTM siRNA)can chock down COX-2 gene at a concentration of 50 nM/1 in human gastric cancer cell lines MKN-45 and SGC-7901.Compared with blank control,the expression of ?-cantenin,CyclinD1 and C-myc,whether in mRNA or protein levels,were significantly decreased(P<0.05).Meanwhile,the protein level of intracellular phospho-GSK-3(3 was significantly higher(P<0.05).No significant difference existed between the two gastric cancer cell lines.Result showed that the cell proliferation of human gastric cancer cell lines MKN-45 and SGC-7901 were suppressed with significant difference(P<0.01)in COX-2 StealthTM siRNA group and no difference of cell proliferation was discovered in other group.From results of flow cytometry,COX-2 Stealth TM siRNA,compared with blank control,made the cell cycle changed after 72h for cell lines MKN-45,SGC-7901.Cells in G1 phase decreased,while the G2/M phase has increased significantly(P<0.05).This resulted in a large number of cell arrested at the G2/M phase.But there were no significant difference in cell apoptosis(P>0.05)for MKN-45,SGC-7901 cell lines between control and COX-2 Stealth TM siRNA group after cells transfected 72h.Conclusion 1.For human gastric cell lines MKN-45 and SGC-7901,the right amount of PGE2 not only can obviously increase the expression,both in mRNA and protein,of ?-catenin,C-myc and CyclinD1 which are key molecule of Wnt/?-catenin signaling pathway,but also enhance cell proliferation activity significantly.2.COX-2 StealthrTM siRNA transfection targetingly interfere with COX-2 gene and can significantly restrain expression of COX-2 gene in transcription and protein levels in human gastric cell lines MKN-45 and SGC-7901.3.When COX-2 gene keeps in silencing by means of targeting COX-2 siRNA transfection,it is obviously reduce for the expression(transcription and protein)of ?-catenin,C-myc and CyclinD1 in human gastric cell lines MKN-45 and SGC-7901.Also,COX-2 gene keeps in silencing can lead to cell proliferation going down markedly of cell lines MKN-45 and SGC-7901.4.COX-2 plays a regulatory role in the expression of Wnt/?-catenin signaling pathway and this role reflect affecting cell proliferating activity in human gastric cell lines MKN-45 and SGC-7901.5.COX-2 may contribute to change the protein level of phospho-GSK-3? to realize the regulation for expression of Wnt/?-catenin signaling pathway.6.The targeting COX-2 siRNA transfection can cause the cell cycle change,but not sesult in cell apoptosis.7.The targeting COX-2 RNA interference has provided some experimental evidence and theoretical foundation for COX-2 gene therapy. |
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