慢病毒介导的RNA干扰下调TSC2表达对白血病U937细胞系的影响及作用机制  

Effect of TSC2 gene expression downregulation by lentivirus induced RNA interference on U937 cell line and its mechanism

作　　者：徐智芳[1] 刘海霞[1] 覃艳红[1] 陈秀花[1] 任方刚[1] 张耀方[1] 常建梅[1] 张娜[1] 胡晋军[1] 王宏伟[1] 

机构地区：[1]山西医科大学第二医院血液病研究所、血液病分子诊疗山西省重点实验室,太原030001

出　　处：《中华血液学杂志》2017年第7期612-617,共6页Chinese Journal of Hematology

基　　金：国家自然科学基金（81200390）;中国博士后科学基金（20100471583）

摘　　要：【摘要】目的研究下调TSC2基因表达对白血病U937细胞系的生物学作用及其对mTOR通路活性的影响。方法选择TSC2高表达的U937细胞系，通过慢病毒介导的RNA干扰技术下调TSC2基因表达；采用CCK-8比色法、细胞集落形成实验和流式细胞术检测其对细胞增殖、分化和凋亡的影响；采用Westernblot法和实时荧光定量PCR（RQ-PCR）法检测TSC2表达下调对mTOR通路蛋白表达及活性的影响。结果TSC2基因表达降低能够促进U937细胞的增殖和集落形成（P〈0．05）；能够使U937细胞G．期[（52．53±3．75）％对（75．10±4．33）％，t=6．829，P=0．002]比例明显降低，G2／M期[（22．43±1．00）％对（15．47±1．20）％，t=-5．581，P=-0．019]、S期[（25．03±4．34）％对（14．33±0．91）％，t=-5．413，P=0．013]比例升高；对细胞分化和细胞凋亡没有明显影响（P〉0．05）。TSC2基因表达下调后，mTOR活性升高，磷酸化的4EBP1和S6KI蛋白活性升高，而AKT蛋白活性没有明显变化；与细胞增殖相关的基因cyclinD1、c—myc表达升高，PTEN基因表达升高，P27KIP基因和凋亡相关基因BCL-XL的表达没有明显的改变。结论TSC2基因表达下调可以通过调节mTOR通路活性促进白血病细胞的增殖。Objective To investigate the effect of biology and mTOR pathway activity of down- regulated TSC2 gene expression on U937 leukemia cells. Methods Gene expression was down-regulated by lentivirus induced RNA interference on TSC2 high expressed U937 cell line; the proliferation, apoptosis and differentiation were detected by CCK-8 assay, colony formation assay and flow cytometry; the gene expression level and protein kinase activity were detected by qRT-PCR and Western blot. Results Down- regulated expression of TSC2 gene promoted U937 cell proliferation and colony formation ability （P〈 0.05）. The proportion in G0/G1 phase of TSC2 down-regulated U937 cell was much lower than that of the control cells [ （52.53±3.75）% vs （75.10±4.33）%, t=-6.829, P=0.002], the S phase [ （22.43± 1.00）% vs （ 15.47±1.20）%, t=-5.581, P=0.019] and G]M phase [ （25.03±4.34）% vs （14.33±0.91）%, t=-5.413, P= 0.013] was remarkably higher than that of the control cells （P〈0.05）. There were no statistically significant differences in cell apoptosis and differentiation （P 〉 0.05）. Down-regulation of TSC2 led to the increased activity of roTOR, 4EBP1 and S6K1, but did not influence the activity of AKT. The expressions of proliferation related cyclinD1, c-myc and PTEN were also up-regulated after TSC2 silenced, but the expressions of P27KIP and BCL-XL were not changed. Conclusion Downregulation of TSC2 could promote the proliferation ofU937 cells through up-regulation ofmTOR activity.

关 键 词：基因 TSC2 ROTOR 白血病 信号通路 

分 类 号：R733.7[医药卫生—肿瘤]