Effect of Ezrin overexpression on pancreatic cancer cell line Panc-1

目的 探讨Ezrin过表达对胰腺癌细胞系Panc-1生物学行为的影响.方法 用过表达载体pcb6-Ezrin稳定转染Panc-1细胞,并用流式细胞仪检测细胞周期,CCK-8法检测细胞生长曲线,扫描电镜观察细胞表面形态及表面突起的变化,用未包被及包被Matrigel胶的Transwell小室分别检测细胞的运动和侵袭能力,并用免疫印迹法检测信号相关蛋白Erk1/2的变化.结果 过表达Ezrin后其蛋白的表达明显升高,Panc-1细胞表面的细胞突起、微绒毛数量明显增加,运动及侵袭能力也明显增加,但对细胞的体外增殖和细胞周期无明显的影响.Ezrin的过表达能够激活Erk1/2的表达.结论 Ezrin蛋白对胰腺癌细胞的细胞突起、表面微绒毛的形成、细胞骨架及细胞的运动和侵袭发挥着重要的作用.因此,Ezrin可能在胰腺癌的进展中发挥着重要的作用,Erk1/2信号转导途径在这一过程中发挥着重要的作用.     

基因沉默ILK对胰腺癌细胞Panc-1细胞增殖、凋亡与侵袭的影响

目的探讨基因沉默整合素连接激酶(ILK)对胰腺癌细胞(Panc-1)细胞增殖、凋亡与侵袭的影响。方法培养胰腺癌Panc-1细胞,构建ILK-specific shRNA慢病毒载体后对Panc-1细胞进行转染,qPCR与Western Blot方法验证干扰基因片段的有效性,MTT实验检测转染后胰腺癌细胞增殖能力的变化,流式细胞仪检测转染后胰腺癌细胞周期的变化,Transwell实验检测转染后胰腺癌细胞侵袭能力的改变。结果成功构建siRNA-ILK慢病毒载体,转染组细胞的ILK mRNA及蛋白表达明显低于空病毒组及阴性对照组。基因沉默ILK的胰腺癌Panc-1细胞增殖能力显著降低,停滞在G0/G1的细胞数显著增多,G2/M期细胞数明显减少,侵袭能力显著降低(P0.01)。结论基因沉默ILK能抑制胰腺癌细胞Panc-增殖与侵袭能力,并促进凋亡。     

ECT2通过调控PI3K/AKT信号通路对胰腺癌细胞恶性行为、糖酵解及TH细胞分化的影响

目的 探讨上皮细胞转化序列2(ECT2)通过调控PI3K/AKT信号通路对胰腺癌细胞恶性行为、糖酵解及TH细胞分化的影响。方法 RT-qRCR检测人脐静脉内皮细胞系HUVEC及胰腺癌细胞中ECT2 mRNA表达，将胰腺癌Panc-1细胞株分为胰腺癌组（Panc-1细胞株正常培养）、NC组（Panc-1细胞株转染ECT2阴性对照）、ECT2 siRNA组（Panc-1细胞株转染ECT2 siRNA）、抑制剂组（Panc-1细胞株转染加入PI3K/AKT抑制剂LY294002,ECT2 siRNA+抑制剂组（Panc-1细胞株转染ECT2 siRNA加入PI3K/AKT抑制剂LY294002），采用RT-qRCR各组细胞中ECT2 mRNA表达；Transwell法检测各组Panc-1细胞侵袭能力；划痕实验检测迁移；流式细胞仪检测各组细胞IFN-γ及IL-4表达；免疫印迹分别检测糖酵解代表蛋白及PI3K/AKT表达。结果 胰腺癌组、NC组、ECT2 siRNA组、抑制剂组及ECT2 siRNA+抑制剂组的ECT2 mRNA比较分别为1.00±0.00、0.95±0.03、0.41±0.08...     

五味子乙素通过Wnt/β-catenin信号通路调节人胰腺癌细胞增殖、凋亡和侵袭

目的 研究五味子乙素对人胰腺癌细胞Panc-1细胞增殖,凋亡和侵袭的调控作用及对Wnt/β-catenin信号通路的影响.方法 不同、浓度的五味子乙素(12、24、48μg/ml)处理胰腺癌Panc-1细胞后,采用CCK8法检测Panc-1细胞增殖变化,Transwell法检测细胞侵袭情况,流式细胞术检测细胞凋亡的变化...     

膜-细胞骨架连接分子Ezrin与肿瘤转移

Ezrin是ERM(Ezrin/Rad ixin/Moesin)家族的重要成员,是真核细胞膜蛋白-细胞骨架的桥梁分子。参与细胞形态学,细胞黏附、运动、细胞骨架的重塑及信号转导过程。Ezrin在不同肿瘤组织中的表达异常,推测它可能参与肿瘤细胞的侵袭转移,但表达的不一致提示Ezrin在不同肿瘤的进展和转移中发挥着不同的作用。     

Survivin特异性siRNA诱导胰腺癌细胞凋亡的研究

目的 观察survivin特异性siRNA对胰腺癌细胞株Panc-1细胞增殖和凋亡的影响.方法 体外构建Survivin特异性siRNA表达载体p-Survivin-siRNA,转染胰腺癌细胞株Panc-1细胞系,RT-PCR和Western印迹法检验其对胰腺癌细胞株Panc-1细胞的RNA干扰(RNAi)效果.采用MTT法分析其对细胞增殖的影响,流式细胞仪检测其对细胞周期的影响.同时,采用Western印迹法检测半胱氨蛋白酶 3(caspase 3)的表达变化.结果 p-survivin-siRNA表达质粒高效而特异地剔降胰腺癌细胞株Panc-1细胞中survivin的表达,抑制肿瘤细胞增殖(P<0.01),阻断胰腺癌细胞株Panc-1细胞在G1期.随着survivin基因被沉默,caspase 3表达升高(P<0.01).结论 靶向survivin基因的siRNA表达载体可以显著抑制胰腺癌细胞株Panc-1细胞增殖,促进细胞凋亡.     

凋亡抑制蛋白XIAP和促凋亡因子Smac在胰腺癌化疗抵抗中的作用

目的探讨x-相关凋亡抑制蛋白（XIAP）和促凋亡因子Smac在胰腺癌细胞化疗抵抗中的作用，以及转染胞浆表达型Smac基因靶向下凋XIAP对化疗药物诱导的胰腺癌细胞凋亡的影响。方法应用流式细胞术检测顺铂、5-FU介导的Panc-1、BXPC-3的凋亡率及胞浆染色分析细胞XIAP表达变化，Western blot分析XIAP、Smac、Caspase-3表达水平；构建pEGFP-NI／Smac真核表达载体并转染胰腺癌Panc-1细胞，流式细胞术检测转染Smac基因前后Panc-1细胞的凋亡敏感性。结果与BXPC-3细胞相比，Panc-1对顺铂或5-FU介导的凋亡具有较强抵抗性，Western blot分析显示Panc-1细胞高表达XIAP，在化疗药物作用下化疗敏感细胞BXPC-3胞浆内XIAP水平下降明显多于Panc-1细胞，而且凋亡的BXPC-3细胞释放入胞浆内的成熟Smac蛋白水平明显高于Panc-1细胞。转染胞浆表达型Smac基因至化疗抵抗Panc-1细胞，可明显下调其XIAP表达水平，促进效应Caspase-3分子活化，显著提高顺铂、5-FU诱导的细胞凋亡率。结论胰腺癌细胞XIAP的表达水平下调与其化疗敏感性有关，XIAP是克服化疗抵抗的重要靶分子，而上调Smac活性蛋白的胞浆表达作为一种有效调节信号，通过拮抗XIAP的凋亡抑制作用协同化疗药物促进胰腺癌细胞凋亡。     

SIRT1通过调节FOXO1/RAB7信号通路促进低氧诱导的胰腺癌细胞自噬

目的:探讨SIRT1对低氧诱导的胰腺癌细胞自噬的影响及FOXO1/RAB7信号通路在其中的作用。方法:利用Western blot和免疫荧光法检测SIRT1在低氧诱导的自噬胰腺癌细胞核内的表达情况。将下调SIRT1基因的小干扰RNA和过表达SIRT1的质粒转染到胰腺癌Panc-1细胞中,敲减和过表达SIRT1基因;激光共聚焦显微镜追踪双色LC3荧光蛋白表达,Western blot实验检测自噬相关蛋白LC3、p62及FOXO1/RAB7信号通路的蛋白表达情况。通过生物信息学GEPIA网站关联性分析预测SIRT1与FOXO1的相互作用,并进一步利用免疫共沉淀技术检测SIRT1与FOXO1蛋白的相互作用。结果:SIRT1在低氧诱导的胰腺癌细胞核内表达增加。敲减SIRT1的表达可以引起胰腺癌细胞自噬受抑制。过表达SIRT1能够增强Panc-1细胞中FOXO1和RAB7的蛋白表达水平(P0.05),而当SIRT1表达下调时,FOXO1和RAB7表达受到抑制(P0.05)。SIRT1与FOXO1蛋白存在相互作用。结论:SIRT1可能参与胰腺癌细胞自噬的调控,其机制可能与FOXO1/RAB7信号通路有关。     

Rab11过表达促进人膀胱癌细胞的增殖和侵袭

目的探讨Rab11过表达对膀胱癌细胞增殖、侵袭的影响。方法在BIU-87细胞系中转染Rab11过表达质粒,用CCK8、细胞周期检测及基质胶侵袭实验分析了Rab11对细胞增殖、细胞周期和侵袭的影响,并用Western blot及RT-PCR方法检测了细胞周期相关因子cyclin D1、cyclin E及侵袭相关因子MMP9的蛋白和m RNA的表达变化。结果Rab11过表达能促进膀胱癌细胞的增殖及侵袭,并能促进细胞周期相关因子cyclin D1、cyclin E及侵袭相关因子MMP9的蛋白和m RNA的表达。结论Rab11可能作为癌蛋白参与了膀胱癌的发病机制,成为治疗膀胱癌的靶点。     

周期蛋白E促进结直肠癌SW1116细胞增殖和侵袭

目的 探究周期蛋白E高表达对结直肠癌SW1116细胞增殖和侵袭的影响。方法 将SW116细胞分为周期蛋白E-过表达组、对照组与空载体组，免疫组化法检测各组细胞周期蛋白E的表达；实时定量PCR法检测周期蛋白E mRNA的表达；四甲基偶氮唑盐实验法(MTT)观察细胞增殖能力；流式细胞仪检测细胞周期；Transwell实验检测细胞侵袭能力。结果 与对照组与空载体组相比，周期蛋白E过表达组SW116细胞的周期蛋白E mRNA表达明显升高；在24h和48h时的细胞存活率明显升高；G₀/G₁期细胞比例明显升高，G₂/M期细胞比例明显降低；穿膜细胞数明显增加。结论 周期蛋白E促进结直肠癌SW1116细胞的增殖与侵袭。     

survivin反义寡核苷酸在体外诱导胰癌细胞凋亡

目的：探讨survivin反义核酸对胰腺癌细胞株Panc-1细胞凋亡的影响.方法：用脂质体瞬时转染法介导survivin反义核苷酸处理胰腺癌Panc-1细胞后,MTT试验测定转染细胞的相对存活率,RT-PCR检测survivin mRNA表达,琼脂糖凝胶电泳分析其对Panc-1细胞的凋亡诱导作用.结果：转染survivin反义核酸的Panc-1细胞增殖明显受抑制,与对照进行比较,具有显著性差异（P＜0.05）;经琼脂糖凝胶电泳,转染survivin反义核酸的Panc-1细胞可见到DNA梯形条带,而对照细胞未见到.结论：survivin反义核酸能够诱导Panc-1细胞凋亡.     

KGF-induced motility of breast cancer cells is dependent on Grb2 and Erk1,2

Breast cancer metastasis is directly associated with breast cancer cell motility. Using a cell culture wounding model, we have demonstrated that keratinocyte growth factor (KGF) enhanced the motility of estrogen receptor-positive breast cancer cells. However, the mechanisms by which KGF enhanced motility of breast cancer cells are not known. In the present study, we report that KGF-induced motility requires intact tyrosine kinase signaling since genistein, a tyrosine kinase inhibitor, led to decreased motility of breast cancer cells mediated by KGF. Using cDNA microarrays, we previously found that KGF increased the expression ofGrb2mRNA by 2-- the involvement of Grb2 in KGF-induced motility. Down-regulation of Grb2 protein expression inhibited KGF-induced motility. Since Grb2 is known to regulate Erk1,2 and Akt kinase activities we determined whether these downstream proteins may be vital to KGF-induced motility. Inhibiting the activation of Erk1,2 by PD98059 suppressed KGF-induced motility whereas inhibiting the activation of Akt by wortmannin did not affect KGF-induced motility. In conclusion, these results indicate that KGF mediated signal transduction employs Grb2 to transduce the tyrosine kinase signals resulting in the activation of Erk1,2 and breast cancer cell motility.     

Ezrin promotes invasion and metastasis of pancreatic cancer cells

Background  

Pancreatic cancer has a high mortality rate because it is usually diagnosed when metastasis have already occurred (microscopic and gross disease). Ezrin plays important roles in cell motility, invasion and tumor progression, and it is especially crucial for metastasis. However, its function in pancreatic cancer remains elusive.     

Identification and characterization of motility stimulating factor secreted from pancreatic cancer cells: role in tumor invasion and metastasis

Cell motility is an important factor in the process of invasion and metastasis of tumor. In this study, the relationship between cell motility and experimental metastatic potential was examined using two human pancreatic cancer cell lines, SW1990 and PANC-1. Serum-free conditioned medium from the highly metastatic cell line SW1990 was found to contain a factor that stimulated the migration of and induced a fibroblast-like morphological change in the weakly metastatic cell line PANC-1. Preincubation of PANC-1 cells with SW1990 conditioned medium (SW-C.M.) induced liver metastasis following splenic injection of PANC-1 cells in nude mice, although no liver metastasis was observed without pretreatment of SW-C.M. This factor, temporarily termed PDMF (pancreatic cancer-derived motility factor) is a heparin non-binding protein having a molecular weight of 40 kDa calculated by gel-filtration HPLC which acts not only chemotactically but also chemokinetically, and also acts mainly in a paracrine fashion. However, this factor had no effect on the proliferation of PANC-1 cells; it therefore appears to be a so-called motility factor. Only TGF-b1 and IL-6 were recognized in the SW-C.M. among cytokines thought to stimulate cell motility. These cytokines stimulated the motility of PANC-1 cells, but differed from PDMF in the neutralizing test with antibody against these cytokines. Results of characterization and preliminary purification suggest that this factor may be a novel motility factor. The above findings suggest that this motility factor may play an important role in the invasion and metastasis of pancreatic cancer, and complete purification of it will be useful in elucidating the mechanism of progression of cancer and designing a strategy for inhibition of invasion and metastasis of pancreatic cancer.     

Hsulf-1 regulates growth and invasion of pancreatic cancer cells

BACKGROUND: Hsulf-1 is a newly identified enzyme with arylsulphatase activity that can regulate the sulphation state of cell-surface heparan sulphate proteoglycans (HSPGs). In vitro overexpression of this enzyme in pancreatic cancer cells decreases responsiveness to fibroblastic growth factor-2, as Hsulf-1 is up regulated in primary pancreatic adenocarcinoma. AIM: To further analyse the functions of the Hsulf-1 enzyme in vitro and in vivo with respect to growth, invasion and tumorigenicity. METHODS AND RESULTS: Transfection of Panc-1 pancreatic cancer cells with a full-length Hsulf-1 expression vector resulted in increased invasiveness and adhesiveness. An in vivo xenograft nude mouse tumour model showed a markedly reduced growth potential of Hsulf-1-expressing Panc-1 cells, which correlated with a considerably lower proliferation rate. Hsulf-1-positive nude mouse tumours showed better development of interstitial matrix structures, with increased blood vessel density in these tumours. In an orthotopic model, Hsulf-1-positive tumours exhibited enhanced local invasiveness. In human primary pancreatic cancers there was strong staining for sulphated HSPGs, which was markedly reduced in metastatic tissue samples. CONCLUSION: Hsulf-1-mediated desulphation of HSPGs reduces the growth ability of Panc-1 pancreatic cancer cells, but increases the basal invasiveness of these cells, suggesting an important role of this enzyme in pancreatic cancer progression.     

miR-183 inhibits the metastasis of osteosarcoma via downregulation of the expression of Ezrin in F5M2 cells

Osteosarcoma is the most common primary malignancy of bone in teenagers and approximately 30% of patients develop lung metastasis, which is the leading cause of mortality. Recent studies suggest that the Ezrin protein is correlated with the metastatic potential of several malignant tumors. In our study, ectopic overexpression of miR-183 repressed the expression levels of Ezrin and significantly inhibited the motility and invasion of osteosarcoma cells. This suggests that miR-183 may possibly play a tumor suppressor role in the metastasis of osteosarcoma by downregulating Ezrin expression levels. These findings show that through inhibition of Ezrin expression levels, miR-183 is significantly involved in cell migration and invasion of osteosarcoma.     

Ezrin的原核表达及与其相互作用蛋白的研究

目的体外筛选人胰腺癌细胞中与Ezrin蛋白相互作用的蛋白。方法提取人胰腺癌Panc-1细胞总RNA,RT-PCR法扩增Ezrin基因。将扩增得到的Ezrin基因片段和pET15b质粒转化E．coli DH5α,获得pET15b-Ezrin重组质粒,双酶切后测序鉴定。将测序正确的重组质粒转化至E．coliB L21-CodonPlus（DE3）-RIL,以异丙基硫代-B-D-半乳糖苷（IPTG）诱导表达,对表达产物进行Ni^2＋-NTA亲和层析和分子筛纯化后,行SDS-PAGE、MALDI—TOF—MS／MS质谱分析和蛋白质印迹鉴定。裂解Panc-1细胞,获得总蛋白提取液,采用Pull—down方法体外筛选与Ezrin重组蛋白相互作用的蛋白,并对筛选出的目的蛋白进行双向凝胶电泳和MALDI—TOF—MS／MS质谱鉴定。结果RT-PCR扩增出的Ezrin基因片段（1761bp）与理论DNA表达片段大小一致,测序鉴定显示克隆的Ezrin基因序列与GenBank报道序列相符。SDS—PAGE、质谱分析和蛋白质印迹分析显示纯化后Ezrin重组蛋白相对分子质量为69400,与质谱分析的相对分子质量结果相符,为带有His-6标签的特异蛋白。双向凝胶电泳和质谱鉴定结果共筛选出可与Ezrin重组蛋白相互作用的7种蛋白,其分别为RNA—binding motif protein39（RBM39）、Transgelin（TAGLN）、Albumin（ALBU）、Formin—1（FMN1）、Rho GTPases（RHGBA）、Tetratricopeptide repeat protein 16（TTC16）、Syntaxin—binding protein 1（STXBPI）。结论在大肠杆菌中成功表达、纯化了Ezrin蛋白,并在体外成功筛选出可信的与Ezrin蛋白相互作用的蛋白。     

BRAF and FBXW7 (CDC4, FBW7, AGO, SEL10) mutations in distinct subsets of pancreatic cancer: potential therapeutic targets

The recognition of biologically distinct tumor subsets is fundamental to understanding tumorigenesis. This study investigated the mutational status of the serine/threonine kinase BRAF and the cyclin E regulator FBXW7 (CDC4, FBW7, AGO, SEL10) related to two distinct pancreatic carcinoma subsets: the medullary KRAS2-wild-type and the cyclin E overexpressing tumors, respectively. Among KRAS2-wild-type carcinomas, 33% (3 of 9) contained BRAF V599E mutations; one of which was identified in the pancreatic cancer cell line COLO357. Among 74 KRAS2-mutant carcinomas, no BRAF mutations were identified. Among the KRAS2/BRAF wild-type carcinomas, no mutations within pathway members MEK1, MEK2, ERK1, ERK2, RAP1B, or BAD were found. Using pancreatic cancer microarrays and immunohistochemistry, we determined that 6% (4 of 46 and 5 of 100 in two independent panels) of pancreatic adenocarcinomas overexpress cyclin E. We identified two potential mechanisms for this overexpression including the amplification/gain of CCNE1 gene copies in the Panc-1 and Su86.86 cell lines and a novel somatic homozygous mutation (H460R, in one of 11 pancreatic cancer xenografts having allelic loss) in FBXW7, which was accompanied by cyclin E overexpression by immunohistochemistry. Both BRAF and FBXW7 mutations functionally activate kinase effectors important in pancreatic cancer and extend the potential options for therapeutic targeting of kinases in the treatment of phenotypically distinct pancreatic adenocarcinoma subsets.     

Regulator of calcineurin 1 modulates cancer cell migration in vitro

Metastasis suppressors and other regulators of cell motility play an important role in tumor invasion and metastases. We previously identified that activation of the G protein coupled receptor 54 (GPR54) by the metastasis suppressor metastin inhibits cell migration in association with overexpression of Regulator of calcineurin 1 (RCAN1), an endogenous regulator of calcineurin. Calcineurin inhibitors also blocked cell migration in vitro and RCAN1 protein levels were reduced in nodal metastases in thyroid cancer. The purpose of the current study was to determine directly if RCAN1 functions as a motility suppressor in vitro. Several cancer cell lines derived from different cancer types with different motility rates were evaluated for RCAN1 expression levels. Using these systems we determined that reduction of endogenous RCAN1 using siRNA resulted in an increase in cancer cell motility while expression of exogenous RCAN1 reduced cell motility. In one cell line with a high migratory rate, the stability of exogenously expressed RCAN1 protein was reduced and was rescued by treatment with a proteasome inhibitor. Finally, overexpression of RCAN1 was associated with an increase in cell adhesion to collagen IV and reduced calcineurin activity. In summary, we have demonstrated that the expression of exogenous RCAN1 reduces migration and alters adhesion; and that the loss of endogenous RCAN1 leads to an increase in migration in the examined cancer cell lines. These results are consistent with a regulatory role for RCAN1 in cancer cell motility in vitro.