| Objective: This article aims to investigate whether ursolic acid(UA)inhibits proliferation,migration and invasion of lung cancer cells by regulating miRNA-21 actioning on the PTEN/PI3K/AKT pathway.Methods: Lung cancer A549 was selected as the research object.1.Different concentrations of UA(0,5,10,20,30,40,50μmol/L)were applied to the cells for 24 h,and UA with an IC50 value of 42 μmol/L was detected by CCK-8 for subsequent experiments.2.miRNA microarray was used to detect differentially expressed miRNAs before and after ursolic acid induction,and miRNA-21 was selected for subsequent experiments.3.Cell grouping and transfection,transfection of miRNA-21 inhibitor was miR-21 inhibitor group,transfection of miRNA-21 mimic was miRNA-21 mimic group,transfection-independent sequence was in the miR-NC group,and the blank control group was not transfected.4.A549 cells in logarithmic growth phase were divided into 4 groups.Group A was transfected with miR-21 inhibitor and added with 42 μmol/L UA.Group B was transfected with miR-21 mimic and added with 42 μmol/L UA.Group C A549 cells were cultured in 42 μmol/L UA,and A549 cells in group D were not given intervention.The CCK-8 method was used to detect the proliferation rate of A549 lung cancer cells and miRNA-21 inhibitor and miRNA-21 mimic cells under the action of UA.5.Transwell and scratch test to detect the migration and invasion ability of cells in each treatment group.6.The expression of miRNA-21 and target gene PTEN、PI3K、AKT mRNA in miRNA-21 inhibitor and miRNA-21 mimic cells was detected by PCR.7.Western blotting was used to detect the relative expression of PTEN、PI3K、AKT protein under the action of UA after down-regulating and up-regulating miRNA-21.Results:1.Different concentrations of UA(0,5,10,20,30,40,50 μmol/L)were applied to the cells,With the increase of ursolic acid concentration,The rate of cell proliferation gradually decreased.After ursolic acid was applied to A549 cells for 24 hours,The IC50 value of the ursolic acid was 42 μmol/L.2.The results of miRNA microarray showed that 34 miRNAs in A549 cells changed more than 2 times before and after ursolic acid induction(20 up-regulated and 14 down-regulated).Combined with the detection of miRNA signal values and fold change,miRNA21 was screened for subsequent experiments.3.The relative expression levels of miRNA-21 in miR-21 inhibitor group,miR-21 mimic group,miR-NC group and blank group were 0.35±0.07,2.18±0.12,1.04±0.07,0.96±0.10,miR-21 inhibitor group,miR-21 mimic group was compared with miR-NC group and blank group,P<0.05,which proved successful transfection.4.The cell proliferation rate,the relative width of the 24 h scratch,the number of transmembrane in group inhibitor+UA were 43.33%±8.51%,0.876±0.099,(28.400±7.403)/HP.Group mimic+UA were 66.85%±5.10%,0.445±0.032,(81.400±11.415)个/HP.Group UA were 53.49% ± 9.72%,0.629 ± 0.051,(42.000 ± 7.348)/HP.Group blank were 100.00%±12.31%,0.199±0.038,(129.400±5.030)个/HP.Group UA was compared with the other three groups,P <0.05.5.The PTEN protein,PTEN mRNA,PI3 K protein,PI3 K mRNA,AKT protein and AKT mRNA in group inhibitor+UA were 0.78±0.12,2.9±0.06,0.24±0.08,0.29±0.16,0.17±0.07,0.16±0.16.Group mimic+UA were 0.21±0.12,0.94±0.05,0.82±0.11,1.21±0.04,0.74±0.08,0.87±0.02.Group UA were 0.47±0.09,1.53±0.09,0.50±0.05,0.72±0.09,0.48±0.08,0.48±0.12.Group blank were 0.2±0.11,0.96±0.13,0.76±0.07,1.09±0.05,0.74±0.07,1.03±0.1.Group UA was compared with the other three groups,P <0.05.Conclusion: 1.UA could significantly inhibit the proliferation of lung cancer A549 cells,and with the increase of UA concentration,the cell proliferation inhibition rate increased gradually.2.Ursolic acid can significantly inhibit the migration and invasion of lung cancer A549 cells.3.Ursolic acid may inhibit the proliferation,migration and invasion of lung cancer A549 cells by down-regulating miRNA-21 on the PTEN/PI3K/AKT pathway. |
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