三结构域蛋白28通过上皮-间充质转化对肝癌细胞侵袭迁移的影响

Effect of tripartite motif-containing protein 28 on invasion and migration of hepatocellular carcinoma cells through epithelial-mesenchymal transition

目的观察沉默三结构域蛋白28(TRIM28)表达对肝癌细胞迁移及侵袭能力的影响。方法通过RNA干扰技术(RNAi)构建干扰TRIM28表达的短发卡RNA(shRNA)质粒,对高表达TRIM28的肝癌细胞株HepG2和人高转移肝癌细胞(HCCLM3细胞)对照组及实验组转染shRNA 48 h后,应用实时定量反转录聚合酶链反应(RT-qPCR)检测TRIM28基因沉默效率。应用划痕迁移实验和Transwell侵袭实验检测TRIM28基因沉默对肝癌细胞迁移及侵袭能力的影响,并通过RT-qPCR及蛋白质印迹法(Western blot)检测E-钙黏蛋白(E-cadherin)、N-钙黏蛋白(N-cadherin)、波形蛋白(Vimentin)及β-连环蛋白(β-catenin)表达。使用SPSS 19.0软件进行统计学分析,组间比较采用t检验。结果RT-qPCR结果显示实验组TRIM28表达(HepG20.84±0.02,HCCLM30.69±0.02)在两组肝癌细胞中均低于对照组(HepG21.00±0.05,HCCLM31.00±0.07,t=5.488、7.240,P<0.01),差异有统计学意义。划痕实验结果显示,48 h实验组HepG2和HCCLM3细胞迁移率[(57.83±0.02)%、(21.52±0.05)%]均明显低于对照组[(94.96±0.02)%、(38.10±0.03)%],差异有统计学意义(t=22.971、5.780,P<0.01)。Transwell侵袭实验结果显示,相应实验组穿膜细胞数分别为(57.88±5.24)、(3.33±1.36)个,明显低于对照组(265.52±9.31)、(26.03±3.13)个,差异有统计学意义(t=23.050、13.242,P<0.01)。Western blot结果显示,HepG2和HCCLM3细胞N-cadherin蛋白表达量(0.31±0.06,0.63±0.08,t=4.822、2.951,P<0.05)、Vimentin(0.41±0.03,0.32±0.02,t=2.909、7.944,P<0.05)及β-catenin(0.10±0.03,0.12±0.02,t=2.970、4.157,P<0.05)低于对照组,E-cadherin表达升高(0.25±0.05,0.27±0.07,t=3.242、5.021,P<0.05),差异有统计学意义。RT-qPCR结果显示,实验组E-cadherin表达高于对照组(1.80±0.30,1.40±0.17,t=4.571、3.095,P<0.01),差异有统计学意义,N-cadherin(0.42±0.01,0.84±0.03,t=22.460、8.441,P<0.01)、Vimentin(0.18±0.00,0.84±0.02,t=52.230、6.194,P<0.01)及β-catenin表达低于对照组(0.72±0.07,0.34±0.07,t=2.972、4.174,P<0.01),差异均有统计学意义。结论降低TRIM28表达能抑制肝癌细胞HepG2和HCCLM3的侵袭和迁移能力。

Objective To investigate the effect of interfering with the expression of tripartite motif-containing protein 28(TRIM28)on the migration and invasion of hepatocellular carcinoma(HCC)cells.Methods A short hairpin RNA(shRNA)plasmid interfering with TRIM28 expression was constructed by RNA interference(RNAi),and the TRIM28 gene silencing efficiency was detected by real-time quantitative reverse transcription-polymerase chain reaction(RT-qPCR)at 48 h after transfection of shRNA into the control and experimental groups of HepG2 and HCCLM3 cell lines with high TRIM28 expression.The effects of TRIM28 gene silencing on the migration and invasion of HCC cells were detected by scratch migration assay and transwell invasion assay,and the expression levels of E-cadherin,N-cadherin,vimentin andβ-catenin were detected by RT-qPCR and Western blotting.SPSS 19.0 software was used for statistical analysis.Results RT-qPCR results showed that TRIM28 expression in the experimental group(HepG20.84±0.02;HCCLM30.69±0.02)was lower than that in the control group(HepG21.00±0.05;HCCLM31.00±0.07)in HepG2 and HCCLM3 cell lines(t=5.488,7.240,P<0.01).The wound-healing assay showed that the migration rates of HepG2 and HCCLM3 cells in the experimental group at 48 h[(57.83±0.02)%and(21.52±y0.05)%]were significantly lower than those in the control group[(94.96±0.02)%and(38.10±0.03)%],with the difference being statistically significant(t=22.971,5.780,P<0.01).The Transwell invasion assay showed that the number of penetrating cells in the corresponding experimental group was(57.88±5.24)and(3.33±1.36),which was significantly lower than that in the control group[(265.52±9.31)and(26.03±3.13)],with the difference being statistically significant(t=23.050,13.242,P<0.01).Western blotting results showed that N-cadherin(0.31±0.06,0.63±0.08,t=4.822,2.951,P<0.05),vimentin(0.41±0.03,0.32±0.02,t=2.909,7.944,P<0.05)andβ-catenin(0.10±0.03,0.12±0.02,t=2.970,4.157,P<0.05)expression was decreased and E-cadherin(0.25±0.05,0.27±0.07,t=3.242,5.021,P<0.05)expression was increased in HepG2 and HCCLM3 cells relative to controls.RT-qPCR results showed that E-cadherin(1.80±0.30,1.40±0.17,t=4.571,3.095,P<0.01)expression increased,and N-cadherin(0.42±0.01,0.84±0.03,t=22.460,8.441,P<0.01),vimentin(0.18±0.00,0.84±0.02,t=52.230,6.194,P<0.01)andβ-catenin(0.72±0.07,0.34±0.07,t=2.972,4.174,P<0.01)expression decreased in the experimental group as compared with the control group.Conclusion Down-regulating the expression of TRIM28 can inhibit the invasion and migration of HepG2 and HCCLM3 cells.