人β防御素3在HPV相关宫颈癌变中的表达调控及促癌机理研究

Human β defensin 3 (hBD3) is an endogenous anti-microbial peptide, which has been found to have a potential to promote tumorigenesis in the epithelial tumors recently. How this happened is still largely unknown. Our previous studies have revealed the expression of hBD3 increased significantly in cervical cancer, related with regulation of high-risk human papillomavirus (HPV) infection. It was also confirmed that hBD3 could promote growth and migration of cervical cancer cells. Based on those data obtained, we propose to investigate the expression regulation and tumor-promoting mechanisms of hBD3 in HPV-related cervical cancer in this project. Firstly, immunohistochemistry and in-situ hybridization will be performed on more clinical tissue samples to confirm the expression increase of hBD3 is related with cervical carcinogenesis and HPV infection status. RNA interference and plasmid transformation will be carried out to manipulate the expression of hBD3 in cervical cancer cells in order to study the tumor-promoting activities of hBD3 in vitro and in vivo. Further, HPV E6/p53 and E7/pRb transforming pathways will be intervened to investigate the mechanisms how HPV regulates hBD3 expression. Downstream signal pathways of hBD3 would be firstly screened by high-throughput antibody microarrays, followed by the detailed analysis of time-dependence and dose-dependence of the regulation of hBD3 on its target signal factors. Inhibition of signal pathways will be employed to examine the contributions of these pathways to the tumor-promoting activities of hBD3. This project is the first systematic study of upstream and downstream pathways of tumor-promoting mechanisms of hBD3, which would be helpful to characterize hBD3 as potential biomarker and therapy target of human tumors.

内源性抗菌肽人β防御素3（hBD3）在上皮肿瘤中的促癌活性是近年来的新发现，机理尚不明确。我们的前期研究表明hBD3在宫颈癌组织中表达明显升高，与HPV的调控相关；在细胞水平上能够促进宫颈癌细胞增殖和迁移。基于此，本项目拟以HPV相关宫颈癌变为疾病模型研究hBD3的表达调控和促癌机理。利用免疫组化和原位杂交进一步证实临床标本中hBD3表达与宫颈癌变及HPV感染的关联；采用质粒转染及RNA干扰技术干预hBD3表达，研究hBD3在细胞水平和小动物模型中的促癌活性；通过干预HPV E6/p53和E7/pRb的转化通路，研究HPV对hBD3表达的调控机制；利用高通量抗体芯片筛选hBD3的信号通路，对hBD3调控靶点信号分子机制进行时效量效分析，通过通路抑制手段明确hBD3促癌活性对相关通路的依赖性，对hBD3的促癌信号通路做以全面系统阐述。为探索hBD3作为潜在的肿瘤标记物和治疗靶点做出有益尝试。

内源性抗菌肽人β防御素3（hBD3）在上皮肿瘤中的促癌活性是近年来的新发现，机理尚不明确。我们的前期研究表明hBD3在宫颈癌组织中表达明显升高，与HPV的调控相关；在细胞水平上初步发现hBD3能够促进宫颈癌细胞增殖和迁移。基于此，本项目的研究以HPV相关宫颈癌变为疾病模型研究hBD3的表达调控和促癌机理。首先利用免疫组化在约100例宫颈组织中证实临床标本中hBD3表达与宫颈癌变呈正相关趋势；随后利用慢病毒转染技术在HPV阳性宫颈癌细胞系中过表达hBD3，研究hBD3表达量升高后对宫颈癌细胞恶性表型的影响。我们的结果表明hBD3高表达会显著促进宫颈癌细胞的增殖和生长，促进了宫颈癌细胞迁移浸润的能力；但并未提高宫颈癌细胞的抗凋亡能力。随后的通路筛选结果表明，hBD3能够提高NF-kappaB通路的活性；通过siRNA敲低p65的活性后，hBD3促肿瘤活性消失，证实了hBD3通过提高NF-kappaB通路的活性来促进宫颈癌发生。小动物实验表明，hBD3高表达的宫颈癌细胞系接种裸鼠后，成瘤能力和肿瘤生长速率都显著高于对照细胞系。我们的研究首次系统的研究了hBD3在HPV相关宫颈癌中的表达水平改变及其对肿瘤细胞的影响，并初步探讨了其机制，为探索hBD3作为潜在的肿瘤标记物和治疗靶点做出有益尝试。