Table1_M6A-Mediated Upregulation of LINC00106 Promotes Stemness and Metastasis Properties of Hepatocellular Carcinoma via Sponging Let7f.DOC

Background: Hepatocellular carcinoma (HCC) cells exhibit the stemness property, which makes the patient with HCC prone to tumor recurrence and metastasis. Despite the prominent regulatory role of long non-coding RNAs (lncRNAs) in tumor stemness, the roles and molecular mechanisms of LINC00106 in HCC are poorly understood.
Methods: LINC00106, let7f and periostin expression levels in tissue specimens and cell lines were assessed through qRT-PCR and immunohistochemistry (IHC). Various in vivo and in vitro assays, namely sphere/colony formation, proportion of side population cells (SP%), invasion, migration, western blot, and murine xenograft model were employed for assessing the stemness and metastatic properties of HCC cells. Luciferase reporter assays, RNA-seq, RNA pull-down, RNA immunoprecipitation (RIP) were conducted to clarificate the target gene and analyze the underlying mechanisms.
Results: LINC00106 was prominently upregulated in tissues and cell lines of HCC. Patients having a high LINC00106 level exhibited a poor outcome. Under in vivo and in vitro conditions, the stemness and metastatic properties of HCC cells were augmented by LINC00106. Additionally, LINC00106 was found to sponge let7f to upregulate periostin, which lead to the activation of periostin-associated PI3K-AKT signaling pathway. Moreover, m6A methylation was found to cause LINC00106 upregulation while maintaining LINC00106 RNA transcript stability.
Conclusion: m6A methylation triggers the upregulation of LINC00106, which promotes the stemness and metastasis properties in HCC cells by sponging let7f, thereby resulting in periostin activation. The findings indicate the potential of LINC00106 as a diagnostic marker and therapeutic target for HCC.

研究背景：肝细胞癌（Hepatocellular carcinoma, HCC）细胞具有干细胞特性，这使得肝癌患者极易出现肿瘤复发与转移。尽管长链非编码RNA（long non-coding RNAs, lncRNAs）在肿瘤干细胞特性调控中发挥着重要作用，但LINC00106在肝细胞癌中的具体作用及分子机制仍尚不明确。
研究方法：通过实时定量逆转录聚合酶链反应（quantitative real-time polymerase chain reaction, qRT-PCR）与免疫组织化学（immunohistochemistry, IHC）检测组织标本及细胞系中LINC00106、let7f及骨膜蛋白的表达水平。采用多种体内外实验手段，包括球体/集落形成实验、侧群细胞比例（side population cells, SP%）检测、侵袭实验、迁移实验、蛋白质印迹法以及小鼠异种移植模型，以评估肝癌细胞的干细胞特性与转移能力。通过荧光素酶报告基因实验、RNA测序（RNA-seq）、RNA下拉实验以及RNA免疫沉淀（RNA immunoprecipitation, RIP），明确其靶基因并解析潜在分子机制。
研究结果：LINC00106在肝细胞癌组织及细胞系中显著上调。LINC00106高表达的肝癌患者预后不良。体内外实验均证实，LINC00106可增强肝癌细胞的干细胞特性与转移能力。此外，LINC00106可通过分子海绵作用结合let7f，从而上调骨膜蛋白的表达，进而激活骨膜蛋白相关的PI3K-AKT信号通路。同时，研究发现m6A甲基化可诱导LINC00106上调，并维持其RNA转录本的稳定性。
研究结论：m6A甲基化介导LINC00106上调，后者通过海绵吸附let7f上调骨膜蛋白、激活PI3K-AKT信号通路，进而促进肝癌细胞的干细胞特性与转移能力。本研究结果表明，LINC00106有望成为肝细胞癌的诊断标志物及治疗靶点。