CircRERE调节miR-128-3p/WEE1轴促进人多发性骨髓瘤细胞系增殖、迁移和侵袭

目的 探究环状RNA RERE(circRERE)调节微小RNA(miR)-128-3p/丝氨酸/苏氨酸蛋白激酶(WEE1)轴对人多发性骨髓瘤(MM)细胞系增殖、迁移和侵袭的影响。方法 体外培养从健康受试者外周血中分离的正常浆细胞(nPCs)和人MM细胞系(U266、RPMI-8226、NCI-H929、LP-1)。RT-qPCR检测circRERE、miR-128-3p表达;Western blot检测WEE1表达。转染RPMI-8226细胞后,将其分为对照组(control)(未进行转染)、sh-circRERE组、sh-NC组、miR-128-3p inhibitor组、inhibitor-NC组、sh-circRERE+inhibitor-NC组、sh-circRERE+miR-128-3p inhibitor组,噻唑蓝(MTT)实验和5-乙炔基-2’脱氧尿嘧啶核苷(EdU)免疫荧光染色检测细胞增殖;划痕愈合实验、Transwell小室法检测细胞迁移、侵袭;双荧光素酶报告基因实验证实miR-128-3p与circRERE或WEE1的靶向作用关系。结果 与nPCs比较,MM细胞(...     

ABCG2单抗联合NPs-PTX体外抑制MMCSCs作用初探

目的:研究ABCG2单抗联合Fe3O4纳米紫杉醇(NPs-PTX)对人多发性骨髓瘤(Multiple myeloma,MM)癌干细胞(Cancer Stem Cells,CSCs)体外抑制作用。方法:经免疫磁珠分选方法,从人MM细胞系NCI-H929和RPMI8226细胞中分选出CD138-CD34-细胞,采用CCK-8检测、流式细胞仪分析法观察ABCG2单抗联合NPs-PTX对MM CSCs增殖、迁徙的抑制作用和其对凋亡及细胞周期的影响。结果:ABCG2单抗联合NPs-PTX能有效的抑制MM CSCs增殖,抑制率达80%,与单用NPs-PTX比较(55%),差异有显著性(P0.05);ABCG2单抗联合NPs-PTX组迁徙率为0.6%,较NPs-PTX组(1.7%),差异有极显著性意义(P0.01);ABCG2单抗联合NPs-PTX能有效诱导MM CSCs凋亡,凋亡率达43.15%,与单用NPs-PTX比较(18.35%),差异有显著性(P0.05),并使MM CSCs发生G/M期阻滞。结论:ABCG2单抗联合NPs-PTX具有体外抑制人MM CSCs作用,其机制可能与其通过诱导细胞凋亡,发生细胞周期阻滞有关。     

阿霉素诱导下多发性骨髓瘤细胞中自噬和氧化应激的相互作用

 目的: 探讨阿霉素(doxorubicin,DOX)诱导对多发性骨髓瘤细胞系RPMI-8226细胞内自噬和活性氧簇(reactive oxidative species,ROS)生成的影响及其相互作用关系。方法: 不同浓度阿霉素诱导RPMI-8226细胞24 h,采用Western blot技术检测细胞内beclin 1、LC3等自噬相关蛋白的表达水平。采用DCFH-DA荧光染色法检测RPMI-8226细胞内ROS的水平,荧光显微镜采集图像。采用氧自由基清除剂N-乙酰半胱氨酸(N-acetyl-L-cysteine,NAC)及tempol处理RPMI-8226细胞后,通过Western blot技术检测阿霉素诱导下细胞内beclin 1、LC3等蛋白的表达水平。采用自噬抑制剂3-甲基腺嘌呤(3-methyladenine,3-MA)处理PRMI-8226细胞后,检测阿霉素诱导下细胞内ROS和凋亡的表达水平。结果: RPMI-8226细胞内beclin 1和LC3Ⅱ/LC3Ⅰ表达水平呈阿霉素剂量依赖性增加,当阿霉素诱导浓度达2 mg/L时,与对照组比较显著增加(P<0.05)。采用2 mg/L阿霉素处理RPMI-8226细胞,通过荧光显微镜观察,ROS水平较对照组明显增加。氧自由基清除剂NAC和tempol处理RPMI-8226细胞后,beclin 1和LC3Ⅱ/Ⅰ的表达水平较阿霉素处理组下降。采用自噬抑制剂3-MA处理细胞后,RPMI-8226细胞内ROS和凋亡的水平较阿霉素处理组及对照组增加。结论: 阿霉素能增加RPMI-8226细胞内自噬和ROS的生成,抑制自噬能增加阿霉素诱导下ROS和凋亡的水平,抑制ROS后能减少阿霉素诱导下多发性骨髓瘤细胞中的自噬。     

过表达Twist1 促进结直肠癌SW620 细胞获得多药耐药性及机制研究

目的:探讨过表达Twist1对结直肠癌SW620细胞多药耐药性(MDR)的影响及其可能的机制。方法:利用过表达Twist1的慢病毒和阴性对照病毒转染结直肠癌SW620细胞,分别为实验组(SW620-Twist1)和对照组(SW620-NC),采用RT-qPCR和Western blot技术检测两组细胞Twist1基因及耐药基因ABCB1、ABCG2 mRNA和蛋白表达; CCK8法测定5-氟尿嘧啶(5-FU)和奥沙利铂(OXA)对两组细胞在24、48及72 h生长抑制率的影响;流式细胞仪检测两组细胞的肿瘤干细胞标记物CD133、CD44表达差异。结果:SW620-Twist1组与SW620-NC组相比,Twsit1基因mRNA和蛋白显著升高,差异具有统计学意义(P0. 01),提示过表达Twist1基因的SW620细胞系构建成功; 5-FU和OXA两种药物在48、72 h时对细胞的抑制率均明显降低(48 h,P0. 05; 72 h,P0. 01); ABCB1、ABCG2基因mRNA及蛋白的表达量均明显升高(P0. 01); CD133及CD44表达量均显著增高(P0. 01)。结论:过表达Twist1可促进直肠癌SW620细胞耐药指标ABCB1、ABCG2表达,使肿瘤细胞获得MDR,该现象可能与促进干性获得有关。     

DIS3表达调控对人骨髓瘤细胞集落形成及内皮细胞成管能力的影响

目的:探究DIS3表达调控对人骨髓瘤细胞集落形成及内皮细胞成管能力的影响及相关机制。方法:选取人骨髓瘤细胞株NCI-H929、RPMI-8226和U266为研究对象,分别设计并构建DIS3基因过表达载体和DIS3-siRNA。3种细胞实验均分成5组:对照(control)组、siRNA阴性对照(siRNA-NC)组、siRNA-DIS3组、空载体(empty vector)组以及DIS3过表达(over-DIS3)组。平板集落形成实验检测各组细胞的集落形成能力;Western blot检测缺氧诱导因子1α(HIF-1α)和HIF-3α蛋白表达的变化;RT-qPCR及Western blot检测血管形成相关基因Ang1、Ang2及VEGF-A在mRNA和蛋白水平上的表达;Matrigel法检测各组细胞培养上清液对人脐静脉内皮细胞(HUVECs)管形结构生成能力的影响。结果:下述检测指标的变化趋势在NCI-H929、RPMI-8226和U266 3种细胞中存在相似性:与空载体组比较,DIS3过表达组的细胞集落形成能力被显著抑制(P0.05);而与siRNA-NC组比较,siRNA-DIS3显著增强细胞的集落形成能力(P0.01)。DIS3过表达降低了HIF-1α和HIF-3α蛋白的表达(P0.05),敲减DIS3表达则显著促进了HIF-1α和HIF-3α蛋白的表达(P0.05)。此外,DIS3过表达显著降低了Ang1和Ang2及VEGF-A的mRNA和蛋白水平(P0.05),而siRNA-DIS3则显著促进了Ang1和Ang2及VEGF-A的表达(P0.05)。与空载体组比较,DIS3过表达组细胞培养上清液显著抑制了HUVECs的成管能力(P0.01);而与siRNA-NC组比较,siRNA-DIS3组细胞培养上清液则显著提高了HUVECs的成管能力(P0.05)。结论:DIS3过表达能显著抑制人骨髓瘤细胞的集落形成能力及HUVECs的成管能力,这可能与其对HIF-1α和HIF-3α蛋白表达的调控密切相关。     

集缩素NCAPG对非小细胞肺癌增殖和凋亡的影响及其可能机制

目的 探讨非小细胞肺癌细胞中集缩素NCAPG表达对A549与H1299细胞增殖、凋亡的影响及其机制.方法 构建shRNA-NCAPG慢病毒载体转染A549及NCI-H1299细胞,实时定量PCR法(qRT-PCR)及免疫印迹法(Western blot)验证两种细胞中NCAPG表达情况.应用CCK-8方法检测对照组及下...     

EFNB2对膀胱癌细胞增殖、侵袭及迁移的影响及其机制

目的 研究EFNB2对膀胱癌细胞增殖、凋亡、侵袭和迁移等生物学功能的影响，探讨其作为免疫治疗的可能性。方法 以膀胱癌细胞系T24、5637、J82和UM-UC-3细胞为研究对象，选用人膀胱上皮永生化细胞系SV-HUC-1细胞作为阴性对照。通过慢病毒载体转染shRNA-EFNB2进行敲低实验，然后分别采用qRT-PCR和Western blot检测细胞中EFNB2的mRNA和蛋白表达水平；CCK8和克隆形成实验检测细胞增殖活力；流式细胞术检测细胞周期和细胞凋亡率；Transwell和细胞划痕实验检测细胞侵袭和迁移能力；Western blot检测Bcl2、Bax、Vimentin和E-cadherin的蛋白表达水平。结果 与人膀胱上皮永生化细胞系SV-HUC-1细胞相比，膀胱癌细胞系中EFNB2的表达水平显著增高,其中以T24细胞表达最高(P<0.01)。敲低T24细胞系EFNB2基因表达后，T24细胞增殖活力显著下降(P<0.05)；细胞周期虽然尚未有显著变化，但细胞凋亡率显著增高(P<0.05)，并且抗凋亡蛋白Bcl2表达显著下降（P<0.05）,促凋亡蛋白B...     

miR-330抑制乳腺癌细胞增殖及其临床病理学意义

目的探讨miR-330对乳腺癌细胞增殖功能的影响,验证miR-330调节HER-2基因的表达,分析乳腺癌患者组织中miR-330表达与临床病理特征的关系。方法采用细胞计数、MTT、软琼脂克隆形成实验验证miR-330对乳腺癌细胞增殖功能的影响;Western blot、qRT-PCR实验验证miR-330调节HER-2基因的表达;qRT-PCR法检测临床48例乳腺癌组织样本miR-330的表达水平,分析其与各临床病理特征的关系。结果细胞计数实验、MTT实验、软琼脂克隆形成实验证明miR-330抑制乳腺癌细胞BT474的生长、增殖和软琼脂克隆形成;Western blot、qRT-PCR实验证明miR-330能够显著抑制HER-2基因的表达;乳腺癌组织中miR-330的低表达与肿瘤直径(P=0.004)、HER-2表达水平(P=0.010)具有显著相关性,与其他病理参数包括患者年龄、淋巴结转移、组织学分级、临床分期、ER、PR水平等无显著相关性(P0.05)。结论 miR-330能抑制乳腺癌增殖、生长,乳腺癌组织中miR-330的表达水平与肿瘤大小、HER-2表达呈明显负相关,miR-330抑制HER-2基因的表达,HER-2很可能介导miR-330对乳腺癌细胞的抑制作用。     

miR-186介导YAP1调控乳腺癌细胞增殖、迁移和侵袭

目的 探讨miR-186介导的YAP1对乳腺癌细胞增殖、迁移和侵袭的影响。方法 采用qRT-PCR和Western blot法检测miR-186、YAP1蛋白在正常乳腺细胞MDA-kb2及人乳腺癌细胞MDA-MB-231中的表达；利用Lipofectamine 2000试剂将miR-186 mimic转染至乳腺癌细胞，荧光显微镜下观察转染效率；采用CCK-8法检测乳腺癌细胞的增殖能力；细胞划痕实验检测细胞的迁移能力；Transwell细胞侵袭实验检测细胞的侵袭能力；Western blot法检测YAP1蛋白表达。双荧光素酶报告基因实验检测miR-186与YAP1的靶向关系。结果 与正常乳腺细胞相比，乳腺癌细胞中miR-186表达量显著减少(P<0.01),YAP1蛋白表达量显著增加(P<0.01)。与对照组相比，miR-186过表达可明显降低乳腺癌细胞的增殖、迁移和侵袭能力(P<0.05),同时降低了YAP1蛋白表达(P<0.01)。miR-186和野生型YAP1载体共转染细胞的荧光素酶活性显著降低(P<0.01)。结论 miR-186在乳腺癌细胞中表达下...     

人肺癌NCI-H446细胞生成肿瘤干细胞球

目的 探索人肺癌NCI-H446细胞系干细胞球体的培养,并鉴定其生物学特性.方法 用无血清培养基培养人肺癌NCI-H446细胞得到肿瘤细胞球.将肿瘤细胞球传代扩增,并用含血清培养基培养促使其分化;四甲基偶氮唑盐(MTT)比色检测肿瘤球和普通NCI-H446细胞的增殖能力,并将肿瘤球和普通NCI-H446细胞分别植入裸鼠皮下,观察肿瘤形成;Transwell侵袭实验检测肿瘤球和普通NCI-H446细胞的侵袭能力的不同;流式细胞术检测CD133、CD44在肿瘤球和普通NCI-H446细胞中的表达,并筛选细胞表面标志物.结果 在无血清培养基中,人肺癌NCI-H446细胞可以形成少量的肿瘤细胞球,并显示很强的自我更新和增殖能力,在含血清环境中能够诱导肿瘤球分化而贴壁生长;在动物实验中,接种5×105个细胞时,肿瘤球细胞较普通NCI-H446细胞显示更强的致瘤能力;在侵袭实验中,肿瘤球细胞的侵袭能力高于普通NCI-H446细胞;干细胞标志物CD133及CD44在肿瘤球细胞的表达较普通NCI-H446细胞明显增高.结论 人肺癌细胞NCI-H446中存在癌干细胞,且可以通过无血清培养、分离和富集.     

Small-molecule inhibition of proteasome and silencing by vascular endothelial cell growth factor-specific siRNA induce additive antitumor activity in multiple myeloma

Angiogenesis plays an important role in the pathogenesis and progression in multiple myeloma (MM), and MM cells secrete vascular endothelial growth factor (VEGF), which further promotes proliferation of the tumor cells. Therefore, we evaluated the anti-myeloma effect of VEGF small interfering RNA (siRNA) silencing in MM cells and whether it can be augmented by the additional application of bortezomib directed against the 26S proteasome. After transfection with VEGF siRNA, we observed a reduction of VEGF expression in all studied cell lines: OPM-2, RPMI-8226, INA-6, Jurkat, Raji, and Karpas-299, as well as in cells of MM and lymphoma patients. VEGF siRNA significantly induced apoptosis and inhibited proliferation in OPM-2 cells (P<0.0001), RPMI-8226 (P<0.0001), and INA-6 (P<0.01) versus controls. Cotreatment with VEGF siRNA and bortezomib in MM cells resulted in an exaggerated inhibition of proliferation and induction of apoptosis compared with VEGF siRNA or bortezomib alone (P<0.001). In addition, the combination of VEGF siRNA and bortezomib significantly (P<0.01) reversed multidrug resistance gene 1-dependent resistance of MM cells. Our data suggest that small-molecule inhibition of proteasome and silencing by VEGF-specific siRNA may be associated with an additive antitumor activity and might be a suitable target for new, therapeutic strategies using RNA interference in MM.     

Human myeloma cells and their strong stimulating capacity in ''one-way'' mixed lymphocyte reaction: a comparative study with leukaemic B lymphoid cells.

Cultured human myeloma cells (ARH-77, RPMI-8226 and U-266), like leukaemic B lymphoid cells, consistently exerted a strong stimulating capacity on allogeneic lymphocytes in the 'one-way' mixed lymphocyte reaction. An optimal stimulation was seen when a 1:1 ratio or 1:2 ratio of responding cell:stimulating cells of each cell line was utilized. The stimulating capacity of ARH-77 or RPMI-8226 cells was significantly diminished when a 1:4 ratio of responding cells:stimulating cells was utilized. Fresh bone marrow cells containing more than 80% plasma cells from a patient with multiple myeloma, on the other hand, failed to exert the stimulating capacity on two occasions. The striking difference between cultured myeloma cells and fresh plasma cells is that the Ia-like antigen is present on cultured myeloma cells, and this antigen is absent on fresh plasma cells. The relationship between the Ia-like antigen and the stimulating capacity in 'one-way' mixed lymphocyte reaction is discussed.     

1800 MHz长期电磁辐射对NIH/3T3细胞增殖能力的影响

目的探讨长期1800 MHz电磁辐射对NIH/3T3细胞增殖能力的影响。方法采用1800MHz电磁辐射,SAR值2 W/kg,间断暴露方式(5 min on/10 min off),5 h/d。对暴露60 d和138 d的细胞,进行平板克隆和软琼脂克隆实验,并采用ELISA法检测端粒酶的表达。结果平板克隆实验中,60 d暴露组细胞的克隆形成率显著低于假暴露组(P0.05),而138 d暴露组细胞的克隆形成率与假暴露组无明显差异。软琼脂克隆实验中,60 d暴露组和假暴露组细胞均未见克隆形成,而138 d暴露组克隆形成率显著高于假暴露细胞。端粒酶检测结果,60 d暴露组细胞的端粒酶表达量与假暴露组无显著差异,而138 d暴露组细胞的端粒酶表达量显著高于假暴露组(P0.05)。结论 1800 MHz电磁辐射长期暴露至60 d,有损NIH/3T3细胞的增殖能力,而更长期的暴露后(138 d)细胞转化增殖能力增强。     

不同IL-15转染对NCI-H446 HLA-Ⅰ和HLA-Ⅱ表达及NK敏感性的影响

目的探讨不同形式IL-15基因转染对人小细胞肺癌细胞株NCI-H446细胞HLA的Ⅰ类和Ⅱ类分子表达及NK杀伤敏感性的影响。方法在本室已构建的3种细胞模型（转染IL-15成熟肽基因的NCI-H446／IL-15mp细胞、转染原型IL-15基因的NCI-H446／IL-15细胞和转染以IL-2信号肽替代IL-15信号肽的改型IL-15基因的NCI-H446／IL-2sp-IL-15mp细胞）基础上,以野生株细胞为对照,流式细胞分析仪（Fluorescence-activated cell Sorter,FACS）分析这3种细胞HLA的Ⅰ类和Ⅱ类分子表达,四甲基偶氮唑蓝（MTT）法测定它们对NK杀伤的敏感性。结果野生株NCI-446细胞表面阳性表达HLA的Ⅰ类和Ⅱ类分子;转染IL-15成熟肽基因或改型IL-15基因后,这两种分子的表达均被抑制为阴性;转染原型IL-15基因后,这两种分子的表达均被显著上调。5名不同健康志愿者的外周血单个核细胞（PBMC）对野生株NCI-H446细胞的平均NK杀伤率最低,对NCI-H446／IL-15、NCI-H446／IL-15mp和NCI-H446／IL-2sp-IL-15mp细胞的平均NK杀伤率依次升高。结论转染不同形式IL-15基因对NCI-H446细胞的免疫生物学特性影响不同。转染IL-15成熟肽基因或改型IL-15基因,抑制NCI-H446细胞表面HLA的Ⅰ类和Ⅱ类分子表达;转染原型IL-15基因,上调这两种分子的表达;转染不同形式IL-15基因均可增强NCI-H446细胞对NK杀伤的敏感性,但程度不同;以改型IL-15者最高,IL-15成熟肽者次之,原型IL-15者最低。     

Dysregulation of the Receptor Activator of NF-κB Ligand and Osteoprotegerin Production Influence the Apoptosis of Multiple Myeloma Patients’ Bone Marrow Stromal Cells Co-Cultured with Myeloma Cells

The interaction of multiple myeloma (MM) cells and bone marrow stromal cells (BMSCs) induces profound changes in the bone marrow environment, influencing osteoclastogenesis and MM cell survival. Differences in receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) production in BMSCs derived from MM patients and control subjects and the apoptosis of BMSCs and MM cells in co-cultures of both cell types were examined. RANKL and OPG expressions were examined by ELISA and semiquantitative RT-PCR. Apoptosis of BMSCs after contact with RPMI8226 and U266 cells was measured by flow cytometry and the level of ALP activity by the spectrophotometric method. OPG production by BMSCs was significantly inhibited after direct contact with RPMI8226 cells. Production of soluble RANKL was enhanced and the increase was more significant in the BMSCs of the MM patients than in those of the controls. In co-cultures of BMSCs and MM cells, significant apoptosis was detected with a concomitant decrease in ALP activity. This apoptosis decreased significantly in the presence of RANK-Fc, an antagonist of RANKL. Disturbances in the RANKL/OPG system are more profound in the BMSCs of MM patients than in those of control subjects after direct contact with RPMI8226 cells. Moreover, direct contact with RPMI8226 and U266 cells induces apoptosis of BMSCs which is mediated by an overproduction of RANKL.     

miR-182 contributes to cell adhesion-mediated drug resistance in multiple myeloma via targeting PDCD4

miR-182 is a well-described oncogenic miRNA playing a crucial role in the development of many malignancies. However, the role of miR-182 in multiple myeloma (MM) remains unclear. Here, we demonstrate that adhesion of H929 and MM.1S cells to fibronectin could induce miR-182 expression and decrease PDCD4 expression. Furthermore, miR-182 was found to negatively regulate PDCD4 expression in H929 and MM.1S cells. In addition, PDCD4 down-regulation was required for cell adhesion-mediated drug resistance (CAM-DR). Intriguingly, miR-182 up-regulation could promote CAM-DR in H929 and MM.1S cells. Moreover, miR-182 up-regulation and PDCD4 down-regulation enhanced AKT phosphorylation at Ser473 in both H929 and MM.1S cells. Our data suggest that cell adhesion-mediated miR-182 up-regulation and PDCD4 down-regulation may confer drug resistance via enhancing AKT phosphorylation at Ser473.