[缺氧诱导因子-1α对宫颈癌HeLa细胞系的体外影响]

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

作者信息

Cheng Yan-xiang, Pu De-min, Liu Rong, Li Tian, Yin Ling, Ma Ding

机构信息

Department of Obstetrics and Gynecology, Tongji Hospital of Tongji Medical College, Huazhong Science and Technology University, Wuhan 430032, China.

出版信息

Zhonghua Fu Chan Ke Za Zhi. 2007 Aug;42(8):551-4.

PMID:17983496

Abstract

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.

摘要

目的

探讨缺氧诱导因子-1α（HIF-1α）对宫颈浸润癌发生发展的直接影响及其可能的分子机制。

方法

构建靶向HIF-1α的重组反义真核表达载体，并转染至培养的人宫颈癌HeLa细胞系，以降低HIF-1α的表达，观察其对细胞增殖、凋亡、侵袭以及HIF-1α下游基因表达（包括血管内皮生长因子（VEGF）、葡萄糖转运蛋白1（GLUT1）和多药耐药基因1（MDR1））的影响。采用化学方法CoCl₂诱导生长细胞的缺氧环境。细胞分为6组，分别为NN组（正常未转染组）、NI组（正常无效转染组）、NT组（正常转染组）、HN组（缺氧未转染组）、HI组（缺氧无效转染组）和HT组（缺氧转染组）。采用噻唑蓝（MTT）法、流式细胞术和Transwell法分别评估细胞增殖、侵袭和凋亡情况，采用逆转录聚合酶链反应（RT-PCR）法检测HIF-1α、VEGF、GLUT1和MDR1的基因表达。

结果

CoCl₂诱导缺氧后，HN组（或HI组）与NN组（或NI组）相比，HIF-1α基因表达变化不明显（P＞0.05），但VEGF、GLUT1和MDR1的表达均增强，细胞总体增殖加快，凋亡受抑制[（11.46±0.28）%对（29.27±0.18）%，（15.77±0.49）%对（31.13±0.08）%]，穿膜行为增强[（37±12）%对（26±7）%，（40±9）%对（28±5）%]，差异均有统计学意义（P＜0.05）。相反，转染pcDNA3.0/HIF-1α后HIF-1α基因表达下降（NT组：0.05±0.12，HT组：0.04±0.16），差异均有统计学意义（P＜0.05）。