血管内皮细胞生长因子受体KDR基因靶向RNA干扰质粒的构建

Construction of KDR-targeting RNA interference recombinant plasmids

目的:应用RNA干扰技术设计构建针对血管内皮细胞生长因子受体KDR的小干扰RNA,并观察脂质体转染肺癌细胞A549后的干扰效果。方法:实验于2005-03/2006-01在沈阳医学院生物化学及分子生物学教研室完成。①设计针对KDR编码区有短发夹结构的3条mRNA序列,经退火成互补双链,克隆到pGCsi.H1/neo/GFP载体中构建3个重组质粒,分别命名为KDR-siRNA1、KDR-siRNA2和KDR-siRNA3。②设立5组:小干扰RNA组,分别转染KDR-siRNA1、KDR-siRNA2和KDR-siRNA3;阳性对照组,转染pGCsi.H1/neo/siGFP,该质粒载体中的插入序列为针对绿色荧光蛋白的小干扰RNA,不干扰待研究的内源性基因;阴性对照组,转染pGCsi.H1/neo/GFP/NON,该载体为不干扰任何内源性基因的小干扰RNA;空白对照组,转染pGCsi.H1/neo/GFP空载体;正常对照组,不进行任何转染。③对重组质粒进行酶切鉴定、DNA测序分析;脂质体法转染质粒至肺癌A549细胞株后,实时定量PCR检测KDRmRNA的水平变化;细胞计数法绘制细胞生长曲线。结果:①小干扰RNA表达载体的鉴定:KDR-siRNA1、KDR-siRNA2和KDR-siRNA3表达载体用限制性内切酶NdeⅠ和SmaⅠ进行单酶切后,均产生约713bp、5480bp和2403bp、3790bp两个片段,与预期结果相同。测序结果与设计的编码相应短发夹状KDR-小干扰RNA的寡核苷酸序列一致,证明KDR-小干扰RNA真核表达载体构建成功。②KDR-小干扰RNA对A549细胞中KDRmRNA水平的影响:与阳性对照组、阴性对照组、空白对照组和正常对照组的A549细胞相比,KDR-siRNA1,2,3表达载体转染后的A549细胞KDR基因表达水平均明显受到抑制,抑制率分别为64%、81%和72%,其中以KDR-siRNA2抑制作用最为明显。③KDR-小干扰RNA对A549细胞生长的影响:阳性对照组、阴性对照组、空白对照组、正常对照组的A549细胞生长趋势较为一致,且生长速度均明显高于转染3种KDR-小干扰RNA表达载体的A549细胞,从接种第2天开始差异有显著性意义(t=15.29～17.65,P均<0.01)。结论:血管内皮细胞生长因子受体KDR靶向RNA干扰重组质粒构建成功,该载体能有效抑制肺癌A549细胞KDR基因表达与细胞增殖。

AIM： To design a short hairpin RNA interference of vascular endothelial cell growth factor （VEGF） receptor-KDR, and investigate its effect on transfecting lung cancer cell line A549. METHODS： The experiment was performed at the Department of Biochemistry and Molecular Biology of Shenyang Medical College from March 2005 to January 2006. （1）Three DNA sequences with short hairpin structures were designed for the KDR encoding region, and then cloned into pGCsi.H1/neo/GFP vector to construct three recombinant plasmids, namely KDR-siRNA1, KDR-siRNA2 and KDR-siRNA3. （2）There were five groups in the experiment including siRNA group that transfected three recombinant plasmids; positive control group that transfected pGCsi.H1/neo/siGFP; negative control group that was used to transfect pGCsi.H1/neo/GFP/NON; blank control group that was used to transfect pGCsi.H1/neo/GFP empty vector; normal control group with no transfection. （3）Enzyme digestion and DNA sequencing were used to testify three recombinant plasmids. Lung cancer cell line A549 was transfected with the recombinant plasmids via Lipofectamine 2000. The KDR mRNA level was detected by real-time PCR and the cell growth curves were drawn by cell counting. RESULTS： （1）Three KDR-siRNA expression vectors were digested by Nde Ⅰ and Sma Ⅰ and produced two fragments of 713 bp, 5 480 bp and 2 403 bp, 3 790 bp, respectively, which were congruous with expectation. The sequencing results were in accordance with that of the designed oligonucleotides that encoded corresponding KDR-siRNA. It indicated that KDR-siRNA expression vectors were constructed successfully. （2）KDR gene expressions in A549 which were transfected by the three KDR-siRNA expression vectors were all significantly decreased compared with that of A549 cells transfected by positive and negative vectors, especially the group transfected by KDR-siRNA2. The inhibition rates were 64%, 81% and 72%, respectively. （3）The cell growth tendency of A549 transfected by positive and negative vectors was almost the same with that of A549 in blank and normal control groups, but it was prominently faster than that of A549 transfected by three KDR-siRNA expression vectors. The differences were statistical significant from the second day of culture （t =15.29-17.65, P〈 0.01）. CONCLUSION： KDR-targeting RNA interference recombinant plasmids were successfully constructed, which could decrease the KDR gene expression effectively and inhibit cell proliferation in lung cancer cell A549.