Nck1与子宫颈鳞癌微血管生成的关系及分子机制

A study on the association of Nck1 with the angiogenesis of cervical squamous cell carcinoma and on the related mechanism

目的探讨Nck1表达与子宫颈鳞癌微血管生成的关系及其分子机制。方法采用免疫组化MaxVision法检测50例子宫颈鳞癌组织和15例慢性子宫颈炎组织中Nck1蛋白的表达。采用CD34内皮标记癌组织的微血管密度(microvascular density,MVD)并根据Weinner的标准计数。分别通过表达质粒p CMV2-Nck1和Nck1-shRNA转染子宫颈鳞癌细胞获得Nck1基因过表达和基因沉默;应用Western blot法检测细胞蛋白的表达;ELISA法检测细胞上清液中VEGF的蛋白含量。利用Matrige tube formation assay检测内皮细胞的管腔形成能力。结果 Nck1的表达水平在子宫颈炎组织(3 769. 84±2 236. 0)、子宫颈上皮内瘤变(cervical intraepithelial neoplasia,CIN)Ⅰ(13 703. 18±1 712. 36)、CINⅡ(35 033. 84±6 516. 05)和CINⅢ(55 966. 19±9 583. 82)以及浸润型子宫颈鳞癌组织(138 329. 1±97 391. 4)各组间比较呈显著升高(P <0. 05),且子宫颈鳞癌组织中NCK表达与MVD正相关(r=0. 586,P <0. 05)。Si Ha细胞Nck1基因转染和RNA干扰分别引起VEGF表达上调和下降(P均<0. 05),相应的癌细胞上清液刺激下的内皮细胞管腔形成能力也分别显著高于和低于对照组(P均<0. 05)。PAK1特异性抑制剂能显著抑制Nck1介导的VEGF表达上调。结论 Nck1促进子宫颈鳞癌的微血管诱生能力,其分子机制与Nck1激活子宫颈鳞癌PAK1信号从而上调VEGF的表达有关。

Purpose To investigate the association of Nck1 expression with the angiogenesis of cervical squamous carcinoma and the related molecular mechanism. Methods Immunohistochemistry MaxVision was used to detect the expression of Nck1 in 50 cases of cervical squamous carcinoma tissues and 15 cases of chronic cervicitis tissues. The microvascular density( MVD)was assessed by labeling endothelia with CD34-antibody and counted according to Weinner ’s standard. Cervical squamous carcinoma cells were transfected with the Nck1 gene overexpressing plamid( p CMV-Nck1) and the Nck1-shRNA to obtain Nck1 gene overexpression and gene silencing. The level of protein expression in cells was assessed by Western blotting. The VEGF protein content in the cellular supernatant was measured with ELISA( enzyme-linked immuno sorbent assay). The tube formation capacity of endothelial cells was assessed by in vitro Matrigel tube formation assay. Results The expression level of Nck1 was 3 769. 84 ± 2 236. 0 in cervicitis tissues,13 703. 18 ±1 712. 36 in CIN Ⅰ,35 033. 84 ± 6 516. 05 in CIN Ⅱ,55966. 19 ± 9 583. 82 in CINⅢ and 138 329. 1 ± 97 391. 4 in invasive cervical squamous cell carcinoma. There was a significant increase along each groups successively( P < 0. 05). Nck1 showed a high expression level in the cervical squamous carcinoma tissues and was was positively correlated to the cancer MVD.Transient transfection and RNAi of Nck1 gene significantly increased and decreased the expression of VEGF in the Si Ha cells,respectively. The HUVECs treated with the corresponding supernatant of these two groups of cancer cells also showed significantly higher and lower tube formation capacity,respectively. The specific inhibitor of PAK1 signaling,IPA3,could significantly inhibit Nck1-mediated upregulation of VEGF in the SiHa cells transfected with Nck1 gene. Conclusion Nck1 promotes the angiogenesis-inducing capacity of cervical squamous carcinoma through the activation of the PAK1 signaling which upregulates the expression of VEGF in the cancer cells.