gas2calpain2轴维持白血病细胞生长-gas 2 cal pai 2 axis maintains leukemia cell growth.docx

中文摘要 GAS2-Calpain2 轴维持白血病细胞的生长sh919﹑sh1206（分别以 shGAS2#1 和 shGAS2#2 表示，分别包含特异性沉默 GAS2 的 shRNA 核苷酸序列）至 THP-1 和 Jurkat 细胞，GAS2 的 mRNA 和蛋白表达抑制 率均＞60%；沉默 GAS2 显著增强 THP-1 和 Jurkat 细胞的 Calpain 的活性并抑制细 胞增殖和集落生成能力；（4）GAS2 沉默显著抑制 THP-1 细胞的裸鼠皮下成瘤能力；（5）通过免疫共沉淀证明了在 Jurkat 和 THP-1 细胞中 GAS2 特异性结合 Calpain2 而非 Calpain1，在 THP-1 细胞中同时沉默 GAS2 和 Calpain2 能够逆转单 独沉默 GAS2 诱发的生长抑制；（6）在 CML 干/祖细胞中沉默 GAS2 显著抑制它们的集落生成能力。 结论：GAS2 在多种人类白血病包括慢性髓细胞白血病（CML）、急性淋巴细胞白血病（ALL）、急性髓细胞白血病（AML）均异常高表达。沉默 GAS2 在体内 和体外均能显著抑制 THP-1 和 Jurkat 的细胞增殖并增强 Calpain 活性，沉默 GAS2 也抑制 CML 患者 CD34+细胞的集落生成能力。GAS2 特异结合 Calpain2 而非 Calpain1，Calpain2 参与 GAS2 沉默诱发的生长抑制。综上，我们证实 GAS2-Calpain2 维持白血病细胞的生长。关键词：GAS2；Calpain2；GASDN；免疫共沉淀作者：孙丽丽 指导教师：赵昀GAS2-Calpain2 轴维持白血病细胞的生长英文摘要GAS2-Calpain2 axis contributes to the growth of leukemic cellsAbstractObjective: GAS2 (Growth arrest specific 2) modulates cell cycle, apoptosis and Calpain activity. The GAS2-Calpain axis has dual functions in malignant transformation. However, the GAS2 expression and the role of GAS2-Calpain2 axis in leukemic cells remain unclear. This study aims to investigate the role and the mechanism of GAS2-Calpain2 axis in leukemic cells.Methods:Q-RT-PCR (Quantitative Real-time PCR) and Calpain activity assay were used to measure mRNA expression of GAS2 and Calpain activity in human leukemic cells;Western blot and immunofluorescence were performed to examine GAS2 expression and localization in leukemic cells;Small hairpin RNA (shRNA) against GAS2 was delivered with lentiviral vector to THP-1 and Jurkat cells, the control and GAS2 silenced cells were assessed for their proliferation, colony–forming cell capacities and Calpain activity;Tumorigenic abilities were compared between control and GAS2 silenced THP-1 cells, when they were injected subcutaneously into nude mice;Co-immunoprecipitation was performed to detect the interaction between endogenous GAS2 and Calpain2 in both THP-1 and Jurkat cells;The effect of GAS2 silence on the colony-foming cell capacities of chronic