细胞外信号调节激酶及蛋白激酶B信号通路在微RNA-21促进胆管癌细胞侵袭转移中的作用

摘要

目的 观察细胞外信号调节激酶1/2(ERK1/2)及蛋白激酶B(Akt)信号通路在微RNA-21(miR-21)促进胆管癌细胞侵袭转移中的作用.方法 采用实验研究方法.体外培养胆管癌细胞QBC939细胞,通过构建合成无关序列、miR-21 mimics和miR-21 inhibitor并转染到细胞中,将细胞分为4组:cell组:自然生长细胞,21-NC组:转染无关序列,21-M组:转染miR-21 mimics,21-Ⅰ组:转染miR-21 inhibitor.另取21-M组细胞进一步分为2组,分别加入20 μmol/L LY294002和10 μmol/L U0126处理48 h,用于后续实验.检测指标:(1) RT-qPCR检测miR-21在各组胆管癌细胞中的表达.(2) Werstern blot检测各组胆管癌细胞第10号染色体缺失的张力同源性磷酸酶(PTEN)、ERK及Akt蛋白的相对表达量.(3)划痕实验检测各组胆管癌细胞迁移能力.(4) Transwell实验检测各组胆管癌细胞迁移与侵袭能力的影响.正态分布的计量资料以(x)±s表示,两组均数比较采用t检验,多组均数比较采用方差分析,组间两两比较采用Bonferroni检验,重复测量数据采用重复测量方差分析.结果 (1) miR-21在cell组、21-NC组、21-M组及21-Ⅰ组中的相对表达量分别为1.010±0.010、0.980±0.050、4.900±0.350、0.260±0.010,4组比较,差异有统计学意义(F=78.23,P＜0.05).21-NC组miR-21表达分别与cell组比较,差异均无统计学意义(P＞0.05).21-M组miR-21表达较cell组增加,21-Ⅰ组较cell组减少,21-M组和21-Ⅰ组分别与cell组比较,差异均有统计学意义(P＜0.05).(2) cell组、21-NC组、21-M组、21-Ⅰ组细胞PTEN蛋白相对表达量分别为0.360±0.020、0.400±0.030、0.140±0.010、0.680±0.110,ERK蛋白相对表达量分别为0.045±0.126、0.470±0.140、0.460±0.060、0.440±0.110,p-ERK蛋白相对表达量分别为0.310±0.020、0.380±0.040、0.590±0.060、0.160±0.010,Akt蛋白相对表达量分别为0.400±0.010、0.390±0.080、0.410±0.090、0.380±0.070,p-Akt蛋白相对表达量分别为0.440±0.110、0.510±0.120、0.980±0.150、0.190±0.010,4组细胞PTEN、p-ERK、p-Akt蛋白相对表达量比较,差异均有统计学意义(F=10.23,12.78,18.11,P＜0.05),与cell组比较,21-NC组PTEN、ERK、p-ERK、Akt、p-Akt表达,差异均无统计学意义(P＞0.05);而21-M组与cell组比较,PTEN表达减少,p-ERK和p-Akt表达增加,差异有均有统计学意义(P＜0.05);21-Ⅰ组与cell组比较,PTEN表达增加,而p-ERK和p-Akt表达降低,差异有统计学意义(P＜0.05).(3)cell组、21-M组、miR-21+ LY294002组、miR-21+U0126组细胞6～48 h迁移率变化范围为12.0％±3.0％～23.0％±5.0％,21.0％±4.0％～43.0％±7.0％,6.0％±1.0％～18.0％±4.0％,9.0％±2.0％～26.0％±6.0％.与cell组比较,21-M组细胞迁移能力各时间点迁移率更高,两组迁移率变化趋势比较,差异有统计学意义(F=16.23,P＜0.05).miR-21+ LY294002组、miR-21+ U0126组迁移率较21-M组下降,3组迁移率趋势比较,差异有统计学意义(F=25.21,P＜0.05),3组迁移率变化趋势与时间交互效应,具有统计学意义(F=35.31,P＜0.05).(4) cell组、21-M组、miR-21+ LY294002组、miR-21+U0126组迁移细胞数分别为(198±32)个、(248±39)个、(187±23)个、(174±28)个,4组比较,差异有统计学意义(F=8.48,P＜0.05);21-M组与cell组比较,差异有统计学意义(=4.13,P＜0.05);miR-21+LY294002组、miR-21+U0126组迁移细胞数较21-M组下降,3组比较,差异有统计学意义(F=21.98,P＜0.05).cell组、21-M组、miR-21+ LY294002组、miR-21+ U0126组侵袭细胞数分别为(102±22)个、(211±36)个、(55±9)个、(67±13)个,4组比较,差异有统计学意义(F=11.32,P＜0.05);21-M组与cell组比较,差异有统计学意义(t=6.67,P＜0.05);miR-21+ LY294002组、miR-21+ U0126组侵袭细胞数较21-M组下降,3组比较,差异有统计学意义(F=36.23,P＜0.05).结论 ERK及Akt信号通路参与了miR-21促进胆管癌细胞侵袭迁移,PTEN可能介导了miR-21通过ERK及Akt通路促进胆管癌侵袭转移的过程.%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.