摘要
目的研究人白细胞相关免疫球蛋白样受体1(LAIR-1)对Janus激酶2(JAK2)V617F突变的人急性髓系白血病HEL细胞JAK/信号转导子与转录激活子(STAT)和磷脂酰肌醇3激酶/蛋白激酶B/哺乳动物雷帕霉素靶蛋白(PI3K/AKT/mTOR)信号通路的调节作用,以及对细胞增殖和凋亡的影响。方法采用反转录PCR和基因测序鉴定JAK2 V617F突变;应用免疫共沉淀和Western blot法鉴定LAIR-1募集的蛋白酪氨酸磷酸酶(PTP)种类;采用CCK-8法检测HEL细胞的增殖;采用异硫氰酸荧光素标记的膜联素Ⅴ/碘化丙啶(annexinⅤ-FITC/PI)双标记结合流式细胞术检测HEL细胞的凋亡率;采用Western blot法检测JAK/STAT和PI3K/AKT/mTOR通路蛋白酪氨酸磷酸化水平及细胞周期蛋白D1(cyclin D1)、Bcl2相关X蛋白(BAX)和B细胞淋巴瘤因子2(Bcl2)的蛋白表达。结果在JAK2 V617F突变的HEL细胞中,LAIR-1与其配体胶原蛋白结合后可募集含Src同源域2磷酸酶2(SHP-2);LAIR-1可以下调HEL细胞JAK2、STAT1、STAT3、STAT5、AKT和mTOR的蛋白酪氨酸磷酸化水平,并能够显著抑制cyclin D1和Bcl2的表达,而对BAX的表达水平未见显著影响;LAIR-1能够明显抑制HEL细胞的增殖,促进HEL细胞凋亡。结论在JAK2 V617F突变的人白血病HEL细胞中,LAIR-1可通过募集SHP-2抑制JAK/STAT和PI3K/AKT/mTOR信号通路的活化,进而抑制HEL细胞的增殖,促进细胞凋亡。
Objective To investigate the role of human leukocyte-associated immunoglobulin-like receptor-1(LAIR-1)in the regulation of Janus kinase/signal transducers and activators of transcription(JAK/STAT)and phosphatidylinositol 3 kinase/protein kinase B/mammalian target of rapamycin(PI3K/AKT/mTOR)signaling pathways in human acute myeloid leukemia HEL cells carrying the JAK2 V617F mutation,along with its effects on cell proliferation and apoptosis.Methods The JAK2 V617F mutation was identified using reverse transcription PCR and gene sequencing.The protein phosphatase(PTP)recruited by LAIR-1 was determined through co-immunoprecipitation and Western blot analysis.The proliferation of HEL cells was detected by CCK-8 assay.The apoptosis rate of HEL cells was detected by flow cytometry with annexinⅤ-FITC/PI labeling.Western blot analysis was employed to assess the phosphorylation status of proteins involved in the JAK/STAT and PI3K/AKT/mTOR pathways,as well as the expression levels of cyclinD1,B cell lymphoma 2(Bcl2),and Bcl2 associated X protein(BAX).Results In HEL cells containing the JAK2 V617F mutation,LAIR-1 was observed to recruit SH2-containing protein tyrosine phosphatase 2(SHP-2)upon binding with its ligand collagen.Moreover,LAIR-1 downregulated the tyrosine phosphorylation levels of JAK2,STAT1,STAT3,STAT5,AKT and mTOR and significantly reduced the expression of cyclin D1 and Bcl2,while having no effect on the expression of BAX.In addition,LAIR-1 exhibited a significantly inhibitory effect on cell proliferation and promoted apoptosis in HEL cells.Conclusion In HEL cells with JAK2 V617F mutation,LAIR-1 can inhibit the activation of JAK/STAT and PI3K/AKT/mTOR signaling pathways by recruiting SHP-2,thereby inhibiting the proliferation of HEL cells and promoting cell apoptosis.
作者
樊翠
张娅薇
杨蕊
吴肖婕
周嘉迪
薛江楠
FAN Cui;ZHANG Yawei;YANG Rui;WU Xiaojie;ZHOU Jiadi;XUE Jiangnan(Department of Immunology of Basic Medical College,Binzhou Medical University,Yantai 264000;Department of Ultrasonography,Linyi Traditional Chinese Medical Hospital,Linyi 276000;Central Laboratory of Binzhou People’s Hospital,Binzhou 256610;Department of laboratory Medcine,Shannxi University of Chinese Medicine,Xianyang 712000,China)
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2024年第3期207-214,共8页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金(81273200)。
