姜黄素对骨髓瘤细胞迁移和侵袭能力的影响及其机制研究

 目的：探讨姜黄素对骨髓瘤细胞迁移侵袭能力的影响及其机制。方法：shRNA表达质粒沉默含IQ模序的RasGTP酶活化蛋白1(IQ motif-containing GTPase-activating protein 1,IQGAP1)后转染RPMI8226细胞,Western blotting法检测RPMI8226-shIQGAP1组、RPMI8226-shRNA阴性对照组和未转染RPMI8226组细胞IQGAP1表达;不同浓度姜黄素作用于各组细胞后,Transwell迁移实验和Matrigel侵袭实验检测细胞的迁移及侵袭力,RT-PCR法检测姜黄素作用后IQGAP1 mRNA的表达,Western blotting检测姜黄素对各组细胞IQGAP1蛋白表达的影响。结果：使用shRNA表达质粒沉默IQGAP1后,RPMI8226细胞IQGAP1表达减少;RPMI8226-shIQGAP1组较RPMI8226-shRNA 阴性对照组和未转染RPMI8226 组细胞迁移及侵袭力下降,姜黄素可降低RPMI8226-shRNA 阴性对照组和未转染RPMI8226组细胞迁移及侵袭力,无浓度相关性,不同浓度的姜黄素对RPMI8226-shIQGAP1组作用后细胞的迁移及侵袭力无明显变化。对于RPMI8226-shRNA阴性对照组及未转染RPMI8226组,姜黄素作用后IQGAP1 mRNA及蛋白的表达下降。结论：姜黄素通过抑制IQGAP1的表达降低骨髓瘤细胞的迁移力和侵袭力。     

丙戊酸钠对RPMI8226和U266细胞增殖及IL-6/JAK/STAT信号通路的影响

 目的：探讨丙戊酸钠(valproic acid sodium, VPA)对人骨髓瘤RPMI8226和U266细胞增殖及IL-6/JAK/STAT信号通路的作用。方法： RPMI8226和U266细胞经不同浓度VPA处理12及24 h后进行实验,MTT法检测VPA对细胞增殖的影响,流式细胞术检测VPA处理后的细胞周期及凋亡率,ELISA法检测培养细胞上清IL-6的浓度,半定量RT-PCR检测VPA作用后RPMI8226和U266细胞中STAT3、STAT5、Bcl-xL、Mcl-1、c-Myc、CCND1和VEGF mRNA表达水平。Western blotting检测JAK2和STAT5总蛋白及磷酸化蛋白表达水平。结果：(1) 10、20及40 μmol/L VPA处理RPMI8226和U266细胞12 h及24 h后,细胞存活率值显著下降,与各对照组比较均有显著差异（P<0.05）,各对照组之间无显著差异。(2) VPA处理RPMI8226和U266细胞 24 h后,G1/G0期细胞增多,S期细胞减少,细胞凋亡率增加,且凋亡率与VPA的作用剂量相关。(3) VPA处理RPMI8226和U266细胞24 h后,上清IL-6浓度与VPA浓度呈负相关。(4) RPMI8226和U266细胞组在40 μmol/L VPA处理后其STAT3、STAT5、Bcl-xL、Mcl-1、c-Myc、CCND1和VEGF mRNA表达减低,与未处理组比较有显著差异。(5) 与对照组比较,VPA处理组p-JAK2、JAK2、p-STAT5和STAT5蛋白表达均显著降低。结论：(1) VPA对多发性骨髓瘤细胞有体外抑制作用; (2) VPA对骨髓瘤细胞株的体外抑制可能是通过调节IL-6/JAK/STAT信号通路来实现的。     

褐藻糖胶对RPMI8226细胞血管生成的影响

 目的: 探讨褐藻糖胶对多发性骨髓瘤RPMI 8226细胞血管生成的影响及可能的作用机制。方法: 体外培养多发性骨髓瘤RPMI 8226细胞及人血管内皮细胞EA.hy 926细胞,采用MTT法检测褐藻糖胶对RPMI 8226细胞活力的影响;流式细胞术检测细胞周期及凋亡率;ELISA法检测褐藻糖胶处理RPMI 8226细胞后的培养液上清中VEGF的含量;小管形成实验观察褐藻糖胶处理RPMI 8226细胞后的培养液上清对EA.hy 926细胞诱导小管形成能力的影响;Western blot检测HIF-1α、VEGF、p-AKT和p-ERK1/2的蛋白水平。结果: 褐藻糖胶对RPMI 8226细胞活力的抑制作用呈浓度及时间依赖性;褐藻糖胶作用RPMI 8226细胞72 h后,细胞周期被阻滞于G₁期,细胞凋亡率呈浓度依赖性明显增高,各组均高于对照组(P<0.05);ELISA法结果显示褐藻糖胶处理72 h后的细胞培养液上清中VEGF的含量明显减少,与对照组相比,处理组上清中VEGF的含量下降(P<0.05);内皮细胞形成小管数目与面积随着褐藻糖胶的浓度升高而减小,100 mg/L 组差异显著(P<0.05);Western blot结果显示HIF-1α、VEGF、p-AKT和p-ERK1/2的蛋白水平与褐藻糖胶浓度呈负相关(P<0.05)。结论: 褐藻糖胶减少骨髓瘤细胞自分泌VEGF,减弱血管内皮细胞形成小管的能力,降低HIF-1α和VEGF的蛋白水平,这可能与抑制AKT和ERK1/2的磷酸化有关。     

Jagged1调控STAT3信号通路影响多发性骨髓瘤细胞的生长和凋亡

目的:研究Jagged1对多发性骨髓瘤细胞生长和凋亡的影响及其机制。方法:多发性骨髓瘤细胞U266转染Jagged1小干扰RNA和小干扰RNA阴性对照,RT-q PCR和Western blot检测细胞中的Jagged1水平,以不做转染的细胞为空白对照,台盼蓝染色,绘制细胞生长曲线,MTT检测细胞活力,流式细胞术检测细胞凋亡,Western blot检测细胞中信号转导及转录激活因子3(STAT3)、磷酸化STAT3(p-STAT3)和Bax的蛋白表达水平。用STAT3信号通路抑制剂AG490处理细胞,检测细胞中p-STAT3水平。AG490处理下调Jagged1表达后的U266细胞,MTT检测细胞活力,流式细胞术检测细胞凋亡。结果:与空白对照组细胞相比,转染Jagged1小干扰RNA后细胞中Jagged1的mRNA和蛋白水平下降(P0.05),而转染小干扰RNA阴性对照的细胞中Jagged1的mRNA和蛋白水平没有变化。与不做转染的细胞相比,敲减Jagged1表达的U266细胞生长速度降低,细胞活力降低,细胞凋亡率升高(P0.05)。与不做转染的细胞相比,敲减Jagged1表达的U266细胞中Bax水平升高,p-STAT3水平下降(P0.05)。AG490处理后的U266细胞p-STAT3水平下降,STAT3信号通路激活受阻。AG490可以促进下调Jagged1诱导的U266细胞凋亡,抑制细胞活力。结论:敲减Jagged1表达通过抑制STAT3信号通路促进多发性骨髓瘤细胞凋亡,抑制细胞生长。     

重组人血管内皮抑素对人多发性骨髓瘤细胞RPMI 8226增殖和相关蛋白表达的影响

 目的： 研究重组人血管内皮抑素（恩度）在体外对人多发性骨髓瘤细胞株RPMI 8226细胞增殖、细胞周期及细胞相关蛋白表达的影响。方法： 用CCK-8检测恩度对RPMI 8226细胞增殖的影响;流式细胞术检测凋亡和细胞周期的改变;Western blotting检测凋亡相关蛋白Bcl-2和caspase-3表达变化;以实时定量PCR和Western blotting检测RPMI 8226细胞血管细胞粘附因子 1（VCAM-1）、白细胞介素 6（IL-6）和血管内皮生长因子（VEGF）mRNA以及蛋白表达变化;ELISA检测细胞上清液中细胞因子IL-6和VEGF水平。结果： 恩度对RPMI 8226细胞有增殖抑制作用,250 mg/L恩度对RPMI 8226细胞 72 h增殖抑制率为（59.5±5.6）%（P<0.05）,细胞G1期比例增加（P<0.05）,但对细胞凋亡及凋亡相关蛋白Bcl-2和caspase-3表达无显著影响;经恩度作用后RPMI 8226细胞 VCAM-1表达下降（P<0.05）,IL-6和VEGF表达及分泌降低（均P<0.05）。结论： 恩度能通过改变细胞周期变化抑制RPMI 8226细胞增殖,并能减少VCAM-1表达及IL-6、VEGF分泌,对细胞凋亡无明显改变。     

用siRNA研究IQGAP1基因调控STAT3信号对脑胶质瘤细胞生物学特性的影响

目的:探讨敲减IQGAP1基因表达对脑胶质瘤细胞侵袭、迁移及免疫抑制的影响及机制。方法:将人脑胶质瘤U251细胞随机分为空白组、阴性对照组和si-IQGAP1组,AG490作为STAT3信号通路抑制剂。转染48 h后,MTT法检测细胞活力;Western blot检测IQGAP1、血管内皮生长因子(VEGF)、转化生长因子β1(TGF-β1)、STAT3和p-STAT3的蛋白水平;Transwell小室检测细胞的侵袭和迁移能力。结果:si-IQGAP1-1组和si-IQGAP1-2组的IQGAP1蛋白表达均显著低于空白组(P0.05)。与空白组比较,si-IQGAP1组和AG490组的细胞活力、侵袭能力和迁移能力均显著降低,VEGF、TGF-β1和p-STAT3的蛋白水平均显著降低(P0.05)。与AG490组比较,AG490+si-IQGAP1组的细胞活力、侵袭能力和迁移能力均显著降低,VEGF和TGF-β1的蛋白表达均显著降低(P0.05)。结论:敲减IQGAP1基因表达可降低脑胶质瘤细胞的侵袭和迁移能力,减弱细胞免疫抑制因子VEGF和TGF-β1的表达,机制与下调STAT3信号通路有关。     

Twist1在结直肠癌多药耐药中的作用机制

目的探讨Twist1在结直肠癌多药耐药中的作用机制。方法建立人结直肠癌耐奥沙利铂细胞株(SW620/OxR),构建带G418抗性的Twist1-shRNA质粒转染SW620/OxR细胞作为实验组,同时,设置转染带G418抗性阴性对照质粒的SW620/OxR细胞作为阴性对照组,并在转染成功后用G418筛选建立稳定转染细胞株。采用实时荧光定量PCR和Western blot法检测SW620/OxR细胞中Twist1基因和蛋白的表达,采用流式细胞术间接法检测细胞中Twist1、p-STAT3、P-gp、Survivin蛋白变化,Western blot法检测两组细胞Twistl、STAT3、p-STAT3蛋白表达变化。结果 Twist1-shRNA干扰质粒能够明显降低SW620/OxR细胞Twist1 mRNA及蛋白水平。且实验组的E-cadherin表达水平明显降低,vimentin表达水平明显升高。采用流式细胞术检测肿瘤细胞凋亡,阴性对照组细胞凋亡率为5.08%,实验组则明显增高至30.69%。流式细胞术间接法检测实验组中Pgp、Survivin、p-STAT3及Twist1阳性率分别为5.25%、1.93%、2.51%、3.58%,阴性对照组中P-gp、Survivin、p-STAT3及Twist1阳性率分别增加至21.98%、22.36%、19.42%、53.68%,二者差异有统计学意义(P0.01)。实验组Twist1、STAT3、p-STAT3蛋白相对表达量明显低于阴性对照组,差异有统计学意义(P0.01)。结论 Twist1可能是结直肠癌多药耐药机制中的关键因子,其可能通过逆向调控STAT3信号通路发挥作用。     

HMGCS1通过MAPK通路调控多发性骨髓瘤细胞增殖和药物敏感性研究

目的 探讨HMGCS1对于多发性骨髓瘤U-266、RPMI8226细胞株增殖及药物敏感性的影响，并进一步研究其作用机制。方法 实时荧光定量PCR(q-PCR)检测33例多发性骨髓瘤患者及33例健康供者骨髓标本HMGCS1的相对表达水平。构建U-266细胞过表达组：mimics-control(空白对照组)、mimics-NC(阴性对照组)、mimics-HMGCS1(HMGCS1过表达组);构建RPMI8226细胞敲低组：inhibitor-control(空白对照组)、inhibitor-NC(阴性对照组)、inhibitor-HMGCS1(HMGCS1敲低组),实时荧光定量PCR(q-PCR)及Western blot检测感染效果，CCK8法检测细胞的增殖能力及对硼替佐米的药物敏感性，Western blot检测HMGCS1对MAPK信号通路中MEK、p-MEK、ERK、pERK蛋白表达水平的影响。结果 多发性骨髓瘤骨髓标本中HMGCS1 mRNA表达水平较健康供者显著升高(P<0.05)。mimics-HMGCS1组中HMGCS1蛋白及mRNA表达量较mimics-NC组、...     

干扰HDAC1通过调控STAT3信号通路影响皮肤鳞癌细胞凋亡

目的:探讨干扰组蛋白去乙酰化酶1(HDAC1)对皮肤鳞癌细胞凋亡的影响。方法:皮肤鳞癌细胞A431分别转染HDAC1小干扰RNA(HDAC1 si RNA)和小干扰RNA阴性对照(si RNA NC),RT-PCR和Western blot检测转染后细胞中HDAC1的表达水平,MTT检测细胞活力,流式细胞术检测细胞凋亡,Western blot检测信号转导及转录激活因子3(STAT3)、磷酸化STAT3(p-STAT3)和cleaved caspase-3的蛋白水平。同时用STAT3信号通路抑制剂作用于转染HDAC1 si RNA的A431细胞,MTT法检测细胞活力,流式细胞术检测细胞凋亡,Western blot检测STAT3、p-STAT3和cleaved caspase-3的蛋白水平。结果:HDAC1 si RNA能够抑制A431细胞中HDAC1的m RNA和蛋白表达。干扰HDAC1表达后细胞活力和细胞中p-STAT3水平下降,而细胞凋亡率和细胞中cleaved caspase-3水平升高。STAT3信号通路抑制剂作用后,转染HDAC1 si RNA的A431细胞活力及p-STAT3水平下降,细胞凋亡率及细胞中cleaved caspase-3水平升高。结论:干扰HDAC1表达可能通过调控STAT3信号通路抑制皮肤鳞癌细胞活力,促进皮肤鳞癌细胞凋亡。     

TCRVβ7.1基因转染对肝癌细胞刺激的外周血单个核细胞ERK信号通路和IFN-γ表达的影响

目的： 研究肝癌特异性识别基因TCRVβ7.1表达重组体转染外周血单个核细胞(PBMCs)后对T细胞细胞因子表达的影响和信号通路的激活作用。方法: 以肝癌特异性T细胞受体Vβ7.1转染PBMCs,流式细胞仪检测TCRVβ7.1表达量;Western blotting检测ERK1/2表达量及磷酸化水平（p-ERK）的改变;用ELISA法检测白细胞介素-4（IL-4）、γ-干扰素（IFN-γ）的表达量。结果: TCRVβ7.1基因转染健康人PBMCs并得到有效表达,转染TCRVβ7.1基因的PBMCs 与肝癌细胞共培养后ERK蛋白磷酸化水平明显高于未转染组(P<0.01)。p-ERK1/2水平与T细胞激活有关。ELISA结果表明,转染PBMCs细胞与肝癌细胞共培养后,IFN-γ水平明显高于未转染组,而IL-4无明显改变。结论: TCRVβ7.1转染PBMCs与肝癌细胞共培养后,ERK信号通路被激活,IFN-γ表达增高。     

Targeting the IL-6 pathway in multiple myeloma and its implications in cancer-associated gene hypermethylation

Aberrant methylation of tumor suppressor genes (TSG) is an important epigenetic event in cancer, including multiple myeloma (MM). Interleukin-6 (IL-6), which plays a significant role in the pathogenesis of MM, also regulates DNA methylation. However, attempts to bring IL-6 blockade to the clinic have had limited success. We hypothesize that IL-6 regulation of hypermethylation may be an important pathway leading to rational chemotherapeutic/anti-IL-6 combinations. We first studied the correlation of IL-6 expression and dependence in MM cell lines: U266B1, RPMI8226, and KAS6/1. We confirmed that KAS6/1 is IL-6-dependent whereas U266B1 and RPMI8226 cells are IL-6-independent and that blocking IL-6 inhibited the growth of U266B1 (36% inhibition; p<0.05) and KAS6/1 (68% inhibition; p<0.01), but not the RPMI8226 cells. Using RT-PCR, we showed that U266B1 cells express IL-6, but RPMI8226 and KAS6/1 cells do not. This IL-6 expression pattern correlates with the anti-IL-6 inhibition findings. To correlate IL-6 sensitivity with hypermethylation of TSG, we investigated promoter methylation of CDH1 and DcR1. We found that the promoter of DcR1 and CDH1 is methylated in U266B1 cells and un-methylated in RPMI8226 cells. Furthermore, the DcR1 promoter was un-methylated in KAS6/1 cells. These data support our hypothesis that an IL-6-dependent pathway may regulate hypermethylation of TSG in MM. Newer chemotherapeutic agents that affect methylation are being studied in combination with IL-6 blockade.     

染料木黄酮通过抑制VEGF表达抑制人口腔癌TCA8113细胞增殖

 目的: 观察染料木黄酮对人口腔癌TCA8113细胞增殖的影响,并探讨其作用机制。方法: MTT法、细胞计数法及集落形成实验检测细胞增殖;蛋白质免疫印迹检测血管内皮生长因子(VEGF)、细胞外信号调节激酶(ERK)及p-ERK的蛋白水平。结果: 染料木黄酮能显著抑制TCA8113细胞的增殖,其抗增殖活性具有浓度依赖性;染料木黄酮还可剂量依赖性地降低VEGF、ERK及p-ERK的蛋白水平;VEGF的表达可被ERK特异性抑制剂U0126所抑制;VEGF受体拮抗剂阿西替尼及U0126均显著抑制TCA8113细胞的增殖。结论: 染料木黄酮可抑制人口腔癌TCA8113细胞的增殖,其机制可能与其抑制ERK表达及活化,进而抑制VEGF的表达有关。     

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.     

miRNA-181a对多发性骨髓瘤细胞增殖和迁移的作用

目的:通过沉默人多发性骨髓瘤细胞株RPM18226中miRNA-181a的表达,观察RPM18226细胞的增殖、迁移和细胞周期的变化,并探讨其可能的作用机制。方法:实时荧光定量PCR检测多发性骨髓瘤患者和健康者血清标本中miRNA-181a的表达水平;转染miRNA-181a inhibitor后,CCK-8法与集落形成实验检测细胞的增殖能力,划痕实验检测细胞的迁移能力,流式细胞术检测细胞的周期变化,Western blot法检测cyclin D1、p-PI3K和pAkt蛋白水平的变化。结果:多发性骨髓瘤患者血清中miRNA-181a呈高表达,显著高于正常人;转染miRNA-181a inhibitor后,RPM18226细胞的存活率和集落形成能力下降,迁移能力降低,G_0/G_1期细胞比例明显减少,S期细胞比例增多,cyclin D1蛋白表达显著低于正常对照组,PI3K和Akt的磷酸化水平明显低于正常对照组。结论:沉默miRNA-181a的表达能够抑制RPM18226细胞的增殖,并降低细胞的迁移能力,可能与细胞周期及PI3K/Akt信号通路有关。     

Streptolysin-O reversible permeabilisation is an effective method to transfect siRNAs into myeloma cells

RNA interference (RNAi) has been shown to be a valuable tool to specifically target gene expression in a number of organisms becoming an indispensable weapon in the arsenal in functional genomics. In this study, we demonstrate that streptolysin-O (SLO) reversible permeabilisation is an efficient method to deliver small interfering RNAs (siRNAs) to hard-to-transfect human myeloma cell lines. We used published, pre-validated siRNAs for ERK2 and non-silencing siRNA control. We transfected siRNAs into human myeloma cell lines using SLO reversible permeabilisation method. Flow cytometry and western blot analysis were performed to assess the effect of SLO on transfection efficiency and ERK2 knockdown. These experiments demonstrate that SLO reversible permeabilisation method is an efficient and easy-to-use method to deliver siRNAs into human myeloma cell lines. Optimised SLO permeabilisation method showed to transfect >80% of JIM-3, H929, RPMI8226 and U266 cells, with minimal effect on cell viability (<10%) and cell cycle. Equally important, SLO permeabilisation induced a substantial knockdown of ERK2 at the protein level. These studies demonstrate that reversible SLO permeabilisation can successfully be applied to hard-to-transfect human myeloma cell lines to effectively silence genes.