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[慢病毒介导的RNA干扰下调TSC2基因表达对U937细胞系的影响及其机制]

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

作者信息

Xu Z F, Liu H X, Tan Y H, Chen X H, Ren F G, Zhang Y F, Chang J M, Zhang N, Hu J J, Wang H W

机构信息

Department of Hematology, the Second Hospital of Shanxi Medical University, Shanxi Key Laboratory of Molecular Diagnosis and Treatment of Blood Diseases, Taiyuan 030001, China.

出版信息

Zhonghua Xue Ye Xue Za Zhi. 2017 Jul 14;38(7):612-617. doi: 10.3760/cma.j.issn.0253-2727.2017.07.012.

Abstract

To investigate the effect of biology and mTOR pathway activity of down-regulated TSC2 gene expression on U937 leukemia cells. 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. Down-regulated expression of TSC2 gene promoted U937 cell proliferation and colony formation ability (<0.05) . The proportion in G(0)/G(1) phase of TSC2 down-regulated U937 cell was much lower than that of the control cells [ (52.53±3.75) % (75.10±4.33) %, =6.829, =0.002], the S phase [ (22.43±1.00) % (15.47±1.20) %, =-5.581, =0.019] and G(2)/M phase [ (25.03±4.34) % (14.33±0.91) %, =-5.413, =0.013] was remarkably higher than that of the control cells (<0.05) . There were no statistically significant differences in cell apoptosis and differentiation (>0.05) . Down-regulation of TSC2 led to the increased activity of mTOR, 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. Downregulation of TSC2 could promote the proliferation of U937 cells through up-regulation of mTOR activity.

摘要

探讨下调TSC2基因表达对U937白血病细胞生物学行为及mTOR信号通路活性的影响。采用慢病毒介导的RNA干扰技术下调高表达TSC2基因的U937细胞株基因表达;运用CCK-8法、集落形成实验及流式细胞术检测细胞增殖、凋亡及分化情况;采用qRT-PCR及Western blot检测基因表达水平及蛋白激酶活性。下调TSC2基因表达可促进U937细胞增殖及集落形成能力(<0.05)。TSC2基因下调的U937细胞G(0)/G(1)期比例显著低于对照细胞[(52.53±3.75)% (75.10±4.33)%,t=6.829,P=0.002];S期[(22.43±1.00)% (15.47±1.20)%,t=-5.581,P=0.019]及G(2)/M期[(25.03±4.34)% (14.33±0.91)%,t=-5.4�13,P=0.013]比例显著高于对照细胞(<0.05)。细胞凋亡及分化差异无统计学意义(>0.05)。下调TSC2可导致mTOR、4EBP1及S6K1活性增强,但不影响AKT活性。TSC2沉默后,增殖相关的cyclinD1、c-myc及PTEN表达上调,但P27KIP及BCL-XL表达未改变。下调TSC2可通过上调mTOR活性促进U937细胞增殖。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/eba2/7342282/940aef1599ff/cjh-38-07-612-g001.jpg

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