摘要目的:探讨下调极光激酶(AURK)B对 CALR突变MARIMO细胞增殖、凋亡的影响，以及相关基因和信号通路的变化情况。 方法:选择来源于1例68岁 CALR突变阳性骨髓增殖性肿瘤(MPN)女性患者的MARIMO细胞为研究对象。采用慢病毒介导的短发夹RNA(shRNA)转染MARIMO细胞，并根据shRNA不同，将该细胞分为sh-AURKA、sh-AURKB和sh-无关序列对照(CTRL)组。采用倒置荧光显微镜观察各组MARIMO细胞株，采用流式细胞术(FCM)检测其绿色荧光蛋白(GFP)阳性率。通过FCM、5-乙炔基-2′-脱氧尿嘧啶核苷(EdU)/4′，6-二脒基-2-苯基吲哚二盐酸盐(DAPI)染色、Annexin V/PE试剂对sh-AURKB和sh-CTRL组MARIMO细胞增殖、凋亡情况进行分析。采用RNA转录组测序(RNA-Seq)技术分析sh-AURKB和sh-CTRL组MARIMO细胞差异表达基因(DEG)的富集情况，对其进行基因本体(GO)功能注释和京都基因与基因组百科全书(KEGG)通路富集分析，筛选显著DEG。通过实时荧光定量-PCR(RT-qPCR)和蛋白质印迹法对DEG的mRNA和蛋白质相对表达水平进行验证。定量资料的2组比较，采用 t检验。3组总体比较，采用单因素方差分析；两两比较，采用最小显著差异(LSD)法。本研究遵循的程序符合2013年修订版《世界医学协会赫尔辛基宣言》要求。 结果:①转染72 h后，sh-AURKB组MARIMO细胞数量较sh-CTRL组减少[ (0.62±0.03)×10 5比(12.44±0.08) ×10 5]，差异有统计学意义( t＝257.20， P<0.000 1)。与sh-CTRL组相比，sh-AURKB组处于S期的MARIMO细胞比例降低[(33.37±0.75)%比(42.77±0.91)%]，处于G2-M期的MARIMO细胞比例增高[(27.83±0.69)%比(17.90±0.40)%]，细胞凋亡比例增高[(16.47±0.70)%比(2.75±0.13)%]，并且差异均有统计学意义( t＝13.83， P＝0.000 2； t＝23.78， P<0.000 1； t＝33.28， P<0.000 1)。② RNA-Seq检测结果显示，与sh-CTRL组相比，sh-AURKB组MARIMO细胞中有2 787个基因表达上调，2 420个基因表达下调。③ sh-AURKB和sh-CTRL组DEG主要注释于细胞内区域、细胞内膜边界细胞器、膜边界细胞器、细胞内细胞器部分、细胞器部分等GO节点。KEGG富集分析结果显示，共89个通路显著富集。对相关通路进一步筛选结果显示，2组显著DEG为 NDUFV1，NPRL2，MAP2K4，CPEB1。④ RT-qPCR结果显示，sh-AURKB组的 NPRL2、MAP2K4、CPEB1mRNA相对表达水平显著低于sh-CTRL组，差异有统计学意义(LSD- t＝14.13、9.13、3.10， P<0.000 1、<0.000 1、＝0.021)。蛋白质印迹法检测结果显示，与sh-CTRL组相比，sh-AURKB组NDUFV1/GAPDH蛋白条带灰度值比值增高，NPRL2/GAPDH、CPEB1/GAPDH、MAP2K4/GAPDH和p-MAP2K4/GAPDH降低，并且差异均有统计学意义(LSD- t＝22.60、12.09、7.48、20.48、8.22， P<0.000 1、<0.000 1、＝0.000 3、<0.000 1、＝0.000 2)。 结论:AURKB参与 CALR突变MARIMO细胞的增殖、分化及凋亡等过程，而下调AURKB可引起相关基因表达水平发生显著变化。DEG主要富集在细胞代谢、细胞周期及凋亡相关通路。

abstractsObjective:To investigate the effects of down-regulation of aurora kinase (AURK) B on the proliferation and apoptosis of CALR-mutated MARIMO cells, as well as changes in related genes and signalling. Methods:MARIMO cells derived from a 68-year-old female patient with CALR mutation-positive myeloproliferative neoplasms(MPN) were selected for the study. Lentivirus-mediated short hairpin RNA (shRNA) was used to transfect MARIMO cells, and the cells were categorized into sh-AURKA group, sh-AURKB group and control shRNA (sh-CTRL) group according to the difference of shRNA. The MARIMO cell lines in each group were observed by inverted fluorescence microscope, and their green fluorescent protein (GFP) positivity was detected by flow cytometry (FCM). The reproduction and apoptosis of MARIMO cells in sh-AURKB group and sh-CTRL group were analyzed by FCM, 5-ethynyl-2′-deoxyuridine (EdU)/4′, 6-diamidino-2-phenylindole dihydrochloride (DAPI) staining, and Annexin V/PE reagent. The enrichment of differentially expressed genes (DEG) in sh-AURKB group and sh-CTRL group was analyzed by RNA sequencing (RNA-Seq), and gene ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were carried out to screen for significant DEG. The relative expression of mRNA and proteins of DEG were validated by real-time fluorescence quantitative-PCR (RT-qPCR) and Western blotting. Relative expression levels were verified. The comparison of measurement data between two groups was performed by t test. The overall comparison among 3 groups were conducted by one-way analysis of variance, pairwise comparisons between groups were conducted by least-significant difference (LSD) test. This study was in line with World Medical Association Declaration of Helsinki revised in 2013. Results:① The number of MARIMO cells 72 h after transfection was reduced in sh-AURKB group compared with sh-CTRL group [ (0.62±0.03) ×10 5vs (12.44±0.08) ×10 5], and the difference was statistically significant ( t=257.20, P<0.000 1).Compared with sh-CTRL group, the proportion of MARIMO cells in the S-stage in sh-AURKB group was decreased [(33.37 ± 0.75)% vs (42.77 ± 0.91)%], and the proportion of MARIMO cells in G2-M stage was increased [(27.83 ± 0.69)% vs (17.90 ± 0.40)%], the proportion of apoptosis was increased [(16.47 ± 0.70)% vs (2.75 ± 0.13)%], all the differences were statistically significant ( t=13.83, P=0.000 2; t= 23.78, P<0.000 1); t=33.28, P<0.000 1). ② RNA-Seq assay showed that compared to sh-CTRL group, there were 2 787 genes up-regulated and 2 420 genes down-regulated in MARIMO cells in sh-AURKB group. ③ The DEG of sh-AURKB group and sh-CTRL group were mainly annotated in GO nodes such as intracellular region, intracellular membrane border organelle, membrane border organelle, intracellular organelle part, organelle part, etc.. KEGG enrichment analysis showed that a total of 89 signal pathways were significantly enriched. Further screening of DEG-related pathways showed that the 2 groups of significant DEG were NDUFV1, NPRL2, MAP2K4, and CPEB1. ④ RT-qPCR results showed that the relative mRNA expression levels of NPRL2, MAP2K4 and CPEB1 in sh-AURKB group were lower than those in sh-CTRL group, and the differences were statistically significant (LSD- t=14.13, 9.13, 3.10; P<0.000 1, <0.000 1, =0.021). Western blotting results showed that compared with SH-CTRL group, the protein strip grayscale value of NDUFV1/GAPDH in sh-AURKB group increased, while NPRL2/GAPDH, CPEB1/GAPDH, MAP2K4/GAPDH and p-MAP2K4/GAPDH reduced, and the differences were statistically significant (LSD- t=22.60、12.09、7.48、20.48、8.22, P<0.000 1、<0.000 1、=0.000 3、<0.000 1、=0.000 2) Conclusions:AURKB is involved in the proliferation, differentiation and apoptosis of CALR mutant MARIMO cells, and down-regulation of AURKB could induce significant changes in gene expression levels. DEG are mainly enriched in the cell metabolism, cell cycle and apoptosis related pathways.