HIF-1α基因沉默对人肺腺癌细胞A549裸鼠移植瘤生长及survivin表达的影响

Effect of HIF-1α Gene Silencing on the Growth of Human Lung Adenocarcinoma Cell A549 in Nude Mice and Survivin Expression

目的观察缺氧诱导因子-1α(HIF-1α)基因沉默对肺癌的抑瘤效应和survivin表达的影响。方法将实验用肺腺癌A549细胞分为空白对照组、无意义序列转染组和实验组,接种裸鼠,建立裸鼠荷瘤模型,观测HIF-1α-MiRNA对裸鼠皮下移植瘤的生长抑制作用和对survivin表达的影响。采用免疫组化SP法,检测HIF-1α和survivin蛋白在裸鼠移植瘤中的表达。采用逆转录聚合酶链反应(RT-PCR)法检测HIF-1α和survivin mRNA的表达。采用原位末端标记(TUNEL)法检测肿瘤组织中细胞凋亡。结果实验组裸鼠肿瘤的生长速度较空白对照组、无意义序列转染组明显减慢(P<0.05)。接种60 d后处死裸鼠,实验组裸鼠的肿瘤重量为(0.59±0.28)g,明显轻于空白对照组[(1.90±0.18)g]和无意义序列转染组[(1.66±0.24)g,P<0.01]。实验组肿瘤组织中HIF-1αmRNA和蛋白的相对表达量分别为(0.45±0.04)%和(24.56±5.83)%,survivin mRNA和蛋白的相对表达量分别为(0.32±0.02)%和(29.08±3.99)%,均明显低于空白对照组、无意义序列转染组(P均<0.05)。但实验组肿瘤组织中凋亡细胞的数量显著高于对照组。结论以HIF-1α为靶点的基因治疗,可能通过下调靶基因survivin的表达促进肺癌细胞凋亡,发挥抑制肿瘤生长的作用。

Objective To observe the Effect of HIF-1α gene silencing on the growth of human lung adenocarcinoma cell A549 in nude mice and survivin expression.Methods Human lung cancer cell 1ine A549 cells were divided into 3 groups：mock control group,control group transfected with scrambled sequence plasmid and experimental group transfected with HIF-1α-MiRNA eukaryotic expression plasmid.Cultured cells of the three groups were inoculated in nude mice to establish lung cancer bearing nude mice.HIF-1α RNAi assay was performed to evaluate the tumor-suppressive effect of HIF-1α silencing on lung cancer-bearing nude mice.Immunohistochemistry assay was done to detect the protein expression of HIF-1α and Survivin.RT-PCR was adopted to detect the mRNA expression of HIF-α and survivin.TUNEL stainin was used to detect apoptosis in tumor cells.Results The tumor growth in experimental group was significantly slower than that in the three control groups（P0.05）.0n the 60th day after transplantation.the tumor weight in the experimental group was（1.90±0.18）g,significantly lower than in the control group transfected with scrambled sequence plasmid（1.66±0.24）g in the mock group and control group without handed（1.90±0.18）g（P0.01）.In the experimental group.the gene and protein 1evels of HIF-1α were（0.45±0.04）%and（25.46±5.83）% and the levels of survivin were（0.32±0.02）%and（29.08±3.99）%,respectively.Both indicators in HIF-1α and survivin were lower than that in the two control groups（P0.05）.but the apoptotic cells were much more numerous in the experimental group than that in matched control groups.Conclusion The gene therapy by Mi RNA targeted silencing of HIF-1α may downregulate its downstream genes survivin expression,therefore,to promote lung cancer cell apoptosis and exert a tumor-suppressing effect in vivo.