靶向HIF-1α小干扰RNA对裸鼠MiaPaCa2细胞移植瘤生长的影响

Effect of Small Interference RNA Targeting HIF-1α Mediated by rAAV on MiaPaCa2 Human Pancreatic Cancer Cells Grown as Xenograft Tumors in Athymic Mice

目的:初步探讨重组腺相关病毒(rAAV)介导的靶向缺氧诱导因子-1α(HIF-1α)小干扰RNA对裸鼠Mia-PaCa2人胰腺癌细胞移植瘤生长的影响。方法:首先构建rAAV介导的以HIF-1α为靶基因的RNA干扰表达载体,即rAAV-siHIF。将rAAV-hrGFP、rAAV-siHIF分别转染对数生长的MiaPaCa2细胞,在乏氧条件下进行培养,应用Real-Time PCR、Western Blot和ELISA法观察其对MiaPaCa2细胞HIF-1α mRNA、蛋白和VEGF表达的影响。将MiaPaCa2细胞接种于裸鼠皮下,形成荷瘤小鼠后随机分别肌注ddH2O(空白对照组)、rAAV-hrGFP(空载病毒组)和rAAV-siHIF(干扰病毒组),30d后处死荷瘤裸鼠,取出移植瘤并测量肿瘤的大小及肿瘤的质量,应用免疫组化染色观察各组肿瘤标本HIF-1α、VEGF、CD34的表达并计数微血管密度(MVD)。结果:体外实验发现,干扰病毒组MiaPaCa2细胞HIF-1α mRNA和蛋白的表达以及VEGF的分泌低于空白对照组(均P<0.01)。体内实验发现,干扰病毒组裸鼠移植瘤的生长和HIF-1α、VEGF及CD34的表达也低于空白对照组(P<0.01或P<0.05)。而空载病毒组与空白对照组相比,体内外实验结果差别均无统计学意义(均P>0.05)。结论:rAAV介导的靶向HIF-1α小干扰RNA有抑制裸鼠MiaPaCa2细胞移植瘤生长的作用,其机制可能是通过抑制移植瘤的血管生成而实现的。

Objective： To study the effect of recombinant adeno-associated virus （rAAV） vector carrying small interference RNA （siRNA） targeting hypoxia inducible factor 1α （HIF-1α） on MiaPaCa2 human pancreatic cancer cells grown as xenograft tumors in athymic mice. Methods： A cassette encoding siRNA targeting HIF-1α mediated by rAAV was constructed, namely rAAV-siHIF. In vitro, rAAV-hrGFP and rAAV-siHIF which were used separately were transfected to exponentially growing MiaPaCa2 cells under hypoxic condition. We examined the expression of HIF-1α mRNA and protein, the secretion of VEGF in MiaPaCa2 cells with Real-time PCR, Western Blot, and ELISA respectively. In vivo, MiaPaCa2 cells were inoculated subcutaneously on the back of nude mice. Nude mice with xenograft tumor were randomly divided into three groups and were injected with ddH20, rAAV-hrGFP, rAAV-siHIF, respectively. After 30 days, all mice were sacrificed and the tumors were dissected. The size and weight of tumors were tested. The expressions of HIF-1α, VEGF and CD34 were examined by immunohistochemistry and counted micro-vessel density （MVD）. Results： In vitro, the expression of HIF-1α mRNA and protein as well as the secretion of VEGF in MiaPaCa2 cells in rAAV-siHIF group were lower than those in control group （P 〈 0.01）. In vivo, the growth of nude mice xenograft tumor and the expression of HIF-1α, VEGF and CD34 in rAAV-siHIF group were slower and lower than those in control group （P 〈 0.01 or P 〈 0.05）. There was no significant difference between control group and rAAV-hrGFP group in vitro or in vivo （P 〉 0.05）. Conclusion： rAAV-siHIF can suppress the growth of nude mice MiaPaCa2 xenograft tumor. The possible reason is that angiogenesis of xenograft tumor is stopped by rAAV-siHIF.