Linc00887调控PKM2可变剪接变体表达介导BCSCs特性的机制研究

Breast cancer stem cells (BCSCs) are responsible for the recurrence and chemoresistance of breast cancer. Alternative splicing (AS) is a key approach to regulate gene function and make gene diversity. LncRNAs play essential roles in regulating gene expression. Whether LncRNAs participate in AS-mediated stemness of BCSCs? What is the regulation mechanism? These remain to be clarified. We found that Linc00887, splice variant PKM2 and splicing factor hnRNPA1 were highly expressed in BCSCs, and silencing Linc00887 reduced the stemness of BCSCs and the expression of PKM2. And we also found that Linc00887 had binding sites with PKM pre-mRNA and hnRNPA1, respectively. It is suggested that Linc00887 specifically binds to PKM pre-mRNA to recruit hnRNPA1 to up-regulate PKM2 expression to mediate the stemness of BCSCs. This project aims to confirm the key binding sites between Linc00887 and PKM pre-mRNA, to elucidate that hnRNPA1 can promote the expression of PKM2 variants, to confirm that Linc00887 specifically binds to PKM pre-mRNA and recruits hnRNPA1 to regulate AS, and to further clarify the mechanism of Linc00887 regulating AS-mediated BCSCs. This study will provide a new idea and a new target for deep researching the mechanism of maintaining the BCSCs characteristics.

BCSCs是乳腺癌复发耐药根源。可变剪接（AS）是基因功能调节和多样化关键方式。LncRNA是调控基因表达重要分子，是否参与AS介导BCSCs干性？调控机制如何？待阐明。申请者发现：BCSCs中Linc00887、剪接体PKM2和剪接因子hnRNPA1高表达；沉默Linc00887降低BCSCs干性和PKM2表达，Linc00887与PKM pre-mRNA和hnRNPA1有结合位点。提示Linc00887募集hnRNPA1特异结合PKM pre-mRNA调控PKM2表达介导BCSCs干性。本项目旨在明确Linc00887与PKM pre-mRNA关键结合位点，阐释hnRNPA1介导PKM2变体表达作用，确认Linc00887募集hnRNPA1特异结合PKM pre-mRNA调控AS方式，阐明Linc00887调控AS介导BCSCs干性机制。为研究BCSCs干性维持新机制与新靶点提供依据。

乳腺癌肿瘤干细胞（BCSCs）特性与糖酵解密切相关，非编码RNA（ncRNA）参与调节糖酵解与BCSCs干性维持，但LINC00887在乳腺癌中是否通过调控BCSCs糖酵解水平影响干性具体机制仍未明确。本研究发现与亲本细胞相比，LINC00887在BCSCs中高表达，；与乳腺癌组织样本相比，具有CD44+干细胞表型的乳腺癌患者组织中LINC00887高表达且预后不良。沉默/过表达LINC00887，同步降低/促进BCSCs糖酵解水平与干性表型。在体移植瘤验证沉默LINC00887降低BCSCs体内成瘤能力。hnRNPA1可通过结合PKM pre-mRNA触发可变剪切事件产生PKM2异构体激活糖酵解过程。我们发现hnRNPA1在BCSCs及具有干细胞表型的CD44+患者组织中高表达且预后不良。在线软件预测hnRNPA1可直接集合PKM pre-mRNA；通过RNA pull down试验证实hnRNPA1与PKM pre-mRNA9号外显子上游内含子-400~-396位UAGGG直接结合。此外，我们还发现过表达hnRNPA1上调PKM2与糖酵解水平。RIP试验证实LINC00887与hnRNPA1结合，且促进hnRNPA1稳定。恢复试验发现沉默LINC00887同时过表达hnRNPA1不影响LINC00887表达，但上调了hnRNPA1、PKM2、CD44表达、促进BCSCs糖酵解水平与干性表型。综上所述，LINC00887通过hnRNPA1-PKM2-糖酵解途径调节BCSCs干性。为揭示BCSCs干性形成新机制与新靶点提供依据。

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