BAG3 蛋白Ser187位点磷酸化诱导人甲状腺癌FRO 细胞上皮间质转化

Bcl-2相关抗凋亡蛋白3（Bcl-2 associated athanogene 3,BAG3）是BAG家族的重要成员,调节肿瘤细胞的粘附、迁移和侵袭,促进恶性肿瘤的复发和转移.本室前期工作证明,PKCδ可催化BAG3的Ser187位点磷酸化.本文研究BAG3蛋白磷酸化修饰对甲状腺癌FRO 细胞EMT表型转化的影响.稳定转染野生型WT-BAG3、模拟磷酸化型S187D-BAG3、阻碍磷酸化型S187A-BAG3 FRO细胞后,观察细胞形态的变化.结果显示,稳定转染模拟磷酸化型S187D-BAG3引起甲状腺癌FRO细胞呈现明显的间质细胞形态.实时PCR 和Western印迹,结果显示,稳定表达S187D-BAG3显著上调间质细胞标记物N-cadherin和波形蛋白mRNA与蛋白质在FRO细胞的表达,但下调上皮细胞标记物E-cadherin的mRNA和蛋白质的表达.同时,免疫荧光结果显示,稳定过表达S187D-BAG3的FRO细胞,E-cadherin和β-catenin出现向核周的内化.本文结果提示,BAG3蛋白磷酸化修饰可诱导甲状腺癌FRO细胞上皮间质转化.     

整合素β2促进人乳腺癌细胞MCF-7的迁移,侵袭与粘附

本研究主要目标为探讨整合素β2 (ITGB2)的高表达对人乳腺癌细胞MCF-7迁移,侵袭与粘附能力的影响。本研究首先构建了ITGB2过表达质粒,实验设阴性对照组(pcDNA-3.1+)与ITGB2基因过表达组(pcDNA-3.1+/ITGB2)。ITGB2过表达质粒转染MCF-7细胞后,采用逆转录PCR与Western blotting方法分别检测ITGB2 mRNA转录水平与蛋白翻译水平;流式细胞术检测细胞周期的改变;划痕实验检测细胞横向迁移能力;Transwell小室实验检测细胞纵向迁移能力及侵袭能力;人脐静脉血管内皮细胞(HUVEC)粘附实验检测癌症细胞与血管内皮细胞之间的粘附能力,Western blotting实验检测侵袭相关指标MMP9,整合素经典通路中FAK蛋白磷酸化水平的改变。研究结果表明:转染ITGB2过表达质粒后,MCF-7细胞中ITGB2的m RNA水平(p<0.01)与蛋白水平(p<0.05)均显著增高;流式细胞术实验中,实验组S期的细胞所占比例与对照组无明显差异;划痕实验与Transwell小室实验中,实验组的迁移侵袭能力显著性增强;人脐静脉血管内皮细胞粘附实验中,实验组乳腺癌细胞与血管内皮细胞的粘附能力强于对照组(p<0.05);且Western blotting结果显示MMP9和p-FAK蛋白水平明显上升。由以上结果可得出结论,过表达ITGB2后会增强人乳腺癌细胞MCF-7的迁移、侵袭与粘附能力,而对其增殖能力无明显影响。     

莪术油对Hela细胞迁移及侵袭的初步实验研究

目的:探讨莪术油对宫颈癌细胞系Hela细胞迁移和侵袭的影响。方法:宫颈癌细胞系Hela细胞培养后分实验组及对照组,实验组以125μg/m L浓度的莪术油作用48 h,对照组细胞不进行任何处置。细胞划痕实验检测莪术油对Hela细胞迁移的影响;Transwell小室实验检测莪术油对Hela细胞侵袭的影响。结果:0 h处理后实验组、对照组划痕宽度无统计学差异(P0.05);48 h后实验组划痕宽度明显大于对照组的划痕宽度(4.33±0.58 m vs 2.17±0.29 m,P0.05)。实验组48 h后的穿透细胞数明显少于对照组(26.2±1.3个vs 62.2±2.3个,P0.05)。结论:莪术油可抑制宫颈癌细胞的迁移与侵袭。     

BMP9对人肺腺癌A549细胞侵袭、迁移的影响及机制的研究

BMP9属于TGF-β超家族的成员,参与多种细胞的增殖、分化、凋亡、侵袭、转移过程。以人肺腺癌细胞A549作为目的细胞,采用腺病毒体外感染方式,外源性高表达BMP9。RTPCR及Western blot检测重组细胞中BMP9的表达,通过细胞划痕实验、Transwell侵袭实验检测AdBMP9细胞侵袭及迁移改变,RT-PCR及Western blot检测感染BMP9腺病毒后IL-6的mRNA和蛋白表达;Western blot检测PI3K/Akt信号通路中总Akt和磷酸化Akt蛋白的表达。结果显示:与对照组细胞相比,感染BMP9腺病毒后,A549中BMP9 mRNA和蛋白表达明显升高;实验组划痕愈合率由对照的(85.4±2.1)%与(86.5±3.4)%上升至(97.4±2.6)%(P0.05);实验组穿膜细胞数由对照的(115.5±13.1)个与(123.3±14.9)个上升至(224.3±24.6)个(P0.05);与对照组细胞相比,AdBMP9组IL-6的表达上调,磷酸化Akt蛋白表达上调。该研究表明,BMP9可能通过上调IL-6的表达,激活PI3K/Akt信号通路,促进人肺腺癌A549的侵袭、迁移。     

BMP9对人肺腺癌AS49细胞侵袭、迁移的影响及机制的研究

BMP9属于TGF-β超家族的成员,参与多种细胞的增殖、分化、凋亡、侵袭、转移过程。以人肺腺癌细胞A549作为目的细胞,采用腺病毒体外感染方式,外源性高表达BMP9。RT-PCR及Westernblot检测重组细胞中BMP9的表达,通过细胞划痕实验、Transwell侵袭实验检测AdBMP9细胞侵袭及迁移改变,RT-PCR及Westernblot检测感染BMP9腺病毒后IL-6的mRNA和蛋白表达：Westemblotg检测NPI3K／Akt信号通路中总Akt和磷酸化Akt蛋白的表达。结果显示：与对照组细胞相比,感染BMP9腺病毒后,A549中BMP9mRNA和蛋白表达明显升高;实验组划痕愈合率由对照的（85．4±2．11％与（86．5±3．4）％上升至（97．4±2．6）％（P〈0．05）;实验组穿膜细胞数由对照的（115．5±13．1）个与（123．3±14．9）个上升至（224．3±24．6）个（P〈0．05）;与对照组细胞相比,AdBMP9组IL-6的表达上调,磷酸化Akt蛋白表达上调。该研究表明,BMP9可能通过上调IL-6的表达,激活P13K／Akt信号通路,促进人肺腺癌A549的侵袭、迁移。     

重组人IL-18-EGF的双顺反子表达及其对NK细胞和肝癌细胞的影响

构建重组人IL-18-EGF肿瘤靶向分子双顺反子原核表达系统,研究重组人IL-18-EGF融合蛋白对人自然杀伤细胞（natural killer cell,NK细胞）和人肝癌细胞（SMMC-7721）的影响。构建重组人IL-18-EGF原核双顺反子表达系统pET28a（＋）-proIL-18-EGF-Caspase-4/BL21,重组蛋白经纯化后,作用于NK细胞,应用CCK-8法和ELISA试剂盒分别检测NK细胞的增殖情况和IFN-γ的分泌量。Cy3荧光标记IL-18-EGF检测融合蛋白与肿瘤细胞表面EGFR的结合情况。IL-18-EGF与NK细胞共同孵育24 h后,取培养上清液作用于人肝癌细胞SMMC-7721,分别使用细胞划痕实验和Transwell小室实验检测IL-18-EGF对肝癌细胞迁移和侵袭能力的影响。实验结果显示：重组人IL-18-EGF能加快NK细胞的增殖,促进NK细胞分泌IFN-γ;IL-18-EGF能与肿瘤细胞表面EGFR特异性结合;细胞划痕实验中,重组人IL-18-EGF组空白区域的抗填充能力高于对照组;Transwell小室实验中,12,24,48 h时IL-18-EGF组细胞穿膜数分别为94.6±2.9、101.8±4.0和116.2±4.5,均显著低于相应时间的对照组（分别为128.6±8.5、133.0±7.5和138.8±5.4）（P〈0.05）。以上结果表明,IL-18-EGF对人肝癌细胞SMMC-7721的迁移和侵袭能力有明显的抑制作用,能提高机体的免疫能力,有可能作为辅助药物运用于肝癌的治疗。     

TRPC3对上皮性卵巢癌细胞株迁移和侵袭的影响

目的:探讨瞬时受体电位通道C3(TRPC3)对人卵巢癌细胞迁移、侵袭能力的影响。方法:采用蛋白免疫印迹法和实时荧光定量PCR法分别检测卵巢癌细胞株SKOV3、ES-2和HEY-T30中TRPC3蛋白和m RNA的表达水平。通过Transwell迁移实验(不含Matrigel胶的Transwell小室)和Transwell侵袭实验分别检测卵巢癌细胞株SKOV3、ES-2和HEY-T30的迁移、侵袭能力。结果:在SKOV3、ES-2和HEY-T30三种卵巢癌细胞株中,ES-2中TRPC3的蛋白和m RNA表达均显著高于其他两株(P0.05)。Transwell迁移实验和Transwell侵袭实验显示卵巢癌细胞株ES-2的迁移、侵袭能力均显著高于其他两种细胞株(P0.05)。结论:瞬时受体电位通道C3(TRPC3)在ES-2人卵巢癌细胞中高表达,并可能促进人卵巢癌细胞的迁移、侵袭。     

NID1促进人卵巢癌细胞OVCAR-3侵袭转移及分子机制的研究

Nidogen-1(NID1)是新发现的一个候选卵巢癌诊断标志物,该研究旨在初探其在卵巢癌细胞中的生物学功能。首先构建稳定过表达外源NID1的人卵巢癌细胞系OVCAR-3,然后采用划痕实验和Transwell迁移和侵袭实验检测细胞的迁移侵袭能力,并通过荧光定量PCR和Western blot检测细胞中上皮–间质转化(epithelial-mesenchymal transition,EMT)相关蛋白和ERK/MAPK通路蛋白的表达情况。结果显示,与空载细胞相比,稳定过表达NID1的OVCAR-3细胞其划痕愈合能力、细胞迁移和侵袭能力均明显增强。较之空载细胞的上皮细胞样外形,稳定过表达NID1的OVCAR-3细胞呈间质细胞样外形,其上皮细胞标志分子E-cadherin表达下调,间质细胞标志分子(包括Vimentin和N-cadherin)和EMT相关转录因子Twist-2表达上调。此外,稳定过表达NID1的OVCAR-3细胞中ERK1/2的磷酸化水平升高,经ERK/MAPK通路的抑制剂U0126下调ERK1/2的磷酸化水平后其Ecadherin、Vimentin、N-cadherin和Twist-2表达水平出现逆转。这些结果提示,NID1可能通过激活ERK/MAPK通路促进卵巢癌细胞的EMT过程,进而增强其侵袭转移的能力。     

温热抑制人胃癌SGC7901细胞增殖、迁移

目的探讨温热对人胃癌SGC790l细胞增殖、迁移的影响。方法对照组常温(37℃)下培养人胃癌SGC790l细胞,实验组43℃水浴加热0.5h、1h、2h、3h后培养24h,采用倒置显微镜观察胃癌细胞的形态结构变化;Hoechst-33258荧光染色观察细胞核的变化;四甲基偶氮唑盐比色法(MTT)检测细胞增殖抑制;细胞划痕愈合实验观察温热对胃癌细胞的运动迁移能力的影响;体外细胞侵袭实验(Transwell实验)观察温热对胃癌细胞侵袭能力的影响。结果温热后细胞明显皱缩、变圆及细胞漂浮,3h大部分细胞漂浮;荧光染色显示温热后部分细胞核内出现浓染致密的颗粒块状荧光,胞核固缩、染色质高度凝聚和碎裂;MTT实验提示温热可明显抑制SGC790l细胞生长;细胞划痕实验发现SGC790l细胞温热1h、2 h后细胞迁移距离均明显小于对照组,温热3h后细胞基本未发生迁移;Transwell实验提示SGC790l细胞温热后细胞侵袭能力明显下降。结论温热对胃癌SGC790l细胞具有明显的杀伤作用,温热可明显抑制胃癌SGC790l细胞增殖和侵袭迁移能力。     

沉默BCAT1对肺癌细胞增殖、迁移及侵袭能力的影响

目的:研究BCAT1在肺癌细胞A549的增殖、迁移及侵袭能力中的作用。方法:通过小干扰RNA(si RNA)沉默A549细胞中BCAT1的表达,细胞分为对照组(Con)、BCAT1基因沉默组(si RNA-BCAT1)和si RNA阴性对照组(si RNA-NC)。利用Western blot检测si RNA对BCAT1的沉默效果;划痕愈合实验检测沉默BCAT1后A549细胞迁移能力的改变;Transwell小室侵袭实验检测沉默BCAT1后A549细胞侵袭能力的变化;MTT实验检测沉默BCAT1对A549细胞增殖能力的影响。结果:与Con组相比,si RNA-BCAT1组的BCAT1蛋白表达明显降低(P0.05),细胞划痕愈合率明显降低(P0.05),能够穿膜的细胞数明显减少(P0.05),而Con组和si RNA-BCAT1组细胞的增殖能力比较差异无明显统计学意义(P0.05)。结论:沉默BCAT1抑制A549细胞的迁移和侵袭能力,而对其增殖能力无影响。     

VEGF is essential for the growth and migration of human hepatocellular carcinoma cells

Vascular endothelial growth factor (VEGF) plays a crucial role in tumor angiogenesis. VEGF induces new vessel formation and tumor growth by inducing mitogenesis and chemotaxis of normal endothelial cells and increasing vascular permeability. However, little is known about VEGF function in the proliferation, survival or migration of hepatocellular carcinoma cells (HCC). In the present study, we have found that VEGF receptors are expressed in HCC line BEL7402 and human HCC specimens. Importantly, VEGF receptor expression correlates with the development of the carcinoma. By using a comprehensive approaches including TUNEL assay, transwell and wound healing assays, migration and invasion assays, adhesion assay, western blot and quantitative RT-PCR, we have shown that knockdown of VEGF165 expression by shRNA inhibits the proliferation, migration, survival and adhesion ability of BEL7402. Knockdown of VEGF165 decreased the expression of NF-κB p65 and PKCα while increased the expression of p53 signaling molecules, suggesting that VEGF functions in HCC proliferation and migration are mediated by P65, PKCα and/or p53.     

Protein kinase Cα promotes cell migration through a PDZ-dependent interaction with its novel substrate discs large homolog 1 (DLG1)

Protein scaffolds maintain precision in kinase signaling by coordinating kinases with components of specific signaling pathways. Such spatial segregation is particularly important in allowing specificity of signaling mediated by the 10-member family of protein kinase C (PKC) isozymes. Here we identified a novel interaction between PKCα and the Discs large homolog (DLG) family of scaffolds that is mediated by a class I C-terminal PDZ (PSD-95, disheveled, and ZO1) ligand unique to this PKC isozyme. Specifically, use of a proteomic array containing 96 purified PDZ domains identified the third PDZ domains of DLG1/SAP97 and DLG4/PSD95 as interaction partners for the PDZ binding motif of PKCα. Co-immunoprecipitation experiments verified that PKCα and DLG1 interact in cells by a mechanism dependent on an intact PDZ ligand. Functional assays revealed that the interaction of PKCα with DLG1 promotes wound healing; scratch assays using cells depleted of PKCα and/or DLG1 have impaired cellular migration that is no longer sensitive to PKC inhibition, and the ability of exogenous PKCα to rescue cellular migration is dependent on the presence of its PDZ ligand. Furthermore, we identified Thr-656 as a novel phosphorylation site in the SH3-Hook region of DLG1 that acts as a marker for PKCα activity at this scaffold. Increased phosphorylation of Thr-656 is correlated with increased invasiveness in non-small cell lung cancer lines from the NCI-60, consistent with this phosphorylation site serving as a marker of PKCα-mediated invasion. Taken together, these data establish the requirement of scaffolding to DLG1 for PKCα to promote cellular migration.     

Overexpression of Hsc70 promotes proliferation,migration, and invasion of human glioma cells

Migration and invasion are often recognized as the main reasons for the high recurrence and death rates of glioma and limit the efficacy of surgery and other antitumor therapies. In this study, we found over activation of heat shock cognate protein 70 (Hsc70) in human glioma specimens, which was closely related to glioma grade. We investigated whether Hsc70 induced the migration and invasion of glioma cells. Wound healing and transwell migration assay were used to determine the migration and invasion ability of human glioma U251 and U87 cells, in which the expression of Hsc70 was knocked down by small interfering RNA. Western blot analysis was performed to determine the expression of FAK-Src signaling in malignant glioma cells. The results showed that Hsc70 deficiency significantly retarded migration and invasion and reduced the phosphorylation of FAK, Src, and Pyk2 in U251 and U87 cells. Overall, our results indicate that the migration and invasion capacity of human brain glioma cells is at least partly induced by Hsc70-dependent activation of FAK-Src signaling.     

Extracellular signal-regulated kinase/mitogen-activated protein kinase regulates actin organization and cell motility by phosphorylating the actin cross-linking protein EPLIN

Extracellular signal-regulated kinase (ERK) is important for various cellular processes, including cell migration. However, the detailed molecular mechanism by which ERK promotes cell motility remains elusive. Here we characterize epithelial protein lost in neoplasm (EPLIN), an F-actin cross-linking protein, as a novel substrate for ERK. ERK phosphorylates Ser360, Ser602, and Ser692 on EPLIN in vitro and in intact cells. Phosphorylation of the C-terminal region of EPLIN reduces its affinity for actin filaments. EPLIN colocalizes with actin stress fibers in quiescent cells, and stimulation with platelet-derived growth factor (PDGF) induces stress fiber disassembly and relocalization of EPLIN to peripheral and dorsal ruffles, wherein phosphorylation of Ser360 and Ser602 is observed. Phosphorylation of these two residues is also evident during wound healing at the leading edge of migrating cells. Moreover, expression of a non-ERK-phosphorylatable mutant, but not wild-type EPLIN, prevents PDGF-induced stress fiber disassembly and membrane ruffling and also inhibits wound healing and PDGF-induced cell migration. We propose that ERK-mediated phosphorylation of EPLIN contributes to actin filament reorganization and enhanced cell motility.     

Arginine vasopressin controls p27(Kip1) protein expression by PKC activation and irreversibly inhibits the proliferation of K-Ras-dependent mouse Y1 adrenocortical malignant cells

The neurohypophyseal hormone arginine vasopressin (AVP) is a classic mitogen in many cells. In K-Ras-dependent mouse Y1 adrenocortical malignant cells, AVP elicits antagonistic responses such as the activation of the PKC and the ERK1/2 mitogenic pathways to down-regulate cyclin D1 gene expression, which induces senescence-associated β-galactosidase (SA-βGal) and leads to cell cycle arrest. Here, we report that in the metabolic background of Y1 cells, PKC activation either by AVP or by PMA inhibits the PI3K/Akt pathway and stabilises the p27(Kip1) protein even in the presence of the mitogen fibroblast growth factor 2 (FGF2). These results suggest that p27(Kip1) is a critical signalling node in the mechanisms underlying the survival of the Y1 cells. In Y1 cells that transiently express wild-type p27(Kip1), AVP caused a severe reduction in cell survival, as shown by clonogenic assays. However, AVP promoted the survival of Y1 cells transiently expressing mutant p27-S10A or mutant p27-T187A, which cannot be phosphorylated at Ser10 and Thr187, respectively. In addition, PKC activation by PMA mimics the toxic effect caused by AVP in Y1 cells, and inhibition of PKC completely abolishes the effects caused by both PMA and AVP in clonogenic assays. The vulnerability of Y1 cells during PKC activation is a phenotype conditioned upon K-ras oncogene amplification because K-Ras down-regulation with an inducible form of the dominant-negative mutant H-RasN17 has resulted in Y1 cells that are resistant to AVP's deleterious effects. These data show that the survival destabilisation of K-Ras-dependent Y1 malignant cells by AVP requires large quantities of the p27(Kip1) protein as well as phosphorylation of the p27(Kip1) protein at both Ser10 and Thr187.     

人肝内胆管癌细胞PRL-3 的表达与侵袭转移研究

目的:探讨PRL-3在人肝内胆管癌侵袭转移中的作用.方法:利用小RNA技术干扰肝内胆管癌细胞株PRL-3表达,并采用细胞划痕实验和Transwell体外侵袭实验评价PRL-3对肝内胆管癌细胞侵袭转移能力的影响.结果:RT-PCR和Western blot结果均显示转染PRL-3特异性siRNA-2组PRL-3表达明显降低(P＜0.05).PRL-3 siRNA-2组在划痕培养24h后划痕区域宽度占初始划痕区域宽度的百分比为(62.12±6.28)％,阴性对照组为(23.88±2.55)％,空白对照HCCC-9810组为(21.20±6.07)％.PRL-3siRNA-2组细胞的划痕两端距离相比明显较宽,分别与阴性对照组细胞和空白对照HCCC-9810组细胞相比均有统计学意义(P＜0.05),后两者无显著性差异(P＞0.05).Transwell体外侵袭实验结果显示,PRL-3 SiRNA-2组细胞侵袭能力明显减弱,穿膜细胞数为(19.40±2.30)个/HP,明显少于阴性对照组(64.00±2.73)个/HP和正常HCCC-9810组(67.20±3.l1)个/HP,差异有显著性(P＜0.05);正常HCCC-9810组和阴性对照组无明显差异(P＞0.05).结论:PRL-3特异性siRNA能够抑制肝内胆管癌细胞HC-CC-9810中内源性PRL-3的表达,并可以明显抑制肝内胆管癌细胞的迁移侵袭能力.     

Chk1反义寡核苷酸联合顺铂抑制卵巢癌侵袭转移的分子机制

目的:探究Chk1反义寡核苷酸(CHK1-ASODN)单独或联合顺铂(DDP)对卵巢癌细胞系SKOV-3侵袭转移能力的影响,并阐明其可能的分子机制。方法:体外培养人卵巢癌细胞系SKOV-3,CHK1-ASODN单独或联合DDP处理48 h后,划痕实验检测细胞迁移能力;Transwell实验检测细胞侵袭能力;显微镜下观察细胞上皮或间质表型特征;Western blot及实时定量PCR技术分别检测上皮间质转化(EMT)特异性标志物(E-cadherin、N-cadherin)以及EMT关键调控分子ZEB1的蛋白及m RNA的表达水平。结果:与对照组相比较,CHK1-ASODN单独或联合DDP均能显著抑制SKOV-3细胞的迁移及侵袭(P0.05);细胞表现为间质化表型;E-cadherin的表达显著升高(P0.05),而N-cadherin的表达则显著降低(P0.05);ZEB1的表达显著降低(P0.05)。结论:CHK1-ASODN单独或联合DDP下调ZEB1的表达进而逆转EMT可能是其抑制卵巢癌侵袭转移的重要机制之一。     

长链非编码RNA SNHG3对人乳腺癌细胞MCF-7增殖、迁移与侵袭的影响 *

目的:探讨长链非编码RNA SNHG3对人乳腺癌细胞MCF-7增殖、迁移与侵袭的影响。方法:构建SNHG3过表达质粒,实验分别设置阴性对照组(pcDNA-3.1+)与SNHG3基因过表达组(pcDNA-3.1+/SNHG3)。将MCF-7细胞转染对照组质粒和SNHG3过表达质粒,采用实时定量PCR 方法检测 SNHG3 mRNA 转录水平,Western blot 检测MMP9及EMT相关蛋白质水平;集落形成实验检测MCF-7细胞增殖能力;划痕愈合实验检测MCF-7细胞横向迁移能力; Transwell 小室实验检测MCF-7细胞纵向迁移能力及侵袭能力。结果:过表达SNHG3后,MCF-7细胞中SNHG3的mRNA水平显著增高(P<0.001);MCF-7细胞的体外增殖能力明显增加(P<0.01),迁移(P<0.01)与侵袭能力(P<0.001)也显著增强,实时定量PCR, Western blot 结果显示SNHG3可激活EMT相关通路。结论:过表达SNHG3可能通过激活EMT通路促进乳腺癌MCF-7细胞的增殖,迁移与侵袭。