慢病毒载体介导HIF-1α RNAi对人胰腺癌细胞Patu8988相关基因表达的影响

Effects of lentiviral vector-mediated RNA interference of hypoxia inducible factor-1α on the expression of related genes in human pancreatic cancer Patu8988 cells

目的探讨慢病毒表达载体介导HIF-1α RNA干扰（RNAi）对人胰腺癌细胞Patu8988HIF-1α和Glut-1表达的影响。方法构建针对HIF-1α基因的RNAi慢病毒表达载体LV-RNAi-HIF-1α乏氧条件下体外培养4h，转染LV—RNAi—HIF-1α的Patu8988细胞为实验组；转染空病毒载体和未转染任何病毒载体Patu8988细胞分别为阴性对照组和空白对照组。采用荧光定量RT—PCR和Western印迹法检测Patu8988细胞HIF-1α表达情况，采用RT—PCR法检测转染LV—RNAi—HIF-1α后Patu8988细胞Glut-1的表达情况。采用SPSS17．0软件行单因素方差分析和两样本t检验分析各组之间的差异。结果构建的慢病毒表达载体LV—RNAi—HIF-1α，在常氧和乏氧状态下致HIF-1α mRNA表达分别下降65．1％（0．209／0．321）和80．6％（0．791／0．982）（t=10．52和15．24，均P〈0．05），阴性对照组分别为0．6％（0．002／0．321）和7．2％（0．07I／0．982）（t=5．26和7．38，均P〈0．05）；乏氧状态下实验组、阴性对照组和空白对照组HIF—1α蛋白的表达分别为0．159±0．010、0．745±0．012和0．711±0．023，差异有统计学意义（F=35．52，t=6．72和10．56，均P〈0．05），实验组较其他2组表达下降；乏氧条件下实验组Glut-1mRNA表达（0．040±0．003）较阴性对照组（0．054±0．003）和空白对照组（0，062±0．004）均有明显下降（F=35．28，t=5．94和8．55，均P〈0．01）。结论通过构建LV—RNAi—HIF—1α慢病毒表达载体沉默HIF-1α基因表达，可降低Patu8988胰腺癌细胞Glut-1mRNA表达。

Objective To investigate the inhibitory effect of the lentiviral vector （LV）-mediated RNA interference （RNAi） targeting HIF-1α on the expression of HIF-1α and Glut-1 in human pancreatic cancer Patu8988 ceils. Methods The RNAi targeting HIF-1α was combined to LV, and transfected into Patu8988 cells. The Patu8988 cells transfected with the empty vector and exposed to 0.5% 02 for 4 h served as hypoxia negative control, the Patu8988 cells not transfected with vector and exposed to 0.5% 02 for 4 h as hypoxia blank control, and the Patu8988 cells transfected with LV-RNAi-HIF-1α and exposed to 0.5% 02 for 4 h as experimental group. The expression of HIF-1α was measured by RT-PCR and Western blot respectively. The expression of Glut-1 was measured by RT-PCR. Each group was compared according to one- way analysis of variance and two-sample t test. Results After transfection with LV-RNAi-HIF-1α, HIF-1α mRNA expression decreased by 65.1% （0.209/0.321） and 80.6% （0.791/0.982） （t = 10.52, 15.24, both P〈0.05） under normoxia and hypoxia conditions, meanwhile with the empty vector, HIF-1α mRNA expression decreased by 0.6% （0.002/0.321） and 7.2% （0.071/0.982） （t=5.26, 7.38, both P〈0. 05）. Under hypoxia conditions, the protein of HIF-1α in experimental group cells （0.159±0.010） was down-regulated obviously compared to the negative control group （0.745±0.012） and the blank control group （0.711±0.023） （F= 35.52, t = 6.72, 10.56, all P〈0.05）. The expression of Glut-1 mRNA in experimental group cells （0.040±0.003） decreased obviously compared to the negative control group （0.054±0.003） and blank control group （0.062±0.004） （F=35.28, t=5.94, 8.55, all P〈0.01）. Conclusion Gene silencing of HIF-1α using LV-mediated RNAi can inhibit the expression of HIF-1α and decrease the expression of Glut-1 mRNA in Patu8988 cells. [ Key words ] Pancreatic neoplasms ; Tumor cells, euhured ; Gene expression ; Hypoxia-inducible factor 1, alpha subunit; RNA interference