Huang Qing Bo, Ma Xin, Zhang Xu, Liu Shang Wen, Ai Qing, Shi Tao Ping, Zhang Yu, Gao Yu, Fan Yang, Ni Dong, Wang Bao Jun, Li Hong Zhao, Zheng Tao
Department of Urology/State Key Laboratory of Kidney Diseases, Chinese PLA General Hospital/PLA Medical School, Beijing, China.
PLoS One. 2013 Jun 27;8(6):e67294. doi: 10.1371/journal.pone.0067294. Print 2013.
Endothelial DLL4 plays an important role in controlling of tumor angiogenesis, which is required for tumor invasive growth and metastasis. However, the regulation of DLL4 in clear cell renal cell carcinoma (ccRCC) has not yet been systematically elucidated.
We performed bioinformatical analysis to explore miRNAs targeting DLL4. miR-30a was selected as a representative to validate its functional association in endothelial cell. Then, the expressions of DLL4 and mature miR-30a from 90 cases of ccRCC and 28 cases of nonmatched adjacent non-tumor tissues were measured by quantitative real-time PCR. Finally, the expression of miR-30a was correlated with DLL4 expression, tumor features (metastatic condition and microvessel density), and patient metastasis-free survival. The univariate and multivariate analyses were performed to select the risk factors associated with hematogenous metastasis, respectively.
miR-30a negatively regulated DLL4 and inhibited the proliferation and migration of endothelial cells. DLL4 was up-regulated in ccRCC and further increased in hematogenous metastatic cases, while miR-30a was down-regulated in tumor tissues and further decreased in hematogenous metastatic ccRCC (student t test, all p<0.05). Additionally, expression of miR-30a was inversely correlated with expression of DLL4 and microvessel density (linear correlation analysis, both p<0.05). Low-level miR-30a also indicated a higher probability of developing metastasis (log-rank test, p = 0.010). Most importantly, miR-30a expression was an independent predictor of ccRCC hematogenous metastasis by the univariate analysis and binary logistic regression model (both p<0.05).
Down-regulated miR-30a in ccRCC was associated with tumor hematogenous metastasis through increasing microvessel density by targeting angiogenesis-specific DLL4.
内皮细胞中的DLL4在控制肿瘤血管生成中起重要作用，而肿瘤血管生成是肿瘤侵袭性生长和转移所必需的。然而，DLL4在肾透明细胞癌（ccRCC）中的调控尚未得到系统阐明。
我们进行了生物信息学分析以探索靶向DLL4的微小RNA（miRNA）。选择miR-30a作为代表来验证其在内皮细胞中的功能关联。然后，通过定量实时PCR检测90例ccRCC和28例不匹配的相邻非肿瘤组织中DLL4和成熟miR-30a的表达。最后，将miR-30a的表达与DLL4表达、肿瘤特征（转移情况和微血管密度）以及患者无转移生存期相关联。分别进行单因素和多因素分析以选择与血行转移相关的危险因素。
miR-30a负向调节DLL4并抑制内皮细胞的增殖和迁移。DLL4在ccRCC中上调，在血行转移病例中进一步增加，而miR-30a在肿瘤组织中下调，在血行转移的ccRCC中进一步降低（学生t检验，所有p<0.05）。此外，miR-30a的表达与DLL4的表达和微血管密度呈负相关（线性相关分析，两者p<0.05）。低水平的miR-30a也表明发生转移的可能性更高（对数秩检验，p = 0.010）。最重要的是，通过单因素分析和二元逻辑回归模型，miR-30a表达是ccRCC血行转移的独立预测因子（两者p<0.05）。
ccRCC中miR-30a下调通过靶向血管生成特异性的DLL4增加微血管密度，与肿瘤血行转移相关。
