CAJ | 학술논문

目的构建人膜联蛋白A3(annexinA3)基因RNA干扰(miRNA)表达载体,体外研究其对人胆囊癌细胞SGC-996的增殖及凋亡作用.方法构建4组针对annexinA3的pcDNATM 6.2-GW/EmGFPmiR/miRNA表达载体和1组阴性对照序列.脂质体介导瞬时转染人胆囊癌细胞株SGC-996.用RT-PCR和Western印迹检测转染前后annexinA3基因的表达.挑选干扰效率最强的一组表达载体,以MTT法和流式细胞仪检测经miRNA干扰annexinA3对人胆囊癌细胞SGC-996的体外作用.结果经测序鉴定,annexinA3-miRNA载体构建成功,转染人胆囊癌细胞SGC-996后荧光定量PCR和westem印迹检测显示:annexinA3基因表达及蛋白表达明显降低(P＜0.05);干扰后细胞体外增殖缓慢,增殖明显受抑(P＜0.05).annexinA3基因干扰对细胞周期无明显影响,但能明显增加胆囊癌细胞的凋亡(P＜0.05).结论 AnnexinA3-miRNA表达载体能有效抑制胆囊癌SGC-996细胞annexinA3的表达,抑制肿瘤细胞生长,引起胆囊癌细胞凋亡.AnnexinA3可能是胆囊癌治疗的一个分子靶点.
목적 구건인막련단백A3(annexinA3)기인RNA간우(miRNA)표체재체,체외연구기대인담낭암세포SGC-996적증식급조망작용.방법 구건4조침대annexinA3적pcDNATM 6.2-GW/EmGFPmiR/miRNA표체재체화1조음성대조서렬.지질체개도순시전염인담낭암세포주SGC-996.용RT-PCR화Western인적검측전염전후annexinA3기인적표체.도선간우효솔최강적일조표체재체,이MTT법화류식세포의검측경miRNA간우annexinA3대인담낭암세포SGC-996적체외작용.결과 경측서감정,annexinA3-miRNA재체구건성공,전염인담낭암세포SGC-996후형광정량PCR화westem인적검측현시:annexinA3기인표체급단백표체명현강저(P＜0.05);간우후세포체외증식완만,증식명현수억(P＜0.05).annexinA3기인간우대세포주기무명현영향,단능명현증가담낭암세포적조망(P＜0.05).결론 AnnexinA3-miRNA표체재체능유효억제담낭암SGC-996세포annexinA3적표체,억제종류세포생장,인기담낭암세포조망.AnnexinA3가능시담낭암치료적일개분자파점.
Objective To construct a recombinant interfering RNA(miRNA)plasmid vector targeting annexinA3 and observe its impact on the growth and apoptosis of human gallbladder cancer cell line SGC-996.Methods Four small fragments of miRNA and one negative control were sequenced and cloned into vector pcDNATM 6.2-GW/EmGFPmiR.The recombinant plasmids were then transfected into gallbladder cancer cell line SGC-996 by positive liposomal transfection.The transcription and translation level of annexinA3 expression were detected by RT-PCR and Western Blot.Meanwhile, the proliferation and apoptosis of transfected tumor cells were determined by MTT and flow cytometry.Results The recombinant plasmids containing annexinA3 miRNA were successfully constructed.AnnexinA3 expression was significantly knocked down after miRNA plasmid transfection.Transfection of annexinA3 gene miRNA could effectively inhibit the proliferation of gallbladder cancer cell and promote apoptosis.Conclusion AnnexinA3 gene silenced by miRNA can induce the apoptosis of human gallbladder cancer cell line SGC-996.The process might serve as a potential approach for cancer gene therapy.