The Effect Of Zhi Zhen Recipe On Invasion And Migration In Human Gastric Cancer Cell Lines By Regulating The MiRNA-30a/COX-2 Signaling

Objective: In China,the incidence and mortality of gastric cancer are in the second.A large numbers of studies show that miRNAs by regulating the expression of target genes may be an important mechanism for the development of gastric cancer.On the basis of our previous study that Zhi Zhen Recipe can improve colorectal cancer multidrug resistance,we consider from the miR-29c–mediated COX-2 axis aspect to further explore the effect of Zhi Zhen Recipe on human gastric cancer cells’ invasion and migration.Method:(1)Constructing COX-2 gene 3’UTR region luciferase reporter plasmid,and performing luciferase reporter assay to determine miR-30 a can targert COX-2 3’UTR and inhibit its luciferase activity;(2)Real-time PCR and Western blot was used to detect the effect of miR-30 a on COX-2 mRNA and protein expression;(3)Transwell assay was used to detect the effect of miR-30 a on human GC cells’ invasive and migrative ability;(4)CCK-8 assay was used to detect the Inhibition rate of Zhi Zhen Recipe for 48 h in human GC cells;(5)Transwell assay was used to detect the effect of Zhi Zhen Recipe on human GC cells’ invasive and migrative ability;(6)Real-time PCR and Western blot was used to detect the effect of Zhi Zhen Recipe on miR-30 a,COX-2 mRNA and protein expression.Results: 1.In 293 T cells,the luciferase activity of COX-2 3’UTR was decreased by transfecting with miR-30 a mimics when compared with miRNA control(P<0.01).In human GC cells,the luciferase activity of COX-2 3’UTR was decreased by transfecting with miR-30 a,miR-30 d,miR-30 e when compared with miR-67(P<0.01)and co-transfection with miR-30a/d/e led to a significantly decresase of luciferase activity when compared with miR-30a(P<0.01).2.In human GC cells,the expressive level of COX-2 mRNA and protein was down-regulated by transfecting with miR-30 a,miR-30 d,miR-30 e when compared with miRNA control(P <0.01)and co-transfection with miR-30a/d/e led to a significantly decresase of COX-2 mRNA and protein expression when compared with miR-30a(P<0.01).Furthermore,transfection with miR-30a/d/e inhibitor led to a increase of COX-2 mRNA and protein expression.3.In human GC cells,compared with miR-67,transfected with miR-30 a mimics displayed a lower invasive and migrative rate(P<0.01)and co-transfected miR-30a/d/e led to a significantly suppress of invasion and migration(P<0.01)when compared with miR-30 a.While cells transfected with miR-30 a inhibitor promoted invasion and migration compared with miR-67(P<0.01).4.Zhi Zhen Recipe’s alcohol extract can inhibit the growth of human GC cells MKN45 and its IC50,IC20,IC10 for 48 h respectively is 301.97μg/mL,120.79μg/mL,60.39μg/mL.5.The number of invasive and migrative human GC cells was reduced after treated with three various concentration(60,120,300μg/mL)of Zhi Zhen Recipe for 48 h compared with the control group(No treatment)(P<0.01),which in a dose dependent manner.6.MiR-30 a expression was up-regulated in human GC cells after treated with three various concentration(60,120,300μg/mL)of Zhi Zhen Recipe for 48 h when compared with the control group(No treatment)(P<0.01)and in a dose-dependent manner.The expressive level of COX-2 mRNA and protein was down-regulated in human GC cells after treated with three various concentration(60,120,300μg/mL)of Zhi Zhen Recipe for 48 h when compared with the control group(No treatment)(P<0.01)and in a dose-dependent manner.Conclusion: 1.MiR-30 a can target regulating COX-2 expression and suppress human GC cells invasion and migration.3.Zhi Zhen Recipe can suppress human GC cells invasion and migration,its mechanism may be related to regulate the miRNA-30a/COX-2 Signaling.