摘要
目的:研究miR-125b在胃癌SGC7901/VCR细胞多药耐药形成中的作用。方法:运用miRNA实时定量PCR方法检测miR-125b在SGC7901/VCR细胞与母代SGC7901细胞中的表达差异,运用Western blot检测SGC7901/VCR细胞与母代SGC7901细胞抗凋亡蛋白BCL2、MCL1的表达差异,分别构建BCL2、MCL1 3’UTR荧光素酶报告质粒验证miR-125b的靶基因,在耐药细胞株中瞬时转染miR-125b模拟物以检测上调miR-125b对SGC7901/VCR细胞BCL2、MCL1蛋白表达及多药耐药性的影响,并运用流式细胞术检测转染后耐药细胞对长春新碱诱导凋亡的影响。结果:miR-125b在SGC7901/VCR细胞中呈低表达,抗凋亡蛋白BCL2、MCL1在SGC7901/VCR细胞中呈高表达,荧光素酶报告实验证实BCL2、MCL1是直接受miR-125b调控的靶基因,在耐药株中上调miR-125b显著抑制BCL2、MCL1蛋白表达水平,显著增加细胞对长春新碱、顺铂、阿霉素、依托泊苷的敏感性,并显著增加细胞对长春新碱诱导的凋亡。结论:miR-125b靶向抑制BCL2、MCL1表达可增加胃癌SGC7901/VCR细胞对多种化疗药物的敏感性。
Objective：To investigate the possible role of microRNA-125b（miR-125b） in the development of multidrug resistance（MDR） in human gastric cancer cell line SGC7901 / VCR.Methods：Using quantitative real-time PCR analysis and Western blot to detect the expression difference of miR-125b and anti-apoptotic protein BCL2 and MCL1 between multidrug-resistant human gastric cancer cell line SGC7901 / vincristine（VCR） and its parental SGC7901 cell line,respectively.BCL2 and MCL1 3’-untranslated re-gion-based luciferase reporter plasmids were constructed to testify the target genes of miR-125b.Transient transfection of miR-125b mimic was used to up-regulate the expression level of miR-125b in SGC7901 / VCR cells and the effect of miR-125b on the expression of BCL2,MCL1 and the MDR phenotype was observed.Flow cytometry was used to detect VCR-induced apoptosis of the SGC7901 / VCR cells after transfection.Results：miR-125b was low expressed in multidrug-resistant human gastric cancer cell line SGC7901 / VCR,and the downregulation of miR-125b was concurrent with the overexpression of antiapoptotic genes BCL2,MCL1 in SGC7901 / VCR cells compared with the parental SGC7901 cell line.The luciferase activity of BCL2 and MCL1 3’-untranslated region-based re-porters constructed in SGC7901 / VCR cells suggested that BCL2 and MCL1 were the common target genes of the miR-125b.Overex-pression of miR-125b sensitized SGC7901 / VCR cells to anticancer drugs of VCR,CDDP,ADR and VP-16.Overexpression of miR-125b also inhibited the expression of BCL2,MCL1 and sensitized SGC7901 / VCR cells to VCR-induced apoptosis.Conclusion：miR-125b plays a role in the development of MDR in human gastric cancer cell line,at least in part,by modulation of apoptosis via tar-geting BCL2 and MCL1.
出处
《南京医科大学学报（自然科学版）》
CAS
CSCD
北大核心
2011年第6期777-782,共6页
Journal of Nanjing Medical University(Natural Sciences)
基金
国家自然科学基金资助项目(30840095)
江苏省2009年度普通高校研究生科研创新基金(CX09B-262Z)
