lncRNA在肢端黑素瘤中的功能和作用机制研究

Acral melanoma is a kind of malignant neoplasm of skin with high mortality and strong ability to invade. It is a main type of melanoma which often occurs in Chinese. The abnormal expression of oncogenes and tumor suppressor genes induced by single nucleotide mutation, insertion, deletion, gene amplification and loss is involved in the pathogenesis of acral melanoma. LncRNA is a type of new-found non-coding RNA which functions by regulating gene expression at different level through interaction with nucleonic acid and proteins. Recent studies have shown that the aberrant expression of lncRNA is close related to breast cancer, colon cancer and non-acral melanoma, participating in proliferation, migration and invasion of cancer cells. However, the function and mechanism of lncRNA in acral melanoma remain elusive. According to the results of exome sequencing in acral melanoma, we have already screened non-coding RNA with single nucleotide mutation, insertion and deletion, suggesting these mutant non-coding RNA may participate in the development of acral melanoma. To seek out specific lncRNA in acral melanoma, we plan to establish expression profile of specific lncRNA with combination of lncRNA and mRNA microarray and RNA sequence followed by identification in large samples. Meanwhile, the biological function of the specific lncRNA will be evaluated in vitro and in vivo. Moreover, the mechanism of specific lncRNA will also be further clarified through bioinformatics prediction and experiments in vitro. By doing so, we expect to find out one or more critical lncRNA and provide new idea for acral melanoma therapy.

肢端黑素瘤是我国人群好发的皮肤恶性肿瘤。基因变异引起癌基因或抑癌基因的异常表达参与肢端黑素瘤的发生。lncRNA是新发现的具有基因调控功能的非编码RNA，它通过与核酸或蛋白分子相互作用在基因表达的不同水平发挥功能。lncRNA的异常表达与乳腺癌、结肠癌和非肢端黑素瘤的发病密切相关。然而，lncRNA在肢端黑素瘤中的功能和作用机制尚不清楚。根据本研究组已有的肢端黑素瘤外显子测序的结果，我们已筛选出外显子发生点突变或插入缺失突变的ncRNA，说明这些异常的ncRNA可能参与肢端黑素瘤的发生。为了进一步找到肢端黑素瘤特异性lncRNA， 本研究拟采用lncRNA和mRNA表达谱芯片及RNA测序技术建立肢端黑素瘤特异性lncRNA表达谱并进行大样本验证，评价目标lncRNA在肢端黑素瘤中的生物学功能，然后通过生物信息学和体外实验相结合进一步发现其可能的作用机制，有望为肢端黑素瘤的治疗提供新思路。

黑素瘤是致死性最强的皮肤肿瘤，其中肢端黑素瘤是我国人群好发的皮肤肿瘤类型，目前，黑素瘤的发病机制尚未完全阐明。长链非编码RNA（lncRNAs）是一类具有基因调控功能的非编码RNA，已有研究表明lncRNAs的异常表达参与肿瘤的发生发展。然而，lncRNAs在肢端黑素瘤中的功能和作用机制尚不清楚。本研究采用lncRNA和mRNA表达谱芯片筛选在肢端黑素瘤和色素痣组织中差异表达的lncRNAs和mRNAs；对它们进行一系列生物信息学分析，包括差异mRNAs的KEGG和GO分析、构建lncRNAs-mRNAs共表达网络、lncRNAs靶基因和转录因子预测等；对差异lncRNAs的表达水平及lncRNAs-mRNAs对的表达水平用定量PCR的方法在组织和细胞中进行验证；根据验证结果，我们在黑素瘤细胞中对所选lncRNAs和mRANs的肿瘤相关的生物学功能和相应的机制进行研究。结果显示， 在肢端黑素瘤和色素痣组织中显著差异表达的lncRNAs共有1646个，主要为基因间lncRNAs和反义lncRNAs，1号、2号染色体分布的差异LncRNA最多；生物信息学分析结果显示差异表达的lncRNAs和mRNAs所参与的信号通路以Hippo信号途径、多能干细胞调节途径、赖氨酸降解、抗原提呈和加工、细胞因子及受体相互作用、NK细胞介导的细胞毒性、吞噬体、癌症途径、B细胞受体信号途径最显著，参与的生物过程以天然免疫和适应性免疫反应、免疫效应过程、免疫激活、免疫反应激活的细胞表面受体信号途径、白细胞和淋巴细胞介导的免疫反应及免疫系统的调节、I型干扰素信号和细胞粘附最显著；用定量RT-PCR的方法对所选的表达差异最显著的8个lncRNA进行验证，结果显示有7个与芯片的检测结果一致；同时，对有共表达关系的lncRNAs-mRNAs的表达水平在组织和细胞系中进行验证，结果与芯片中的表达趋势一致；体外功能研究发现lncRNA CD27-AS可能通过调节CD27的表达促进黑素瘤的发生发展，另外还发现HMHA1蛋白在黑素瘤发生发展中的促癌作用。综上，本项目的研究表明lncRNAs是黑素瘤治疗的一个潜在的靶点。