人U6基因POI Ⅲ启动子表达反义VEGF cDNA抑制SMMC-7721肝癌细胞VEGF表达的观察

Inhibition of VEGF Expression in Human Hepatoma Cell Line SMMC-7721 by Antisense VEGF RNA Transcribed from U6 Gene POI Ⅲ Promoter

由 RNA聚合酶启动子在细胞内转录高浓度反义 RNA是抑制靶蛋白的一种有效手段 ,有报道 POI 启动子转录小分子 RNA存在效率不高 ,带有较多的非特异性序列等缺点 ,为了克服这些问题 ,表达反义 VEGF RNA的人 U6基因表达盒对人肝癌细胞株 SMMC- 772 1 VEGF表达的抑制作用进行了研究 .首先 PCR扩增 2 0 0 bp VEGF c DNA以正、反向插入人 U6 sn RNA.通过测序证实反向插入的正确性 .采用细胞原位杂交 ,RNA酶保护分析 ,Northern印迹来证实反义 RNA表达的情况 ,利用 RT- PCR方法研究了其对人肝癌细胞株 SMMC- 772 1 VEGF表达的抑制效果 .细胞原位杂交结果显示 U6启动子转录产物主要分布于细胞核内 ,细胞浆内亦有表达 ,RNA酶保护分析显示 U6基因 POI 启动子能高表达所需大小反义 RNA,Northern印迹结果显示脂质体 lipo-fectamine介导含 U6基因 POI 启动子的质粒转染人肝癌细胞株 SMMC- 772 1后 1 2 h即有表达且可持续表达 6 d. RT- PCR证实 U6基因 POI 启动子转录的反义 VEGF RNA能有效抑制SMMC- 772 1细胞 VEGF的 m RNA表达 .已有的研究结果揭示 POI

It is a useful way to inhibit the expression of target gene by antisense RNA transcribed by RNA polymerase in cells.Some studies show that there are some drawbacks,when small therapeutics RNA transcript by RNA plymerase Ⅱ,such as ineffective intracellular expression,and many unnecessary RNA sequence.To overcome these problems,the inhibitory effects on the expression of VEGF of human hepatoma cell line SMMC 7721 by cassette based on the human U6 snRNA promoter were tested.Firstly,the PCR\|amplified 200 bp VEGF cDNA was inserted into the human U6 snRNA cassette with sense or antisense orientation.The antisense clone was verified by DNA sequence analysis.Through in situ hybridization,RNase protection analysis and Northern blot,the expression of antisense VEGF RNA was detected.The efficacy of inhibition was verified by RT PCR.From the results of in situ hybridization,it was showed that antisense transcripts by U6 promoter located in the nuclear,and also had some cytoplasmic staining.The RNase protection analysis showed that antisense VEGF RNA could be efficiently transcribed by human U6 snRNA promoter.The results of Northern blot suggested that the expression of antisence VEGF RNA could be detected within 12 hours post\|transfection and was still readily detectable 6 days post\|transfection.The RT\|PCR results showed that SMMC\|7721 cells transduced with antisense vector expressed less VEGF mRNA than did tumor cells transduced with the sense vector alone.These results demonstrate that expression of antisense VEGF transcribed by human U6snRNA promoter can decrease the expression of VEGF mRNA in transduced SMMC\|7721 cells and this may be efficiently used in antiangiogenesis of hepatoma.