低氧诱导因子1α对子宫颈癌细胞生物学行为的影响

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CoCl2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（HI）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、72h时的活细胞数分别为0．053±O．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。

Influence of hypoxia inducible factor-1alpha on cervical cancer cell line HeLa in vitro

OBJECTIVE: To explore the direct influence of hypoxia inducible factor-1alpha (HIF-1alpha) on the development of invasive cervical cancer and the possible molecular mechanism. METHODS: Recombinant antisense targeting HIF-1alpha eukaryotic expression vector was constructed and transfected into cultured human cervical cancer cell line HeLa to reduce the expression of HIF-1alpha and its effect on cell proliferation, apoptosis, invasion and the cascade downstream gene expression of HIF-1alpha, including vascular endothelial growth factor (VEGF), glucose transport 1 (GLUT1) and multidrug resistance 1 (MDR1) genes was observed. The chemical method using CoCl(2) to induce hypoxia environment of growing cell was performed. Cells 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-1alpha, VEGF, GLUT1 and MDR1. RESULTS: After induction of hypoxia by CoCl(2), the change of gene expression of HIF-1alpha 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-1alpha was companied by declined gene expression of HIF-1alpha (NT: 0.05 +/- 0.12, HT: 0.04 +/- 0.16), and all the variations were significant (P < 0.05). CONCLUSIONS: HIF-1alpha 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 upregulation of its downstream genes. Suppression of HIF-1alpha expression in vitro can inhibit cervical cancer cell line.