NPM1基因突变对急性白血病细胞系体外侵袭能力的影响

核仁磷酸蛋白基因(nucleophosmin,NPM1)突变是目前急性髓系白血病发生突变率最高的基因改变,与白血病的发生发展密切相关。为探讨NPM1突变对白血病细胞体外侵袭能力的影响,将载体pEGFPC1-NPM1-mA转染THP-1白血病细胞系,筛选稳定表达NPM A型突变蛋白(NPM1-mA)的白血病细胞株(THP-1-mA)。通过transwell迁移实验、Matrigel侵袭实验以及细胞粘附实验来观察THP-1-mA细胞体外浸润转移能力的改变。结果发现,THP-1-mA细胞的体外迁移能力和侵袭能力明显高于亲代THP-1细胞;此外,THP-1-mA细胞对纤维连接蛋白的粘附能力也显著高于THP-1细胞。因此,我们的研究结果提示,NPMI突变可增强白血病细胞的体外侵袭能力,这有利于进一步明确NPM1突变基因在白血病细胞恶性转化中的调控作用。     

核仁磷酸蛋白基因突变对K562白血病细胞分化的影响

核仁磷酸蛋白基因（nucleophosmin,NPM1）突变在急性髓系白血病的发生发展中发挥着重要作用,而与白血病分化阻滞的关系尚未完全阐明。为探讨NPM1基因突变对白血病细胞体外分化的影响,将携带NPM1 A型突变（NPM1-mA）的表达质粒载体pEGFPC1-NPM1-mA转染白血病K562细胞系,构建稳定表达NPM1-mA蛋白的细胞株（K562 mA）,同时设立野生型NPM1转染组（K562 wt）、空载体转染组（K562 C1）和未处理组（K562）为对照。利用豆蔻酰佛波醇乙酯（PMA）诱导各组细胞分化,瑞氏–吉姆萨染色观察细胞分化的形态改变,计算诱导分化率;相差显微镜计数贴壁细胞数量;流式细胞术分析细胞表面分化抗原CD41的表达。结果显示,PMA作用72 h后,与对照组相比,K562 mA组细胞的诱导分化率及贴壁细胞数明显降低（P〈0.05）;同时,CD41的表达受到显著抑制（P〈0.01）。提示NPM1基因突变能够阻滞白血病细胞系K562的体外分化。     

NPM1突变基因调控K562白血病细胞侵袭表型的相关机制

目的探讨CXCR4、Angs在NPM1突变参与调控的白血病细胞浸润转移中的作用,以期进一步明确NPM1突变在白血病浸润转移中的调控机制。方法通过基因转染构建稳定表达NPM1突变蛋白的K562白血病细胞株（K562-mA）。qRT—PCR检测各组细胞CXCR4、Ang-1／2的mRNA表达水平;Western免疫印迹和流式细胞仪分别检测细胞CXCR4总蛋白和膜蛋白的表达。结果建立了稳定表达NPM突变基因的K562-mA细胞株。与未处理组和空载体转染组相比,K562-mA细胞CXCR4的mRNA和蛋白表达水平显著增高;Ang-1mRNA表达水平明显降低、Ang-2mRNA表达水平明显增高。结论CXCR4、Ang-1／2可能在MPM1突变调控白血病细胞的浸润转移中发挥重要作用。     

NPM1突变基因通过MMPs参与调控白血病细胞体外侵袭

核仁磷蛋白基因(nucleophosmin,NPM1)突变是目前急性髓系白血病(AML)中突变率最高的基因改变,在白血病的发生发展过程中发挥重要的调控作用。为探讨NPM1突变参与调控白血病髓外浸润的分子机制,将表达质粒pEGFPC1-NPM1-mA转染THP-1细胞系,筛选稳定表达NPM1突变蛋白的白血病细胞株(THP-1-mA)。利用RT-PCR及Western blot分析了THP-1-mA细胞与亲代细胞间MMP-2、MMP-9、TIMP-1、TIMP-2表达水平的差异。结果显示,具有体外高侵袭能力的THP-1-mA组细胞MMP-2的mRNA水平和蛋白水平均明显高于两对照组,而MMP-9 mRNA表达水平虽有所增高,但蛋白表达水平却明显降低。同时,与空载体转染组和未处理组细胞相比,THP-1-mA组细胞TIMP-2的mRNA水平和蛋白水平表达显著降低,差异具有统计学意义;TIMP-1表达水平无明显改变。提示MMP-2及其抑制剂TIMP-2在NPM1突变参与调控的白血病细胞髓外浸润中可能发挥重要作用。     

下调RALA表达抑制人白血病K562细胞迁移和侵袭

目的 研究小干扰RNA（siRNA）抑制v-ral 白血病致病因子RALA基因表达对人白血病K562细胞迁移和侵袭的影响.方法 利用LipofectamineTM 2000将化学合成的RALA siRNA转染体外培养的K562细胞,Real-time PCR检测细胞内RALA mRNA的表达水平;Western印迹检测细胞内RALA蛋白的表达水平;Boyden趋化小室实验检测细胞体外迁移和侵袭能力.结果与随机对照组相比,转染48 h后,RALA siRNA显著下调K562细胞内RALA mRNA和蛋白的表达（P＜0.05）.与随机对照组相比,转染RALA siRNA的K562细胞迁移和侵袭能力显著降低（P＜0.05）.结论 癌基因RALA在人白血病K562细胞迁移和侵袭过程中发挥重要作用,通过siRNA下调RALA的表达可抑制K562细胞迁移和侵袭能力.     

TGF-β信号通路抑制剂LY364947对急性髓系白血病细胞增殖、凋亡和侵袭的影响

该文旨在探讨抑制TGF-β信号通路对人急性髓系白血病(acute myeloid leukemia,AML)细胞体外增殖、凋亡和侵袭能力的影响。使用不同浓度TGF-β信号通路抑制剂LY364947处理AML细胞系(KG1a、OCI-AML3)后,采用CCK-8实验检测细胞体外增殖能力;流式细胞术检测细胞周期分布及凋亡情况; Western blot检测细胞周期调控因子Cyclin D1/p21、凋亡相关蛋白Bcl-2/Bax以及上皮细胞间质转化相关蛋白E-cadherin、N-cadherin和vimentin的表达; Transwell实验测定AML细胞迁移及侵袭能力的变化。结果显示:LY364947作用后,白血病细胞生长明显受抑制;细胞周期阻滞在G1期,伴有Cyclin D1表达下调和p21表达上调;细胞凋亡率增加,同时细胞抗凋亡蛋白Bcl-2的表达水平下降,促凋亡蛋白Bax表达增高;细胞体外迁移和侵袭能力减弱。此外, E-cadherin表达增高, N-cadherin和vimentin表达下降。该研究结果提示,抑制TGF-β信号通路能够抑制白血病细胞的体外增殖,诱导细胞凋亡,降低细胞迁移及侵袭能力。     

干扰PKM2对人白血病细胞增殖和凋亡的影响及潜在机制

目的:探讨糖酵解酶丙酮酸激酶M2型(PKM2)对人白血病细胞体外增殖和凋亡的影响及潜在机制。方法:将靶向PKM2的慢病毒载体转染人K562细胞株(sh PKM2组),同时设立空载体转染组为对照(Vector组)。采用qRT-PCR和Western blot技术分别检测Vector组和sh PKM2组PKM2 mRNA和蛋白的表达以及自噬标志物的变化; CCK-8实验检测细胞体外增殖能力;流式细胞术检测细胞周期和凋亡情况; Western blot检测凋亡相关蛋白Bax、Bcl-2表达水平。结果:在稳定干扰PKM2后,PKM2的mRNA(t=11. 58,P=0. 000 3)和蛋白水平(t=11. 88,P=0. 000 3)均明显降低。与Vector组比较,shPKM2组细胞体外增殖能力显著降低(F=118. 87,P 0. 000 1)。同时,干扰PKM2可使K562细胞周期阻滞在G1期,细胞凋亡率显著增加(t=37. 23,P 0. 000 1);使促凋亡蛋白Bax表达增加(t=15. 36,P=0. 000 1)、抗凋亡蛋白Bcl-2表达降低(t=9. 965,P=0. 000 6)。此外,干扰PKM2可减弱K562细胞自噬水平,表现为LC3Ⅱ降低(t_(LC3Ⅱ)=10. 32,P_(LC3Ⅱ)=0. 000 5),而p62水平增加(t_(p62)=14. 59,P_(p62)=0. 000 1)。还发现自噬诱导剂能逆转shPKM2引起的白血病细胞体外增殖能力减弱(F=96. 32,P 0. 000 1)。结论:以上结果表明干扰PKM2可抑制人白血病K562细胞的体外增殖并促进其凋亡,其机制可能与PKM2介导的自噬活性降低有关,提示PKM2可能作为白血病诊疗的一个潜在靶点。     

蜕皮激素诱导MIIP基因的表达对肝癌细胞增殖迁移的影响

目的:探讨蜕皮激素诱导迁移侵袭抑制蛋白基因(migration and invasion inhibitory protein,MIIP)基因的表达对肝癌细胞SK-Hep-1增殖和迁移能力的影响。方法:采用PCR扩增MIIP基因,连入T载体测序正确后,插入到蜕皮激素可诱导真核表达载体pIND中。将pIND-MIIP和含蜕皮激素受体基因的表达载体pVgRXR以脂质体转染法转染到SK-Hep-1中,经G418和Zeocin双抗生素筛选获得稳定转染细胞株。蜕皮激素Ponasterone A诱导后,采用Western blot检测MIIP表达,CCK-8实验检测细胞增殖,划痕实验检测细胞迁移。结果:蜕皮激素诱导MIIP表达上调后,SK-Hep-1细胞的增殖能力显著下降(P0.05),细胞的迁移能力明显减弱。结论:建立了MIIP基因可诱导表达系统,证实在肝癌细胞SK-Hep-1中MIIP过表达可抑制细胞的增殖及迁移能力。     

SPARC基因过表达对卵巢癌淋巴结高转移细胞生物学特性的影响

该研究旨在探讨富含半胱氨酸的酸性分泌蛋白基因(secreted protein acidic and rich in cysteine gene,SPARC)过表达对卵巢癌淋巴结高转移细胞(SKOV3-PM4)生物学特性的影响。构建SPARC基因的慢病毒表达载体并转染SKOV3-PM4细胞,Real-time PCR和Western blot验证转染后的表达效率,激光共聚焦免疫荧光进行蛋白的细胞定位,细胞计数法和集落形成实验测定细胞增殖能力,流式细胞仪检测细胞周期,Transwell小室实验测定细胞体外侵袭、迁移能力。实验结果显示,SPARC蛋白存在于核周及胞质;过表达SPARC基因后,SKOV3-PM4细胞增殖受到明显抑制(P0.05);细胞周期检测结果显示,各期改变无明显差异;体外侵袭、迁移实验结果显示,SKOV3-PM4细胞侵袭、迁移能力显著降低(P0.05)。实验结果表明,SPARC基因在卵巢癌淋巴结转移中可能发挥抑癌基因的生物学作用。     

长链非编码RNA SPRY4-IT1对髓母细胞瘤增殖及侵袭转移的影响

目的：探讨长链非编码RNA SPRY4-IT1（LncRNA）对髓母细胞瘤增殖及侵袭转移的影响。方法：髓母细胞瘤Daoy细胞分为对照组和si-SPRY4-IT1组,分别利用脂质体Lipofectamine 2000将阴性对照荧光序列和SPRY4-IT1-siRNA转入细胞中,采用Real-time PCR检测转染后各组细胞中SPRY4-IT1的表达情况,CCK-8实验及平板克隆形成实验检测细胞增殖能力的变化,以细胞体外侵袭、迁移实验分别检测细胞侵袭、迁移能力的变化,Western blot检测SPRY4-IT1对基质金属蛋白酶（MMP）-2和MMP-9蛋白的影响。结果：si-SPRY4-IT1组SPRY4-IT1 mRNA表达水平、细胞体外增殖能力、细胞侵袭、迁移能力、细胞MMP-2蛋白表达均较对照组明显降低（P<0.05）,而MMP-9蛋白表达未见明显变化。结论：干扰长链非编码RNA SPRY4-IT1在髓母细胞瘤Daoy细胞中的表达能显著抑制细胞的增殖、侵袭和迁移能力。     

Nucleophosmin Mutants Promote Adhesion,Migration and Invasion of Human Leukemia THP-1 Cells through MMPs Up-regulation via Ras/ERK MAPK Signaling

Acute myeloid leukemia (AML) with mutated nucleophosmin (NPM1) has been defined as a unique subgroup in the new classification of myeloid neoplasm, and the AML patients with mutated NPM1 frequently present extramedullary infiltration, but how NPM1 mutants regulate this process remains elusive. In this study, we found that overexpression of type A NPM1 gene mutation (NPM1-mA) enhanced the adhesive, migratory and invasive potential in THP-1 AML cells lacking mutated NPM1. NPM1-mA had up-regulated expression and gelatinolytic matrix metalloprotease-2 (MMP-2)/MMP-9 activity, as assessed by real-time PCR, western blotting and gelatin zymography. Following immunoprecipitation analysis to identify the interaction of NPM1-mA with K-Ras, we focused on the effect of NPM1-mA overexpression on the Ras/Mitogen-activated protein kinase (MAPK) signaling axis and showed that NPM1-mA increased the MEK and ERK phosphorylation levels, as evaluated by western blotting. Notably, a specific inhibitor of the ERK/MAPK pathway (PD98059), but not p38/MAPK, JNK/MAPK or PI3-K/AKT inhibitors, markedly decreased the cell invasion numbers in a transwell assay. Further experiments demonstrated that blocking the ERK/MAPK pathway by PD98059 resulted in reduced MMP-2/9 protein levels and MMP-9 activity. Additionally, NPM1-mA overexpression had down-regulated gene expression and protein production of tissue inhibitor of MMP-2 (TIMP-2) in THP-1 cells. Furthermore, evaluation of gene expression data from The Cancer Genome Atlas (TCGA) dataset revealed that MMP-2 was overexpressed in AML patient samples with NPM1 mutated and high MMP-2 expression associated with leukemic skin infiltration. Taken together, our results reveal that NPM1 mutations contribute to the invasive potential of AML cells through MMPs up-regulation via Ras/ERK MAPK signaling pathway activation and offer novel insights into the potential role of NPM1 mutations in leukemogenesis.     

Antizyme1基因转染对K562细胞增殖与凋亡的影响

研究鸟氨酸脱羧酶抗酶蛋白对人红白血病K562细胞增殖、三氧化二砷（ As2O3）诱导凋亡时的影响。方法: 定点突变技术构建缺失frameshift位点的pEGFP-N1-AZ1-mutation重组表达载体。脂质体法转染K562细胞,通过G418筛选获得稳定表达antizyme1的K562pAZ1m细胞系。采用不同浓度的As2O3处理细胞,通过MTT法检测细胞增殖,流式细胞术分析细胞周期及凋亡变化。并通过RT-PCR方法检测antiyme1转染对cyclin D1和survivin基因表达的影响。结果：获得稳定表达antizyme1的K562-AZ1m细胞株后,其增殖能力明显减慢。CyclinD1基因表达降低,细胞主要停滞于G0/G1期。在 As2O3的诱导作用下,细胞凋亡增多,survivin基因表达降低。结论：AZ1基因能够抑制K562细胞增殖,通过对cyclinD1的负调控使细胞周期停滞于G0/G1期。并可能通过下调survivin表达来加强 As2O3对其的诱导凋亡作用     

Protective effect of Bosutinib with caspase inhibitors on human K562 cells

IntroductionCancer therapy has become increasingly focused on molecularly targeted medications. Despite the fact that multi-cytotoxic medication regimens have proven to be highly effective, many investigations in targeted treatments have focused on a single agent. The precise molecular mechanism of action of second-generation BCR–ABL tyrosine kinase inhibitors, which includes different targets and pathways, can help rationalize therapy in chronic myelogenous leukemia (CML) and other diseases affected by BCR–ABL tyrosine kinase inhibitors (TKIs).AimThe purpose of this study was to analyze if bosutinib (BOS) combined with Boc-D-FMK effectively suppressed proliferation and induced apoptosis in K562 cells to a lesser extent, implying that bosutinib is an effective leukemia treatment and that its combination with Boc-D-FMK is a mild chemotherapeutic agent against leukemia.MethodsIn this study, bosutinib was obtained together with other materials to perform a cell culture experiment with human cell lines, as well as additional drug treatment. Furthermore, cell viability (MTT assay) and flow cryometry such as viability and cell cycle assays are performed. The target profile of the dual SRC/ABL inhibitor bosutinib was studied in this study as a first kinase inhibitor to target K562 cells, which has recently been linked to the proliferation of myelogenous leukaemia cells, these results suggest the effectiveness of inhibitory activity on cell viability/proliferation, alone generated a potent value of 250 nM (39.27 ± 1.17) for 48 h as optimal dose.ResultsThe cytotoxic effect of bosutinib on the K562 cell line was assessed in vitro using the MTT assay, and the cytotoxicity was further clarified using cell viability and cell cycle assays. Guava Cell Assay software validated the activation of apoptosis. Sub-G1, G0/G1, S, and G2/M phases are depicted. Cell cycle research revealed that K562 cells treated with bosutinib accumulated much more in the sub-G1 phase, which was later validated by a drop peak at the G2/M phase.ConclusionIn conclusion, the nature of bosutinib's reduction of cancer cell growth may open the door to future research into the development of green synthesis medicines, particularly for cancer treatment.     

Involvement of nPKC-MAPK pathway in the decrease of nucleophosmin/B23 during megakaryocytic differentiation of human myelogenous leukemia K562 cells

Human myelogenous leukemia K562 cells were induced to undergo megakaryocytic differentiation by long-term treatment with phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA). The protein level of nucleophosmin/B23 (NPM/B23), a nucleolar protein, was substantially decreased upon TPA treatment. In this study, we found that the proteasome inhibitors blocked the decrease of NPM/B23 protein in response to TPA, suggesting the proteasomes were involved in the downregulation of NPM/B23 upon megakaryocytic differentiation. To investigate the signaling pathway in the downregulation of NPM/B23 during early TPA-induced megakaryocytic differentiation of K562 cells, K562 cells were treated with TPA in the presence of the PKC isozyme-selective inhibitors, GF109203X and Gö 6976, or MEK1 inhibitor, PD98059. The decrease of NPM/B23 protein in the TPA-treated K562 cells was blocked by GF109203X but not by Gö 6976, suggesting the involvement of novel PKCs in the downregulation of NPM/B23 during TPA-induced megakaryocytic differentiation of K562 cells. The application of MEK1 inhibitor PD98059 upon TPA treatment blocked the TPA-induced decrease of NPM/B23 protein and aborted the megakaryocytic differentiation but not to break through the cell growth arrest. Unlike NPM/B23, the degradation of nucleolin in the TPA-treated K562 cells could not be blocked by PD98059 while the TPA-induced megakaryocytic differentiation was abrogated. The decrease of NPM/B23 protein seems to be more correlated with the novel PKC-MAPK-induced megakaryocytic differentiation than another nucleolar protein, nucleolin. Taken together, our results indicated that novel PKC-MAPK pathway was required for the decrease of NPM/B23 during TPA-induced megakaryocytic differentiation.     

氯化高铁血红素诱导K562细胞红系分化对其细胞周期的影响

细胞周期的测量是细胞增殖动力学的研究基础。通过添加30μmol·L-1氯化高铁血红素(Hemin)诱导人慢性髓系白血病K562细胞红系分化,利用5-溴脱氧尿嘧啶核苷(BrdU)与7-AAD双染的方法检测Hemin诱导的K562红系分化细胞对细胞周期各期比例的影响,未诱导的K562细胞周期各期比例作为对照,检测发现Hemin诱导的K562红系分化细胞对其细胞周期相对值无明显影响。应用BrdU间隔染色结合流式细胞术的方法,通过分析BrdU间隔染色后BrdU阳性细胞群的动态变化规律,从而推算出K562红系分化细胞的倍增时间及细胞周期各期时长。根据测量结果发现,未诱导的K562细胞总倍增时间约为20 h,与通过生长曲线公式法计算倍增时间的结果相符,Hemin诱导的K562细胞的细胞周期倍增时长约为23 h。Hemin诱导的K562红系分化细胞较未诱导的K562细胞倍增时间与各期时长无明显差异。因此,Hemin诱导K562细胞红系分化对其细胞周期绝对值及相对值均无明显影响。     

敲除LSD1基因对人慢性髓系白血病K562细胞周期的影响

为了探讨敲除LSD1基因后抑制人慢性髓系白血病 K562细胞增殖的原因,使用前期CRISPR/Cas9技术构建的人慢性髓系白血病 K562 LSD1基因敲除株,通过细胞凋亡Annexin V/PI（碘化丙啶）双染色、细胞PI染色以及流式细胞术技术,探究敲除LSD1基因后,K562细胞的凋亡水平是否改变,细胞周期是否受到影响。结果表明敲除LSD1基因后K562细胞被阻滞在G0/G1期,进入DNA复制期的细胞变少,因此导致细胞增殖速度减慢;通过细胞凋亡Annexin V/PI双染色并分析早期以及晚期凋亡细胞总比例,显示敲除LSD1基因后,不影响K562细胞的凋亡。研究结果表明,敲除LSD1基因后人慢性髓系白血病 K562细胞的增殖受到抑制,这是由于K562细胞增殖周期发生了改变,进入DNA复制期和分裂期的细胞减少;而与细胞凋亡水平的变化无关。