成骨细胞龛通过上调白血病细胞IL-1的表达促进白血病干细胞增殖的研究

Bone Marrow Osteoblasts Promotes the Proliferation Leukemia Stem Cell by Up-regulating Interleukin-1

目的:探索骨髓微环境对白血病干细胞的调控机制,阐明成骨细胞龛是否通过上调白血病细胞中白细胞介素-1(IL-1)的表达,促进白血病细胞的增殖及自我更新。方法:获取AML1-ETO9a(AE9a)小鼠白血病模型的小鼠骨内膜和骨髓腔的白血病细胞,进行转录组测序(RNA-seq),利用GSEA对转录组差异基因进行功能富集分析;实时荧光定量PCR检测并验证与成骨细胞共培养后,AE9a小鼠白血病细胞中IL-1的表达。同时采用实时荧光定量PCR测定43例急性髓系白血病(AML)患者中IL-1的表达;通过集落培养体系检测在与成骨细胞共培养后及加入IL-1β,AE9a小鼠白血病细胞的集落形成能力。结果:RNA-seq及GSEA分析结果显示,在AE9a白血病小鼠,骨内膜区域的白血病细胞中表达上调的基因,显著富集于炎症通路相关分子,其中IL-1α和IL-1β在骨内膜区的白血病细胞中的表达均显著高于骨髓腔内的白血病细胞(IL-1α:P<0.001, IL-1β:P<0.001)。与成骨细胞共培养后,AE9a小鼠白血病细胞IL-1α和IL-1β表达升高,分别是未与成骨细胞共培养的2.5倍和3.5倍,共培养后集落形成数量显著增加(P<0.001)。体外培养加入IL-1β后,AE9a小鼠白血病细胞集落生成数量显著增加(P<0.01)。在AML患者骨髓细胞中,高表达CD34的患者骨髓单个核细胞同时高表达IL-1。结论:成骨细胞龛通过上调白血病细胞IL-1的表达,促进白血病细胞的增殖和自我更新。白血病患者骨髓IL-1的表达与CD34相关。

Objective:To explore the extrinsic regulation mechanism of bone marrow microenvironment in leukemia cells,and investigate the promoting effect of osteoblast niche on the proliferation and self-renewal of leukemia stem cell by up-regulating the expression of interleukin-1(IL-1)in leukemia cell. Methods:The gene expression profiles on leukemia cells derived from AE9a mouse bone marrow endosteum and central bone marrow were determined by RNA sequencing and gene set enrichment analysis(GSEA). Quantitative real-time PCR(qRT-PCR)was used to detect the expression of IL-1 in AE9a mouse leukemia cells co-cultured with or without osteoblasts in vitro. In addition,qRT-PCR was also used to determine the expression of IL-1 in bone marrow mononuclear cell(BMMNC)from43patients with acute myeloid leukemia(AML). For leukemia cells co-cultured with osteoblasts or treated with IL-1β,colony forming ability of AE9a leukemia cells was determined by colony formation assay. Results:In AE9a leukemia mouse,RNA-seq data and GSEA showed that the enrichment of the upregulated genes in leukemia cells located in endosteum fell into inflammatory response gene set,among them,IL-1α and IL-1β were significantly higher expressed in AE9a leukemia cells that located osteoblast niche(IL-1α:P< 0.001,IL-1β:P< 0.001). After AE9a leukemia cells were co-cultured with osteoblasts in vitro,the expression of IL-1α and IL-1β in leukemia cells were increased by2.5and3.5times respectively. In colony formation assay,the number of colonies was increased significantly after leukemia cells were cocultured with osteoblasts(P< 0.001). In addition,when AE9a leukemia cells were treated with IL-1β,the number of colonies was also increased significantly(P< 0.01). In AML patients,BMMNC with high percentage of CD34positive cells exhibited higher level of IL-1 expression. Conclusion:Osteoblast niche can promote leukemia cell proliferation and self-renewal through up-regulating the expression of IL-1in leukemia cells. In AML patients,the expression level of IL-1was correlated to the percentage of CD34positive cells in BMMNC.