Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology, Center for Human Genome Research, Cardio-X Institute, Huazhong University of Science and Technology, Wuhan 430074, China.
Division of Medical Oncology, Puai District of Huangshi Central Hospital, Wuhan, China.
Br J Cancer. 2014 Feb 18;110(4):1034-44. doi: 10.1038/bjc.2013.817. Epub 2014 Jan 14.
The purpose of this study was to confirm that RRM2 as a novel target of HPVE7 involved in cervical cancer angiogenesis.
Gene expression was analysed by RT-qPCR, western blot and immunohistochemistry in cervical cancer tissue and cell lines. Luciferase reporter assay was used to determine the activities of various RRM2 promoters. Secreted VEGF was measured by ELISA. RRM2-mediated capillary tube formation induced by HPVE7 in cervical cancer cells were evaluated using human umbilical vein endothelial cells in vitro. ROS induced by RRM2 in cercal cancer cells was confirmed by flow cytometry. The growth of cervical cancer cell overexpression RRM2 was examined by nude mouse xenograft.
RRM2 as a novel downstream target for HPVE7 was upregulated by it at the transcriptional level through the E7-pRb interaction and binding of E2F to the RRM2 promoter region. Immunohistochemical analysis showed that the level of RRM2 positively correlated with the HPVE7 level in human cervical cancer. Functionally, overexpression of RRM2 enhanced the expression of HIF-1α and VEGF via activation of the ERK1/2 signalling pathway in cervical cancer cells, and significantly associated with increased microvessel densities in cervical cancer tissues. In vitro, HPVE7 stimulated RRM2-dependent capillary tube formation by HUVECs, and RRM2-enhanced angiogenesis was VEGF dependent. RRM2-activated ERK1/2 pathway was mediated through production of ROS. In the xenograft mouse model, overexpression of RRM2 in cervical cancer cells enhanced tumour growth as well as microvessel densities.
HPVE7 induces upregulation of RRM2, which then promotes cervical carcinogenesis via ROS-ERK1/2-HIF-1α-VEGF-induced angiogenesis. Thus, the inhibition of RRM2 activity may be a novel therapeutic strategy for human cervical cancer.
本研究旨在证实 HPV E7 作为 RRM2 的一个新靶点，参与宫颈癌的血管生成。
采用 RT-qPCR、western blot 和免疫组化分析宫颈癌组织和细胞系中的基因表达。采用荧光素酶报告基因检测分析各种 RRM2 启动子的活性。采用 ELISA 法测定分泌型 VEGF。采用体外人脐静脉内皮细胞检测 HPV E7 诱导的 RRM2 介导的宫颈癌细胞毛细血管形成。采用流式细胞术证实 RRM2 在宫颈癌细胞中诱导的 ROS。通过裸鼠异种移植检测过表达 RRM2 的宫颈癌细胞的生长。
RRM2 作为 HPV E7 的一个新的下游靶标，通过 E7-pRb 相互作用和 E2F 结合到 RRM2 启动子区域，在转录水平上被其上调。免疫组化分析显示，RRM2 的水平与人宫颈癌中 HPV E7 的水平呈正相关。功能上，过表达 RRM2 通过激活宫颈癌细胞中的 ERK1/2 信号通路增强 HIF-1α 和 VEGF 的表达，并与宫颈癌组织中微血管密度的增加显著相关。体外，HPV E7 刺激 HUVEC 中 RRM2 依赖性毛细血管形成，而 RRM2 增强的血管生成依赖于 VEGF。RRM2 激活的 ERK1/2 途径是通过产生 ROS 介导的。在异种移植小鼠模型中，过表达 RRM2 的宫颈癌细胞增强了肿瘤生长和微血管密度。
HPV E7 诱导 RRM2 的上调，进而通过 ROS-ERK1/2-HIF-1α-VEGF 诱导的血管生成促进宫颈癌的发生。因此，抑制 RRM2 活性可能是人类宫颈癌的一种新的治疗策略。
