| 殷杰，杨晓燕，虞佳，李倩，向琼，唐惠芳，雷小勇.miR-122表达载体的构建及其对Bcl-xL,Bcl-2基因的抑制作用.[J].中南医学科学杂志.,2013,41(1):13-16. |
| miR-122表达载体的构建及其对Bcl-xL,Bcl-2基因的抑制作用 |
| The miR-122 Expression Vector Construction and theDown-regulation of Bcl-xL,Bcl-2 Gene Expression |
| 投稿时间：2012-09-05 |
| DOI： |
| 中文关键词: miRNA miR-122 BEL-7402/5-FU Bcl-xL Bcl-2 耐药 |
| 英文关键词:miRNA miR-122 pSilencer 3.1-H1 neo Bcl-xL Bcl-2 drug resistance |
| 基金项目:国家自然基金(30900625);湖南省教育厅项目(08A059). |
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| 中文摘要: |
| 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。 |
| 英文摘要: |
| ObjectiveTo 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.MethodsThe miR-122 mature sequences were composited manually and inserted into the pSilencer 3.1-H1 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 transfected,and the empty vector transfectant is the cells where the empty vector was transfected.The miR-122 expression were detected 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 untreated cells,miR-122 and empty vector transfectants by Real time fluorescent quantitative RT-PCR.ResultsmiR-122 expression 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.ConclusionThese 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. |
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