CAJ | 학술논문

目的观察RNA干扰(RNAi)技术抑制上皮细胞黏附分子(EP-CAM)的表达后对胆囊癌GBC-SD细胞增殖、凋亡的影响.方法针对EP-CAM基因的不同位点构建4个微小RNA(miRNA)重组质粒,转染GBC-SD细胞,逆转录一聚合酶链反应(RT-PCR)和Western blot检测转染48 h后EP-CAM mRNA及蛋白的表达水平,选用干扰效率最强的质粒再转染GBC-SD细胞,噻唑蓝(MTT)比色法检测细胞增殖的变化,流式细胞仪检测细胞凋亡的变化.结果 4个miRNA重组质粒构建成功;RT-PCR显示干扰质粒-1、-2、-3、-4组EP-CAM mRNA的表达量分别为0.26±0.02、0.79±0.03、0.37±0.02、0.45±0.04均低于空白对照组0.94±0.03及阴性对照组0.92±0.03(P＜0.01);Western blot显示,干扰质粒-1、-2、-3、-4组EP-CAM蛋白的表达量分别为0.22±0.03、0.71±0.03、0.32±0.02、0.41±0.04均低于空白对照组0.85±0.03及阴性对照组0.84±0.04(P＜0.01);选用效果最佳的干扰质粒-1转染GBC-SD细胞后,MTY法显示干扰组细胞增殖活性受到抑制,转染48 h后最显著,抑制率为39%,流式细胞仪检测显示3组细胞的凋亡率差异无统计学意义(P＞0.05).结论针对EP-CAM的miRNA重组质粒能特异地抑制EP-CAM基因表达,抑制GBC-SD细胞的增殖.
목적 관찰RNA간우(RNAi)기술억제상피세포점부분자(EP-CAM)적표체후대담낭암GBC-SD세포증식、조망적영향.방법 침대EP-CAM기인적불동위점구건4개미소RNA(miRNA)중조질립,전염GBC-SD세포,역전록일취합매련반응(RT-PCR)화Western blot검측전염48 h후EP-CAM mRNA급단백적표체수평,선용간우효솔최강적질립재전염GBC-SD세포,새서람(MTT)비색법검측세포증식적변화,류식세포의검측세포조망적변화.결과 4개miRNA중조질립구건성공;RT-PCR현시간우질립-1、-2、-3、-4조EP-CAM mRNA적표체량분별위0.26±0.02、0.79±0.03、0.37±0.02、0.45±0.04균저우공백대조조0.94±0.03급음성대조조0.92±0.03(P＜0.01);Western blot현시,간우질립-1、-2、-3、-4조EP-CAM단백적표체량분별위0.22±0.03、0.71±0.03、0.32±0.02、0.41±0.04균저우공백대조조0.85±0.03급음성대조조0.84±0.04(P＜0.01);선용효과최가적간우질립-1전염GBC-SD세포후,MTY법현시간우조세포증식활성수도억제,전염48 h후최현저,억제솔위39%,류식세포의검측현시3조세포적조망솔차이무통계학의의(P＞0.05).결론 침대EP-CAM적miRNA중조질립능특이지억제EP-CAM기인표체,억제GBC-SD세포적증식.
Objective To observe the effect of epithelial cell adhesion molecule (EP-CAM) gene silencing by RNA interfering (RNAi) technology on the proliferation, apoptosis of GBC-SD cells. Methods Recombinant plasmid pcDNA? 6. 2-GW/EraGFPmiR-EPCAM-l, -2, -3 and -4 targeting human EP-CAM gene were respectively transfected into GBC-SD cells by lipofectamine? 2000 after DNA sequencing. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were used to detect the mRNA and protein expression of EP-CAM and adopted to select the recombinant plasmid which showed the most optimal inhibition effct. After tranfection by pcDNA?6. 2-GW/EmGFPmiR-EPCAM-l with the highest inhibition rate, cell proliferation was analyzed by methyl thiazol tetrazolium (MTT) assay, and apoptosis was assayed by flow cytometry. Results Four EP-CAM-targeted microRNA (miRNA)-l, -2, -3 and -4 were successfully inserted into the plasmid vector pcDNA?6. 2-GW/EmGFPmiR, and the coding sequences of the obtained miRNA were consistent with the designed fragments. RT-PCR results showed that the mRNA expression of EP-CAM gene in GBC-SD cells transfected with recombinant plasmid-1 ,-2,-3 and -4 were 0. 26 ± 0. 02, 0. 79 ± 0. 03, 0. 37 ± 0. 02, 0.45 ± 0.04 respectively, which was significantly lower than that in blank and negative control groups (0. 94 ± 0. 03, 0. 92 ± 0. 03, respectively, P ＜ 0. 01). Western blotting revealed that the relative expression of EP-CAM protein in GBC-SD cells transfected with recombinant plasmid-1, -2, -3 and 4 was 0. 22 ±0. 03, 0.71 ±0.03, 0.32 ±0.02, and 0.41 ±0.04 respectively, which was significantly lower than that in blank and negative control groups (0. 85 ±0.03, 0. 84 ±0.04, respectively,P＜0. 01). After transfection with pcDNA?6. 2-GW/EmGFPmiR-EPCAM-l, the proliferation ability of the cells was significantly inhibited and the inhibition rate was 39% after 48 h. As compared with the control groups, the difference was significant (P ＜ 0. 05). However cell apoptosis had no significant change ( P ＞ 0. 05 ). Conclusion The recombinant plasmid pcDNA? 6. 2-GW/EmGFPmiR miRNA targeting human EP-CAM gene could effectively down-regulate EP-CAM expression and inhibit proliferation of GBC-SD cells in vitro.