DataSheet_3_RNA Demethylase ALKBH5 Prevents Lung Cancer Progression by Regulating EMT and Stemness via Regulating p53.zip

BackgroundAlthough N6-methyladenosine (m6A) RNA methylation is the most abundant reversible methylation of mRNA, which plays a critical role in regulating cancer processing, few studies have examined the role of m6A in nonsmall-cell lung cancer-derived cancer stem-like cells (CSCs). MethodsCSCs were enriched by culturing NSCLC cells in a serum-free medium, and stem factors, including CD24, CD44, ALDH1, Nanog, Oct4, and Sox2 were detected by Western blot. ALKBH5 expression was measured by employing a tissue array. Global m6A methylation was measured after ALKBH5 knockdown. Malignances of CSCs were detected by performing CCK-8 assay, invasion assay, cell cycle analysis, and tumor formation in vitro and in vivo. Resultsm6A demethylase ALKBH5 is highly expressed in CSCs derived from NSCLC. Knockdown of ALKBH5 increased global m6A level, and also increased E-cadherin, decreased stem hallmarkers, Nanog and Oct4, and inhibited stemness of CSCs. In lung carcinoma, ALKBH5 is found to be positively correlated with p53 by using Gene Expression Profiling Interactive Analysis (GEPIA) online tool. P53 transcriptionally regulates ALKBH5 and subsequently regulates the global m6A methylation level. Knockdown of p53 or inhibition of p53’s transcriptional activity by addition of its specific inhibitor PFT-α decreased expression of ALKBH5 and CSCs’ malignancies, including proliferation, invasion, and tumor formation ability, indicating that p53 may partially regulate CSC’s malignancies via ALKBH5. Furthermore, we also found p53 transcriptionally regulates PRRX1, which is consistent with our previous report. ConclusionCollectively, our findings indicate the pivotal role of ALKBH5 in CSCs derived from NSCLC and highlight the regulatory function of the p53/ALKBH5 axis in modulating CSC progression, which could be a promising therapeutic target for NSCLC.研究背景 尽管N6-甲基腺嘌呤（N6-methyladenosine, m6A）RNA甲基化是mRNA上最丰富的可逆甲基化修饰，在调控癌症进程中发挥关键作用，但目前鲜有研究探讨m6A在非小细胞肺癌（non-small-cell lung cancer, NSCLC）来源的肿瘤干细胞样细胞（cancer stem-like cells, CSCs）中的作用。 研究方法 本研究通过无血清培养基培养非小细胞肺癌细胞以富集肿瘤干细胞样细胞，采用蛋白质印迹法（Western blot）检测CD24、CD44、ALDH1、Nanog、Oct4及Sox2等干细胞标志物；利用组织芯片（tissue array）检测ALKBH5的表达水平；在ALKBH5敲低后检测整体m6A甲基化水平；通过CCK-8实验、侵袭实验、细胞周期分析以及体内外成瘤实验检测肿瘤干细胞样细胞的恶性生物学行为。 研究结果 m6A去甲基化酶ALKBH5在非小细胞肺癌来源的肿瘤干细胞样细胞中高表达。敲低ALKBH5可提升整体m6A甲基化水平，同时上调E-钙粘蛋白（E-cadherin）的表达，下调干细胞标志物Nanog与Oct4的水平，并抑制肿瘤干细胞样细胞的干性。通过基因表达谱交互分析（Gene Expression Profiling Interactive Analysis, GEPIA）在线工具分析发现，在肺癌组织中ALKBH5与p53呈正相关。p53可转录调控ALKBH5的表达，进而调控整体m6A甲基化水平。敲低p53或使用其特异性抑制剂PFT-α抑制p53的转录活性，均可降低ALKBH5的表达以及肿瘤干细胞样细胞的恶性生物学行为，包括增殖、侵袭及成瘤能力，这表明p53可通过ALKBH5部分调控肿瘤干细胞样细胞的恶性表型。此外，本研究还发现p53可转录调控PRRX1，这与我们此前的研究报道一致。 研究结论 综上，本研究结果揭示了ALKBH5在非小细胞肺癌来源的肿瘤干细胞样细胞中的关键作用，并凸显了p53/ALKBH5轴在调控肿瘤干细胞样细胞进程中的调控功能，该轴有望成为非小细胞肺癌的潜在治疗靶点。