FAP和Rho 家族蛋白在卵巢癌侵袭迁移中的作用

目的：明确FAP 是否通过RhoA/ROCK、Rac1-GTP 通路发挥促增殖、侵袭和迁移作用。方法：用MTT 实验,Transwell 实验 和迁移实验检测FAP、RhoA/ROCK、Rac1-GTP 对卵巢癌细胞系HO-8910PM 的增殖,侵袭和迁移的影响。结果：1、MTT 法,迁移和 侵袭实验证实用Y-27632 抑制RhoA/ROCK 途径能够促进卵巢癌细胞的增殖、迁移和侵袭,与FAP 联合作用时促进作用增强。 2、MTT 法, 迁移和侵袭实验证实NSC23766 抑制Rac1 途径能够抑制卵巢癌细胞的增殖、迁移和侵袭,与FAP 联合作用使FAP 的 促进作用减弱。结论：1、RhoA/ROCK 通路抑制HO-8910PM 细胞增殖、迁移和侵袭;Rac1-GTP 促进HO-8910PM 细胞增殖、迁移 和侵袭。2、FAP不是通过RhoA/ROCK而是通过Rac1-GTP 信号通路在HO-8910PM细胞发挥促增殖、迁移和侵袭作用的。     

FAP-alpaha在胞外信号传递中与整合素的关系

目的：研究成纤维激活蛋白（Fibroblast Activation Protein,FAP）在促进卵巢癌细胞发生侵袭、迁移、增殖过程中与整合素α3β1、尿激酶型纤溶酶原激活剂受体（uPAR）的关系。方法：1）．免疫共沉淀共同检测整合素α3β1、uPAR在HO-8910PM上是否为二聚体。2）．transwell侵袭实验、迁移实验检测抑制整合素α3β1、uPAR后,卵巢癌细胞系HO-8910PM的侵袭迁移能力;3）．抑制整合素α3β1、uPAR后,再给予FAPet对HO-8910PM侵袭、迁移和增殖的影响。结果：1）．整合素α3β1、uPAR在HO-8910PM细胞外同一个位置表达;2）．抑制整合素α3β1能够明显抑制HO-8910PM的侵袭、迁移、增殖能力,并且可以抑制FAP对肿瘤的作用。3）．PAI—1抑制uPAR后,HO—8910PM的侵袭、迁移、增殖无明显变化,同时对FAP也无明显作用。结论：整合素α3β1和uPAR在HO-8910PM是一个复合体,FAPα,在细胞外是通过整合素α3β1传递信号进入细胞内而不是通过uPAR,整合素α3β1是通过uPAR与肿瘤细胞相连接。     

FAPα在胞外信号传递中与整合素的关系

目的:研究成纤维激活蛋白(Fibroblast Activation Protein,FAP)在促进卵巢癌细胞发生侵袭、迁移、增殖过程中与整合素α3β1、尿激酶型纤溶酶原激活剂受体(uPAR)的关系。方法:1).免疫共沉淀共同检测整合素α3β1、uPAR在HO-8910PM上是否为二聚体。2).transwell侵袭实验、迁移实验检测抑制整合素α3β1、uPAR后,卵巢癌细胞系HO-8910PM的侵袭迁移能力;3).抑制整合素α3β1、uPAR后,再给予FAPα对HO-8910PM侵袭、迁移和增殖的影响。结果:1).整合素α3β1、uPAR在HO-8910PM细胞外同一个位置表达;2).抑制整合素α3β1能够明显抑制HO-8910PM的侵袭、迁移、增殖能力,并且可以抑制FAP对肿瘤的作用。3).PAI-1抑制uPAR后,HO-8910PM的侵袭、迁移、增殖无明显变化,同时对FAP也无明显作用。结论:整合素α3β1和uPAR在HO-8910PM是一个复合体,FAPα在细胞外是通过整合素α3β1传递信号进入细胞内而不是通过uPAR,整合素α3β1是通过uPAR与肿瘤细胞相连接。     

小鼠胚胎对人卵巢癌细胞生物学行为影响的体外研究

探讨体外共培养环境中小鼠胚胎对人卵巢癌细胞HO8910PM的影响.通过小鼠胚胎与肿瘤细胞体外共培养模型观察小鼠胚胎对肿瘤细胞的形态及生长行为的影响,Annexin V-EGFP/PI原位检测与小鼠胚胎共培养后肿瘤细胞的凋亡情况,MTT粘附实验、transwell迁移及侵袭实验研究与小鼠胚胎共培养后的人卵巢癌细胞的粘附性、迁移性及侵袭性的变化.共培养时小鼠胚胎能够侵入人卵巢癌细胞并推开肿瘤细胞形成自己的生长空间,激光共聚焦显微镜下见胚胎周围的肿瘤细胞凋亡增加,与对照组比较共培养后的HO8910PM肿瘤细胞的粘附性、迁移性及侵袭性均显著降低(P0.05、P0.01).结果表明体外共培养体系中小鼠胚胎能够侵袭肿瘤细胞,促进人卵巢癌细胞的凋亡,并使其粘附性、迁移及侵袭相关恶性行为降低.     

OPN对卵巢癌Hey细胞增殖、迁移和侵袭能力的影响

目的:探讨骨桥蛋白(Osteopontin,OPN)对卵巢癌Hey细胞增殖、迁移和侵袭能力的影响及其可能涉及的分子机制。方法:选择卵巢癌细胞株Hey、HO8910、A2780和正常卵巢上皮细胞IOSE80,采用Western blot检测OPN蛋白的表达情况。对OPN表达量相对较高的Hey细胞株的OPN基因敲减或过表达,运用CCK-8、平板克隆实验、细胞划痕、Transwell侵袭实验等方法研究OPN对细胞增殖、迁移及侵袭能力的影响,采用Western blot检测Wnt/β-catenin通路相关蛋白β-catenin、CyclinD1、c-myc的表达情况。结果:OPN在卵巢癌细胞株Hey、HO8910、A2780内均有表达,且表达量均高于正常卵巢上皮细胞IOSE80。si RNA-OPN转染卵巢癌Hey细胞沉默OPN表达,CCK-8和平板克隆实验显示沉默OPN能够降低Hey细胞的增殖能力,划痕实验和Transwell侵袭实验显示下调OPN可降低Hey细胞的迁移和侵袭能力,Wnt/β-catenin通路相关蛋白β-catenin、CyclinD1、c-myc表达下降。ex-OPN转染Hey细胞使OPN表达升高,与对照组相比,OPN过表达能够增强Hey细胞的增殖、迁移和侵袭能力,并且Wnt/β-catenin通路相关蛋白β-catenin、CyclinD1、c-myc表达升高。结论:OPN能够促进卵巢癌细胞的增殖、迁移及侵袭能力,该作用可能通过促进Wnt/β-catenin信号通路实现。     

Survivin和RhoA双基因沉默对卵巢癌细胞的增殖与侵袭影响

目的：探讨RNA干扰（RNAi）双向沉默Survivin和RhoA基因表达对人卵巢癌H08910PM细胞增殖、凋亡及侵袭能力的影响。方法：构建Survivin和RhoA基因的串联microRNA干扰载体（Survivin-RhoA-microRNA）,通过脂质体介导转染人卵巢癌H08910PM细胞作为实验组,对照组为空载体转染组。转染48小时后,RT—PCR检测Survivin和RhoA的mRNA表达,Western．Blot检测Survivin和RhoA的蛋白质表达;利用MTT法、Transwell小室体外侵袭实验及流式细胞术检测转染后卵巢癌细胞的增殖、侵袭及凋亡情况。结果：Survivin—RhoA—microRNA明显抑制了H08910PM细胞中Survivin和RhoA基因mRNA和蛋白的表达：转染48小时后,实验组SurvivinmRNA和RhoAmRNA表达抑制率分别为68．82％、90．23％,Survivin和RhoA蛋白表达抑制率分别为63．91％、88．47％;MTT分析显示实验组细胞OD490的值为0．706±0．177,较对照组（1．172±0．241）明显降低,差异有统计学意义（P〈0．05）;流式细胞术检测实验组细胞凋亡率为36．41±3．34％,较对照组（2．92±0．78％）明显升高（P〈0．01）;实验组细胞侵袭百分比为12．56±6．17％,较对照组（32．96±5．14％）明显降低（P〈0．05）。结论：成功构建Survivin和RhoA串联microRNA干扰载体（Survivin．RhoA．microRNA）。Survivin和RhoA双基因沉默显著抑制了卵巢癌H08910PM细胞的增殖和侵袭能力,并增加其凋亡率,两者具有协同作用,为双靶点治疗卵巢癌提供了新的思路和策略。     

姜黄素对A549肺癌细胞增殖、侵袭和迁移的抑制作用及与Keap1/Nrf2信号通路的关系

目的探究姜黄素对肺癌A549细胞增殖、侵袭和迁移的抑制作用及其机制。方法通过MTT法检测姜黄素对A549细胞增殖能力的影响;通过流式细胞术测定姜黄素对A549细胞凋亡的调节作用;通过Transwell试验,观察姜黄素对肺癌A549细胞的侵袭和迁移能力的影响;采用Western blot实验和RT-PCR实验,观察姜黄素对其Keap1/Nrf2信号通路的调节作用。结果增殖实验、凋亡实验和Transwell实验显示,姜黄素能够显著性抑制癌细胞的增殖、侵袭和迁移,并能够促进其凋亡。Western blot检测显示姜黄素能够显著抑制Keap1和Nrf2表达;RT-PCR实验结果显示姜黄素能够显著抑制Keap1mRNA和Nrf2 mRNA表达。结论姜黄素可能通过抑制Keap1/Nrf2信号通路蛋白的表达而抑制肺癌A549细胞的增殖、迁移和侵袭,并促进其凋亡。     

RhoA在上皮性卵巢癌细胞中的表达与侵袭性的相关性

培养上皮性卵巢癌细胞H08910PM、H08910及正常卵巢上皮细胞HOSEA 3种细胞.对3种细胞分别采用免疫细胞化学法检测RhoA的表达、Transwell小室体外侵袭试验测定体外侵袭能力、与人脐静脉内皮细胞HUVEC建立细胞共培养系统测定血管形成能力.结果表明,RhoA在H08910PM细胞中表达最强,在H08910中次之,在HOSEA中最弱;3种细胞的侵袭能力以H08910PM最强,H08910较弱,HOSEA无侵袭能力;H08910PM的血管形成能力最强,H08910其次,HOSEA无血管形成能力(P<0.01).Pearson相关分析结果显示,RhoA表达水平分别与细胞侵袭能力及血管形成能力呈正相关(P<0.05).RhoA在上皮性卵巢癌细胞中表达水平越高,细胞体外侵袭及血管形成能力随之更强.RhoA可能作为重要因子参与上皮性卵巢癌的侵袭转移过程.     

促卵泡激素结合片段通过P13K／Akt—cyclinD1通路抑制卵巢癌细胞增殖活性

目的：研究促卵泡激素（FSH）结合片段对卵巢上皮性细胞癌细胞株hey增殖活性的影响。方法：应用卵巢上皮性细胞癌细胞株hey作为实验对象,分别加入FSH、FSH结合片段、FSH＋FSH结合片段。用四甲基偶氮唑蓝（MTT）法检测卵巢癌hey细胞的生长状况,Westernblot检测cyclinD1、Akt、pAkt分子的表达。结果：FSH对卵巢癌hey细胞的生长有明显的促进作用,细胞生长活性提高21％。FSH结合片段对卵巢癌细胞的生长有明显抑制作用,对细胞平均抑制率为22％,且能下调cyclinD1的表达,在2．5×10^-9Mol／L达70％。FSH结合片段对FSH有竞争抑制作用,细胞抑制率为18％,能抑制FSH上调cyclinD1的作用,抑制率为61％。FSH能上调pAkt的表达,对Akt的袁达没有明显影响,在40mlU／ml的浓度时pAkt／Akt上调率达224％。FSH结合片段对Akt的表达没有明显影响,但能明显下调pAkt的表达,且呈时间依赖性（在15分钟时达66％）和剂量依赖性（在2．5×10^-9Mol／L达31％）。FSH结合片段能抑制FSH上调pAkt的作用,抑制率为80％。结论：FSH结合片段可通过抑制FSH诱导的P13FdAkt-cycl—inD1信号通路进而抑制上皮性卵巢癌hey细胞的增殖活性,     

卵巢癌细胞中RhoA和NF-κB表达与癌细胞增殖、侵袭能力的关系

目的:探讨卵巢癌细胞中RhoA和NF-κB表达与癌细胞增殖、侵袭能力的关系。方法:采用RT-PCR和Western blot检测卵巢癌细胞A2780、SW626、Skow3和正常卵巢上皮HOSEA中RhoA和NF-κB的表达;MTT法测定A2780、SW626、Skow3的增殖能力;Transwell小室体外侵袭实验检验细胞侵袭能力。结果:三种卵巢癌细胞中RhoA与NF-κB的表达无差异,但均高于正常卵巢上皮HOSEA中表达(P0.05);MTT实验结果显示在24 h、48 h、72 h、96 h、120 h时间点,过表达RhoA组和NF-κB组吸光光度值大于对照组(P0.05);RhoA转染后,A2780、SW626、Skow3、HOSEA 4种细胞的穿膜细胞数测值分别是33.56±12.42、56.21±9.53、25.53±11.89、0,说明SW626侵袭力最强,HOSEA无侵袭力(P0.05),不同细胞间侵袭力存在差异,且具有统计学意义(P0.05)。NF-κB转染后,A2780、SW626、Skow3、HOSEA 4种细胞的穿膜细胞数分别为22.53±12.57、33.90±10.12、45.91±9.02、0,其中穿膜细胞数最多的是Skow3细胞,不同细胞间穿膜细胞数比较差异具有统计学意义(P0.05)。结论:卵巢癌细胞中RhoA与NF-κB表达增加,可增强细胞增殖能力,并使细胞具有侵袭性。     

ROCKⅠ／Ⅱ基因下调对血管平滑肌细胞迁移及增殖的影响

目的：探讨ROCK亚型ROCKⅠ和ROCKⅡ对血管平滑肌细胞（A7r5）迁移及增殖的影响。方法：利用Western blot技术检测ROCKⅠ和ROCKⅡ蛋白在A7r5细胞中的表达水平;利用siRNA技术使ROCKⅠ和ROCKⅡ基因表达分别下调,并检测基因下调后蛋白表达水平;利用Boyden小室法,观察ROCKⅠ和ROCKⅡ基因下调后及ROCK特异抑制剂Y-27632对PDGF诱导的A7r5细胞迁移的影响;使用MTT法检测ROCKⅠ和ROCKⅡ基因下调后对A7r5细胞生长曲线的影响。结果：ROCKⅠ和ROCKⅡ在A7r5细胞中的蛋白表达水平不同,ROCKⅡ较ROCKⅠ的表达水平高4倍;通过对A7r5细胞进行ROCKⅠ和ROCKⅡsiRNA转染,使二者蛋白表达水平分别下调83.4％和94.7％;基因表达下调后,ROCKⅠ明显抑制了PDGF诱导的A7r5细胞的迁移,而ROCKⅡ无明显影响,Y-27632也抑制了A7r5细胞的迁移;ROCKⅠ和ROCKⅡ基因下调后对A7r5细胞生长曲线的影响无明显差别。结论：ROCKⅠ在血管平滑肌细胞迁移过程中起主导作用,ROCKⅠ和ROCKⅡ对血管平滑肌细胞的增殖作用无明显差异。     

Polarization and Migration of Hematopoietic Stem and Progenitor Cells Rely on the RhoA/ROCK I Pathway and an Active Reorganization of the Microtubule Network

Understanding the physiological migration of hematopoietic progenitors is important, not only for basic stem cell research, but also in view of their therapeutic relevance. Here, we investigated the role of the Rho kinase pathway in the morphology and migration of hematopoietic progenitors using an ex vivo co-culture consisting of human primary CD34⁺ progenitors and mesenchymal stromal cells. The addition of the Rho kinase inhibitor Y-27632 led to the abolishment of the uropod and microvillar-like structures of hematopoietic progenitors, concomitant with a redistribution of proteins found therein (prominin-1 and ezrin). Y-27632-treated cells displayed a deficiency in migration. Time-lapse video microscopy revealed impairment of the rear pole retraction. Interestingly, the knockdown of ROCK I, but not ROCK II, using RNA interference (RNAi) was sufficient to cause the referred morphological and migrational changes. Unexpectedly, the addition of nocodazole to either Y-27632- or ROCK I RNAi-treated cells could restore their polarized morphology and migration suggesting an active role for the microtubule network in tail retraction. Finally, we could demonstrate using RNAi that RhoA, the upstream regulator of ROCK, is involved in these processes. Collectively, our data provide new insights regarding the role of RhoA/ROCK I and the microtubules in the migration of stem cells.     

Hypoxia promotes HO-8910PM ovarian cancer cell invasion via Snail-mediated MT1-MMP upregulation

The molecular mechanisms of ovarian cancer cell invasion under hypoxia remain unclear. Here we employed a 3D collagen model and chick chorioallantoic membrane (CAM) invasion assay to explore the influence of hypoxia on ovarian cancer cell invasion. Hypoxia (both 1% O₂ and CoCl₂ 150 and 250 µM) induced HO-8910PM ovarian cancer cell invasion in 3D collagen and collagenolysis determined by hydroxyproline. Pretreatment with a hypoxia inducible factor-1α inhibitor, YC-1, or MMP inhibitor, GM6001, significantly inhibited 3D collagen invasion and degradation and cell proliferation. Hypoxia stimulated both mRNA and protein expressions of membrane-type 1 matrix metalloproteinase (MT1-MMP) and promoted MT1-MMP translocation to the cell surface in an YC-1 sensitive manner. MT1-siRNA transfection inhibited hypoxia-induced invasion, proliferation, and collagen degradation of cells in 3D collagen. Hypoxia stimulated Snail mRNA and protein expression as well as translocation to nucleus in an YC-1 sensitive manner. Overexpression of Snail with a recombinant plasmid in HO-8910PM cells resulted in an enhanced invasion in 3D collagen. Transfection with Snail-specific siRNA significantly decreased MT1-MMP expression and 3D collagen invasion. Hypoxia-treated cells significantly broke the upper CAM surface of 11-day-old chick embryos and infiltrated interstitial tissue, completely blocked in the presence of YC-1 or GM6001, or after MT1-MMP siRNA or Snail siRNA transfection. Together, these data suggest that hypoxia promotes HO-8910PM ovarian cancer cell traffic through 3D matrix via Snail-mediated MT1-MMP upregulation, a possible molecular mechanism of ovarian cancer cell invasion under hypoxia.     

Functional Expression of TWEAK and the Receptor Fn14 in Human Malignant Ovarian Tumors: Possible Implication for Ovarian Tumor Intervention

The aim of this current study was to investigate the expression of the tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) and its receptor fibroblast growth factor-inducible 14 (Fn14) in human malignant ovarian tumors, and test TWEAK’s potential role on tumor progression in cell models in-vitro. Using immunohistochemistry (IHC), we found that TWEAK and its receptor Fn14 were expressed in human malignant ovarian tumors, but not in normal ovarian tissues or in borderline/benign epithelial ovarian tumors. High levels of TWEAK expression was detected in the majority of malignant tumors (36 out of 41, 87.80%). Similarly, 35 out of 41 (85.37%) malignant ovarian tumors were Fn14 positive. In these malignant ovarian tumors, however, TWEAK/Fn14 expression was not corrected with patients’ clinical subtype/stages or pathological features. In vitro, we demonstrated that TWEAK only inhibited ovarian cancer HO-8910PM cell proliferation in combination with tumor necrosis factor-α (TNF-α), whereas either TWEAK or TNF-α alone didn’t affect HO-8910PM cell growth. TWEAK promoted TNF-α production in cultured THP-1 macrophages. Meanwhile, conditioned media from TWEAK-activated macrophages inhibited cultured HO-8910PM cell proliferation and invasion. Further, TWEAK increased monocyte chemoattractant protein-1 (MCP-1) production in cultured HO-8910PM cells to possibly recruit macrophages. Our results suggest that TWEAK/Fn14, by activating macrophages, could be ovarian tumor suppressors. The unique expression of TWEAK/Fn14 in malignant tumors indicates that it might be detected as a malignant ovarian tumor marker.     

ROCK inhibitor Y-27632 suppresses dissociation-induced apoptosis of murine prostate stem/progenitor cells and increases their cloning efficiency

Activation of the RhoA/ROCK signaling pathway has been shown to contribute to dissociation-induced apoptosis of embryonic and neural stem cells. We previously demonstrated that approximately 1 out of 40 Lin(-)Sca-1(+)CD49f(high) (LSC) prostate basal epithelial cells possess the capacities of stem cells for self-renewal and multi-lineage differentiation. We show here that treating LSC cells with the ROCK kinase inhibitor Y-27632 increases their cloning efficiency by 8 fold in an in vitro prostate colony assay. Y-27632 treatment allows prostate colony cells to replate efficiently, which does not occur otherwise. Y-27632 also increases the cloning efficiency of prostate stem cells in a prostate sphere assay and a dissociated prostate cell regeneration assay. The increased cloning efficiency is due to the suppression of the dissociation-induced, RhoA/ROCK activation-mediated apoptosis of prostate stem cells. Dissociation of prostate epithelial cells from extracellular matrix increases PTEN activity and attenuates AKT activity. Y-27632 treatment alone is sufficient to suppress cell dissociation-induced activation of PTEN activity. However, this does not contribute to the increased cloning efficiency, because Y-27632 treatment increases the sphere-forming unit of wild type and Pten null prostate cells to a similar extent. Finally, knocking down expression of both ROCK kinases slightly increases the replating efficiency of prostate colony cells, corroborating that they play a major role in the Y-27632 mediated increase in cloning efficiency. Our study implies that the numbers of prostate cells with stem/progenitor activity may be underestimated based on currently employed assays, supports that dissociation-induced apoptosis is a common feature of embryonic and somatic stem cells with an epithelial phenotype, and highlights the significance of environmental cues for the maintenance of stem cells.     

Rho kinases regulate corneal epithelial wound healing

We have previously shown that Rho small GTPase is required for modulating both cell migration and proliferation through cytoskeleton reorganization and focal adhesion formation in response to wounding. In the present study, we investigated the role of Rho kinases (ROCKs), major effectors of Rho GTPase, in mediating corneal epithelial wound healing. Both ROCK 1 and 2 were expressed and activated in THCE cells, an SV40-immortalized human corneal epithelial cell (HCEC) line, in response to wounding, lysophosphatidic acid, and heparin-binding EGF-like growth factor (HB-EGF) stimulations. The ROCK inhibitor Y-27632 efficiently antagonized ROCK activities without affecting Rho activation in wounded HCECs. Y-27632 promoted basal and HB-EGF-enhanced scratch wound healing and enhanced cell migration and adhesion to matrices, while retarded HB-EGF induced cell proliferation. E-cadherin- and beta-catenin-mediated cell-cell junction and actin cytoskeleton organization were disrupted by Y-27632. Y-27632 impaired the formation and maintenance of tight junction barriers indicated by decreased trans-epithelial resistance and disrupted occludin staining. We conclude that ROCK activities enhance cell proliferation, promote epithelial differentiation, but negatively modulate cell migration and cell adhesion and therefore play a role in regulating corneal epithelial wound healing.