Xu Yangfan, Wang Huixue, Li Fang, Heindl Ludwig M, He Xiaoyu, Yu Jie, Yang Jie, Ge Shengfang, Ruan Jing, Jia Renbing, Fan Xianqun
Department of Ophthalmology, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
Shanghai Key Laboratory of Orbital Diseases and Ocular Oncology, Shanghai, China.
Front Cell Dev Biol. 2019 Dec 20;7:350. doi: 10.3389/fcell.2019.00350. eCollection 2019.
Long non-coding RNAs (lncRNAs) have been identified as crucial regulators in many human cancers. Many lncRNAs show aberrant expression in cancer, and some of them play critical roles in tumor proliferation, invasion, and metastasis. However, the regulatory functions of lncRNAs in melanoma progression remain to be elucidated. We utilized the Real-time PCR methodology to determine the expression of LINC-PINT in melanoma cell lines. To evaluate the effect of LINC-PINT on tumorigenesis of melanoma, we used Cell Counting Kit-8 (CCK8) and colony formation assay. Flow cytometry assay was used to detect the function of LINC-PINT on cell cycle status. PINT-interacting proteins were identified by chromatin isolation using RNA purification (ChIRP). Microarray assay and bioinformatics analysis were used to find the potential target genes of LINC-PINT and the status of LINC-PINT target gene candidate was verified using chromatin immunoprecipitation assay (ChIP). LINC-PINT plays a role in suppressing the tumorigenicity of melanoma, which was further determined by xenograft model assay. LINC-PINT was significantly downregulated in melanoma tissues and cell lines. The overexpression of LINC-PINT in tumor cells resulted in significant tumor growth reduction and migration inhibition in A375, Mum2B and CRMM1 cells. Results based on the xenograft model were further consistent with the findings that LINC-PINT impeded growth and metastasis of melanoma cells. Microarray assay and bioinformatics analysis indicated that CDK1, CCNA2, AURKA, and PCNA were potential targets of LINC-PINT. In conclusion, LINC-PINT inhibits the tumorigenicity of melanoma through recruiting EZH2 to the promoter of its target genes, leading to H3K27 trimethylation and epigenetic silencing of target genes. LINC-PINT may serve as a novel diagnostic and therapeutic target for melanoma.
长链非编码RNA（lncRNAs）已被确认为多种人类癌症中的关键调节因子。许多lncRNAs在癌症中表现出异常表达，其中一些在肿瘤增殖、侵袭和转移中发挥关键作用。然而，lncRNAs在黑色素瘤进展中的调节功能仍有待阐明。我们利用实时PCR方法来确定LINC-PINT在黑色素瘤细胞系中的表达。为了评估LINC-PINT对黑色素瘤肿瘤发生的影响，我们使用了细胞计数试剂盒-8（CCK8）和集落形成试验。流式细胞术检测用于检测LINC-PINT对细胞周期状态的作用。通过RNA纯化的染色质分离法（ChIRP）鉴定与PINT相互作用的蛋白质。使用微阵列分析和生物信息学分析来寻找LINC-PINT的潜在靶基因，并使用染色质免疫沉淀试验（ChIP）验证LINC-PINT靶基因候选物的状态。LINC-PINT在抑制黑色素瘤的致瘤性中发挥作用，这通过异种移植模型试验进一步得到证实。LINC-PINT在黑色素瘤组织和细胞系中显著下调。在肿瘤细胞中过表达LINC-PINT导致A375、Mum2B和CRMM1细胞中的肿瘤生长显著减少和迁移抑制。基于异种移植模型的结果进一步与LINC-PINT阻碍黑色素瘤细胞生长和转移的发现一致。微阵列分析和生物信息学分析表明，CDK1、CCNA2、AURKA和PCNA是LINC-PINT的潜在靶标。总之，LINC-PINT通过将EZH2募集到其靶基因的启动子上，导致H3K27三甲基化和靶基因的表观遗传沉默，从而抑制黑色素瘤的致瘤性。LINC-PINT可能作为黑色素瘤的一种新型诊断和治疗靶点。
