白藜芦醇通过拮抗hPXR对P-gp的影响

目的研究白藜芦醇通过拮抗hPXR对P-gp基因(MDR1)、蛋白表达及活性的影响。方法在LS174T细胞中,采用瞬时共转染报告基因实验研究白藜芦醇对PXR介导的MDR1的转录调节作用,并进一步应用Real-Time定量PCR和Western blot方法检测白藜芦醇作用24 h后对利福平诱导的P-gp基因和蛋白变化的影响,罗丹明转运实验考察P-gp活性的变化。结果双荧光素酶报告基因检测结果显示,25和50μmol.L-1白藜芦醇可通过拮抗PXR将利福平对MDR1的诱导作用由4.70倍分别降至1.76倍和0.69倍(P<0.01),在过表达hPXR的LS174T细胞中,50μmol.L-1白藜芦醇可以将利福平诱导的MDR1 mRNA水平由1.8倍降至1.3倍(P<0.05),Western blot结果表明白藜芦醇也可降低利福平诱导的P-gp表达。此外,罗丹明转运实验显示,25和50μmol.L-1白藜芦醇可以将利福平抑制的累积量由77.7%升至91.7%和95.1%(P<0.05),表明白藜芦醇可降低利福平诱导的P-gp活性。结论白藜芦醇可以通过拮抗PXR而影响P-gp的基因、蛋白表达及活性。     

Sp1反义寡核苷酸抑制Jurkat T细胞hTERT表达而抑制端粒酶活性(英文)

目的:研究转录因子Sp1反义寡核苷酸(ODN)对Jurkat T细胞端粒酶活性和端粒酶催化亚基hTERT表达的影响。方法:设计Sp1反义ODN并用脂质体转染细胞,用端粒酶PCR-ELISA方法检测端粒酶活性;用逆转录PCR方法检测Sp1和hTERT mRNA水平,用Western blot检测蛋白水平。结果:sp1反义ODN(1μmol/L)明显抑制Jurkat T细胞Sp1 mRNA和蛋白表达,抑制率分别为44.8％(P<0.05)和57％(P<0.01),并抑制hTERT mRNA表达,抑制率为43.7％(P<0.01);当浓度从0.25到2.0μmol/L,Sp1反义ODN对端粒酶活性抑制率从27.1％到64.6％,呈明显的剂量依赖性关系。结论:Sp1反义寡核苷酸通过抑制hTERT mRNA表达而抑制端粒酶活性。     

人参皂苷F1通过激活孕烷X受体诱导CYP3A4的表达

目的探索人参皂苷F1(ginsenoside F1)是否通过激活孕烷X受体(PXR)实现对CYP3A4基因表达及酶活性的诱导作用。方法利用本实验室构建的PXR-CYP3A4稳定转染Hep G2工程细胞株结合荧光素酶报告基因技术,检测人参皂苷F1对PXR的转录激活效应;用不同浓度的人参皂苷F1处理LS174T细胞,并通过Q-PCR和酶活性试剂盒检测CYP3A4的mRNA表达和酶活性变化。结果不同浓度人参皂苷F1作用于LS174T细胞以后,可以浓度依赖性地诱导CYP3A4 mRNA水平的表达,并且增强其酶活性;同时,PXR-CYP3A4稳定转染Hep G2工程细胞株结合荧光素酶报告基因检测结果亦表明,人参皂苷F1能够浓度依赖性地增强PXR的转录激活效应。结论本研究揭示了人参皂苷F1可诱导CYP3A4的基因表达并增强其酶活性,这一过程可能与人参皂苷F1对孕烷X受体的激活有关。     

山姜素激活孕烷X受体和上调CYP3A4 mRNA转录的研究

目的探讨山姜素能否通过活化孕烷X受体(PXR)诱导CYP3A4的转录表达及对CYP3A4 mRNA的实际诱导作用。方法在人结肠癌LS174T细胞中,用瞬时共转染报告基因实验研究山姜素(1～50μmol.L-1)对PXR介导的CYP3A4基因的转录激活;用荧光定量RT-PCR方法检测其对CYP3A4 mRNA的实际诱导。结果 10μmol.L-1浓度山姜素能够通过活化PXR诱导CYP3A4的转录(1.63倍);10和20μmol.L-1山姜素能够明显上调CYP3A4 mRNA(2.28倍和1.65倍)的表达。结论 PXR途径也许是山姜素诱导CYP3A4基因表达的调控因素之一。     

银杏内酯B通过激活孕烷X受体诱导CYP3A4的表达

目的研究银杏内酯B(ginkgolide B)对CYP3A4的诱导作用,进一步验证是否通过激活孕烷X受体实现对CYP3A4 mRNA和蛋白水平的诱导表达。方法用不同浓度银杏内酯B处理LS174T细胞,通过Q-PCR法检测CYP3A4 mRNA表达变化,进一步利用本实验室构建的PXRCYP3A4稳定转染HepG2工程细胞株,结合荧光素酶报告基因技术,显示检测银杏内酯B对PXR的转录激活活性的影响。蛋白质免疫印迹法检测CYP3A4蛋白水平表达的变化;利用siRNA技术敲低PXR的mRNA表达,检测在PXR低表达的条件下银杏内酯B对CYP3A4 mRNA和蛋白水平表达影响。结果银杏内酯B可以浓度依赖性的使CYP3A4基因及蛋白表达水平上调,报告基因结果显示,银杏内酯B能够浓度依赖性的增强PXR的转录激活活性,在PXR低表达的条件下,银杏内酯B对CYP3A4诱导作用明显低于正常表达组PXR。结论以上表明银杏内酯B通过激活PXR受体,促进其转录激活活性进而诱导CYP3A4表达上调,同时对PXR自身的表达无明显影响。     

多药耐药相关蛋白在乳腺癌患者外周血单个核细胞中的表达研究

目的检测健康人和乳腺癌患者外周血单个核细胞中多药耐药相关蛋白(multidrug resistance-associated protein,MRP)基因的表达水平,探讨MRP在乳腺癌患者中的可能作用。方法抽取20例健康人和50例乳腺癌患者外周血,运用ELISA法检测2组实验对象血清中肿瘤坏死因子α(TNF-α)、白介素-8(IL-8)、白介素6(IL-6)细胞因子的含量;提取外周血中单个核细胞(Peripheral blood monouclear cells,PBMCs),后用荧光实时定量PCR和Western blot技术检测BCRP和MRP2/3/4/5 mRNA和相应蛋白表达情况。结果 ELISA法检测乳腺癌患者外周血血清中TNF-α、IL-8、IL-6细胞因子的含量明显高于健康人对照组(P<0.05);荧光实时定量PCR检测BCRP和MRP5的mRNA表达水平高于健康人对照组(P<0.05),其余MRP没有显著性差别(P>0.05);Western blot检测蛋白表达与mRNA结果一致。结论乳腺癌患者外周血中的促炎因子含量的升高与BCRP和MRP5的表达调控有一定关系,具体相关机制有待进一步研究。     

贲门癌组织中多药耐药基因和多药耐药相关蛋白基因表达的临床研究

目的 了解多药耐药基因(MDR1)和多药耐药相关蛋白基因(MRP)在贲门癌组织中的表达及其临床意义。方法 采用逆转录-多聚酶链反应(RT-PCR)。观察46侧贲门癌及癌旁组织中MDR1和MRP的表达。结果 癌组织中MDR1和MRP表达的阳性率分别为63%和50%,均高于癌旁组织(P<0.05);贲门癌术前化疗者MDR1 mRNA和MRP mRNA表达水平显著高于未化疗者(P<0.05);中低分化肿瘤MDR1和MRP两基因mRNA表达水平高于高分化肿瘤(P<0.05)。结论 贲门癌组织中具有内源和获得性耐药性;MDR1和MRP表达与肿瘤的TNM分期无关,其高表达状态可预示肿瘤组织的分化不良。     

白英乙醇提取物诱导人肺癌SPC-A-1细胞凋亡的实验研究

目的:探讨白英乙醇提取物对人肺癌SPC-A-1细胞凋亡及凋亡相关基因fas和caspase-3表达的影响。方法:采用体外培养人肺癌SPC-A-1细胞,随机分为正常对照组、白英乙醇提取物处理组(浓度分别为2.5、5、10mg.L-1)和阳性对照组(顺铂);药物处理48h后,用MTT法检测细胞增殖抑制率,转移酶介导脱氧尿苷三磷酸缺口末端标记法检测细胞凋亡率,半定量反转录-聚合酶链反应检测fas和caspase-3mRNA表达水平。结果:与正常对照组比较,白英各浓度组细胞抑制率显著上升(P<0.001),细胞凋亡率明显升高(P<0.05或P<0.01),fas和caspase-3mRNA表达显著上升(P<0.05或P<0.01)。结论:白英乙醇提取物可能通过上调fas和caspase-3基因表达,诱导细胞凋亡,从而抑制SPC-A-1细胞增殖。     

瑞香狼毒水提取物对肺腺癌细胞株耐药及凋亡的影响

目的探讨瑞香狼毒水提取物对肺腺癌细胞株A549和耐药细胞株A549/DDP细胞凋亡率及耐药基因表达的影响。方法用不同浓度的瑞香狼毒水提取物、顺铂分别及联合处理人肺腺癌细胞株A549和A549/DDP,用MTT法检测肺癌细胞凋亡率、实时荧光定量PCR检测多药耐药(multidrug resistance protein1,MRP1)基因mRNA水平以及用Westernblot检测多药耐药蛋白1(multidrug resistance protein1,MRP1)的表达水平。结果不同浓度瑞香狼毒水提取物作用相同时间或相同浓度作用不同时间对A549和A549/DDP的细胞抑制率差异有统计学意义(P<0.05);瑞香狼毒处理前后A549/DDP细胞株与A549细胞株相比,对顺铂的敏感性差异有统计学意义(P<0.05);同时两细胞株与瑞香狼毒和顺铂联合孵育后MRP1mRNA水平及MRP1蛋白表达水平差异有统计学意义(P<0.05)。结论瑞香狼毒水提取物有明显抗肿瘤作用,能增强肺癌耐药细胞株对DDP的敏感性,对肺癌耐药具有一定的逆转作用。     

Hsa-miR-340对膀胱癌T24细胞增殖及凋亡的影响

目的探讨和研究hsa-miR-340表达上调对人膀胱癌T24细胞增殖、凋亡能力的影响。方法化学合成hsa-miR-340模拟物,瞬时转染膀胱癌T24细胞,以实时荧光定量聚合酶链反应(RT-PCR)检测hsamiR-340在膀胱癌T24细胞中相对表达量,确定能够转染成功并有较高的转染率后,分别通过四甲基偶氮唑蓝(MTT)法和流式细胞计数仪,分析转染hsa-miR-340模拟物前后对膀胱癌T24细胞的增殖及凋亡能力的影响。结果①hsa-miR-340模拟物可在膀胱癌T24细胞中表达,并以RT-PCR验证相对含量较高,差异有统计学意义(P<0.05)。②增殖实验中分别在48h和72h时把实验组与阴性对照组和空白对照组相对比,膀胱癌T24细胞生长增殖明显受抑制且差异有统计学意义(P<0.05),说明hsa-miR-340过表达后,能使T24细胞增殖能力受到有效抑制。③hsa-miR-340模拟物转染T24细胞48h后,以流式细胞仪检测T24细胞凋亡率,分别为实验组(11.37±0.41)%、阴性对照组(6.72±0.32)%、空白对照组(6.62±0.23)%,比较后差异有统计学意义(P<0.05),说明hsa-miR-340过表达使T24细胞的凋亡显著增加。结论 hsa-miR-340可能参与膀胱癌的进展。     

Breast cancer resistance protein BCRP/ABCG2 regulatory microRNAs (hsa-miR-328, -519c and -520h) and their differential expression in stem-like ABCG2+ cancer cells

Recent studies have shown that a number of microRNAs (miRNA or miR) may regulate human breast cancer resistance protein (BCRP/ABCG2), an important efflux transporter responsible for cellular drug disposition, whereas their effects on ABCG2 protein expression are not compared. In this study, we first identified a new proximal miRNA response element (MRE) for hsa-miR-519c within ABCG2 3′-untranslated region (3′UTR) through computational analyses. This miR-519c MRE site was confirmed using dual luciferase reporter assay and site-directed mutagenesis. Immunoblot analyses indicated that ABCG2 protein expression was significantly down-regulated in MCF-7/MX100 cells after transfection with hsa-miR-328- or -519c expression plasmids, and was markedly up-regulated in MCF-7 cells after transfection with miR-328 or -519c antagomir. However, ABCG2 protein expression was unchanged in MCF-7/MX100 cells after transfection with hsa-miR-520h expression plasmids, which was associated with undetectable miR-520h expression. Furthermore, ABCG2 mRNA degradation was accelerated dramatically in cells transfected with miR-519c expression plasmid, suggesting the involvement of mRNA degradation mechanism. Intervention of miR-328 or -519c signaling led to significant change in intracellular mitoxantrone accumulation, as determined by flow cytometry analyses. In addition, we separated RB143 human retinoblastoma cells into stem-like (ABCG2+) and non-stem-like (ABCG2−) populations through immunomagnetic selection, and found that miR-328, -519c and -520h levels were 9-, 15- and 3-fold lower in the ABCG2+ cells, respectively. Our data suggest that miR-519c and -328 have greater impact on ABCG2 expression than miR-520h in MCF-7 human breast cancer cells, and the presence of proximal miR-519c MRE explains the action of miR-519c on shortened ABCG2 3′UTR.     

miR-149抑制结直肠癌细胞迁移的分子机制研究

目的 探讨miR-149对结直肠癌（CRC）细胞增殖、侵袭、迁移及凋亡的影响及其作用机制。方法 实时荧 光定量PCR（q-PCR）方法检测miR-149在CRC细胞SW620、LS174T和人结肠上皮细胞FHC中的表达水平;荧光素酶 分析法验证 STAT3 与 miR-149 之间的靶向结合关系;q-PCR 和 Western blot 检测 miR-149 过表达对 CRC 细胞中 STAT3、p-STAT3 mRNA和蛋白表达的影响。将miR-149 mimics与STAT3过表达质粒单独或者共转染至CRC细胞 中,并分为miR-NC组、miR-149 mimics组、miR-149 mimics+pEGFP/STAT3组。采用CCK-8法、Transwell法、细胞划 痕法、流式细胞术分别检测各分组CRC细胞的增殖、侵袭、迁移和凋亡情况。结果 与正常结肠上皮细胞FHC相比, CRC 细胞 SW620、LS174T 中 miR-149 表达水平受到抑制（P＜0.01）。荧光素酶实验证实,在 CRC 细胞中 STAT3 是 miR-149的一个直接靶基因。过量表达miR-149抑制STAT3、p-STAT3 mRNA和蛋白的表达,降低CRC细胞增殖能 力、侵袭能力以及迁移能力（P＜0.01）。同时,过量表达 miR-149 也可促进 CRC 细胞的凋亡（P＜0.01）。但过表达 STAT3可解除miR-149对CRC细胞增殖、侵袭以及迁移能力的抑制作用。结论 miR-149通过靶向STAT3抑制结直 肠癌细胞的增殖、侵袭与迁移,同时促进细胞的凋亡。     

MiR-24对3T3-L1脂肪细胞分化及脂肪型脂肪酸结合蛋白表达的调节作用

目的探讨microRNAs对脂肪细胞分化过程的调节作用分子机制及其对脂肪特异性基因——脂肪型脂肪酸结合蛋白(fatty acid binding protein,FABP4)表达的影响。方法用microRNAs微阵列分析法筛选和鉴定3T3-L1脂肪细胞分化相关性microRNAs,构建脂肪细胞分化相关性micro-RNAs的高表达质粒,通过脂质体介导转染3T3-L1前体脂肪细胞,观察脂肪相关性microRNAs对脂肪细胞分化过程的影响,Westernblot和RT-PCR分别检测FABP4蛋白及其mR-NA在脂肪细胞分化过程中的表达变化。结果3T3-L1脂肪细胞分化过程中microRNA表达谱发生明显改变,其中35个microRNAs下调,miR-24最明显;17个microRNAs上调,miR-21最明显;用不同脂肪相关性microRNAs转染3T3-L1前体脂肪细胞并高表达后,发现miR-24明显抑制脂肪细胞的分化与成熟,而miR-21则无影响;miR-24明显抑制FABP4蛋白表达,但对其mRNA无影响,miR-21对FABP4的蛋白和mRNA表达都没有影响。结论3T3-L1脂肪细胞分化过程中存在脂肪细胞分化相关性microRNAs;miR-24脂肪细胞分化过程及其脂肪特异蛋白FABP4表达有重要的调节作用。     

LncRNA GAS5靶向miR-103减轻3T3L1脂肪细胞胰岛素抵抗的作用机制 #br#

目的 探究长链非编码RNA（LncRNA）生长抑制特异因子5（GAS5）靶向微小RNA（miR）-103,从而减轻 3T3L1脂肪细胞胰岛素抵抗（IR）的机制。方法 培养并诱导分化3T3L1小鼠前脂肪细胞,油红O染色鉴定细胞分化 情况。建立3T3L1脂肪细胞IR模型。实验分为对照组、模型组、空载体组（转染pEGFP-C1空载体）、GAS5过表达组 （转染 pEGFP-C1-GAS5 载体）、GAS5 过表达+mimic NC 组（转染 pEGFP-C1-GAS5+mimic NC）、GAS5 过表达+miR- 103 mimic组（转染pEGFP-C1-GAS5+miR-103 mimic）。实时荧光定量PCR检测细胞中GAS5、miR-103 mRNA水平; 液体闪烁法检测葡萄糖摄取能力;蛋白质免疫印迹检测细胞中胰岛素受体底物-1（IRS-1）、p-IRS-1、过氧化物酶体 增殖物激活受体γ（PPARγ）、葡萄糖转运蛋白4（GLUT4）、蛋白激酶B（AKT）、p-AKT蛋白表达水平;双荧光素酶鉴定 miR-103与GAS5的靶向位点。结果 诱导后脂肪细胞呈圆形、胞体变大,胞浆丰富,含有大量脂滴,油红染色明显, 呈“指环样”结构,模型构建成功。与对照组比较,模型组、空载体组、GAS5过表达+miR-103 mimic 组细胞中GAS5 mRNA水平、葡萄糖摄取能力、p-IRS-1/IRS-1、PPARγ、GLUT4、p-AKT/AKT蛋白水平降低,细胞中miR-103 mRNA水 平升高（P＜0.05）;与模型组、空载体组比较,GAS5过表达组、GAS5过表达+mimic NC组细胞中GAS5 mRNA水平、葡萄糖摄取能力、p-IRS-1/IRS-1、PPARγ、GLUT4、p-AKT/AKT蛋白水平升高,而miR-103 mRNA水平降低（P＜0.05）; 与GAS5过表达组、GAS5过表达+mimic NC组比较,GAS5过表达+miR-103 mimic组细胞中GAS5 mRNA水平、葡萄糖 摄取能力、p-IRS-1/IRS-1、PPARγ、GLUT4、p-AKT/AKT 蛋白水平降低,miR-103 mRNA 水平升高（P＜0.05）。miR- 103与GAS5存在互补的结合位点并经双荧光素酶靶向关系验证。结论 过表达GAS5后靶向下调miR-103的表达 可减轻3T3L1脂肪细胞IR。     

Integrated mRNA and micro RNA profiling reveals epigenetic mechanism of differential sensitivity of Jurkat T cells to AgNPs and Ag ions

In our previous in vitro study of the toxicity on silver nanoparticles (AgNPs), we observed a dramatically higher sensitivity of Jurkat T cells to AgNPs than to Ag ions, and DNA damage and apoptosis were found to be involved in that toxicity. In this study, to understand underlying mechanism of different sensitivity of Jurket T cells to AgNPs and Ag ions, mRNA microarray and micro RNA microarray were concomitantly conducted on AgNPs and Ag ions exposed Jurkat T cells. Surprisingly only a small number of genes were differentially expressed by exposure to each of the silver (15 altered mRNA by AgNPs exposure, whereas 4 altered mRNA by Ag ions exposure, as determined 1.5-fold change as the cut-off value). miRNA microarray revealed that the expression of 63 miRNAs was altered by AgNPs exposure, whereas that of 32 miRNAs was altered by Ag ions exposure. An integrated analysis of mRNA and miRNA expression revealed that the expression of hsa-miR-219-5p, was negatively correlated with the expression of metallothionein 1F (MT1F) and tribbles homolog 3 (TRIB3), in cells exposed to AgNPs; whereas, the expression of hsa-miR-654-3p was negatively correlated with the expression of mRNA, endonuclease G-like 1 (EDGL1) in cells exposed to Ag ions. Network analysis were further conducted on mRNA-miRNA pairs, which revealed that miR-219-5p–MT1F and –TRIB3 pairs by AgNPs are being involved in various cellular processes, such as, oxidative stress, cell cycle and apoptosis, whereas, miR-654-3p and ENDOGL1 pair by Ag ions generated a much simpler network. The putative target genes of AgNPs-induced miR-504, miR-33 and miR-302 identified by Tarbase 6.0 are also found to be involved in DNA damage and apoptosis. These results collectively suggest that distinct epigenetic regulation may be an underlying mechanism of different sensitivity of Jurkat T cells to AgNPs and Ag ion. Further identification of putative target genes of DE miRNA by AgNPs and Ag ions may provide additional clues for the mechanism of differential toxicity. Overall results suggest that epigenetic mechanism is involved in toxicity of AgNPs and Ag ions in Jurkat T cells.     

Mammary epithelial cell derived exosomal MiR-221 mediates M1 macrophage polarization via SOCS1/STATs to promote inflammatory response

Lactational mastitis seriously alters the normal physiological function of mammary gland and activates the innate immune. Mammary epithelial cells (MECs) secret cytokines and regulate the function of immune system. However, the mechanism MECs mediated crosstalk with immune cells, such as macrophages, during mastitis is unclear. In this study, mouse mammary epithelial cells (HC11), treated with Lipoteichoic acid (LTA), and macrophages (RAW264.7) were used to mimic intercellular communication. Our results showed that exosomal miR-221 level was up-regulated and reached the peak at 12 h after infected by LTA. The expression of miR-211, CD11b protein and TNF-α mRNA were upregulated and the expression of CD206 protein and Arg-1 mRNA were inhibited in RAW264.7 treated with exosomes. In addition, miR-221 mimics and inhibitors enhanced and depressed HC11-derived exosomal miR-221 level, respectively. After treatment of Exo(mimic) in RAW264.7, the expression of CD11b protein and TNF-α mRNA were up-regulated, the expression of CD206 and Arg-1 mRNA were down-regulated. Additionally, Exo(inhibitor) enhanced CD206 protein and Arg-1 mRNA levels and inhibited CD11b protein and TNF-α mRNA levels. Furthermore, SOCS1 was identified to be a target gene of miR-221 by using Luciferase assays. And western blot assays showed that the expression of p-STAT1 and p-STAT3 were elevated and repressed, respectively. Taken together, we suggest that exosomal miR-221 promotes polarization of M1 macrophages via SOCS1, STAT1 and STAT3. And we reveal a novel crosstalk signaling pathway between mammary epithelial cells and macrophages in the process of inflammation.     

Regulation of human pregnane X receptor and its target gene cytochrome P450 3A4 by Chinese herbal compounds and a molecular docking study

The pregnane X receptor (PXR) plays a critical role in the regulation of human cytochrome P450 3A4 (CYP3A4) gene. In this study, we investigated the effect of an array of compounds isolated from Chinese herbal medicines on the activity of PXR using a luciferase reporter gene assay in transiently transfected HepG2 and Huh7 cells and on the expression of PXR and CYP3A4 in LS174T cells. Furthermore, molecular docking was performed to investigate the binding modes of herbal compounds with PXR. Praeruptorin A and C, salvianolic acid B, sodium danshensu, protocatechuic aldehyde, cryptotanshinone, emodin, morin, and tanshinone IIA significantly transactivated the CYP3A4 reporter gene construct in either HepG2 or Huh7 cells. The PXR mRNA expression in LS174T cells was significantly induced by physcion, protocatechuic aldehyde, salvianolic acid B, and sodium danshensu. However, epifriedelanol, morin, praeruptorin D, mulberroside A, tanshinone I, and tanshinone IIA significantly down-regulated the expression of PXR mRNA in LS174T cells. All the herbal compounds tested can be readily docked into the ligand-binding cavity of PXR mainly through hydrogen bond and aromatic interactions with Ser247, Gln285, His407, and Arg401. These findings suggest that herbal medicines can significantly regulate PXR and CYP3A4 and this has important implication in herb-drug interactions.