外泌体microRNAs：肢端黑素瘤预后及干扰素治疗敏感性标志物筛选研究

Acral melanoma is the predominant melanoma subtype in Chinese, which has distinct clinical and epidemiological characteristics and a notably worse prognosis than common cutaneous melanoma. High-dose interferon (IFN) alfa-2b is the only adjuvant therapy for melanoma approved by the US Food and Drug Administration. Several studies have demonstrated that adjuvant therapy for high-risk melanoma patients with IFN alfa-2b improves disease-free survival (DFS), although the impact on overall survival (OS) is controversial. Besides, no association has been shown between outcome and dose or duration of treatment, suggesting that the few patients sensitive to interferon should be identified and sensitive markers for interferon treatment and prognosis are in great need. Recent studies have shown that exosome contain microRNAs (miRNAs), and cells can secrete miRNAs via exosomes and deliver them into recipient cells where the exogenous miRNAs can regulate target gene expression and recipient cell function. It is suggest that exosome microRNA can be used a marker in providing new means for individualized cancer treatments. Via microarray analysis, we identified 48 differentially expressed microRNAs of exosome in the plasma of acral melanoma patients. Among these microRNAs, miR-25-106b was selected for further study, as two of its predicted target genes, STAT1 and STAT2, play critical roles in type I IFN signaling. It has proved that PBMCs isolated from cancer patients exhibit depressed STAT1 phosphorylation in response to IFN-α, suggesting IFN signaling dysfunction. Thus, we well test and verify the potential role of miR-25-106b as sensitive markers for interferon treatment and prognosis of acral melanoma and demonstrate the exact regulatory mechanisms of exosome microRNA in abnormalities in innate immunity.

肢端黑素瘤是中国黑素瘤的高发类型，具有特殊的遗传背景、流行病学特征、发病机制及临床特点。干扰素是黑素瘤术后高危患者的主要辅助治疗药物，在针对肢端黑素瘤的治疗中发现，患者平均受益率低于欧美非肢端黑素瘤，亟待通过大样本临床研究筛选预后及干扰素治疗敏感性标志物以推进个体化治疗。我课题组发现肢端黑素瘤外泌体microRNAs表达谱具有疾病特异性，miR-25/93/106b异常高表达，生物信息学预测提示其可协同抑制STAT1/2，负向调控干扰素信号活化。本项目拟重点验证miR-25/93/106b表达水平与黑素瘤恶性进展的相关性，全面评估其作为肢端黑素瘤预后及干扰素治疗敏感性标志物的可行性，深入研究miR-25/93/106b通过细胞内外不同调控途径，分别靶向STAT1/2及MITF/ BCL2，负向调控干扰素信号活化及直接对抗干扰素促肿瘤凋亡作用的具体分子机制，为制定个体化治疗方案提供重要参考。

肢端黑素瘤是中国黑素瘤的高发类型，具有特殊的遗传背景、流行病学特征、发病机制及临床特点。干扰素是黑素瘤术后高危患者的主要辅助治疗药物，在针对肢端黑素瘤的治疗中发现患者平均受益率低于欧美非肢端黑素瘤，亟待通过大样本临床研究筛选预后及干扰素治疗敏感性标志物以推进个体化治疗。本项目初步证明肢端黑素瘤患者外泌体中miRNAs的表达谱具有疾病特异性，在前期研究基础上进一步扩大样本量验证后发现，重要的miRNA簇miR-25/93/106b中的两个miRNA，miR-25及miR-106b在外泌体中呈现显著高表达，在120例临床标本（60例原发及60例转移标本）的检测中发现，miR-25及miR-106b在黑素瘤患者外泌体中表达随肿瘤的恶性发展进一步增高；外泌体中高表达miR-25或miR-106b，均与较差预后显著相关，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，加入外泌体中miR-25或/和miR-106b表达量分级指标，可显著提高预后判断准确性。将转移黑素瘤细胞系细胞（包括肢端及非肢端黑素瘤）培养上清外泌体与健康人来源的PBMC共孵育，在给予外源性的干扰素α刺激时，细胞内的Jak-STAT信号通路活化不同程度受到抑制，T细胞增殖不同程度收到抑制且细胞膜干扰素受体表达也呈下调趋势。黑素瘤细胞内miR-25表达量显著降低，促进黑素瘤细胞的增殖，从功能学角度进一步验证了流行病学研究结果。