CRIM1通过激活ERK1/2-VEGF信号通路促进子宫颈鳞状细胞癌微血管生成

CRIM1 promotes the angiogenesis of cervical squamous carcinoma by activating the ERK-VEGF signaling

目的探讨CRIM1表达与子宫颈鳞状细胞癌微血管生成的关系及其分子机制。方法采用免疫组化MaxVision法检测并结合光密度分析CRIM1蛋白的组织表达;采用CD34内皮标记及Weinner计数法检测子宫颈鳞状细胞癌组织中的微血管密度(microvascular density,MVD)。通过真核表达质粒pCMV3-CRIM1转染子宫颈鳞状细胞癌SiHa细胞获得CRIM1基因过表达;采用Western blot法检测癌细胞中p-ERK和VEGF的表达,并利用ERK抑制处理癌细胞,观察ERK信号抑制对VEGF表达的影响;通过ELISA法检测细胞上清液中VEGF蛋白含量。分别利用CCK-8实验和Matrigel管腔形成实验检测人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVEC)的增殖和管腔形成能力。结果子宫颈鳞状细胞癌组织中CRIM1表达水平(200039±163274.86)显著高于慢性子宫颈炎组织(28258.0±16606.04,P<0.001),且CRIM1表达与子宫颈鳞状细胞癌MVD呈正相关(r=0.546,P<0.01)。CRIM1基因转染诱导SiHa细胞VEGF表达水平升高(P<0.001),且细胞上清液中VEGF含量也升高(P<0.01)。CRIM1转染后的癌细胞上清液刺激下的内皮细胞增殖能力和管腔形成能力均显著增强。CRIM1基因转染诱导SiHa细胞p-ERK和VEGF蛋白表达上调,而ERK抑制剂(U0126)能显著抑制CRIM1诱导的VEGF上调。人重组蛋白CRIM1直接作用于SiHa细胞后,VEGF蛋白表达及上清液中VEGF含量无明显变化。结论CRIM1促进子宫颈鳞状细胞癌微血管生成,其机制可能与CRIM1激活ERK1/2-VEGF信号通路有关。

Purpose To investigate the association of CRIM1 expression with the angiogenesis of cervical squamous cell carcinoma(CSCC)and the related mechanism.Methods Immunohistochemistry MaxVision in combination with optical denstity analysis was used to detect the expression of CRIM1 in tissues.The microvascular density(MVD)was assessed by labeling endothelia with CD34-antibody and counted according to Weinner’s standard.Overexpression of CRIM1 gene in SiHa cells was acqauired by transfecting the expressing plamid(pCMV3-CRIM1).The protein levels of p-ERK and VEGF in cancer cells were assessed by Western blot.The specific inhibitor U0126 was used to treat cancer cells and thus observed the effect of ERK signaling inhibition on the VEGF expression.The protein content in the cellular supernatant was measured with enzyme-linked immuno sorbent assay(ELISA).The proliferation and tube formation capacity of endothelial cells(HUVEC)were evaluated by CCK-8 assay and in vitro Matrigel tube formation assay,respectively.Results CRIM1 showed a significantly higher expression level in CSCC(200039±163274.86)than that in normal epithelia of chronic cervicitis tissues(28258.0±16606.04,P<0.001)and was positively correlated to the cancer MVD(r=0.546,P<0.01).The proliferation and tubeformation capacity of HUVEC treated with the supernatant from the cancer cells with CRIM1 gene transfection was increased.CRIM1 gene transfection led to increase of p-ERK and VEGF in SiHa cells.The specific inhibitor of ERK1/2 signaling,U0126,could inhibit CRIM1-mediated up-regulation of VEGF in SiHa cells.In addition,exogenous CRIM1 protein couldn’t induce VEGF up-regulation in SiHa cell.Conclusion CRIM1 promotes the angiogenesis of cervical squamous carcinoma,and the mechanism may be related to the CRIM1-mediated activation of ERK1/2-VEGF signal pathway.