摘要
目的探讨低氧诱导因子1α(HIF-1α)对宫颈癌细胞的生物学行为的影响以及其中可能存在的分子机制。方法通过 CoCl_2化学诱导宫颈癌 HeLa 细胞缺氧;构建靶向 HIF-1α的反义真核表达载体、经脂质体介导转染 HeLa 细胞的方法沉默 HIF-1α的表达。将实验细胞分为常氧未转染对照(NN)组、常氧空质粒转染对照(NI)组、常氧转染 pcDNA3.0/HIF-1α质粒(NT)组、缺氧未转染对照(HN)组、缺氧空质粒转染对照(HT)组、缺氧转染 pcDNA3.0/HIF-1α质粒(HT)组。用四甲基偶氮唑蓝法、Transwell 侵袭小室方法观察各组细胞的增殖、侵袭能力的改变,用流式细胞仪检测各组细胞的凋亡率,用 RT-PCR 技术检测各组细胞目的基因 HIF-1α及其靶基因血管内皮生长因子(VEGF)、葡萄糖转运体1(GLUT1)、多药耐药基因1(MDR1)的表达变化。结果 NT 组细胞在培养12、24、48、72 h 时的活细胞数分别为0.053±0.003、0.074±0.004、0.148±0.015、0.192±0.038,而 HT 组分别为0.069±0.003、0.155±0.022、0.224±0.022、0.308±0.069;NT 和 HT 组的细胞增殖受到抑制,NT组与 NN 及 NI 组、HT 组与 HN 及 HI 组间的活细胞数分别比较,差异均有统计学意义(P<0.01)。各组细胞的凋亡率分别是,NN 组(29.27±0.18)%、NI 组(31.13±0.08)%、NT 组(51.11±0.14)%、HN 组(11.46±0.28)%、HI 组(15.77±0.49)%、HT 组(40.05±0.97)%;HT 组与 HN 及 HI 组、NT组与 NN 及 NI 组间分别比较,差异均有统计学意义(P<0.01)。各组细胞的侵袭能力由高到低依次为 HI、HN、NI、NN、HT、NT 组,分别为(40±9)%、(37±12)%、(28±5)%、(26±7)%、(19±7)%、(10±5)%;NT 组与 NN 及 NI 组、HT 组与 HN 及 HI 组间分别比较,差异均有统计学意义(P<0.05)。NT 组细胞 HIF-1α、VEGF、GLUT1、MDR1 mRNA 的相对表达量分别为0.05±0.12、0.09±0.11、0.08±0.15、0.05±0.15,而 HT 组分别为0.04±0.16、0.16±0.16、0.12±0.20、0.20±0.21;NT 组与 NN及 NI 组、HT 组与 HN 及 HI 组间分别比较,HIF-1α、VEGF、GLUT1、MDR1 mRNA 的相对表达量均有降低,差异均有统计学意义(P<0.01)。结论 HIF-1α可能主要通过其下游的靶基因对宫颈癌细胞的恶性生物学行为产生影响,包括抗凋亡、促增殖、增加血液供应和能量供应、耐药等,且体外抑制 HIF-1α的表达对宫颈癌细胞有抑制作用。
Objective To explore the direct influence of hypoxia inducible factor-1α （HIF-1α） on the development of invasive cervical cancer and the possible molecular mechanism. Methods Recombinant antisense targeting HIF-1α eukaryotic expression vector was constructed and transfected into cultured human cervical cancer cell line HeLa to reduce the expression of HIF-1α and its effect on cell proliferation, apoptosis, invasion and the cascade downstream gene expression of HIF-1α, including vascular endothelial growth factor （VEGF）, glucose transport 1 （GLUT1） and multidrug resistance 1 （MDR1） genes was observed. The chemical method using CoCl2 to induce hypoxia environment of growing ceil was performed. Ceils were divided into six groups, NN （normal non-transfected）, NI （normal invalid transfected）, NT （ normal transfected）, HN （ hypoxia non-transfected）, HI （ hypoxia invalid transfected）, and HT （ hypoxia transfected）. Methyl thiazolyl tetrazolium （MTT）, flow cytometry, and Transwell methods were performed to evaluate the proliferation, invasion and apoptosis, and RT-PCR method was used to detect the gene expression of HIF-1α, VEGF, GLUT1 and MDR1. Results After induction of hypoxia by COCl2, the change of gene expression of HIF-1α in HN （or HI） group compared to that in NN （ or NI） group was not obvious （P 〉 0. 05 ）, but expression of VEGF, GLUT1 and MDR1 were all enhanced and overall proliferation was promoted, apoptosis inhibited [ （ 11.46 ± 0. 28）% vs （29. 27 ± 0. 18）% , （ 15.77 ± 0.49）% vs （31.13±0.08）%], and transmembrane behavior enhanced [（37 ±12）% vs （26±7）%, （40 ±9）% vs （28 ±5）% ], and the variations were significant （P 〈0. 05）. On the contrary, transfection with pcDNA3.0/HIF-1α was companied by declined gene expression of HIF-1α （ NT： 0. 05 ± 0. 12, HT： 0.04 ± 0. 16）, and all the variations were significant （ P 〈 0. 05）. Conclusions HIF-1α may participate in malignant biological behaviors of cervical cancer such as anti-apoptosis, accelerating proliferation, increasing supply of blood and energy, increased resistance to chemotherapy through upregnlation of its downstream genes. Suppression of HIF-1α expression in vitro can inhibit cervical cancer cell line.
出处
《中华妇产科杂志》
CAS
CSCD
北大核心
2007年第8期551-554,共4页
Chinese Journal of Obstetrics and Gynecology
