人肝素酶RNAi序列的筛选及鉴定  

Screening and identification of heparanase RNAi sequence

作　　者：熊震[1] 汤旭东[1] 房殿春[1] 陈婷[1] 余松涛[1] 陈陵[1] 罗元辉[1] 杨仕明[1] 

机构地区：[1]第三军医大学西南医院全军消化病研究所,重庆400038

出　　处：《第三军医大学学报》2007年第20期1929-1932,共4页Journal of Third Military Medical University

基　　金：国家自然科学基金(30570841;30200123)~~

摘　　要：目的筛选及鉴定有效人肝素酶(heparanase,Hpa)RNA干扰(RNA interference)序列。方法通过在线网络软件选择3个Hpa RNAi的靶点,再设计1组无关序列作阴性对照。通过基因重组技术,把3个干扰片段及阴性对照片段分别克隆入质粒pGenesile-1,脂质体法稳定转染至HepG2肝癌细胞;经G418抗性筛选后各挑选2个阳性克隆进行扩大培养;Western blot检测不同克隆肝素酶蛋白的表达水平,并进一步采用RT-PCR验证筛选克隆肝素酶mRNA的表达水平。结果通过在线网络软件设计3个RNA干扰序列,分别为Hpa/RNAi-1:GGCTATCTCTTCTGTTCAA,Hpa/RNAi-2:TCCT-GTCCGTCACCATTGA,Hpa/RNAi-3:CTCAGTTGCTCCTGGACTA及阴性对照序列Hpa/RNAi-N:CTACCGTTGTATAGGTGT;测序证实克隆入pGenesil-1载体的3个干扰序列及阴性对照序列与设计序列完全一致;经G418抗性筛选后形成的阳性克隆采用Western blot检测其肝素酶蛋白的表达,结果表明3个干扰序列对HepG2细胞人肝素酶蛋白的表达均有抑制作用,其中Hpa/RNAi-1和Hpa/RNAi-3干扰链的抑制效果最好,分别达到57%和71%;RT-PCR检测结果亦表明Hpa/RNAi-1和Hpa/RNAi-3序列在mRNA水平对肝素酶有较好的抑制效果。结论成功筛选出有效的人肝素酶RANi序列。Objective To screen and identify the efficient heparanase （Hpa） RNAi sequence. Methods We chose three candidate targets of Hpa RNAi through online software and designed a set of independent sequence as negative control group. The three interference fragments and independent sequence were cloned into pGenesile-1 expression vector by gene recombination technique. The recombinant vectors were then transfected into HepG2 liver cancer cell line. After screening by CA18, the expression of heparanase in the selected clones was detected by Western blot, and its mRNA expression level by RT-PCR. Results The three interference sequences designed with online tool respectively was Hpa/RNAi-1 ： GGCTATCTCTTCTGTTCAA ; Hpa/RNAi-2 ： TCCTGTCCGTCACCATTGA; Hpa/RNAi-3： CTCAGTTGCTCCTGGACTA and negative control Hpa/RNAi-N： CTACCGTTGTATAGGTGT; The three interference sequence were cloned into pGenesil-1 vector, respectively. Sequencing analysis confirmed their correctness. Positive clones formed after their stable transfection into HepG2 and G418 screening. Compared with untransfected HepG2 and negative control, Western blot analysis showed heparanase protein in interference sequence transfected HepG2 was significantly down-regulated. Hpa/ RNAi-1 and Hpa/RNAi-3 showed better inhibition effect, with their inhibition efficiency to 57% and 71% respectively; RT-PCR result proved the sequence of Hpa/RNAi-1 and Hpa/RNAi-3 could effectively down-regulate heparanase on mRNA level. Conclusion We successfully screen and identify the efficient heparanase RNAi sequence.

关 键 词：人肝素酶 RNA干扰 SIRNA 肝癌 

分 类 号：Q556.2[生物学—生物化学] Q785