摘要
目的构建miR-122真核表达载体并检测其表达对BEL-7402/5-FU细胞中耐药相关基因Bcl-xL,Bcl-2基因表型的作用。方法人工合成miR-122成熟cDNA序列,以质粒载体pSilencer 3.1-H1 neo为母本,构建miR-122真核表达载体,空载体作为对照,利用脂质体2000和G418筛选稳转至BEL-7402/5-FU细胞中。其中转染miR-122载体的细胞为miR-122转染组,转染空载体的细胞为空载体转染组,通过实时荧光定量RT-PCR检测未转染组、转染空载体组和miR-122转染组细胞中miR-122表达水平。验证miR-122载体是否在细胞中稳定表达。并通过实时荧光定量RT-PCR检测未转染组、空载体转染组和miR-122转染组中耐药相关基因Bcl-xL,Bcl-2的基因表达情况。结果 miR-122真核表达载体在BEL-7402/5-FU细胞中稳定表达,与未转染组和空载体转染组比较,miR-122转染组中Bcl-xL,Bcl-2的基因表达水平显著降低。结论 miR-122可下调耐药相关基因Bcl-xL,Bcl-2的基因表达,MiR-122是降低BEL-7402/5-FU细胞的5-氟尿嘧啶药物敏感性影响的潜在因素。
Objective To construct the miR-122 expression vector and examine the effect of miR-122 expression on Bcl-xL,Bcl-2 drug resistance genes expression in BEL-7402/5-FU cells. Methods The miR-122 mature sequences were composited manually and inserted into the pSilencer 3.1-HI neo Eukaryotic expression vector,which was the construction of miR-122 expression vector. And the empty vector was as a negative control. The vectors were transfected into BEL-7402/5- FU cells by liposome and screened by G418. The miR-122 transfectant is the cells where the miR-122 vector was transfeet- ed,and the empty vector transfeetant is the cells where the empty vector was transfected. The miR-122 expression were de- tected in untreated cells, miR-122 and empty vector transfectants by Real time fluorescent quantitative RT-PCR to validation of miR-122 stable expression in BEL-7402/5-FU cells. The Bcl-xL, Bcl-2 mRNA expression were detected in ontreated cells ,miR-122 and empty vector transfectants by Real time fluorescent quantitative RT-PCR. Results miR-122 expres- sion vector can stably express in BEL-7402/5-FU cells. Compared with empty vector transfectants or untreated cells, Bcl-2 and Bcl-xL mRNA expression in miR-122 transfectants were obviously decreased. Conclusion These results showed that miR-122 have down-regulated Bcl-2 and Bcl-xL gene expression. It will facilitate further studies of the functions of miR-122 in the drug resistance mechanism of hepatocellular carcinoma to 5-FU.
出处
《中南医学科学杂志》
CAS
2013年第1期13-16,共4页
Medical Science Journal of Central South China
基金
国家自然基金(30900625)
湖南省教育厅项目(08A059)
国家自然基金(30900625)
湖南省教育厅项目(08A059)
湖南省重点学科药理学资助
