环状RNA：黑素瘤预后标志物筛选及其在发病中的作用和机制研究

Melanoma is a highly aggressive malignant tumor of the skin, and its staging of the disease has a very important guiding significance for the Melanoma is a highly invasive malignancy of the skin, and the patients of melanoma usually have poor prognosis and high mortality. At present, the staging and prognosis of melanoma patients are often evaluated according to the clinical features like thickness. However, the data from epidemiology analysis indicates that melanoma has a high heterogeneity, and the current methods are not able to evaluate prognosis comprehensively and accurately. Therefore, a novel marker for melanoma prognosis with more accuracy and effectiveness is urgently needed. In our previous study, we found a disease-specific characteristic in the expression profiling of circRNAs in melanoma, in which hsa_circ_000906 was abnormally highly expressed. Bioinformatics analysis suggested that it might inhibit the expression of miR-16 and miR-137, both of which are tumor suppressor miRNAs. Therefore, hsa_circ_000906 may contribute the progression of melanoma and be highly related to prognosis. In the current study, we will first verify the specific expression profile of circRNAs in melanoma and validate the association between aberrantly expressed circRNAs and the staging and progression of melanoma. Moreover, we will evaluate the practicability of the application of circRNAs, especially of hsa_circ_000906, as biomarkers for melanoma prognosis by using survival curve and COX regression model. In addition, we will investigate on the mechanism of hsa_circ_000906 in promoting the progression of melanoma by targeting miR-16 and miR-137 both in vitro and in vivo, which may provide not only insights for melanoma prognosis, but also novel therapeutic targets for melanoma treatment.

黑素瘤是高度恶性的皮肤肿瘤，预后较差，致死率高。目前通过厚度等临床特征进行分期并判断预后。流行病学数据显示黑素瘤具有强异质性，现有方法不能全面准确的判断预后，发现更为准确有效的预后标志物成为亟待解决的科学问题。本课题组发现黑素瘤环状RNA表达谱具有特异性，hsa_circ_000906异常高表达，生物信息学分析提示其可负调控具有抑癌作用的miR-16和miR-137表达，参与黑素瘤恶性进展，并与预后密切相关。本项目拟验证黑素瘤环状RNA表达差异，明确异常表达环状RNA与黑素瘤分期及恶性进展的相关性，通过生存曲线、构建Cox风险模型等方法对 has_circ_000906 等环状RNA作为预后标志物的可行性进行重点分析。通过体内、外实验深入研究has_circ_000906调控miR-16及miR-137，参与黑素瘤恶性进展的具体机制，以期在发现预后标志物的基础上，发现黑素瘤防治的新靶点。

黑素瘤是高度恶性的皮肤肿瘤，预后较差，致死率高。目前通过厚度等临床特征进行分期并判断预后。流行病学数据显示黑素瘤具有强异质性，现有方法不能全面准确的判断预后，发现更为准确有效的预后标志物成为亟待解决的科学问题。本项目初步证明黑素瘤患者组织中环状RNA的表达谱具有疾病特异性，扩大样本量验证后发现，hsa_circ_000906在黑素瘤组织中呈现显著高表达，在200例临床标本（100例I-II期原发组织及100例III-IV期转移组织）的检测中发现，hsa_circ_000906在黑素瘤患者组织中表达水平和黑素瘤恶性程度呈正相关；hsa_circ_000906表达水平越高，病人不良预后越显著，ROC曲线进一步证实，在原有临床数据基础上建立的预后评估模型中，联合组织中hsa_circ_000906表达量进行分级，可显著提高预后判断准确性。在黑素瘤细胞系中过表达hsa_circ_000906，细胞的增殖，迁移能力均明显升高。靶基因预测miR-16及miR-137存在hsa_circ_000906结合位点，过表达hsa_circ_000906之后靶基因miR-16及miR-137表达下调。干涉hsa_circ_000906之后靶基因miR-16及miR-137表达上调。这提示hsa_circ_000906和miR-16及miR-137之间可能存在相互抑制关系。荧光素酶报告实验证实hsa_circ_000906和miR-137/16之间存在直接相互作用。且随着黑素瘤病理分级的增高，miR-16及miR-137的表达水平逐渐降低，与hsa_circ_000906趋势相反，证明hsa_circ_000906和miR-16及miR-137存在负相关。最后通过回复实验证实hsa_circ_000906逆转了miR-16及miR-137对黑素瘤细胞增殖和侵袭的抑制作用。上述结果从功能学角度进一步验证流行病学研究结果，探讨环状 RNA 作为黑素瘤治疗新靶点的可能性，为黑素瘤治疗提供新的思路。