广西医学

目的探讨SMAD家族成员4（SMAD4）通过调控血管内皮生长因子（VEGF）⁃A的表达促进宫颈鳞癌细胞血管生成与肿瘤进展的相关机制。方法（1）构建SMAD4基因沉默模型，将SiHa细胞分为SMAD4敲低基因组（SMAD4⁃down组）及空载对照组（SMAD4⁃down⁃NC组），进行相应慢病毒转染处理后，采用qRT⁃PCR检测两组SiHa细胞中SMAD4的mRNA表达水平，采用Western blot检测两组SiHa细胞中SMAD4和VEGF⁃A 蛋白的表达水平。（2）建立裸鼠皮下移植瘤模型，将成功转染的两组SiHa细胞注射到裸鼠腋下，观察肿瘤形成情况，每5 d测量肿瘤体积并绘制生长曲线，比较两组裸鼠移植瘤体积差异。采用免疫组织化学技术检测两组裸鼠移植瘤组织中VEGF⁃A蛋白的表达水平，同时观察微血管计数指标CD34的表达量以评估微血管密度（MVD）。结果（1）SMAD4⁃down组SiHa细胞中SMAD4 mRNA和蛋白表达水平低于SMAD4⁃down⁃NC组，VEGF⁃A蛋白表达水平高于SMAD4⁃down⁃NC组（P<0.05）。（2）SMAD4⁃down组裸鼠移植瘤体积大于SMAD4⁃down⁃NC组（P<0.05），肿瘤生长趋势快于SMAD4⁃down⁃NC组；SMAD4⁃down组的移植瘤中VEGF⁃A蛋白的表达水平、MVD高于SMAD4⁃down⁃NC组（P<0.05）。结论敲低SMAD4可促进宫颈鳞癌SiHa细胞VEGF⁃A的表达，SMAD4低表达与肿瘤血管生成、癌症转移相关。

Objective To explore the relevant mechanism of SMAD family member 4 (SMAD4)⁃promoted angiogenesis in cervical squamous cell carcinoma and tumor progression via vascular endothelial growth factor (VEGF)⁃A expression regulation. Methods (1) A SMAD4 gene⁃silencing model was constructed. SiHa cells were divided into SMAD4 gene knockdown group (the SMAD4⁃down group) or empty⁃vector control group (the SMAD4⁃down⁃NC group). After the treatment of corresponding lentivirus transfection, qRT⁃PCR was adopted to detect mRNA expression of SMAD4 in SiHa cells of the two groups. Western blot was employed to detect protein expressions of SMAD4 and VEGF⁃A in SiHa cells of the two groups. (2) A subcutaneous xenograft model was constructed in nude mice by injecting the two⁃group successfully transfected SiHa cells into the axilla of the nude mice. Tumor formation was observed, tumor volume was measured every five days to generate growth curves, and the xenograft volume differentiation was compared between nude mice of the two groups. Immunohistochemistry was performed to detect VEGF⁃A protein expression in the xenograft tissues of nude mice from the two groups, and microvascular count indicator CD34 expression was observed to evaluate microvessel density (MVD). Results (1) The expressions of SMAD4 mRNA and protein in SiHa cells of the SMAD4⁃down group were lower than those in the SMAD4⁃down⁃NC group, while the expression of VEGF⁃A protein was higher than that in the SMAD4⁃down⁃NC group (P<0.05). (2) The volume of xenograft in nude mice of the SMAD4⁃down group was larger than that in the SMAD4⁃down⁃NC group (P<0.05), and the tumor growth trend was faster than that in the SMAD4⁃down⁃NC group; in addition, the expression of VEGF⁃A protein and the MVD in the xenograft of the SMAD4⁃down group were higher than those in the SMAD4⁃down⁃NC group (P<0.05). Conclusion Knockdown of SMAD4 can promote the expression of VEGF⁃A in cervical squamous cell carcinoma SiHa cells, and the low expression of SMAD4 is related to tumor angiogenesis and cancer metastasis.

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