Ursolic Acid Down-regulates Survivin And VECF Expression In Thyroid Cancer TPC-1 Cells By Inhibiting HIF-1α

Objective: To investigate the effects of ursolic acid(UA)on the proliferation,apoptosis,migration and invasion of thyroid papillary carcinoma TPC-1 cells and the expression of HIF-1α,Survivin,and VEGF in cells under hypoxic conditions and the effects of drugs Change of expression.Methods:TPC-1 cells were divided into group A: hypoxic control group;group B: UA low concentration group(4μmol/L)and group C: UA high concentration group(8μmol/L).TPC-1 cell culture and drug intervention1.TPC-1 cells were treated with different concentrations of UA(0,2,4,8,16,32μmol/L)for 24 h and 48 h,respectively,and then used the CCK-8 reagent to detect the drug’s ability to inhibit cell proliferation.2.TPC-1 cells were treated with different concentrations of UA(0,4,8μmol/L)for 24 hours,and the apoptosis was detected by flow cytometry.3.TPC-1 cells were treated with different concentrations of UA(0,4,8μmol/L)for 24 hours,and the effect of drugs on cell migration was tested by cell scratch test.4.TPC-1 cells were treated with different concentrations of UA(0,4,8μmol/L)for 24 hours,and the effects of drugs on cell migration and invasion were tested by Transwell test.5.TPC-1 cells were treated with different concentrations of UA(0,4,8μmol/L)for 24 hours,and then stained with indirect immunofluorescence.Flow cytometry was used to detect the expression of HIF-1α,Survivin,and VEGF in the cells.6.TPC-1 cells were treated with different concentrations of UA(0,4,8μmol/L)for 24 hours,and then the m RNA expression of HIF-1α,Survivin,and VEGF in the cells was detected by Real-time PCR.Results:1.The inhibition rate of cell proliferation increased in a concentration-dependent manner under hypoxia,and there was significant difference among the three groups(P < 0.05).2.The results of flow cytometry showed that UA could effectively induce the apoptosis of TPC-1 cells,and with the increase of drug concentration,the apoptosis rate of TPC-1 cells gradually increased(A< B < C).After 24 hours of UA intervention,the apoptosis rates of each group were A(4.73 ± 0.97)%,B(9.03 ± 1.60)%,C(14.50 ± 0.92)%.Through one-way ANOVA,F= 49.50,P < 0.05,the difference between corresponding groups was statistically significant.3.The results of cell migration assay showed that the cell healing ability of TPC-1 cells treated with UA for 24 h and 48 h decreased in a concentration-and time-dependent manner,indicating that TPC-1 cells were affected by UA,and their migration ability decreased in a concentration-and time-dependent manner.There was significant difference among the corresponding groups(P < 0.05).4.The results of Transwell test showed that the cell invasiveness of TPC-1 cells in hypoxic environment decreased in a concentration dependent manner after 24 hours of UA intervention.The corresponding invasiveness of TPC-1 cells in each group was 391.67 ± 21.83 in group A,146.33 ± 7.52 in group B and 96.00 ± 4.58 in group C,respectively.Through the analysis of single factor variance,F=406.50,P<0.05,the difference between the corresponding groups was statistically significant.5.The expression of HIF-1 α,Survivin and VEGF in TPC-1 cells was detected by flow cytometry.The results showed that after 24 hours of UA intervention,HIF-1 α,Survivin and VEGF were all expressed in TPC-1 cells,and the relative expression decreased in a concentration dependent manner.The relative expression of HIF-1 α protein in each group was 5.28 ± 0.13 in group A,3.81 ± 0.12 in group B and 0.67 ± 0.27 in group C,respectively.By one-way ANOVA,F=484.20,P<0.01,the difference between thecorresponding groups was statistically significant.The relative expression of Survivin in the cells of each group was 3.09±0.29 in group A,1.58±0.30 in group B and 0.41±0.21 in group C,respectively.By one-way ANOVA,F=74.33,P<0.01,the difference between the corresponding groups was statistically significant.The relative expression of VEGF protein in each group was 3.23±0.33 in group A,1.68±0.35 in group B and 0.55±0.25 in group C,respectively.Through one-way ANOVA,F=55.31,P<0.01,the difference between the corresponding groups was statistically significant.6.qRT-PCR was used to detect the expression of HIF-1 α,Survivin and VEGF m RNA in the cells of each group.The results showed that the expression of HIF-1 α,Survivin and VEGF m RNA in TPC-1 cells were all expressed in the cells of each group after 24 hours of UA intervention in hypoxia environment,and the relative expression decreased in a concentration dependent manner.The relative expression of HIF-1 α m RNA in each group was 1.00 ± 0.00 in group A,0.79 ± 0.08 in group B and 0.49 ± 0.07 in group C,respectively.By means of one-way ANOVA,F=50.97,P < 0.01,the difference between the corresponding groups was statistically significant.The relative expression of Survivin m RNA in each group was 1.00 ± 0.00 in group A,0.86 ± 0.04 in group B and 0.70 ± 0.07 in group C;the relative expression of VEGF m RNA in each group was 1.00 ± 0.00 in group A,0.78 ± 0.07 in group B and 0.63 ± 0.06 in group C,respectively.Conclusion:1.UA can effectively inhibit the proliferation and promote the apoptosis of thyroid papillary carcinoma TPC-1 cells in micro-oxygen environment.2.UA can effectively inhibit the migration and invasion of TPC-1 cells in microoxygen environment.3.UA can down-regulate the expression of Survivin and VEGF in TPC-1 cells by inhibiting HIF-1α.