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Effect Of Dihydromyricetin On The Proliferation And Invasion Migration Ability And PI3K/Akt Pathway Of Choriocarcinoma Cells

Posted on:2019-06-18Degree:MasterType:Thesis
Country:ChinaCandidate:Y T LeiFull Text:PDF
GTID:2404330566978377Subject:Pathology and pathophysiology
Abstract/Summary:PDF Full Text Request
Chorionic epithelioma(Choriocarcinoma,CC)is a kind of malignant tumor which is caused by excessive invasion and proliferation of trophoblast cells in gestational trophoblastic tumor.In China and Southeast Asian countries of high incidence;although most of the clinical patients with choriocarcinoma can be cured by chemotherapy,but there are still some patients with chemotherapy drug resistance,and the distant metastasis,of which brain metastasis is an important factor of adverse prognosis;the transfer pathway is mainly blood;some patients have serious side effects of chemotherapeutic drugs.,including liver and kidney injury,insomnia,hair loss and so on.The dihydromyricetin was first found in a wild vine of the genus W.T.Wang,a grape of the Ampelopsis grossedentata,belonging to dihydroflavonols.In recent years,it has been found that it plays an important role in inhibiting the occurrence and development of tumor,including inhibiting the proliferation of tumor cells,inhibiting the cycle progression of tumor cells,inducing the apoptosis of tumor cells,increasing the sensitivity of tumor cells to chemotherapeutic drugs and so on.However,there is no literature report on the effect of dihydromyricetin on the proliferation ability and invasion and metastasis ability in the choriocarcinoma cell.In this study,the choriocarcinoma cells JEG-3 and JAR were treated with a certain concentration of dihydromyricetin to investigate if dihydromyricetin has effect on the proliferation of choriocarcinoma cells and whether dihydromyricetin has effect on migration of the JEG-3 and JAR cells by PI3K/Akt signaling pathway;The study provides a basis for enriching the dihydromyricetin anti-cancer on JEG-3 and JAR migration andprovides a new direction for the treatment of choriocarcinoma.Objective:To investigate the effect of dihydromyricetin on the proliferation ability of JEG-3 and JAR cells in choriocarcinoma,by observing the effect of dihydromyricetin on the survival rate of JEG-3 and JAR cells;To investigate the effects and possible mechanisms of dihydromyricetin on JEG-3 and JAR cells PI3K/Akt signaling pathway by observing the effects of dihydromyricetin on the expression of phospatidylinositol 3-kinase p85(PI3K p85),protein kinase B(PKB/Akt),Akt Ser473-point phosphorylation(P-akt(Ser473)),Akt Thr308-point phosphorylation(P-akt(Thr308)),Akt total protein(Akt(pan)),Matrix Metalloproteinase 2(MMP2),Matrix Metalloproteinase 9(MMP9).Methods:1.The effects of dihydromyricetin concentrations of 0?g/ml,20?g/ml,40?g/ml,60?g/ml,80?g/ml on the proliferative ability of JEG-3 and JAR cells in choriocarcinoma were detected by Thiazole Blue(MTT)technique in24 hours and 48 hours.2.The effects of dihydromyricetin concentrations of 0?g/ml,40?g/ml,60?g/ml,80?g/ml on the migration of choriocarcinoma JEG-3 and JAR cells were detected by cell scratching technique and transwell technique.3.The effects of dihydromyricetin concentrations of 0?g/ml,40?g/ml,60?g/ml,80?g/ml on the mRNA and protein expression of MMP2 in choriocarcinoma JEG-3 and JAR cells with 36 hours were detected by Real-time quantitative PCR and Western blot technique.4.The effects of dihydromyricetin concentrations of 60?g/ml on the mRNA expression of PI3 K,Akt and MMP2 in choriocarcinoma JAR cells with 36 hours were detected by Real-time quantitative PCR technique.5.The effects of dihydromyricetin concentrations of 0?g/ml,40?g/ml,60?g/ml on the protein expression of PI3 K p85,P-Akt(Ser473),P-Akt(Thr308),Akt(pan)and MMP9 in choriocarcinoma JEG-3 and JAR cellswith 36 hours were detected by Western blot.6.Using SPSS19.0 for statistical software analysis,the data were expressed by using single factor variance analysis.T-Test was adopted in the comparison between the two groups,and the correlation and regression were analyzed by Pearson correlation and linear regression,and the difference was statistically significant with P<0.05.Results:1.Cell survival rate in each groupAfter treating with concentration of dihydromyricetin 20,40,60,80?g/ml 24 hours,the JEG-3 Cell survival rate respectively were(109.73±4.90)%,(104.43±9.12)%,(94.83±4.36)%,(82.60±7.51)%,(F=10.552,P=0.026),as the concentration increases the survival rate decreases,(P=0.004,r=-0.704);the JAR Cell survival rate respectively were(95.89±7.49)%,(84.25±6.93)%,(63.56±4.56)%,(51.58±4.62)%,(F=125.310,P=0.013)as the concentration increases the survival rate decreases(P<0.001,r=-0.942).After treating for 48 hours,JEG-3 Cell survival rate respectively were(101.56±6.64)%,(91.76±7.85)%,(84.24±13.21)%,(66.42±19.58)%,(F=16.953,P=0.028)as the concentration increases the survival rate decreases(P<0.001,r=-0.854)with Statistically significant;the JAR Cell survival rate respectively were(94.25±2.76)%,(68.53±4.66)%,(42.32±4.15)%,(27.87±4.23)%,(F=299.349,P=0.009);as the concentration increased the survival rate decreases(P <0.001,r=-0.986)with Statistically significant.2.The migration ability of each group of cells:Cell scratches,After treating with concentration of dihydromyricetin0,40,60,80?g/ml 24 hours,the JEG-3 Cell migration rates respectively were(31.80±8.07)%,(19.87±5.48)%,(10.00±8.47)%,(5.83±5.03)%(F=8.345,P=0.008),with Statistically significant.After 24 hours,JAR Cell Migration rate respectively were(16.16±4.03)%,(6.20±3.28)%,(4.10±2.85)%,(0.81±0.81)%(F=14.683,P=0.001),with Statistically significant.Transwell,After treating with concentration of dihydromyricetin 0,20,40,60,80?g/ml 36 hours,the number of JEG-3 cells passing through membrane in each group respectively were(43.67±3.51),(17.00±2.65),(9.33±3.06),(4.00±1.00),(F=125.378,P<0.001),with Statistically significant;And for JAR cells,After 24 hours,number of cells passing through membrane in each group respectively were(129.67±14.50),(83.67±3.22),(37.67±3.79),(18.67±4.04),(F=117.784,P < 0.001),with Statistically significant.Transwell with matrigel,After treating with concentration of dihydromyricetin 0,20,40,60,80?g/ml 72 hours,the number of JEG-3 cells passing through membrane in each group respectively were(69.7±5.10),(27.6±1.52),(14.2±2.63),(3.25±0.95),(P<0.001),with Statistically significant;And for JAR cells,After 48 hours,number of cells passing through membrane in each group respectively were(167.4±12.36),(55.6±4.50),(37.2±5.71),(11.2±0.83)(P < 0.001),with Statistically significant.3.The expression of MMP2 mRNA in each group:After 36 hours,compared with the group of 0 ?g/ml concentration of dihydromyricetin,the expression of MMP2 mRNA in the group of 40,60,80?g/ml were decreased,with Statistically significant,(JEG-3 cells:F=35.225,P< 0.001;JAR cells:F=63.791,P < 0.001),as the concentration increased the expression of MMP2 mRNA decreases,(JEG-3 cells:r=-0.804,P<0.001;JAR cells: r=-0.880,P<0.001),with statistically significant.4.The expression of MMP2 protein in each group:After 36 hours,compared with the group of 0 ?g/ml concentration of dihydromyricetin,the expression of MMP2 protein in the group of 40,60,80?g/ml were decreased,with Statistically significant,as the concentration increased the expression of MMP2 mRNA decreases,(JEG-3:F=589.52,P<0.001;JAR:F=354.69,P<0.001),with Statistically significant.5.The expression of MMP2,PI3 K,Akt mRNA in each group in JAR cells:Compared with the group of 0 ?g/ml concentration of dihydromyricetin,the expression of MMP2,PI3 K,Akt mRNA in the group of 60 ?g/ml were decreased,with Statistically significant,(P<0.05),and the expression level of MMP-2 mRNA changes with the expression of PI3 K mRNA and Akt mRNA.(r=0.957,0.953,P<0.05),with Statistically significant.6.The expression of PI3 K p85,Akt(pan),P-Akt(Ser473),P-Akt(Thr308),MMP9 protein in each group in JEG-3and JAR cells:In JEG-3 cells,compared with the group of 0 ?g/ml concentration of dihydromyricetin,the expression of PI3 K p85 protein in the group of 40,60?g/ml were decreased,with Statistically significant(F=982.43,P <0.001);Akt(pan)protein has no significant changes,(P>0.05);P-Akt(Ser473)were decreased,(F=226.94,P < 0.001),with Statistically significant;P-Akt(Thr308)were increase,(F=285.59,P < 0.001),with Statistically significant;MMP9 were decreased,(F=4763.85,P<0.001),with Statistically significant.In JAR cells,compared with the group of 0 ?g/ml concentration of dihydromyricetin,the expression of PI3 K p85 protein in the group of40,60 ?g/ml were decreased,with Statistically significant(F=832.61,P <0.001);Akt(pan)protein has no significant changes,(P>0.05);P-Akt(Ser473)were decreased,(F=766.54,P < 0.001),with Statistically significant;P-Akt(Thr308)were increase,(F=443.60,P < 0.001),with Statistically significant;MMP9 were decreased,(F=1013.15,P<0.001),with Statistically significant.Conclusion:It can reduce the survival rates of JEG-3 and JAR cells in choriocarcinoma,and can inhibit the expression of invasive migration related factors by regulating the pi3k/akt signaling pathway in JEG-3 and JAR cells,thus inhibiting the invasion and migration of the JEG-3 and JAR cells of the choriocarcinoma,and has a certain concentration dependence.The above regulation mechanism may be one of the factors inhibiting the metastasis of choriocarcinoma by dihydromyricetin.
Keywords/Search Tags:dihydromyricetin, choriocarcinoma, phosphatidylinositol3-kinase, protein kinase B, PI3K/Akt cell signaling pathway, MMP2, MMP9
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