慢病毒PARG-shRNA转染降低大肠癌lovo细胞基质黏附、运动和侵袭能力

目的探讨聚腺苷二磷酸核糖水解酶(PARG)基因沉默对人大肠癌lovo细胞基质黏附、运动和侵袭能力的影响。方法慢病毒PARG-shRNA转染人大肠癌lovo细胞并筛选出稳定沉默PARG基因的lovo细胞株;Western blot法检测PARG、PARP和NF-κB的表达。用细胞基质黏附、运动和侵袭实验观察lovo细胞基质黏附、运动和侵袭能力。结果获得了稳定的PARG基因沉默lovo细胞株,实验组PARP和NF-κB的表达显著降低(P<0.05)。PARG沉默后lovo细胞黏附、运动和侵袭能力降低。和未转染组相比,其黏附、运动和侵袭抑制率分别为25.22%、38.71%和35.29%。结论PARG基因沉默可以降低lovo细胞基质黏附、运动和侵袭能力,此可能与PARG沉默后下调PARP,并下调NF-κB的活性有关。提示PARG在肿瘤侵袭和转移中可能发挥重要作用。     

RAB5A反义RNA对人肺腺癌细胞系侵袭转移影响的体外研究

目的　已知RAB5A基因过表达与人非小细胞肺癌恶性表型形成相关,本研究在此基础上进一步探 讨该基因过表达在人肺腺癌恶性演进中的作用。　方法　将RAB5A反义表达载体稳定转染入高浸润人肺腺癌细 胞系L18和高转移人肺腺癌细胞系95D中,采用重组基底膜侵袭、与基底膜成分黏附能力测定、趋化运动能力测 定、明胶酶分泌与活性检测等体外实验方法观察转染前后细胞生物学特性的改变。　结果　RAB5A反义RNA稳 定转染后L18细胞趋化运动和侵袭基底膜能力显著降低(P<0.05),明胶酶活性检测发现转染后细胞MMP 2的分 泌能力明显减弱;稳定转染后95D细胞趋化运动和侵袭基膜能力显著降低(P<0.05)。　结论　体外实验显示, RAB5A基因过表达对肿瘤细胞的趋化运动能力和侵袭重组基底膜能力起重要作用,进一步提示RAB5A基因过表 达在人肺腺癌的侵袭转移过程中发挥一定作用。     

miR-95对结肠癌细胞系SW620侵袭和迁移的影响及机制

目的探讨小分子非编码RNA-95(miR-95)表达对结肠癌细胞系SW620侵袭、迁移能力的影响及其机制。方法将miR-95干扰慢病毒转入结肠癌细胞系SW620,用qRT-PCR检测细胞中miR-95表达,用Transwell小室和划痕实验检测细胞侵袭和迁移能力;MTT法检测细胞同种、异种细胞黏附能力;用Western印迹检测3组细胞的EMP1,VEGFC和MMP2蛋白表达。结果 SW620转染miR-95干扰慢病毒72 h后,转染率较高;与空病毒组和对照组比较,转染组miR-95的表达降低(P0.05);侵袭和迁移能力减弱(P0.05);同种黏附能力增强(P0.05);异种黏附能力减弱(P0.05);同时转染组EMP1蛋白表达明显增高,VEGFC、MMP2蛋白表达明显降低(P0.05)。结论 miR-95能够促进SW620细胞侵袭和迁移能力,可能是通过EMP1、VEGFC和MMP2实现的。     

Tiaml在鼻咽癌细胞株中的表达及其与侵袭转移的关系

目的 探讨Tiam1对人鼻咽癌细胞株生物学特性的影响.方法 应用脂质体lipofectamine2000法对C666-1、CNE1两种人鼻咽癌细胞株转染Tiam1/C1199HA质粒.逆转录PCR、即时PCR、Western blot方法检测Tiam1转染组与空载体转染组细胞中Tiam1的表达,细胞黏附实验、划痕实验和基质胶侵袭实验检测不同转染组间细胞的黏附、迁移和侵袭能力.结果 C666-1、CNE1细胞Tiam1稳定转染组Tiam1的表达、细胞黏附、迁移及侵袭能力较空载体转染组均有明显增强(P<0.05).结论 Tiam1基因与人鼻咽癌C666-1、CNE1细胞株的侵袭转移有关.     

蛇毒半胱氨酸蛋白酶抑制剂的表达对黑色素瘤B16细胞侵袭与转移的作用

目的探讨蛇毒半胱氨酸蛋白酶抑制剂cystatin（sv-cystatin）在黑色素瘤细胞侵袭与转移中的作用。方法构建真核表达质粒pcDNA3．1／sv-cystatin,采用脂质体法将重组质粒导人小鼠黑色素瘤B16FI细胞。G418筛选抗性克隆,Western blotting法和RT-PCR鉴定sv—cystatin在黑色素瘤细胞中的表达,四甲基偶氮唑盐（MTT）法检测肿瘤细胞体外生长和黏附能力的变化,体外侵袭、运动实验和小鼠实验性肺转移模型分析sv-cystatin表达对黑色素瘤细胞体内、外侵袭力的影响。结果sv-cystatin基因稳定转染后B16F1细胞体外侵袭与运动能力明显降低,其穿膜的细胞数显著低于B16F1／pcDNA3．1空载体组和未转染细胞组（P〈0．01）;sv—cystatin的表达可抑制C57BL／6小鼠肺转移瘤灶的形成;其体外增殖及黏附能力未见明显改变。结论sv—cystatin基因转染可抑制黑色素瘤B16细胞的体内、外侵袭与转移能力。     

三叶因子3在甲状腺乳头状癌TPC 1细胞株黏附中的作用机制

目的:探讨沉默三叶因子3(TFF3)表达对甲状腺乳头状癌(PTC)TPC-1细胞黏附作用的影响。方法:稳定敲低TFF3表达的TPC-1细胞株(shRNA-TFF3组)、转染空载体的TPC-1细胞株(shRNAC组)、TPC-1细胞(TPC-1组)分别进行同质黏附实验、异质黏附实验;qPCR检测TFF3mRNA及WNT/β-catenin信号通路关键分子β-catenin mRNA水平,免疫印迹和免疫细胞化学检测β-catenin、E-cadherin、ALCAM、MMP-9、MMP-2蛋白表达水平。结果:细胞同质黏附实验显示shRNA-TFF3组黏附细胞数显著高于shRNAC组和TPC-1组。细胞异质实验显示shRNA-TFF3组黏附细胞数显著低于shRNAC组和TPC-1组,shRNAC组和TPC-1组同质和异质黏附数差异无统计学意义。qPCR结果显示shRNA-TFF3组TFF3mRNA、β-catenin mRNA水平显著低于shRNAC组和TPC-1组,shRNAC组和TPC-1组TFF3 mRNA、β-catenin mRNA水平差异无统计学意义。免疫细胞化学与免疫印迹结果显示沉默TFF3后ALCAM、MMP-9、MMP-2、β-catenin蛋白表达水平显著降低;E-cadherin蛋白表达水平显著升高,shRNAC组和TPC-1组差异无统计学意义。结论:敲低TFF3可能通过WNT/β-catenin信号通路影响PTC细胞TPC-1的黏附能力。     

蛋白酶激活受体-1促进肺癌细胞侵袭和转移功能的研究

目的研究蛋白酶激活受体1（PAR-1）对人类肺癌细胞侵袭和转移功能的影响。方法采用阳离子脂质体介导法,将正义和反义PAR-1重组质粒“pC／PARls”和“pC／PARlas”分别转染至人肺巨细胞癌低转移株（PLA801C）和高转移株（PLA801D）细胞中;采用逆转录．聚合酶链反应（RT-PCR）和Western印迹检测转染后PAR-1基因和蛋白表达水平变化;通过MTT、软琼脂集落形成、流式细胞仪、细胞-基质黏附和Transwell细胞侵袭实验检测PAR-1对肺癌细胞转移相关功能的影响。结果转染正义和反义PAR-1分别明显上调和下调了PLA801C和PLA801D的PAR-1 mRNA和蛋白表达水平。转染正义PAR-1对细胞的生长和克隆形成具有促进作用;并可明显增强细胞对细胞外基质的黏附和侵袭能力（与空载体对照组比较均P〈0．01）。相反,转染反义PAR-1对细胞模型的生长和克隆形成具有抑制作用;能明显降低细胞的S期和G2／M期（与空载体对照组比较分别为P〈0．05、0．01）、升高G0／G1期所占比例（P〈0．01）;还可使细胞对细胞外基质的黏附力（P〈0．05）和侵袭力明显降低（P〈0．01）。结论正义和反义PAR-1基因能够分别上调和下调PAR-1的表达水平;PAR-1能够影响肺癌细胞的生长和增殖、黏附和侵袭特性。抑制PAR-1的表达可能是一种治疗肺癌的途径。     

βig-h3对肝癌细胞黏附、侵袭和分泌基质金属蛋白酶的影响

目的:研究βig-h3对肝癌细胞SMMC-7721黏附、侵袭和分泌基质金属蛋白酶(MMP)的影响。方法:通过黏附、侵袭及明胶酶谱实验,检测瞬时分别转染βig-h3基因和转染空载体的SMMC-7721细胞的黏附、侵袭能力和分泌MMP的变化。结果:转染βig-h3基因的SMMC-7721细胞的黏附率、侵袭率及MMP-2、MMP-9的分泌量,均明显高于转染空载体的SMMC-7721细胞。结论:βig-h3具有促进肝癌细胞浸润和转移的作用,是肝癌发生发展过程中的促进因子。     

Twist对TNF-α作用后乳腺癌细胞侵袭及上皮间质转化的影响

RNA干扰技术抑制Twist在乳腺癌细胞MDA-MB-231中的表达,观察其沉默对TNF-α作用后乳腺癌细胞侵袭及上皮间质转化(epithelial mesenchymal transition,EMT)的影响。以乳腺癌细胞MDA-MB-231为空白对照,shRNA-NC空载体为阴性对照,构建Twist沉默表达载体,转染乳腺癌细胞MDA-MB-231,qRT-PCR和Western blotting检测转染细胞中Twist mRNA及蛋白质表达水平。建立Twist沉默表达的乳腺癌细胞模型后,用TNF-α分别处理对照组及转染后的乳腺癌细胞,Transwell实验检测细胞侵袭和迁移能力,Western blotting检测细胞EMT相关蛋白波形蛋白(Vimentin)、上皮钙黏附素(E-cadherin)和转移相关蛋白基质金属蛋白酶2(matrix metalloproteinase 2, MMP-2)、基质金属蛋白酶9(MMP-9)的表达。结果显示,下调Twist的乳腺癌细胞经TNF-α处理后,细胞侵袭和迁移能力降低,Vimentin表达减少,E-cadherin表达增加,细胞中MMP-2、MMP-9表达减少,与空白对照及TNF-α作用后的阴性对照相比,差异有统计学意义(P0.05)。因此,下调Twist可降低TNF-α诱导的乳腺癌细胞的侵袭和迁移能力,抑制其EMT。     

胰岛素抗性对肝癌HepG2细胞生物学功能及顺铂敏感性的影响

 目的:研究胰岛素抗性在原发性肝癌细胞中的生物学功能及对顺铂抗癌药物的抗药敏感性的影响。 方法: 利用高浓度的胰岛素连续培养HepG2细胞72 h获得抗胰岛素的HepG2细胞（HepG2/IR），检测HepG2/IR细胞的黏附、迁移和侵袭的能力改变以及对顺铂的敏感性；同时利用流式细胞术测定HepG2和HepG2/IR中胰岛素受体和葡萄糖转运蛋白2的表达情况。 结果: 胰岛素抗性HepG2/IR细胞中葡萄糖消耗量显著降低；胰岛素受体和葡萄糖转运蛋白2表达下调；而HepG2/IR细胞的黏附、迁移和侵袭能力明显增强；同时HepG2/IR细胞对顺铂的敏感性降低；但用吡格列酮处理HepG2/IR细胞后其黏附、迁移和侵袭能力显著减弱。 结论: 胰岛素抗性与HepG2细胞的耐药性、细胞黏附、迁移和侵袭能力密切相关。这有助于解释具有胰岛素抗性的癌症患者对化疗失敏的临床现象。     

Upregulated CD44v9 expression inhibits the invasion of oral squamous cell carcinoma cells.

OBJECTIVES: CD44 is one of the cell surface molecules that play an important role in cancer metastasis. In oral squamous cell carcinoma (OSCC), the downregulation of CD44v9 has been shown to be associated with tumor metastasis. We found that treatment with an anti-CD44v9 antibody enhanced the invasive potential of OSCC cell lines. Based on previous studies and our results, reduced expression of CD44v9 may be correlated with an increased invasive potential, i.e. the overexpression of CD44v9 may inhibit the invasive activity of OSCC cells. METHODS: To study this correlation, we transfected the CD44v9 gene into HSC-4 cells with low CD44v9 expression and examined their invasive potential using the cell culture invasion assay and a three-dimensional culture invasion assay. RESULTS: Overexpression of CD44v9 resulted in a downregulation of the invasive potential of HSC-4 cells. Moreover, CD44v9-transfected cells did not invade reorganized stroma, while parent HSC-4 cells exhibited diffuse invasion into reorganized stroma by the three-dimensional culture invasion assay. CONCLUSIONS: Overall, these findings suggest that the inhibition of the invasive potential by upregulation of CD44v9 expression may be due to enhanced cell-cell adhesion. In our opinion, the upregulation of CD44v9 may be a target for future cancer treatment.     

Osteopontin increases CD44 expression and cell adhesion in RAW 264.7 murine leukemia cells

BACKGROUND: Osteopontin (OPN) is an inducible cell attachment protein which binds alphavbeta3-integrin and CD44 receptors to promote tumor metastasis. We hypothesized that OPN alters expression of its CD44 receptor to promote neoplastic cell migration. METHODS: RAW264.7 cells were stimulated with OPN (0-10 nM) for 0-12 hours to determine the time- and concentration-dependence of CD44 protein and mRNA expression. In selected instances, a competitive ligand for the alphavbeta3-integrin, GRGDSP (50 nM), or an inhibitor of protein synthesis, anisomycin (10 microg/ml), was added. Cell adhesion to hyaluronan was assayed with the crystal violet assay. RESULTS: OPN upregulates plasma membrane total CD44 protein in a concentration-(ANOVA P = 0.001) and time-dependent (ANOVA P = 0.001) fashion. CD44v6 is not altered. Cell adhesion to hyaluronate increases in parallel with CD44 expression. Steady state mRNA levels for CD44 are not altered by OPN. 5 nM OPN increases CD44 protein half-life from 105 +/- 11 minutes to 278 +/- 15 minutes. (P < 0.03) Blockade of either alphavbeta3-integrin ablates the OPN-dependent increase in CD44. CONCLUSIONS: These data indicate that OPN increases plasma membrane CD44 expression and cell adhesion by binding to its alphavbeta3-integrin receptor. We conclude that OPN may promote tumor metastatic behavior by CD44 expression.     

不同缺氧状态对人肝癌HepG2细胞侵袭和转移能力的影响

目的: 探讨不同缺氧状态对人肝癌HepG2细胞黏附、侵袭和转移能力的影响及其可能机制。方法: 不同浓度的低氧处理对数生长期的人肝癌HepG2细胞,采用MTT法、Transwell膜侵袭系统、免疫细胞化学方法、明胶酶谱分析和反转录PCR检测变异型白细胞分化抗原CD44v6、基质金属蛋白酶2(MMP-2)、MMP-9、低氧诱导因子(HIF-1α)和血管内皮生长因子(VEGF)的表达差异。结果: HepG2细胞经低氧处理后,细胞的基质黏附率、穿透基底膜与游走迁移的细胞数均增高,以3%氧浓度最为显著(P<0.05);3%和5%氧处理还可显著促进CD44v6的表达,增加MMP-2和MMP-9的表达,并可上调HIF-1α和VEGF。结论: 适度缺氧能增强人肝癌细胞的黏附、侵袭和转移能力,其机制可能与CD44v6、基质金属蛋白酶、HIF-1α和VEGF的表达改变有关。     

Osteopontin regulates multiple functions contributing to human colon cancer development and progression

Osteopontin (OPN) is a secreted phosphoglycoprotein known to interact with a number of integrin receptors. While increased OPN expression has been reported in a number of human cancers, and its cognate receptors (αv-β3, αv-β5, and αv-β1 integrins and CD44) have been identified, its role in colon cancer development and progression has not been extensively studied. We previously identified, using a combination of gene expression and tissue microarrays, that increased OPN expression is concordant with tumor stage. The current study examined the functional role of OPN in colon cancer progression and metastatic potential. The principal findings of this study were that both endogenous OPN expression (via stable transfection) as well as exogenous OPN (added to culture medium) enhanced the motility and invasive capacity of human colon cancer cells in vitro. OPN appeared to regulate motility though interaction with CD44. OPN expression also reduced intercellular (homotypic) adhesion, an important characteristic of metastatic cancer cells. Stable transfection of four poorly tumorigenic human colon cancer cell lines with OPN also resulted in enhanced tumorigenicity in vivo with increased proliferation and increased CD31 positive microvessel counts, concordant with the degree of OPN expression. Collectively, these results suggest that OPN may affect multiple functional components contributing to human colon cancer progression and solidifies its role in this process. This revised version was published online in July 2006 with corrections to the Cover Date.     

肺腺癌细胞微环境及转移与黏附分子表达的关系

目的研究肺腺癌细胞生长环境及转移性与黏附分子CD44v6和CD29的表达关系。方法将起源相同、转移性不同的两个肺腺癌细胞系AGZY和Anip分别用简便肿瘤多细胞球体(MTS)培养法培养，并设常规单层贴壁细胞培养对照。通过倒置显微镜、扫描及透射电镜观察MTS形成情况，并用免疫组化法分别对MTS及贴壁细胞上CD44v6和CD29表达进行检测。结果MTS培养成功，贴壁细胞与MTS在细胞结构及细胞连接结构上相似，两种MTS在形态及结构上差异无显著性。免疫组化结果显示，CD29在高转移性的Anip细胞及其MTS上呈阳性表达；在低转移性的AGZY细胞及其MTS上阴性表达。CD44v6在Anip和AGZY细胞及MTS上均呈阳性表达，差异无显著性。贴壁细胞与MTS上两种黏附分子表达均无差异。结论成功建立了一种简易制备MTS的方法。细胞生长方式(单层贴壁与MTS)可能不影响CD44v6和CD29的表达。CD29表达可能与肺腺痛转移性相关；CD44v6表达可能与肺腺癌转移无关。     

Expression of c-Met and heparan-sulfate proteoglycan forms of CD44 in colorectal cancer

In colorectal cancer patients, prognosis is not determined by the primary tumor but by the formation of distant metastases. Molecules that have been implicated in the metastatic process are the proto-oncogene product c-Met and CD44 glycoproteins. Recently, we obtained evidence for functional collaboration between these two molecules: CD44 isoforms decorated with heparan sulfate chains (CD44-HS) can bind the c-Met ligand, the growth and motility factor hepatocyte growth factor/scatter factor (HGF/SF). This interaction strongly promotes signaling through the receptor tyrosine kinase c-Met. In the present study, we explored the expression of CD44-HS, c-Met, and HGF/SF in the normal human colon mucosa, and in colorectal adenomas and carcinomas, as well as their interaction in colorectal cancer cell lines. Compared to the normal colon, CD44v3 isoforms, which contain a site for HS attachment, and c-Met, were both overexpressed on the neoplastic epithelium of colorectal adenomas and on most carcinomas. Likewise, HGF/SF was expressed at increased levels in tumor tissue. On all tested colorectal cancer cell lines CD44v3 and c-Met were co-expressed. As was shown by immunoprecipitation and Western blotting, CD44 on these cells lines was decorated with HS. Interaction with HS moieties on colorectal carcinoma (HT29) cells promoted HGF/SF-induced activation of c-Met and of the Ras-MAP kinase pathway. Interestingly, survival analysis showed that CD44-HS expression predicts unfavorable prognosis in patients with invasive colorectal carcinomas. Taken together, our findings indicate that CD44-HS, c-Met, and HGF/SF are simultaneously overexpressed in colorectal cancer and that HS moieties promote c-Met signaling in colon carcinoma cells. These observations suggest that collaboration between CD44-HS and the c-Met signaling pathway may play an important role in colorectal tumorigenesis.     

Evaluation of osteopontin and CD44v6 expression in odontogenic cystic lesions by immunohistochemistry

Odontogenic cysts are common lesions with different biological behavior. Odontogenic keratocysts (OKCs) and calcifying odontogenic cysts (COCs) with ameloblastoma-like epithelium are more aggressive than dentigerous cysts (DCs) and radicular cysts (RCs). Therefore, they were included in the list of odontogenic tumors by WHO. Osteopontin (OPN) is a calcium-binding glycoprotein present in many normal tissues. It plays a role in the migration and invasion of transformed epithelial cells. Binding of OPN to its receptor CD44v6 can enhance cell motility and migration. The purpose of this study was to compare the expression of these markers between odontogenic cysts of varying biological behavior. We examined OPN and CD44v6 expression in tissue sections of 14OKCs, 14COCs, 14RCs and 14DCs by immunohistochemistry. OPN and CD44v6 immunostaining was observed in all lining epithelial cells of the studied lesions with different degrees. The highest level of OPN and CD44v6 expression was found in OKCs, followed by COCs, RCs and DCs. Comparison of both markers among four groups revealed significant differences (P<0.001). Our findings suggest that higher level of OPN and CD44v6 expression in epithelial cells of some lesions such as OKC and COC can explain the local aggressive behavior of them.