Jing Rongrong, Chen Wen, Wang Huimin, Ju Shaoqing, Cong Hui, Sun Baolan, Jin Qin, Chu Shaopeng, Xu Lili, Cui Ming
Center of Laboratory Medicine, Affiliated Hospital of Nantong University, Nantong, China;
Department of Pediatrics, Affiliated Hospital of Nantong University, Nantong, China;
Am J Physiol Gastrointest Liver Physiol. 2015 Nov 1;309(9):G719-29. doi: 10.1152/ajpgi.00078.2015. Epub 2015 Aug 27.
The receptor for advanced-glycation end products (RAGE) is upregulated in various cancers and has been associated with tumor progression, but little is known about its expression and regulation by microRNAs (miRNAs) in esophageal squamous cell carcinoma (ESCC). Here, we describe miR-185, which represses RAGE expression, and investigate the biological role of miR-185 in ESCC. In this study, we found that the high level of RAGE expression in 29 pairs of paraffin-embedded ESCC tissues was correlated positively with the depth of invasion by immunohistochemistry, suggesting that RAGE was involved in ESCC. We used bioinformatics searches and luciferase reporter assays to investigate the prediction that RAGE was regulated directly by miR-185. Besides, overexpression of miR-185 in ESCC cells was accompanied by 27% (TE-11) and 49% (Eca-109) reduced RAGE expression. The effect was further confirmed in RAGE protein by immunofluorescence in both cell lines. The effects were reversed following cotransfection with miR-185 and high-level expression of the RAGE vector. Furthermore, the biological role of miR-185 in ESCC cell lines was investigated using assays of cell viability, Ki-67 staining, and cell migration and invasion, as well as in a xenograft model. We found that overexpression of miR-185 inhibited migration and invasion by ESCC cells in vitro and reduced their capacity to develop distal pulmonary metastases in vivo partly through the RAGE/heat shock protein 27 pathway. Interestingly, in clinical specimens, the level of plasma miR-185 expression was decreased significantly (P = 0.002) in patients with ESCC [0.500; 95% confidence interval (CI) 0.248-1.676] compared with healthy controls (2.410; 95% CI 0.612-5.671). The value of the area under the receiver-operating characteristic curve was 0.73 (95% CI 0.604-0.855). In conclusion, our findings shed novel light on the role of miR-185/RAGE in ESCC metastasis, and plasma miR-185 has potential as a novel diagnostic biomarker in ESCC.
晚期糖基化终末产物受体（RAGE）在多种癌症中表达上调，并与肿瘤进展相关，但关于其在食管鳞状细胞癌（ESCC）中的表达及受微小RNA（miRNA）调控的情况知之甚少。在此，我们描述了抑制RAGE表达的miR-185，并研究了miR-185在ESCC中的生物学作用。在本研究中，我们发现通过免疫组织化学检测，29对石蜡包埋的ESCC组织中RAGE的高表达水平与肿瘤浸润深度呈正相关，提示RAGE参与了ESCC的发生发展。我们利用生物信息学搜索和荧光素酶报告基因检测来研究RAGE是否直接受miR-185调控。此外，在ESCC细胞中过表达miR-185后，RAGE的表达在TE-11细胞中降低了27%，在Eca-109细胞中降低了49%。通过免疫荧光检测在这两种细胞系中进一步证实了RAGE蛋白水平的变化。与miR-185共转染并高表达RAGE载体后，上述效应被逆转。此外，我们利用细胞活力检测、Ki-67染色、细胞迁移和侵袭检测以及异种移植模型研究了miR-185在ESCC细胞系中的生物学作用。我们发现，miR-185过表达在体外抑制了ESCC细胞的迁移和侵袭，并在体内部分通过RAGE/热休克蛋白27通路降低了其发生远处肺转移的能力。有趣的是，在临床标本中，与健康对照（2.410；95%置信区间[CI]0.612 - 5.671）相比，ESCC患者血浆中miR-185的表达水平显著降低（P = 0.002）[0.500；95% CI 0.248 - 1.676]。受试者工作特征曲线下面积值为0.73（95% CI 0.604 - 0.855）。总之，我们的研究结果为miR-185/RAGE在ESCC转移中的作用提供了新的见解，血浆miR-185有潜力作为ESCC的一种新型诊断生物标志物。
