RNA干扰CXCR4表达抑制乳腺癌MDA-MB-231细胞的增殖、黏附和迁移

目的：构建CXC趋化因子受体4（CXC chemokine receptor 4, CXCR4）RNA干扰真核表达载体,研究其对人乳腺癌细胞MDA-MB-231增殖、黏附及迁移能力的抑制作用。方法：构建针对CXCR4的带发夹结构的小RNA干扰序列,并连接到pGCsi-U6-Neo-GFP载体中,转染293T细胞,筛选出干扰效率最高的表达载体。脂质体法转染MDA-MB-231细胞。利用CCK8法、细胞-基质黏附实验和划痕修复实验检测shRNA干扰CXCR4表达对MDA-MB-231细胞增殖、黏附和迁移能力的影响。结果：成功构建CXCR4-shRNA重组质粒,并转染293T细胞,利用RT-PCR及Western blotting检测发现CXCR4沉默效率最高可达81.3%。CXCR4-shRNA转染能显著抑制MDA-MB-231细胞的增殖(P<0.05)以及细胞与细胞外基质的黏附(P<005)。CXCR4-shRNA转染组MDA-MB-231细胞的迁移距离明显低于对照质粒组和空白对照组(P<0.01)。结论：CXCR4-shRNA干扰载体能特异性抑制CXCR4的表达,从而抑制乳腺癌MDA-MB-231细胞的增殖、黏附及迁移。     

SDF-1／CXCR4与肿瘤研究进展

基质细胞衍生因子-1（SDF-1）及其受体CXCR4在促进肿瘤细胞增殖、定向迁移、黏附、降解细胞外基质、血管形成及诱导免疫耐受中发挥着重要作用。文章就SDF-1／CXCR4轴在肿瘤生物学行为中的作用进行简要综述。     

CXCR4 单克隆抗体对新生血管形成的影响

目的：探讨CXC趋化因子受体4（CXCchemokinereceptor4，CXCR4）单克隆抗体（CXCR4mAb）对新生血管形成的影响及其作用机制。方法：以鸡胚绒毛尿囊膜（chorioallantoicmembrane，CAM）血管抑制实验检测CXCR4单克隆抗体对新生血管形成的影响，WesternBlot免疫印迹法检测血管内皮细胞SDF-1蛋白的表达。结果：CXCR4单克隆抗体可明显抑制新生血管的形成，并且使培养的血管内皮细胞SDF-1蛋白表达明显降低：结论：CXCR4mAb可通过SDF-1／CXCR4生物轴抑制新生血管的形成。     

基质细胞衍生因子-1及其受体CXCR4与乳腺癌的关系

引言 乳腺癌是危害妇女健康的主要恶性肿瘤之一。在世界范围内乳腺癌的发病率呈上升趋势,据文献报道,欧美国家每10个妇女约有1人发生乳腺癌。乳腺癌患者的预后与肿瘤的复发、转移密切相关,远处转移己成为制约患者长期生存和改善预后的主要障碍。近年来研究发现基质细胞衍生因子-1（stromal cell-derived factor-1,SDF-1）及其受体CXCR4构成的信号通路与乳腺癌细胞的定向播散和器官特异性转移密切相关。本文就SDF-1及其受体CXCR4的结构、功能以及与乳腺癌的关系作一论述。     

SDF-1/CXCR4在胰腺癌中的表达及与临床病理特征的关系

[目的]探讨基质细胞衍生因子-1（SDF-1）及其受体CXCR4在胰腺癌组织中的表达及与临床病理特征之间的关系。[方法]采用免疫组织化学方法检测37例胰腺癌及10例癌周组织中SDF-1及其受体CXCR4的表达。[结果]胰腺癌组织CXCR4的表达高于癌周组织（P=0．0001）．胰腺癌组织中CXCR4的表达在不同TNM分期及淋巴结是否转移之间有显著差异（P〈0．05）。胰腺癌间质组织SDF—1的表达高于在癌周间质组织中表达（P=0．0007）,胰腺癌间质组织的SDF-1表达在不同TNM分期及淋巴结是否转移之间有显著差异（P〈0．05）。[结论]胰腺癌组织CXCR4的表达与胰腺癌的侵袭转移密切相关,而间质组织SDF-1的表达也可能在此过程中起一定作用。     

CXCR4/SDF-1α对宫颈癌HeLa细胞定向迁移及增殖的影响

 目的了解CXCR4在HeLa细胞的表达情况,并借助细胞培养评价CXCR4/SDF-1α对HeLa细胞定向迁移及增殖的影响。方法CXCR4 mAb免疫染色HeLa细胞。用Transwell侵袭转移模型评价HeLa细胞的迁移情况,其中,上室中加入预先用(或不用)CXCR4单抗预孵育的HeLa细胞,下室中加入含0~100ng/mlSDF-1α的培养基。为评价CXCR4、SDF-1α对HeLa细胞增殖的影响,将HeLa细胞接种于有(无)SDF-1α和(或)CXCR4的低血清环境72h。结果CXCR4在所有HeLa细胞上均有表达。HeLa细胞能顺SDF-1α浓度差定向迁移,且这一作用可被CXCR4 mAb拮抗。SDF-1α能促进He-La细胞在低血清环境中增殖。结论CXCR4/SDF-1α参与了HeLa细胞定向迁移的过程并影响其增殖。     

抑制CXCR4活性对乳腺癌骨转移影响的体内外研究

目的:应用特异性抑制剂AMD3100抑制人乳腺癌骨高转移MDA-MB-231SA-rfp细胞中CXCR4的活性,探讨CX-CR4在乳腺癌细胞体内、外增殖和迁移中的作用和机制。方法:CCK8法和Transwell法检测AMD3100对MDA-MB-231SA-rfp细胞体外增殖和迁移能力的影响。构建MDA-MB-231SA-rfp细胞骨转移裸鼠模型,以不同质量浓度的AMD3100处理后,X线影像观察骨转移情况,进一步利用MicroPET进行半定量分析,并应用H-E染色检测骨转移灶的定位。Western blotting法检测AMD3100对MDA-MB-231SA-rfp细胞和移植瘤转移灶组织中CXCR4蛋白表达的影响。结果:AMD3100能明显抑制MDA-MB-231SA-rfp细胞在SDF-1刺激下的增殖和迁移(P<0.05),较高质量浓度(2 000 ng/ml)的AMD3100效果更明显(P<0.01)。成功构建MDA-MB-231SA-rfp细胞裸鼠乳腺癌转移模型,不同质量浓度AMD3100处理后,小鼠下肢骨骨质破坏程度降低;MicroPET分析发现,对照组、低剂量AMD3100组、高剂量AMD3100组SUVmax值分别为9.44±0.53、5.70±0.25、2.18±0.47(P<0.01);组织病理检测证实为乳腺癌骨转移灶。Western blotting结果显示,AMD3100作用前后MDA-MB-231SA-rfp细胞和骨转移灶标本中CXCR4蛋白表达无明显变化。结论:AMD3100降低CXCR4的活性能抑制乳腺癌MDA-MB-231SA-rfp细胞体外增殖和迁移能力,并能抑制裸鼠体内乳腺癌骨转移灶的形成。     

TLR4信号通过对miR-21的表达调控影响乳腺癌4T1细胞的凋亡和增殖

目的：探讨TLR4信号对乳腺癌4T1细胞株中miR-21表达的影响及调控机制,研究miR-21对乳腺癌细胞凋亡和增殖的影响。方法：体外培养乳腺癌细胞株4T1,以TLR4配体LPS刺激6、12、18、24 h,实时荧光定量PCR检测4T1细胞中miR-21的表达变化。Western blotting 检测TLR4信号活化后4T1细胞中NF-κBp65的表达和磷酸化情况;应用NF-κB抑制剂PDTC预处理30 min,实时荧光定量PCR检测4T1细胞中miR-21的变化。转染miR-21抑制剂和阴性对照后, Annexin-V/PI双标法检测4T1细胞凋亡情况,MTT法检测4T1细胞增殖情况。结果：LPS刺激致TLR4信号活化能够时间依赖性地上调miR-21的表达(18 h时: 207±0.33 vs 1; t=5.61, P=0.03),TLR4信号能够时间依赖性地上调NF-κB的活化,NF-κB抑制剂PDTC能明显抑制TLR4信号诱导的4T1细胞的miR-21上调(0.70±0.10 vs 2.14±0.32; t=-7.357,P=0.002)。与阴性对照组相比,miR-21抑制剂组4T1细胞的凋亡率明显增高\[(24.2±2.4)% vs (14.8±5.1)%; t=2.891, P=0.044\],4T1细胞的增殖能力明显降低(042±0.02 vs 0.55±0.01;t=-8.528, P=0.001)。结论：TLR4信号通路的活化能够上调乳腺癌4T1细胞中miR-21的表达,其机制与NF-κB的活化有关;靶向抑制miR-21能有效促进4T1细胞的凋亡、抑制4T1细胞增殖。     

基质细胞衍生因子-1及其受体CXCR4在白血病中表达的临床意义

 基质细胞衍生因子-1（SDF-1）是早期细胞生长因子,属趋化因子亚家族成员之一。其受体是CXCR4、SDF-1、CXCR4配体受体系统在血细胞的生成、干细胞归巢、血管新生以及白血病细胞的浸润等生理和病理过程中发挥重要作用。研究CXCR4在白血病中的表达及其与白血病浸润的关系,对丰富白血病形态学、免疫学、细胞遗传学（MIC）诊断分型的免疫学指标,为防治白血病浸润、复发而采取相关分子靶向治疗都具有重要意义。     

miR-100抑制乳腺癌细胞迁移的作用和机制

目的:探究miR-100对乳腺癌细胞株MDA-MB-231迁移能力的调节与机制.方法:Real time-PCR检测人正常乳腺上皮细胞MCF-10A和乳腺癌细胞株MDA-MB-231中miR-100的基础表达水平.应用脂质体法将 miR-100 mimic及阴性对照分别转染乳腺癌细胞株MDA-MB-231,通过real time-PCR检测转染后miR-100的表达水平,细胞划痕实验检测过表达miR-100对MDA-MB-231细胞迁移能力的影响,Western blot方法检测slug、snail和E-cadherin等EMT蛋白表达水平的变化.结果:miR-100在乳腺癌细胞株MDA-MB-231中的表达明显低于人正常乳腺上皮细胞MCF-10A.转染miR-100 mimic的乳腺癌细胞株MDA-MB-231的miR-100表达水平明显增高,细胞划痕实验显示过表达miR-100的MDA-MB-231细胞划痕愈合速度明显减慢.过表达miR-100的MDA-MB-231细胞E-cadherin蛋白表达水平明显增加,而slug和snail蛋白表达水平明显降低.结论:miR-100抑制乳腺癌细胞株MDA-MB-231的迁移能力与其上调E-cadherin,下调slug、snail蛋白表达,抑制EMT有关.     

TRAIL Suppresses Human Breast Cancer Cell Migration via MADD/CXCR7

Background: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can specifically induceapoptosis limited to various cancer cells, so this reagent is considered a promising medicine for cancer therapy.TRAIL also exerts effects on non-apoptotic signals, relevant to processes such as metastasis, autophagy andproliferation in cancer cells. However, the mechanisms of TRAIL-regulated non-apoptotic signals are unclear.The purpose of this study was to investigate MADD/CXCR7 effects in TRAIL-mediated breast cancer cellmigration. Materials and Methods: The ability of MADD/CXCR7 to regulate MVP signaling in TRAIL-mediatedbreast cancer cells migration was evaluated by transwell migration assay, quantitative RT-PCR, Westernblotting and knock down experiments. Results: In this study, we found that treatment with TRAIL resultedin induced expression levels of MADD and CXCR7 in breast cancer cells. Knock down of MADD followed bytreatment with TRAIL resulted in increased cell migration compared to either treatment alone. Similarly, throughoverexpression and knockdown experiments, we demonstrated that CXCR7 also positively regulated TRAILinhibitedmigration. Surprisingly, knock down of MADD lead to inhibition of TRAIL-induced CXCR7 mRNAand protein expression and overexpression of CXCR7 lead to the reduction of MADD expression, indicatingthat MADD is an upstream regulatory factor of TRAIL-triggered CXCR7 production and a negative feedbackmechanism between MADD and CXCR7. Furthermore, we showed that CXCR7 is involved in MADD-inhibitedmigration in breast cancer cells. Conclusions: Our work defined a novel signaling pathway implicated in thecontrol of breast cancer migration.     

Adenosine A2A receptor activation reduces brain metastasis via SDF-1/CXCR4 axis and protecting blood-brain barrier

Brain metastasis is a leading cause of death worldwide, but the mechanism involved remains unclear. Stromal cell-derived factor-1 (SDF-1)/C-X-C motif chemokine receptor 4 (CXCR4) signaling has been reported to induce the directed metastasis of cancers, and adenosine A2A receptor activation suppresses the SDF-1/CXCR4 interaction. However, whether A2A receptor activation implicates the SDF-1/CXCR4 signaling pathway and thus modulates brain metastasis remains unclear. In this study, Western blot was performed to evaluate the protein levels. Cell invasion and migration assays were used to estimate the metastasis ability of PC-9 cells. The viability of cells was demonstrated by lactate dehydrogenase and cell proliferation assays. And the findings in vitro were further identified in nude mice. Notably, adenosine A2A receptor activation inhibited the proliferation and viability of PC-9 cells and thus suppressed the brain metastasis. A2A receptor stimulation protected the function of blood-brain barrier (BBB). The suppression of brain metastasis and the protection of BBB by A2A receptor relied on SDF-1/CXCR4 signaling, and treatment using A2A receptor agonist and CXCR4 antagonist protected the nude mice from malignancy metastasis in vivo. Adenosine A2A receptor activation suppressed the brain metastasis by implicating the SDF-1/CXCR4 axis and protecting the BBB.     

MiR-150-5p Suppresses Colorectal Cancer Cell Migration and Invasion through Targeting MUC4

Growing evidence suggests that miR-150-5p has an important role in regulating genesis of various types of cancer. However, the roles and the underlying mechanisms of miR-150-5p in development of colorectal cancer (CRC) remain largely unknown. Transwell chambers were used to analyze effects on cell migration and invasion by miR-150-5p. Quantitative real-time PCR (qRT-PCR), Western blotting and dual-luciferase 3’ UTR reporter assay were carried out to identify the target genes of miR-150-5p. In our research, miR-150-5p suppressed CRC cell migration and invasion, and MUC4 was identified as a direct target gene. Its effects were partly blocked by re-expression of MUC4. In conclusiomn, miR-150-5p may suppress CRC metastasis through directly targeting MUC4, highlighting its potential as a novel agent for the treatment of CRC metastasis.     

胰岛素样生长因子-1受体在乳腺癌组织和正常乳腺组织中的表达

目的　探讨胰岛素样生长因子 1受体 (IGF 1R)在乳腺癌组织和正常乳腺组织中的分布和表达。方法　采用免疫组织化学方法 ,对乳腺癌组织和正常的乳腺组织中IGF 1R的表达情况进行检测分析。结果　乳腺癌组织中IGF 1R的阳性表达率均明显较正常乳腺组织高 (P <0 .0 5 )。结论　由此推断 ,IGF 1R在乳腺癌发生发展过程中发挥着重要作用。IGF 1R有可能成为临床乳腺癌诊断和判断预后的一个重要指标。     

Role of TMPRSS4 Modulation in Breast Cancer Cell Proliferation

Background: TMPRSS4 is a novel Type II transmembrane serine protease found at the surface of the cells andis involved in the development and cancer progression. However, TMPRSS4 functions in breast cancer remain poorunderstand. The present study investigated the function of TMPRSS4 in the breast cancer cells and the potentialmechanistic action underling. Materials and Methods: The lentiviral vectors causing TMPRSS4 down-regulation andover-expression were established and transfected in MDA-MB-468 and MCF-7 cells, respectively. By using the CCK-8 assay, cell proliferation was analyzed. Moreover, western blot was used to detect the expression of certain proteinsrelated to cell apoptosis (Bax and Bcl2) signaling pathway and telomere maintenance (POT1, TPP1, and UBE2D3).Cell cycle and cell apoptosis were also analyzed by using the Flow cytometry analysis. TMPRSS4 expression wasdetected at the mRNA level and protein level by performing qPCR and western blot technique, respectively. Results:TMPRSS4 expression is inhibited in stable transfected MDA-MB-468-shTMPRSS4 cells compared to the controlMDA-MB-468-NC and its expression is up-regulated in stable transfected MCF-7-TMPTSS4 compared to its controlMCF-7-NC. Moreover, TMPRSS4 silencing in breast cancer reduces cells proliferation by promoting cell cycle arrestin G2/M phase, cell apoptosis, and telomere maintenance impairment while the TMPRSS4 overexpression increasescells proliferation through cell apoptosis reduction and telomere maintenance reinforcement associated with insignificantchange in cell cycle progression. Conclusion: TMPRSS4 plays important roles in cancer progression and may beconsidered as a good therapeutic target for cancer gene therapy especially breast cancer.     

MicroRNA-664 Targets Insulin Receptor Substrate 1 to Suppress Cell Proliferation and Invasion in Breast Cancer

A large number of microRNAs (miRNAs) have been previously demonstrated to be dysregulated in breast cancer (BC), and alterations in miRNA expression may affect the initiation and progression of BC. This study showed that miR-664 expression was obviously reduced in BC tissues and cell lines. Resumption of the expression of miR-664 attenuated the proliferation and invasion of BC cells. The molecular mechanisms underlying the inhibitory effects of BC cell proliferation and invasion by miR-664 were also studied. Insulin receptor substrate 1 (IRS1) was identified as a novel and direct target of miR-664. In addition, siRNAmediated silencing of IRS1 expression mimicked the suppressive effects of miR-664 overexpression in BC cells. Rescue experiments demonstrated that recovered IRS1 expression partially antagonized the inhibition of proliferation and invasion of BC cells caused by miR-664 overexpression. Thus, miR-664 may serve as a tumor suppressor in BC by directly targeting IRS1. Moreover, miR-664 downregulation in BC may contribute to the occurrence and development of BC, suggesting that miR-664 may be a novel therapeutic target for patients with BC.