UBE2D3基因过表达对食管癌细胞迁移增殖及顺铂敏感性的影响

Overexpression of UBE2D3 gene in esophageal carcinoma cell line EC109 can influence the proliferation, migration and the sensitivity to cisplatin

目的 构建过表达人泛素交联酶UBE2D3基因的稳定转染食管癌EC109细胞株,并观察过表达后迁移增殖能力及对顺铂化疗敏感性的改变.方法 食管癌EC109细胞株分别转染pEGFP-C1和pEGFP-UBE2D3质粒后,分为阴性对照组（NC组）和过表达组（OE组）,并用G418进行筛选构建稳定转染细胞株.通过实时定量PCR（RT-PCR）法及Western blotting验证过表达效率,用划痕试验检测迁移能力,并用CCK-8法检测细胞增殖,半抑制浓度（IC50）法评价对顺铂的敏感性.结果 相对于亲本细胞,NC组UBE2D3 mRNA表达量为1.010±0.590,OE组为2.026±0.898;NC组UBE2D3蛋白表达量为0.923 ±0.237,OE组为1.520±0.487;NC组UBE2D3 mRNA及蛋白表达水平与空白对照组亲本细胞EC109相近（t=1.005,P＞0.05;t=1.492,P＞0.05）,OE组较亲本细胞组表达高（t=6.682,P＜0.05;t=9.150,P＜0.05）.在12 h NC组的迁移距离为（0.161±0.062） mm,OE组为（0.052±0.007） mm（=4.370,P＜0.05）;在24 h NC组的迁移距离为（0.309 ±0.071） mm,OE组为（0.074 ±0.012） mm（=3.644,P＜0.05）;在12 h和24 h时OE组相对于NC组的迁移率分别为0.236±0.051和0.241±0.037,OE组迁移能力明显较NC组降低;在第1天时OE组相对于NC组的增殖率为0.713±0.220（t=13.466,P＜0.05）,第2天为0.848±0.241（t =27.241,P＜0.05）,第3天为0.742 ±0.233（t=15.107,P＜0.05）.使用顺铂处理后,在第1天时OE组的IC50值相对于NC组为0.356±0.154（t =4.326,P＜0.05）,第2天为0.445±0.098（t =5.870,P＜0.05）,第3天为0.481±0.096（=5.009,P＜0.05）,OE组IC50值明显低于NC组;给予5μg/ml顺铂处理后,在第1、2、3天时OE和NC组相对于未使用顺铂处理时的增殖效率均减慢,其中OE组相对于NC组的增殖率分别为0.606±0.283（t=8.872,P＜0.05）、0.759±0.089（t=16.771,P＜0.05）和0.569±0.574（t =7.885,P＜0.05）,然而在第1、2、3天时OE组的存活率相对于NC组分别为0.831 ±0.323（t=24.226,P＜0.05）、0.875±0.102（t=33.541,P＜0.05）和0.853±0.359（t=28.187,P＜0.05）,顺铂对OE组抑制效率较NC组增加.结论 食管癌细胞EC109中过表达UBE2D3基因,能够抑制其迁移、增殖,增强其对顺铂的敏感性.

Objective To establish a stable transfection of UBE2D3 gene in esophageal carcinoma cell line EC109,and investigate the gene overexpression effects of migration,proliferation,and the sensitivity to cisplatin in the cell line.Methods pEGFP-C1 and pEGFP-UBE2D3 plasmids were transfected into the esophageal carcinoma EC109 strain.G418 screening method was used to select stably transfected cell lines which were named as control group （NC） and overexpression group （OE）,respectively.The expression efficiency was verified by RT-PCR and Western blotting,the migration was evaluated by scratch test,and the proliferation and sensitivity of cells to cisplatin were respectively determined by CCK-8 assay and half maximal inhibitory concentration （IC50）.Results Relative to EC109 cells,the mRNA expression of UBE2D3 of NC group was 1.010 ±0.590,that of OE group was 2.026 ± 0.898.The protein expression of UBE2D3 of NC group was 0.923 ±0.237,OE group was 1.520 ± 0.487,respectively.The expression of mRNA and protein level of UBE2D3 in NC was similar to that in EC109 cells （t=1.005,P ＞0.05;t =1.492,P ＞0.05）,while that in OE was much higher than both above （t =6.682,P ＜ 0.05;t =9.150,P ＜ 0.05）.The migration length of NC group was （0.161 ± 0.062） mm at 12 h,while that of OE group was （0.052 ± 0.007） mm （t =4.370,P ＜ 0.05）.The migration length of NC group was （0.309 ± 0.071） mm at 24 h,compared with OE group was （0.074 ± 0.012） mm （t =3.644,P ＜ 0.05）.At 12 h and 24 h,relative to NC group,the migration length of OE group was 0.236 ±0.051 and 0.241 ±0.037,respectively,which pointed out that the migration of OE was significantly lower than that of NC.For the 1st day,the proliferation of OE was 0.713 ±0.220 compared with that in NC （t =13.466,P ＜ 0.05）,and it was 0.848 ± 0.241 （t =27.241,P ＜ 0.05） and 0.742 ± 0.233 （t =15.107,P ＜ 0.05） at the 2nd day and 3rd day,respectively.The IC50 of OE group was 0.356 ± 0.154 （t =4.326,P ＜ 0.05） relative to that of NC by disposing with cisplatin at the 1 st day,it was 0.445 ± 0.098 （t =5.870,P ＜ 0.05） and 0.481 ± 0.096 （t =5.009,P ＜ 0.05） at the 2nd day and 3rd day,respectively.The IC50 of OE group was less than that of NC group;when deposed with 5 μg/ml cisplatin,both proliferation of OE and NC group was slower than before.At the 1st day,2nd day and 3rd day,the proliferation of OE group was 0.606 ± 0.283 （t =8.872,P ＜ 0.05）,0.759 ± 0.089 （t =16.771,P ＜ 0.05） and 0.569 ± 0.574 （t =7.885,P ＜ 0.05） relative to NC group,respectively.Moreover,relative to NC group,fraction surviving of OE group was 0.831 ±0.323 （t =24.226,P ＜0.05）,0.875 ±0.102 （t =33.541,P ＜0.05） and 0.853 ± 0.359 （t =28.187,P ＜ 0.05） at those time points above,respectively.The proliferation of OE was slower compared with that in NC,and the inhibition efficiency of cisplatin on the proliferation of OE was more significant than that of NC.Conclusion UBE2D3 gene overexpressed in esophageal carcinoma cell line EC109 strain may result in the cell＇s phenotype inhibition of the migration and proliferation and enhance its sensitivity to cisplatin.