乳腺癌患者病理特征与Bcl-2、CXCL13、PAX8表达情况的关系分析

目的分析乳腺癌患者病理特征与B细胞淋巴瘤/白血病-2基因(Bcl-2)、趋化因子配体13(CXCL13)、配对盒基因8抗体(PAX8)表达情况的关系。方法收集2021年1月至2023年1月该院收治的160例乳腺癌患者临床资料。采用免疫组化法对其癌组织与癌旁组织Bcl-2、CXCL13、PAX8表达情况进行检测,并分析3项指标与患者病理特征的关系。结果与癌旁组织比较,癌组织Bcl-2、CXCL13、PAX8阳性率更高,差异有统计学意义(P<0.05)。与雌激素受体(ER)阴性、肿瘤最大径≥3 cm、孕激素受体(PR)阴性患者比较,ER阳性、肿瘤最大径<3 cm、PR阳性患者中Bcl-2高表达占比更高,差异有统计学意义(P<0.05);与无淋巴结转移、Ⅰ~Ⅱ期患者比较,淋巴结转移、Ⅲ~Ⅳ期患者中CXCL13高表达占比更高,差异有统计学意义(P<0.05);与Ⅰ~Ⅱ期、高/中分化、无淋巴结转移患者比较,Ⅲ~Ⅳ期、低分化、有淋巴结转移患者中PAX8高表达占比更高,差异有统计学意义(P<0.05)。ER、PR表达情况与Bcl-2表达情况呈正相关(P<0.05),肿瘤最大径与Bcl-2表达情况呈负相关(P<0.05);临床分期、淋巴结转移情况与CXCL13、PAX8表达情况呈正相关(P<0.05);分化程度与PAX8表达情况呈负相关(P<0.05)。结论乳腺癌患者Bcl-2、CXCL13、PAX8表达情况对疾病的发生和发展具有明显影响,有望成为评估乳腺癌患者病情严重程度的标志物。

血清胸腺活化调节趋化因子、CXC亚家族趋化因子13与弥漫大B细胞淋巴瘤患者化疗疗效和预后的关系研究

目的探讨血清胸腺活化调节趋化因子(TARC)、CXC亚家族趋化因子13(CXCL13)与弥漫大B细胞淋巴瘤(DLBCL)患者化疗疗效和预后的关系。方法选取2017年1月至2020年6月该院收治的285例DLBCL患者(DLBCL组)及同期健康体检者160例(对照组)作为研究对象,DLBCL患者接受利妥昔单抗+环磷酰胺+多柔比星+长春新碱+泼尼松(R-CHOP)相关化疗方案,根据化疗效果分为有效组和无效组。比较DLBCL组与对照组、有效组与无效组血清TARC、CXCL13水平;随访统计DLBCL患者3年总生存期(OS)及无进展生存期(PFS),采用Logistic回归分析DLBCL患者预后及疾病进展的影响因素,并构建回归预测模型,采用受试者工作特征(ROC)曲线分析其预测评估效能。采用Kaplan-Meier生存曲线分析不同水平血清TARC、CXCL13与DLBCL患者3年OS及PFS的关系。结果DLBCL组治疗前血清TARC、CXCL13水平高于对照组(P<0.05)。DLBCL患者化疗有效率为85.26%,无效组治疗前血清TARC、CXCL13水平高于有效组(P<0.05)。多因素Logistic分析结果显示,Ann Arbor分期Ⅲ/Ⅳ期、TARC高表达及CXCL13高表达均是DLBCL患者预后的独立危险因素(P<0.05)。乳酸脱氢酶水平升高、TARC高表达及CXCL13高表达均是DLBCL患者疾病进展的独立危险因素(P<0.05)。以上述影响因素构建回归预测模型,ROC曲线分析显示,预测模型预测预后的曲线下面积(AUC)为0.874,预测疾病进展的AUC为0.911。TARC低表达患者的3年OS、PFS优于高表达患者(P<0.05),CXCL13低表达患者的3年OS、PFS优于高表达患者(P<0.05)。结论DLBCL患者血清TARC、CXCL13水平异常升高,血清TARC、CXCL13低表达的DLBCL患者具有更好的化疗效果及预后。包括血清TARC、CXCL13在内的多因子预测模型对DLBCL患者预后具有较高的评估效能。

ILC2和MDSC及其相关细胞因子IL-13和iNOS在宫颈癌中的表达及其列线图模型的构建和评价

目的:研究2型固有淋巴样细胞(ILC2)和髓源性抑制细胞(MDSC)及其相关细胞因子IL-13和诱导型一氧化氮合酶(iNOS)在宫颈癌(CC)中的表达,并基于其构建CC发病风险的列线图预测模型。方法:采集2022年5月至2024年1月在新疆医科大学第一附属医院手术切除的40例CC组织及100例外周血作为CC组,选取同期收治的30例子宫肌瘤经CC筛查为阴性的宫颈组织和100例正常健康个体外周血作为对照组。用多重免疫荧光技术(mIF)及免疫组化染色(IHC)法检测两组组织中ILC2和MDSC细胞浸润及其相关细胞因IL-13和iNOS的表达;使用流式细胞术和ELISA技术分别检测两组外周血中ILC2和MDSC及IL-13和iNOS的表达差异;通过Person相关性分析评估其相关性;使用单因素和多因素Logistic分析来确定ILC2和MDSC及IL-13和iNOS是否为CC发病的独立危险因素,再利用R软件建立免疫预测模型,使用ROC曲线下面积(AUC值)、Hosmer-Lemeshow检验、校准曲线、临床决策曲线和临床影响曲线来分别评估模型。结果:CC组中ILC2及MDSC及其相关细胞因子IL-13和iNOS均高于对照组(均P<0.05),且其均呈正相关(均P<0.05);经过单因素及多因素Logistic回归分析显示,ILC2、MDSC及IL-13、iNOS均是CC发病的独立危险因素(均P<0.05);基于这些危险因素的CC发病列线图,经过验证提示,该列线图模型具有一定的临床实用价值。结论:ILC2和MDSC及其相关的细胞因子IL-13和iNOS在CC组织及外周血中均呈高表达,基于这些危险因素构建的预测模型具有良好的预测能力和一定的实用性,为CC的早期诊断和治疗提供了一种简便有效的辅助工具。

补肾痹通方联合骨髓间充质干细胞对损伤软骨细胞的保护机制及SOX9、MMP-13表达的影响

目的:探讨补肾痹通方(BSBT)联合骨髓间充质干细胞(BMSCs)对白介素(IL)-1β诱导的损伤软骨细胞的保护机制及性别决定区Y框蛋白9(SOX9)、基质金属蛋白酶13(MMP-13)表达的影响。方法:使用10 ng/ml的IL-1β建立损伤软骨细胞模型,实验分为对照组、模型组(IL-1β)、中药组(IL-1β+BSBT)、干细胞组(IL-1β+BMSCs)和联合组(IL-1β+BSBT+BMSCs);CCK-8法检测各组软骨细胞增殖情况;RT-qPCR法和Western blot法分别检测软骨细胞内SOX9、MMP-13、Ⅱ型胶原α1重组蛋白(COL2A1)、IL-10的基因和蛋白表达;ELISA检测各组细胞培养上清中肿瘤坏死因子-α(TNF-α)、IL-6、胰岛素样生长因子1(IGF-1)、碱性成纤维细胞生长因子(bFGF)的水平。结果:与模型组比较,各组软骨细胞活性显著增强(P<0.01),软骨细胞中的SOX9、COL2A1、IL-10的基因和蛋白水平显著升高(P<0.01),MMP-13的基因和蛋白水平明显降低(P<0.01),软骨细胞培养上清中TNF-α、IL-6的含量显著降低(P<0.01),IGF-1、bFGF的水平升高(P<0.01),其中联合组变化最明显(P<0.05)。结论:BSBT联合BMSCs可有效保护损伤软骨细胞,其机制可能是通过上调SOX9,下调MMP-13,抑制炎症,改善软骨细胞微环境,促进关节软骨的修复与再生。

人类恶性肿瘤靶向治疗的新希望—CDK12/CDK13

细胞周期的更替有赖于细胞周期蛋白依赖性激酶(CDKs),CDKs是重要的蛋白激酶家族,在调节细胞周期和调控基因转录方面具有关键的作用。其中CDK12/CDK13在DNA损伤应答、RNA剪接调控、转录及细胞周期调控等方面至关重要。近年发现CDK12/CDK13在多种癌症中表达异常或发生突变,在癌症发展中扮演着重要角色,已成为近年来研究的热点之一。本篇基于Google Scholar、PubMed和万方数据库,归纳总结了CDK12/CDK13的基本结构、生物学功能、与恶性肿瘤的相关性、以及针对性抑制剂的研究进展进行综述。为后续的靶向治疗新型靶点的研发和机制探讨提供方向,为恶性肿瘤的防治、诊断以及治疗提供新思路。

细胞周期蛋白O调控细胞周期蛋白依赖性激酶13对宫颈癌进展的影响

目的:探讨细胞周期蛋白O(CCNO)通过调控细胞周期蛋白依赖性激酶13(CDK13)进而影响宫颈癌细胞周期进展的分子机制。方法:通过生物信息学方法分析CCNO在不同癌症中的表达情况。利用免疫组化(IHC)检测CCNO在宫颈癌患者组织样本中的表达情况,利用蛋白质免疫印迹(WB)检测CCNO在宫颈癌细胞系中的表达情况。通过WB检测CCNO载体沉默效率,通过细胞克隆、胸腺嘧啶核苷类似物实验检测(EDU)和细胞计数试剂盒-8(CCK8)检测肿瘤样本(NC)和肿瘤沉默CCNO样本(si-CCNO)的细胞增殖情况,通过细胞迁移实验和流式细胞分析实验检测2组细胞迁移情况和细胞周期的变化,最后通过免疫共沉淀(CO-IP)实验检测CCNO是否调控细胞周期蛋白依赖性激酶13(CDK13)的表达。结果:CCNO在宫颈癌患者的组织样本中高表达;与对照组比较,si-CCNO组中CCNO表达量降低(P<0.05);与NC组比较,si-CCNO组的细胞增殖降低(P<0.05),si-CCNO组的细胞迁移数目减少(P<0.05);与NC组比较,si-CCNO组停滞在G1期细胞明显增多(P<0.05);与NC组比较,si-CCNO组的CDK13表达显著降低。结论:沉默CCNO能够抑制宫颈癌细胞的增殖、细胞周期进展和转移能力,并且CCNO能够调控CDK13进而对宫颈癌进展产生影响。

绞股蓝总皂苷对人肾上腺皮质癌SW-13细胞增殖及凋亡蛋白Caspase-8表达的影响

目的探讨绞股蓝总皂苷(Gyp)体外干预对人肾上腺皮质癌(ACC)SW-13细胞增殖及其含半胱氨酸的天冬氨酸蛋白水解酶-8(Caspase-8)表达的影响。方法采用MTT法检测不同浓度的Gyp对SW-13细胞增殖的影响;普通显微镜下观察细胞形态学改变;实时荧光定量PCR检测Gyp干预后Caspase-8表达的变化。结果 Gyp对SW-13细胞增殖产生明显地抑制作用,且呈时间和浓度依赖性;光镜下观察,Gyp干预后SW-13细胞凋亡特征更明显;实时荧光定量PCR结果显示,Gyp干预后SW-13细胞Caspase-8 mRNA表达明显上调(P<0.01)。结论 Gyp可显著抑制人ACC SW-13细胞的增殖,其作用机制可能为通过上调Caspase-8 mRNA的表达来实现。

姜黄素干预肾上腺皮质癌SW-13细胞的miRNA调控机制

目的 探讨姜黄素干预肾上腺皮质癌SW-13细胞的miRNA调控机制。方法 将肾上腺皮质癌SW-13细胞分为对照组和姜黄素干预组,分别加入基础培养基2 mL、50μmol/L姜黄素溶液2 mL,干预24 h后提取总RNA用于小RNA文库构建和测序。将获得的序列进行miRNA注释和新miRNA预测后,筛选差异性表达的miRNA;对差异性表达的miRNA进行靶基因预测,对靶基因进行功能和通路富集分析。使用实时荧光定量PCR鉴定部分差异性表达miRNA的表达量。结果 共筛选出44个差异性表达的miRNA,包括21个表达上调的miRNA和23个表达下调的miRNA,其中预测有靶基因且有功能的miRNA共9个。功能富集分析结果显示,靶基因主要富集在细胞增殖、细胞迁移、细胞分化和凋亡等生物学过程;通路富集分析结果显示,靶基因主要富集在癌症途径、丝裂原活化蛋白激酶信号通路、内质网应激信号通路、细胞周期和凋亡途径等。实时荧光定量PCR结果提示,miR-184、miR-490-3p、miR-766-3p、miR-200b-3p、miR-200c-3p和miR-3622a-5p的表达量与小RNA测序数据结果基本一致。结论 姜黄素可能通过miR-184、miR-490-3p、miR-766-3p、miR-200c-3p、miR-3622a-5p等miRNA参与调控癌症途径、丝裂原活化蛋白激酶信号通路、内质网应激信号通路、细胞周期和凋亡途径等信号通路,以抑制细胞增殖、迁移、分化和凋亡,从而发挥抗肾上腺皮质癌的作用。

氯硝柳胺对肾上腺皮质癌SW-13细胞增殖和凋亡的影响

目的研究氯硝柳胺对人肾上腺皮质癌SW-13细胞株细胞增殖、细胞周期、细胞凋亡和细胞迁移力、侵袭力的影响。方法通过体外培养SW-13细胞,采用CCK8法和i CELLigence实时无标记细胞增殖监测仪检测不同浓度氯硝柳胺对细胞增殖的影响;设对照组和氯硝柳胺组,流式细胞技术检测氯硝柳胺作用后SW-13细胞周期的变化,AO/EB染色法和Annexin V/PI染色流式细胞技术测定细胞凋亡率的变化,Transwell实验和细胞划痕实验检测细胞侵袭、迁移能力的变化,Western blot(WB)法检测β-catenin/LRP6表达的变化。结果不同浓度的氯硝柳胺分别作用SW-13细胞24、48、72、96、120 h后,呈时间和浓度依赖性抑制SW-13细胞增殖(F=157.93,P<0.001);流式细胞仪周期检测结果显示24、48、72 h后,与对照组相比氯硝柳胺组将SW-13细胞周期阻滞在G0/G1期(P=0.001、0.005、0.008);AO/EB染色法和Annexin V/PI染色法结果均显示氯硝柳胺组的凋亡率与对照组相比明显升高(P<0.001);Transwell实验结果示氯硝柳胺组SW-13细胞的侵袭力降低(P<0.001),细胞划痕实验结果显示氯硝柳胺组SW-13细胞的迁移能力下降(P=0.002),WB结果示氯硝柳胺组SW-13细胞β-catenin/LRP6的表达明显降低(P<0.001)。结论氯硝柳胺抑制人肾上腺皮质癌SW-13细胞增殖,阻滞细胞周期,抑制侵袭及迁移能力,促进细胞凋亡;可能与其抑制Wnt/β-catenin信号通路有关。

羟喜树碱对人肾上腺皮质癌SW-13细胞凋亡的影响

目的:研究羟喜树碱对人肾上腺皮质癌SW-13细胞生长抑制和凋亡作用的影响。方法:采用CCK8检测不同浓度羟喜树碱对SW-13细胞增殖的影响;流式细胞仪测定药物作用前后的细胞凋亡情况。结果:不同浓度的羟喜树碱分别作用24,48,72h后,SW-13细胞的生长增殖均受到明显抑制(P<0.01),并呈时间和浓度依赖性;流式细胞术显示SW-13细胞凋亡率随羟喜树碱浓度的增加而升高。结论:羟喜树碱可以显著抑制人肾上腺皮质癌SW-13细胞生长,促进其凋亡。

羟喜树碱对人肾上腺皮质癌SW-13细胞生长增殖的影响

目的观察羟喜树碱对人肾上腺皮质癌SW-13细胞生长增殖的影响。方法体外培养人肾上腺皮质癌SW-13细胞,随机分为干预组和对照组,干预组加入羟喜树碱使其终浓度为0.8μg/ml,对照组不加羟喜树碱,分别培养48 h后,采用台盼蓝拒染法观察羟喜树碱对SW-13细胞生长增殖的影响。结果光镜下可见干预组SW-13细胞数减少,死亡细胞数较多,干预组活细胞率明显低于对照组(P<0.05)。结论羟喜树碱可能具有抑制人肾上腺皮质癌SW-13细胞增殖并促进其死亡的作用。

Interleukin-13 inhibits cytokines synthesis by blocking nuclear factor-κB and c-Jun N-terminal kinase in human mesangial cells

Objective： Monocytes/macrophages, proinflammatory cytokines and chemokines are important in the pathogenesis of glomerulonephritis. Interleukin （IL） -13 has been shown to exert potent anti-inflammatory properties. This study was designed to investigate the effect of IL-13 on the expression of proinflammatory cytokines, chemokines and profibrogenic cytokines and the involved molecular mechanism in cultured human mesangial cells （HMCs）. Methods： The expressions of proinflammatory cytokines, chemokines and profibrogenic cytokines were determined by ribonuclease protection assay （RPA）. Activity of nuclear factor-kappa B （NF-κB） and activa- tor protein-1 （AP-1） was examined by electrophoretic mobility shift assay （EMSA）. NF-κB subunit p65 nuclear transportation and c-Jun N-terminal kinase （JNK） activity were assayed by immunoblot. Results： Recombinant IL-13 inhibited tumor necrosis factor-α （TNF-u）, IL-1α, IL-1β, monocyte chemoattractant protein-1 （MCP-1）, IL-8, and transforming growth factor-β1 （TGF-β1） mRNA expressions in a dose-dependent manner. Lipopoly- sacchorides （LPS） dramatically increased NF-κB DNA binding activity of HMCs, which was inhibited by IL-13 in a dose-dependent manner. LPS-activated NF-κB contained p50 and p65 dimers, but not c-Rel subunit. IL-13 blocked LPS-induced NF-κB subunit p65. LPS stimulated JNK/AP-1 activation, which was inhibited by IL-13 in a dose-dependent manner. Conclusion： IL-13 inhibits proinflammatory cytokines, chemokines, and profibrogenic cytokines synthesis by blocking NF-κB and JNK/AP-1 activation. These observations point to the importance of IL-13 in the modulation of inflammatory processes in the renal glomerulus.

shRNA沉默IGF-Ⅱ基因对人肾上腺皮质癌细胞株SW-13增殖与凋亡的影响

目的探讨shRNA沉默IGF-Ⅱ基因对人肾上腺皮质癌细胞株SW-13增殖与凋亡的影响。方法构建针对人IGF-Ⅱ基因的shRNA慢病毒表达载体,稳定转染人肾上腺皮质癌细胞株SW-13。将实验随机分为3组:空白对照组、阴性对照组、IGF-Ⅱ-shRNA组,应用RT-PCR检测转染后各组IGF-Ⅱ基因mRNA的表达,以MTT比色法、流式细胞术检测转染后SW-13细胞生长增殖、凋亡的情况。结果 IGF-Ⅱ-shRNA组的IGF-Ⅱ基因mRNA表达量明显低于阴性对照组和空白对照组(P均<0.01),IGF-Ⅱ-shRNA组较之其他两组能明显抑制细胞的增殖(P均<0.01),IGF-Ⅱ-shRNA组的细胞凋亡率与其他两组相比显著增加(P均<0.01)。结论靶向IGF-Ⅱ基因的shR-NA可以有效抑制人肾上腺皮质癌细胞株SW-13的生长,并促进其凋亡,IGF-Ⅱ基因可能是肾上腺皮质癌治疗的一个潜在靶点。

Construction of eukaryotic expression vector of human S100A13 gene and its effect on proliferation of human thyroid cancer cell line TT

Objective To investigate the effect of exogenous S100A13 gene overexpression on the proliferation of human thyroid cancer cell line TT.Methods The recombinant ORF of S100A13 tagged with six histidines at the 5' end was subcloned into the pcDNA3.2/V5/GW/D-TOPO vector and sequenced.The eukaryotic expression plasmid pcDNA3.2/V5 /GW/D-S100A13 and empty vector pcDNA3.2/V5/GW/D were transfected into TT cells.The positive clones were selected by G418.The expressions of S100A13 mRNA and protein were detected by real time reverse transcription-polymerase chain reaction(RT-PCR) and Western blot.The effect of S100A13 on cell proliferation and cell cycle was evaluated by cell growth curve,MTT colorimetric assay and flow cytometry.Results S100A13 gene tagged with six histidines at the 5 ' end was confirmed to be inserted into the pcDNA3.2/V5/GW/D vector correctly.TT-S100A13-V5 cells,which over-expressed S100A13,were constructed successfully.TT-S100A13-V5 cells grew much faster than TT-V5 and TT cells(P <0.001).The proportions of both S and G2/M phase cells were significantly higher in TT-S100A13-V5 cells than those in TT-V5 and TT cells(P <0.001).Conclusion The eukaryotic expression vector containing human S100A13 gene has been successfully constructed,which highly expresses S100A13 in TT cells.Exogenous S100A13 gene overexpression accelerates TT cell proliferation and drives the cell cycle progression of TT cells from G0/G1 phase to S and G2/M phases.

IL-13Ra2-and glioma stem cell-pulsed dendritic cells induce glioma cell death in vitro

Objective Gliomas are the most common malignant tumors in the central nervous system.Despite multiple therapies including surgery,chemotherapy,and radiotherapy,the prognosis of patients remains poor.Immunotherapy is an alternative method of treating glioma,and the use of dendritic cell vaccines is one of the promising treatment options.However,there is no specific tumor cell antigen that can trigger dendritic cells(DCs).IL-13Ra2 is a specific antigen expressed in glioma cells;in the current study,we have attempted to explore whether IL-13Ra2 could be the antigen that triggers DCs and to envisage its application as potential therapy for glioma.Methods The expression of IL-13Ra2 was detected in U251 glioma cell lines and primary glioma tissues using different methods.DCs from human blood were isolated and pulsed with recombinant IL-13Ra2,following which the cytotoxicity of these DCs on glioma cells was detected and analyzed.Results About 55.9% human glioma tissue cells expressed IL-13Ra2,while normal brain tissue cells did not show any expression.DC vaccines loaded with IL-13Ra2,glioma cell antigen,and brain tumor stem cell(BTSC) antigen could significantly stimulate the proliferation of T lymphocytes and induce cell death in the glioma tissue.Compared to other groups,DC vaccines loaded with BTSC antigen showed the strongest ability to activate cytotoxic T lymphocytes(CTLs),while the glioma cell antigen group showed no significant difference.Conclusion IL-13Ra2,which is expressed in gliomas and by glioma stem cells,as well as IL-13Ra2 could prove to be potential antigens for DC vaccine-based immunotherapy.

STR分型法鉴定人SW-13、293T及AGS细胞系种内及种间污染

目的检测研究所使用的人肾上腺皮质癌细胞SW-13、人胚肾细胞293T及人胃癌细胞AGS 3种细胞系污染情况,并探索短串联重复序列(short tandem repeat,STR)分型方法检测人类细胞系污染的可靠性。方法人类基因组上的微卫星位点携带着大量STR,每个STR核心序列的重复次数随个体不同存在差异,作为一种DNA标记使得细胞系在DNA水平上具有个性化。通过荧光聚合酶链式反应体系扩增STR位点,检测的数据与DSMZ细胞库进行匹配以对细胞系进行鉴别。结果比对得出SW-13基本匹配,其中20个STR基因位点中只有TH01位点的两个等位基因重复数为7和8,与DSMZ细胞库中此等位基因重复数7和7有差异,其余均一致在可接受范围内。细胞系293T、AGS等位基因的重复数与DSMZ细胞库完全匹配。结论 STR分型检测方法被ATCC、DSMZ等权威机构推崇,且此研究采用20位点检测法涵盖ATCC-9位点及中检院-16位点检测法,该法权威可靠。本所3种细胞系遗传稳定,均无种内或种间污染。

Tinospora crispa extract inhibits MMP-13 and migration of head and neck squamous cell carcinoma cell lines

Objective: To investigate the effect of Tinospora crispa(T. crispa) extract on matrix metalloproteinase 13(MMP-13) expression and cell migration. Methods: The cytotoxicity of T. crispa extract was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay on head and neck squamous cell carcinoma(HNSCC) cell lines. The effect on expression of MMP-13 was analysed by RT-PCR and ELISA. The migration was assessed by wound healing assay. Results: MMP-13 m RNA was highly expressed in the metastatic human HNSCC cell lines, HN22 and HSC-3. T. crispa extract at a concentration of 100.0 μg/m L caused about 50% reduction of cell survival. T. crispa extract at a non-toxic concentration of 12.5, 25.0 and 50.0 μg/m L signii cantly suppressed MMP-13 m RNA expression and secreted MMP-13 in both HN22 and HSC-3. The expression of tissue inhibitors of metalloprotease by HSC-3 cells was attenuated by 25.0 and 50.0 μg/m L of T. crispa extract. Addition of the extract to cells in a wound healing assay showed inhibition of cell migration by HN22 cells. Conclusions: These data suggest that T. crispa could be considered as a potential therapeutic drug to prevent metastasis of HNSCC.

Glycosylation-independent binding to extracellular domains 11-13 of mannose-6-phosphate/insulin-like growth factor-2 receptor mediates the effects of soluble CREG on the phenotypic proliferation of vascular smooth muscle cells

Background The present study aimed to investigate the detailed mode and specific sites for their binding as well as the functional relevance of this binding in the phenotypic proliferation of vascular smooth muscle cells(SMCs). Methods CREG knocked-down SMCs were employed to evaluate the biological activity of wtCREG and mCREG.Expressions of SMC differentiation markers SM myosin heavy chain(SM-MHC),SM-actin,heavy caldesmon and myocardin were determined by Western blotting using specific antibodies. Cellular growth of SMCs was assessed by bromide dewuridine (BrdU) incorporation and cell cycle analysis on fluorescence-activated cell sorting(FACS).A solid-phase binding assay was used to study the binding of CREG to extracellular domains of M6P/IGF2R.The cellular co-localization of the two recombinant CREGs with M6P/IGF2R was detected on SMC surface by immunoprecipitation and immunofluorescence analysis.Results The molecular weight of wtCREG was around 30 kD while that of the mCREG was～25 kD.Treatment of wtCREG with PNGase F reduced its molecular weight from～30 kD to～25 kD,whereas PNGase F treatment had no effect on the molecular weight of mCREG.Both wtCREG and mCREG proteins enhanced SMC differentiation,inhibited BrdU incorporation,and arrested cell cycle progression when added to the culture medium.In CREG knocked-down SMCs,the amount of CREG detected by immunoblotting in M6P/IGF2R immunoprecipitates was significantly reduced when compared to normal cells.Both recombinant CREGs co-immunoprecipitated with M6P/IGF2R, although slightly reduced amount of the mutant CREG was detected in M6P/IGF2R immunoprecipitates.Immunostaining revealed that His-tagged CREGs co-localized with IGF2R on the cell surface in a glycosylation-independent manner.In vitro binding assay showed that CREGs bound to M6P/ IGF2R extracellular domains 7-10 and 11-13 in a glycosylation -dependent and -independent manner,respectively.Further blocking experiments using soluble M6P/IGF2R fragments and M6P/IGF2R neutralizing antibody indicated that the biological activities of recombinant CREGs in SMC growth and the up-regulation of SMC differentiation markers were all abolished by treatment with the M6P/IGF2R neutralizing antibody. However,although the growth inhibitory effect of wtCREG was nearly abolished by D7-10 or D11-13,the effect of mCREG was only reversed by Dll-13,indicating that the binding to domains 11-13 is required for CREG to modulate the proliferation of SMCs.Conclusions These data suggest that solubleCREG proteins can exert their biological function via binding to the extracellular domains 7-10 and 11-13 of cell surface M6P/IGF2R in both a glycosylation-dependent and -independent manner.

Regulation of mitochondrial carrier SLC25A13 on breast cancer cell cycle in vitro

Objective·To investigate the role of mitochondrial solute carrier family 25 member 13(SLC25A13)on breast cancer development.Methods·SLC25A13 mRNA and protein expressions in invasive breast cancer tissues and normal breast tissues were from The Cancer Genome Atlas(TCGA)breast cancer dataset.Survival analysis was conducted online by Kaplan-Meier software.MCF-7 cell line was used for in vitro cell assay.Knockdown of SLC25A13 and sirtuin 2(SIRT2)were conducted by siRNA transfection.Cell viability was measured with trypan blue exclusion.Cell cycle arrest was determined by flow cytometry.The mRNA expression of SLC25A13 and P27 were detected by quantitative PCR.The protein level of SLC25A13,P27 and SIRT2 were detected by Western blotting.Protein half-life of P27 was assessed by Western blotting after cycloheximide treatment.Results·SLC25A13 was up-regulated in invasive breast cancer tissues.High expression of SLC25A13 correlated with poor overall survival and breast cancer recurrence.SLC25A13 knockdown inhibited MCF-7 cell cycle progression.P27 and SIRT2 both accumulated after SLC25A13 knockdown.P27 accumulation resulted from prolonged protein half-life.Knockdown of SIRT2 restored cell cycle arrest as well as P27 accumulation caused by SLC25A13 silencing.Conclusion·High expression of SLC25A13 may promote cell cycle progression via SIRT2 in breast cancer development.

Two potentially specific but relevant patterns of proteomic change Response of SH-SY5Y cells to differentiation with retinoic acid followed by phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate, and susceptibility of differentiated cells to dopamine

Dopamine （DA） exposure at a dose of 100 pmol/L for 24 hours causes oxidative stress in SH-SY5Y cells with induction of neuronal differentiation by retinoic acid （RA,10 pmol/L,72 hours） followed by phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate （TPA,80 nmol/L,72 hours）. However,it remains unclear whether the alteration of phenotype observed in response to oxidative stress is associated with protein regulation in this cellular model for Parkinson＇s disease. The present study detected protein regulation affected by oxidative stress at a proteomic level：selection of differentially altered proteins using two dimensional difference in-gel electrophoresis and identification of these proteins using matrix assisted laser desorption/ionization time-of-flight mass spectrometry. The results demonstrated significant alterations in expression of six proteins in SH-SY5Y cells following the differentiation and fourteen proteins in the differentiated cells following the exposure,exemplified by an increase of tubulin alpha1 in the former but a decrease of tubulin alpha-ubiquitous chain in the latter. These results suggest that two potentially specific but relevant patterns of proteomic change may be produced in SH-SY5Y cells with the induction of differentiation by RA followed by TPA,and in the differentiated cells after DA exposure.