摘要目的 通过观察pGenesil-1肿瘤转移相关基因1(MTA1)对人乳腺癌细胞株MDA-MB-231和MCF-7细胞MTA1基因的沉默效应,了解其对ERα表达及浸润能力的影响.方法 设计合成并构建重组质粒pGenesil-1MTA1,将该质粒分别转染MDA-MB-231和MCF-7细胞,荧光显微镜评估转染效率,RT-PCR检测MTA1和基质金属蛋白酶(MMP)-9 mRNA表达,免疫细胞化学检测ERα和MMP-9表达,Western blot检测ERα蛋白表达,用Boyden小室体外侵袭实验检测转染前后两株细胞侵袭力的变化.结果 成功构建重组质粒pGenesil-1MTA1,并成功转染入MDA-MB-231和MCF-7细胞(平均转染效率分别为82.5%和78.2%).两株细胞MTA1 mRNA表达均受到不同程度的抑制(分别为80.2%和58.7%).经干扰MTA1基因后MDA-MB-231细胞ERα蛋白表达得到逆转,呈阳性表达;MMP-9 mRNA表达下降;细胞体外侵袭力减弱(侵袭指数为27.2%±2.1%),与干扰前(76.3%±2.4%)相比差异有统计学意义(P＜0.05).而MCF-7细胞转染重组质粒并干扰MTA1基因后,ERα蛋白、MMP-9 mRNA表达和体外侵袭能力变化均无统计学意义(P＞0.05).结论 重组质粒pGenesil-1MTA1能有效抑制人乳腺癌细胞株MDA-MB-231和MCF-7细胞MTA1基因表达,ERa在MDA-MB-231细胞得到逆转,表达阳性,其高侵袭能力减弱,使干扰肿瘤转移靶基因成为可能.

abstractsObjective To investigate the inhibitory effect on MTA1 gene by MTA1 short hairpin RNA(shRNA),downstream regulation of expression of Era and invasion of human breast cancer cells.Methods Recombinant plasmid pGenesil-1MTA1 was constructed and transfected into human breast cancer cell lines MDA-MB-231 and MCF-7. Fluorescence microscopy was used to evaluate the efficacy of transfection.The transcription expression of MTA1 and MMP-9 gene were determined by reverse transcription-polymerase chain reaction(RT-PCR),the protein expression of Erα and MMP-9 were determined by immunohistochemical and western blot.The invasion ability was evaluated by Boyden chamber invasive assay.Results Recombinant plasmid pGenesil-1MTA1 was constructed and transfected into MDA-MB-231(82.5%)and MCF-7(78.2%)successfully.After the transfection,MTA1 mRNA was suppressed in both cell lines(80.2%and 58.7%).The expression of Erα protein became positive in transfected MDA-MB-231 cell,and the expression of MMP-9 mRNA were down-regulated.The invasion ability was decreased(27.2%4±2.1)%compared with(76.3%±2.4%),(P＜0.05).In contrast,transfected MCF-7 cells failed to show significant difference in the expression of Erα and MMP-9,without change of the invasion ability(P＞0.05).Conclusions RNA interference can effectively suppress the expression of MTA1 in human breast cancer cells.An induction of Erα protein and suppression of MMP-9 may be related to the decrease of tumor cell invasive ability.RNA interference involving MTA1 gene may provide an effective anti-cancer gene therapy.