ABSTRACT
Objective To observe the effects of extracellular signal-regulated kinase (ERK) 1/2 and protein kinase B (Akt) signal pathway in cholangiocarcinoma cells invasion and migration promoted by microRNA-21 (miR-21).Methods The experimental study was adopeted.QBC939 cholangiocarcinoma cells were cultured in vitro,through constructing and synthesizing unrelated sequence,miR-21 mimics and miR-21 inhibitor which were transfected into cells,and these cells were allocated into 4 groups,including growing naturally cells in the cell group,cells transfected by unrelated sequence in the 21-NC group,cells transfected by miR-21 mimics in the 21-M group and cells transfected by miR-21 inhibitor in the 21-Ⅰ group.Besides,cells in the 21-M group were allocated again into the 2 groups,20 μmol/L LY294002 and 10tμmol/L U0126 were respectively added in order to dispose 48 hours for follow-up experiments.Indicatiors of the test:(1) real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression of miR-21 in each group of cholangiocarcinoma cells.(2) Werstern blot was performed to detect the relative expressions of PTEN,ERK and Akt proteins in each group of cholangiocarcinoma cells.(3) Scarification assay was executed to test the migration of each group of cholangiocarcinoma cells.Transwell experiment was conducted to examine the migration and invasion of each group of cholangiocarcinoma cells.The measurement data with normal distribution were presented by x-s.The means of the 2 groups were compared by the t test.The means among groups were compared by the ANOVA,and pairwise comparison was analyzed by the Bonferroni test.The repeated measurement data were analyzed by the repeated measures ANOVA.Results (1) The relative expression of miR-21 in the cell group,21-NC group,21-M group and 21-Ⅰ group were 1.010 ±0.010,0.980 ± 0.050,4.900 ± 0.350 and 0.260 ± 0.010,respectively,with a statistically significant difference among the 4 groups (F =78.23,P ＜ 0.05),with no statistically significant difference between the 21-NC group and cell group (P ＞0.05).There was increased expression between the 21-M group and cell group,decreased expression between the 21-Ⅰ group and cell group and significant difference between 21-M group or 21-Ⅰ group and cell group (P ＜ 0.05).(2) The relative expressions of PTEN,ERK,p-ERK,Akt and p-Akt proteins in the cell group,21-NC group,21-M group and 21-Ⅰ group were 0.360 ± 0.020,0.400 ± 0.030,0.140 ± 0.010,0.680 ± 0.110 and 0.045 ± 0.126,0.470 ± 0.140,0.460 ± 0.060,0.440 ± 0.110 and 0.310 ± 0.020,0.380 ± 0.040,0.590 ± 0.060,0.160 ±0.010 and 0.400 ±0.010,0.390 ±0.080,0.410 ±0.090,0.380 ±0.070 and 0.440 ±0.110,0.510 ± 0.120,0.980 ± 0.150,0.190 ±0.010,respectively,showing statistically significant differences among the4 groups (F =10.23,12.78,18.11,P ＜ 0.05).There was no significant difference in the relative expressions of PTEN,ERK,p-ERK,Akt and p-Akt proteins between the cell group and 21-NC group (P ＞0.05).Compared with cell group,there was decreased PTEN expression and increased p-ERK and p-Akt expressions in the 21-M group,showing statistically significant differences (P ＜ 0.05).Compared with cell group,there was increased PTEN expression and decreased p-ERK and p-Akt expressions in the 21-Ⅰ group,showing statistically significant differences (P ＜ 0.05).(3) The change of migration rate of cells from 6 hours to 48 hours were from 12.0％ ± 3.0％ to 23.0％ ± 5.0％ in the cell group,from 21.0％ ± 4.0％ to 43.0％ ± 7.0％ in the 21-M group,from 6.0％ ±1.0％ to 18.0％ ±4.0％ in the miR-21 + LY294002 group and from 9.0％ ±2.0％ to 26.0％ ± 6.0％ in the miR-21 + U0126 group,respectively.The migration rate of cells in the 21-M group at each time point was higher than that in the cell group (F =16.23,P ＜0.05).The migration rate of cells in the miR-21 + LY294002 group and miR-21 + U0126 group were lower than that in the 21-M group (F =25.21,P ＜ 0.05),and there was the interaction effects between the change of migration rate of cells of the 3 groups and time,with a statistically significant difference (F =35.31,P ＜ 0.05).(4) The numbers of migration cells in the cell group,21-M group,miR-21 + LY294002 group and miR-21 + U0126 group were 198 ± 32,248 ± 39,187 ±23 and 174 ± 28,respectively,with a statistically significant difference among the 4 groups (F =8.48,P ＜ 0.05) and between the 21-M group and cell group (t =4.13,P ＜0.05).Compared with the 21-M group,the numbers of migration cells in the miR-21 + LY294002 group and miR-21 + U0126 group were decreased (F =21.98,P ＜0.05).The numbers of invasion cells in the cell group,21-M group,miR-21 + LY294002 group and miR-21 + U0126 group were 102 ± 22,211 ± 36,55 ± 9 and 67 ± 13,respectively,showing a statistically significant difference among the 4 groups (F =11.32,P ＜ 0.05) and between the 21-M group and cell group (t =6.67,P ＜ 0.05).Compared with the 21-M group,the numbers of invasion cells in the miR-21 + LY294002 group and miR-21 + U0126 group were decreased (F =36.23,P ＜ 0.05).Conclusion ERK and Akt signal pathway participate in the cholangiocarcinoma cells invasion and migration promoted by miR-21,PTEN could mediate the process of promoting cholangiocarcinoma cells invasion and migration through ERK and Akt signal pathway promoted by miR-21.