CircROR1上调SKP2表达促进黑色素瘤侵袭转移的分子机制研究

How to effectively prevent melanoma metastasis is a clinical problem. Our previous study showed that CircROR1 was overexpressed in melanoma, Silencing CircROR1 can down-regulate the expression of SKP2 to inhibit melanoma cell migration, but its specific regulatory mechanism is unknown. Pre-experiment confirmed that SKP2 is a direct target gene of miR-378c. The prediction software suggests that CircROR1 may adsorb miR-378c and TCF3 may be a transcription factor of miR-378c, In combination with the literature, TCF3 can be degraded by SKP2 ubiquitination. We speculate that "CircROR1 can up-regulate the expression of SKP2 by regulating the positive feedback loop of miR-378c/SKP2/TCF3, thereby promoting melanoma invasion and metastasis". This study intends to firstly explore the specific molecular mechanism of CircROR1 up-regulating SKP2 expression to promote the malignant phenotype of melanoma invasion and metastasis at the molecular and cellular levels, further verification at the animal level;Finally, clinical data and tissue samples are used to evaluate the clinical value of key molecules such as CircROR1, miR-378c and SKP2, in order to provide new targets and new evidence for the individualized intervention of clinical invasion and metastasis of melanoma.

如何有效防控黑色素瘤转移是临床难题。前期研究示CircROR1在黑色素瘤中高表达，沉默CircROR1可下调SKP2的表达来抑制黑色素瘤细胞转移，但其具体调控机制不明。预实验证实SKP2为miR-378c的直接靶基因，预测软件提示CircROR1可能吸附miR-378c，TCF3可能为miR-378c的转录因子，结合文献报道TCF3可被SKP2泛素化降解，我们推测“CircROR1可通过调控miR-378c/SKP2/TCF3正反馈回路来上调SKP2的表达，从而促进黑色素瘤侵袭转移”。本项目拟首先从分子、细胞层面深入探讨CircROR1上调SKP2表达来促进黑色素瘤侵袭转移恶性表型的具体分子机制；在动物水平进一步验证；最后利用临床资料和组织标本等，评估CircROR1、miR-378c及SKP2等关键分子的临床价值，以期为临床上黑色素瘤的侵袭转移个体化干预提供新靶点和新证据。

越来越多的研究显示环状RNA在肿瘤的发生和发展过程中发挥着重要的作用，然而，环状RNA在黑色素瘤侵袭转移中的表达及作用机制尚不清楚。本研究采用RNA干扰、质粒转染、PCR、Western blot、CHIP、RNA-pull down、RIP、动物建模等技术，探讨CircROR1促进黑色素瘤细胞侵袭转移的具体分子机制。.本研究前期芯片结果及临床小样本发现CircROR1在转移性黑色素瘤细胞和组织中高表达。细胞实验及裸鼠移植瘤实验证实CircROR1在体内外促进黑色素瘤细胞的增殖、侵袭转移。敲除CircROR1的mRNA微阵列，qRT-PCR和Western blot证实CCNE1是CircROR1的下游靶基因。临床组织标本证实CCNE1在转移性黑色素瘤中表达最高，且与不良生存预后相关。通过功能挽救实验证实无论是体内还是体外CircROR1可通过调控CCNE1的表达促进黑色素瘤细胞的增殖、侵袭和转移。生信分析发现CCNE1启动子区域富含H3K9乙酰化修饰，TSA处理提高CCNE1的乙酰化水平。通过RNA-pull down和RIP证实CircROR1与KAT2A特异性结合。进一步CHIP-qPCR明确CircROR1使KAT2A结合CCNE1能力增加，导致CCNE1启动子区H3K9乙酰化水平增高，从而诱导CCNE1的转录。.研究结论：我们首次发现CircROR1在转移性黑色素瘤细胞和组织中高表达，过表达CircROR1促进黑色素瘤的增殖侵袭转移，其机制是CircROR1通过KAT2A介导的组蛋白乙酰化促进CCNE1基因表达。我们的研究结果证实了CircROR1在黑色素瘤侵袭转移中的关键作用，为临床上黑色素瘤的侵袭转移个体化干预提供新靶点和新证据。