Linc00681通过miR-16/YAP1及RNF2/PTPN14/pYAP1通路促进恶性黑素瘤转移的机制研究

Metastasis is the primary factor account for the death of malignant melanoma(MM). Our preliminary studies found linc00681 was upregulated in the tissues of metastasis melanoma，and could competitively bind with miR-16, upregulate YAP1 expression, and promote migration and invasion in MM cells. While, YAP1 was down-regulated, and the phosphorylated levels of YAP1 and PTPN14 expression which was proved to be a key regulator of YAP1 phosphorylation were upregulated after silencing Linc00681 expression in MM cells. Further investigation revealed that Linc00681 could interact with RNF2 protein, and silencing RNF2 also upregulated PTPN14 expression. Thus, we hypothesis that Linc00681 promotes invasion-metastasis cascade of MM not only by sponging miR-16 to enhance YAP1 expression in cytoplasma, but also interacting with RNF2 in nucleus to repress PTPN14 expression and YAP1 phosphorylation, and promote YAP1 nuclear transposition. Thus, we will clarify the mechanism of Linc00681 in regulation of YAP1 expression and YAP1 phosphorylation during MM metastasis from cell-tissue-whole organism levels by performing RIP, ChIP, in situ hybridization histochemistry, and animal experiments. Our study will contribute to explicit the molecular mechanisms of metastasis of MM, and identify new therapeutic targets of MM.

肿瘤转移是恶性黑素瘤患者死亡的首要因素。我们前期发现Linc00681在转移性恶性黑素瘤中高表达，并竞争性结合miR-16，上调YAP1表达，促进恶性黑素瘤细胞侵袭转移；敲低Linc00681后，YAP1表达降低，但YAP1磷酸化水平及PTPN14表达增高，既往文献报道PTPN14促进YAP1磷酸化。进一步实验证实Linc00681与RNF2蛋白结合，且沉默RNF2亦上调PTPN14表达。故我们提出假说：Linc00681不仅在胞浆中竞争性吸附miR-16上调YAP1表达；还在胞核中通过结合RNF2蛋白抑制PTPN14转录，阻遏YAP1磷酸化，增强YAP1核转位，促进恶性黑素瘤转移。本研究拟通过RIP、ChIP、原位杂交、动物实验等从细胞-组织-整体层面阐明Linc00681通过调控YAP1表达及磷酸化促进黑素瘤转移的具体机制。本课题将有助于明确恶性黑素瘤转移的分子机制及发现新的治疗靶点。

非编码RNA在恶性黑素瘤中发挥着至关重要的作用，贯穿于肿瘤转移的整个过程。因此，进一步研究非编码RNA 在恶性黑素瘤侵袭转移中的作用，对阐明恶性黑素瘤转移的分子机制、寻找有效的治疗靶点意义重大。课题组检测了Linc00681在恶性黑素瘤组织和皮肤良性痣组织中的表达，探索沉默Linc00681对黑素瘤细胞侵袭和迁移能力的影响及机制，部分结果已发表于《细胞与分子免疫学杂志》上，此部分按项目计划顺利执行。在项目研究过程中，课题组还发现长链非编码RNA Linc00961、LNMAT1及环状RNA等非编码RNA在恶性黑素瘤中发挥着重要作用。同时，申请者获得了陕西省及中央高校基金资助，故课题组在进行Linc00681研究基础上，同时探索了长链非编码RNA-Linc00961在恶性黑素瘤的表达及功能，相关结果分别发表在《细胞与分子免疫学杂志》，继而对Linc00961调控机制做了进一步研究，发现Linc00961在皮肤黑素瘤中发挥抑癌基因作用，可通过海绵吸附mir-367抑制后者的表达，从而抑制恶性黑素瘤细胞迁移及侵袭，相关结果发表于《International Journal of Oncology》杂志上；长链非编码RNA-LNMAT1通过抑制CADM1表达促进恶性黑素瘤侵袭转移，其结果发表在《Frontiers in oncology》杂志上。此外，课题组成员还探索了环状RNA在黑素瘤中的功能，发现circFNDC3B通过抑制miR-147的表达促进黑素瘤侵袭转移，研究结果发表于《西部医学》杂志上；研究发现环状RNA circ-0001073在黑素瘤中低表达，并可抑制黑素瘤细胞的增殖、侵袭及转移，相关成果发表在《现代生物医学进展》杂志上。上述结果为探索非编码RNA在黑素瘤进展过程中作用奠定了理论基础，为寻找黑素瘤治疗靶点做了良好理论支撑。