趋化性细胞因子受体CXCR4基因沉默对乳腺癌细胞体外侵袭和定向肺转移的影响

目的 利用RNA干扰技术下调趋化性细胞因子受体CXCR4基因的表达,探讨其沉默对乳腺癌细胞体外侵袭及肺转移潜能的影响.方法 设计合成CXCR4的特异性短发卡状RNA(shRNA),将其插入至pSilencer载体中,并将重组后的pSilencer质粒载体经脂质体包裹转染乳腺癌MDA-MB-231细胞株,抗性筛选稳定抑制CXCR4表达的永久细胞克隆.应用逆转录聚合酶链反应(RT-PCR)和Western blot法,检测shRNA对细胞内CXCR4基因表达的影响.利用Boyden小室模型检测细胞体外侵袭能力.二苯基溴化四氮唑蓝(MTT)法检测细胞的增殖状况.通过裸鼠尾静脉瘤细胞注射方法,构建肺转移模型,检测CXCR4基因沉默对乳腺癌细胞肺转移能力的影响.结果 成功构建和筛选出CXCR4特异性的shRNA质粒载体,稳定转染CXCR4-shRNA的乳腺癌细胞的CXCR4 mRNA和蛋白的表达较空白对照组明显下调(29.5%±3.8%比69.7%±2.6%,15.4%±1.1%比39.0%±2.4%;均P<0.01),体外侵袭能力减弱,增殖速度减慢,肺转移能力下降.结论 以CXCR4为靶向的shRNA能够有效下调CXCR4基因的表达,降低人乳腺癌细胞体外侵袭、增殖以及肺转移的能力.CXCR4是乳腺癌侵袭和转移过程中的重要调控因子.     

siRNA抑制食管癌EC9706细胞CXCR4基因表达的实验

目的运用RNAi技术沉默食管癌EC9706细胞CXCR4（chemokine receptor4）基因表达,观察其对肿瘤细胞的生长和转移影响。方法设计CXCR4 基因为靶向的siRNA ,构建siRNA表达载体,通过脂质体将siRNA表达载体转入EC9706细胞。荧光定量PCR法检测细胞CXCR4 mRNA表达水平;流式细胞术检测细胞周期; MTT法检测细胞侵袭和增殖的情况。结果构建出CXCR4 基因为靶向的siRNA表达载体pRNAT-U6.2/Lenti-siCX1和pRNAT-U6.2/Lenti -siCX2;荧光定量PCR结果显示,转染pRNAT-U6.2/Lenti-siCX1的EC9706细胞和转染pRNAT-U6.2/Lenti-siCX2的EC9706细胞与对照组相比CXCR4 mRNA的表达水平明显降低（P＜0.01）;细胞生长速度较对照组明显减慢（P＜0.01）;流式细胞仪检测结果显示：转染pRNAT-U6.2/Lenti-siCX1的EC9706S期细胞比例低于对照组(P＜0.05)。结论沉默CXCR4基因表达对食管癌EC9706细胞的生长和侵袭、转移有明显的抑制作用。     

CXCR4-siRNA逆转录病毒载体对宫颈癌Caski细胞CXCR4基因表达的抑制作用

目的: 构建针对趋化因子受体CXCR4的RNA干扰逆转录病毒载体,检测由逆转录病毒介导的RNA干扰对人宫颈癌Caski细胞CXCR4基因表达的抑制作用. 方法: 人工合成CXCR4特异性小干扰RNA(small interfering RNA,siRNA)片段,将其插入带有绿色荧光蛋白(EGFP)的逆转录病毒载体质粒pSOS,测序正确后进行重组,转染PT67细胞包装成病毒,收获病毒上清用其转染Caski细胞,采用实时定量PCR和Western blot分别从mRNA水平和蛋白水平检测其对Caski细胞CXCR4表达的抑制作用. 结果: 成功构建pSOS-siCXCR4逆转录病毒载体,筛选出高效抑制序列,与对照组比较,在Caski细胞的mRNA水平,CXCR4-siRNA在24h、48h和72h对CXCR4 mRNA表达的抑制率分别为29.9%、56.8%和62.8%,而在蛋白水平对CXCR4蛋白的抑制率分别为43.6%、49.6%和62.9%. 结论: pSOS-HUS-siCXCR4干扰载体可以有效抑制Caski细胞中CXCR4表达.     

CXCR4基因RNA干扰慢病毒载体的构建与鉴定

目的：构建人CXCR4基因RNAi（RNA interference,RNAi）慢病毒载体。方法：针对筛选确定的人CXCR4基因RNAi有效靶序列,合成靶序列的Oligo DNA,退火形成双链DNA,与经HpaⅠ和XhoⅠ酶切后的pGCL—GFP载体连接产生短发卡RNA慢病毒载体,PCR筛选阳性克隆,测序鉴定。结果：PCR鉴定与DNA测序证实合成的含CXCR4 shRNA慢病毒载体寡核苷酸链插入正确。结论：成功构建人CXCR4基因RNAi慢病毒载体。     

靶向沉默CXCR4基因抑制肝细胞癌侵袭体外研究

目的:采用RNAi技术构建CXCR4shRNA干扰载体,探讨靶向沉默CXCR4基因表达后对人肝细胞癌增殖和侵袭作用的影响及可能机制。方法:通过蛋白质印迹法和RT-PCR,检测不同分化程度的肝癌细胞株CXCR4表达程度。通过RNAi技术,将CXCR4shRNA干扰载体转染CXCR4高表达的肝癌细胞HepG2,并采用G418筛选出稳定表达株,蛋白质印迹法和RT-PCR验证shRNA对CXCR4基因的靶向沉默效率。以转染空载体细胞为阴性对照组,MTT法检测CXCR4基因沉默后肝癌细胞的增殖能力,Transwell小室侵袭试验检测CXCR4基因沉默后肝癌细胞的侵袭能力,蛋白质印迹法检测MMP-2和MMP-9蛋白表达水平,免疫荧光检测MMP-2在细胞内表达。结果:CXCR4靶向沉默的细胞CXCR4表达受到显著抑制。HepG2、HepG2-Vector和HepG2-CXCR4-细胞CXCR4蛋白相对表达量分别为0.56±0.07、0.54±0.04和0.14±0.05,F=57.42,P<0.001。正常HepG2、HepG2-Vector和HepG2-CXCR4-细胞的CXCR4mRNA相对表达量分别为1.04±0.05、1.05±0.11和0.19±0.03,P<0.001。MTT结果显示,CXCR4基因沉默能明显抑制HepG细胞的增殖。72h时正常HepG2、HepG2-Vector和HepG2-CXCR4-细胞的相对增殖速度分别为1.34±0.05,1.32±0.03和1.14±0.03。Transwell试验显示,10%FBS趋化作用下,HepG2、HepG2-Vector和HepG2-CXCR4-穿膜细胞数分别为85±13、89±17和23±6,差异有统计学意义,F=63.91,P<0.001;SDF-1趋化作用下,HepG2、HepG2-Vector和HepG2-CXCR4-穿膜细胞数分别为168±20、171±24和30±9。蛋白质印迹法显示,相对于正常HepG2细胞(0.83±0.04),HepG2-CXCR4-细胞MMP-2相对表达量(0.31±0.06)明显下降,P<0.001;而HepG2-Vector细胞MMP-2相对表达量(0.85±0.07)改变不明显,P=0.75。HepG2、HepG2-Vector和HepG2-CXCR4-细胞MMP-9相对表达量分别为0.35±0.04、0.33±0.07和0.32±0.06,差异无统计学意义,F=0.23,P=0.79。结论:通过RNAi技术成功靶向干扰CXCR4基因表达,可抑制肝癌细胞增殖和侵袭,可能与其抑制侵袭相关分子MMP-2表达有关。     

AEG-1与CXCR4对乳腺癌脑转移的影响

目的探讨AEG-1与CXCR4基因表达对乳腺癌脑转移的影响。方法对1997～2007年收治的乳腺癌患者进行随访,以发生脑转移的33例患者作为病例组,以未发生脑转移的45例患者作为对照组。通过免疫组化法,对照分析AEG-1及CXCR4对脑转移的影响。结果脑转移组中AEG-1及CXCR4的阳性表达率分别为63.6%和60.6%,与其在对照组中表达（31.1%和33.3%）差异显著（P〈0.05）。logistic回归分析结果显示,AEG-1和CXCR4回归系数分别为1.242和1.545。结论 AEG-1和CXCR4阳性表达是乳腺癌发生脑转移的独立危险因子,AEG-1及CXCR4有望成为针对乳腺癌脑转移的高特异性早期诊断指标及基因治疗靶点。     

脂质体介导CXCR4 RNA干扰对结肠癌细胞SW480迁移与侵袭影响的研究

目的:探讨CXCR4 RNA 干扰对结肠癌细胞株SW480 CXCR4 mRNA和CXCR4蛋白凋亡、迁移和侵袭能力的影响.方法:采用脂质体转导CXCR4 RNA干扰序列sihCXCR4-3,RT-PCR检测CXCR4 mRNA相对含量,蛋白质印迹法检测CXCR4蛋白表达水平,流式细胞术检测细胞凋亡,Transwell小室评价SW480细胞迁移与侵袭能力.结果:sih-CXCR4-3组CXCR4 mRNA的抑制率为81.40％,CXCR4蛋白相对含量为9.62％.早期凋亡率各组间均无差别,晚期凋亡率sihCXCR4-3组为(7.62±0.88)％,高于SW480细胞组(6.41士0 26)％,P=0.0451.迁移实验显示,sihCXCR4-3组平均细胞数为84.25±22.46,低于SW480组(154.63±44.53,P=0.002 6)与NCsihCXCR4组(110.63+ 20.16,P=0.026 6).侵袭实验发现,sihCXCR4-3组平均细胞数为0,低于SW480组(294.43±70.10,P=0.000 0)与NCsihCXCR4(201.43±54.41,P=0.000 0).结论:CXCR4 RNA干扰抑制SW480细胞CXCR4基因与蛋白表达,促进凋亡,降低迁移和侵袭能力.     

骨肉瘤细胞株CXCR4表达与细胞体外侵袭能力的相关性

目的:探讨趋化因子受体CXCR4的表达水平对骨肉瘤细胞株体外侵袭能力的影响.方法:通过单细胞克隆技术,从人骨肉瘤细胞系MG63中获取2株CXCR4不同表达水平的细胞株,免疫组化和RT-PCR检测CXCR4的表达水平,Tran-swell趋化侵袭实验观察不同细胞株趋化侵袭能力的差异,并用CXCR4单克隆抗体对高表达CXCR4的细胞株进行趋化干预.结果:CXCR4蛋白在B8和G5细胞株的表达分别为94.74±17.35和41.86±6.54,CXCR4 mRNA在B8和G5细胞株的表达分别为1.37±0.36和0.44±0.13,差异均有统计学意义,P<0.05.B8细胞株的体外趋化侵袭能力(53.60±15.37)明显强于G5细胞株(19.60±8.20).与CXCR4的表达呈正相关,P值均<0.05.单克隆抗体干预组的侵袭细胞数(21.80±8.53)明显小于未干预组(53.60±15.37),P<0.05.结论:骨肉瘤细胞株高度的体外侵袭能力与CXCR4高表达密切相关并能被特异性抗体抑制.     

RNAi对肝癌细胞RhoA基因表达和侵袭转移影响的研究

目的:探讨RNA干扰(RNAi)技术沉默RhoA基因表达对肝癌细胞HHCC生物学特性的影响。方法:合成RhoA特异性小干扰RNA(siRNA),脂质体介导转染肝癌细胞株HHCC。检测RhoA mRNA和蛋白表达水平和RhoA活性的变化,激光共聚焦显微镜观察细胞骨架变化,Boyden小室测定细胞侵袭性变化。结果:RhoA siRNA能有效抑制HHCC细胞RhoA mRNA和蛋白的表达,致使RhoA活性显著下降(P<0.01),细胞侵袭能力明显降低(P<0.01),侵袭抑制率为26.2%。结论:RhoA siRNA能够显著下调HHCC细胞RhoA的表达和侵袭能力,表明RhoA可影响肿瘤细胞的转移和侵袭能力,抑制其功能可能为肝癌治疗提供新的靶点。     

vMIP-Ⅱ通过 CXCR4拮抗乳腺癌转移作用的初步研究

背景与目的: CXCR4-SDF-1α体系在乳腺癌靶向转移中具有重要作用,已证明多种 CXCR4拮抗剂对乳腺癌转移有抑制作用.本研究拟探讨作为 CXCR4封闭因子的病毒巨噬细胞炎症蛋白Ⅱ( viral macrophage inflammatory protein-Ⅱ , vMIP-Ⅱ)对乳腺癌细胞株 MCF 7转移相关因素的影响.方法: MTT法检测不同浓度 vMIP-Ⅱ刺激下 MCF-7的增殖效应;软琼脂集落形成实验评价克隆形成率;粘附和趋化实验观察 vMIP-Ⅱ对不同转移阶段 MCF-7细胞的作用.结果:(1)系列浓度的 vMIP-Ⅱ处理细胞 72h后,细胞没有表现出增殖效应( P >0.05). (2)vMIP-Ⅱ以浓度依赖的方式抑制细胞集落的形成, 50、 100、 500和 1 000 ng /ml vMIP-Ⅱ作用后,细胞的琼脂集落抑制率在 18.2%～ 54.6%之间. (3)经 300 ng/ml 的 vMIP-Ⅱ处理不同时间( 0 min、 30 min、 2 h 和 6 h)后,细胞在 2 h对纤维连接素( FN)和 Matrigel的粘附都达到了抑制高峰. (4)在趋化实验中, 500 ng/ml vMIP-Ⅱ组穿膜细胞数( 24± 10)比对照组( 60± 9)要低( P< 0.05).结论: MCF 7的克隆形成率与 vMIP-Ⅱ的刺激浓度呈负相关. vMIP-Ⅱ能降低 MCF-7对 FN和 Matrigel的粘附和有效抑制 MCF-7 对人肺蛋白粗提物的靶向性趋化作用.     

The role of CXCR3 and CXCR4 in colorectal cancer metastasis

Chemokines and their receptors play key roles in leukocyte trafficking and are also implicated in cancer metastasis. We previously demonstrated that forced expression of CXCR3 promotes colon cancer metastasis preferentially to the draining lymph nodes (LNs), with poor prognosis. Using clinical colorectal cancer (CRC) samples, here, we show that expressions of CXCR3 and CXCR4 are significantly higher in metastatic foci within LNs and liver compared to primary tumors, whereas ligands for CXCR3 and CXCR4 are not. We also have demonstrated that some human CRC cell lines constitutively express both CXCR3 and CXCR4, and that activation of CXCR3 strengthens the CXCR4‐mediated cell migration in vitro in a synergistic manner. By constructing SW620 cell lines with reduced expression of CXCR3 and/or CXCR4 using microRNA, we investigated in vivo metastatic activities in a mouse rectal transplantation model. Six weeks after inoculation, CXCR3‐, CXCR4‐, and CXCR3/CXCR4 double‐knockdowns significantly reduced metastasis to LNs, liver and lungs, compared to the control (p < 0.05). Importantly, its suppressive effect on LN metastasis was significantly stronger in CXCR3‐ and CXCR3/CXCR4 double‐knockdowns. In addition, CXCR3‐ and CXCR3/CXCR4 double‐knockdowns significantly decreased the dissemination of cancer cells to liver and lungs, even after 2 weeks. These results indicate that targeting CXCR3 and CXCR4 can be a promising therapy against CRC metastasis.     

逆转录病毒介导重组CXCR4对前列腺癌细胞侵袭力的影响

目的：构建CXCR4基因重组逆转录病毒表达载体pLEGFP—CXCR4,并转染PC-3细胞,探讨趋化因子受体CXCR4对人前列腺癌细胞体外侵袭能力的影响。方法：分离健康人外周血单个核细胞,提取总RNA,以其为模板,RT—PCR法扩增CXCR4,获取CXCR4基因全长序列,装入带有绿色荧光蛋白（GFP）的逆转录病毒质粒pLEGFP—N1,用脂质体法转染PC-3细胞,采用实时定量PCR和Western blotting观察CXCR4表达情况,并通过体外细胞一基质粘附试验和Transwells小室检测肿瘤细胞体外侵袭能力的变化。结果：成功构建pLEGFP—CXCR4载体,转染人前列腺癌细胞72h后,CXCR4mRNA及蛋白质水平明显上调,细胞的体外侵袭力明显增强,增殖活性增强。结论：CXCR4转染能够明显提高CXCR4基因mRNA及蛋白水平,提高人前列腺癌细胞体外侵袭能力。     

Dissemination of hepatocellular carcinoma is mediated via chemokine receptor CXCR4

In different tumour entities, expression of the chemokine receptor 4 (CXCR4) has been linked to tumour dissemination and poor prognosis. Therefore, we evaluated, if the expression of CXCR4 exerts similar effects in human hepatocellular carcinoma (HCC). Expression analysis and functional assays were performed in vitro to elucidate the impact of CXCL12 on human hepatoma cells lines. In addition, expression of CXCR4 was evaluated in 39 patients with HCC semiquantitatively and correlated with both, tumour and patients characteristics. Human HCC and hepatoma cell lines displayed variable intensities of CXCR4 expression. Loss of p53 function did not impact on CXCR4 expression. Exposure to CXCL12 mediated a perinuclear translocation of CXCR4 in Huh7/Hep3B cells and increased the invasive potential of Huh7 cells. In HCC patients, CXCR4 expression significantly correlated with progressed local tumours (T-status; P=0.006), lymphatic metastasis (N-status; P=0.005) and distant dissemination (M-status; P=0.009), as well as with a decreased 3-year-survival rate (P=0.01). In summary, strong expression of CXCR4 is significantly associated with progressed hepatocellular cancer.     

Effect of Lentivirus-induced shRNA Silencing CXCR4 Gene on Proliferation and Apoptosis in Human Esophageal Carcinoma Cell Line Eca109

OBJECTIVE To discuss the application of the slow virus-induced short-hairpin RNA (vshRNA) to silence the expression of CXCR4 in EsCa cell lines Eca109, and observe the effect of silencing CXCR4 on the proliferation and apoptosis of Eca109 cells in vitro. METHODS The expression plasmid of vshRNA targeting CXCR4 was constructed, with a concurrent construction of negative vshRNA expression plasmid, and without targeting any known mRNA. Real-time quantitative PCR and Western blot assay were used to determine the change of CXCR4 expression in the post-transfected EsCa cell Eca109, and MTT assay was conducted to detect the change of proliferation in EsCa Eca109 cell after silencing the CXCR4. The .ow cytometry was used to detect the change of the cell cycle and apoptosis in the post-silenced EsCa Eca109 cell in di. erent groups. RESULTS The transfection rate was respectively (87.3 ± 1.2)% and (90.1 ± 1.4)% in the CXCR4- RNAi-LV (silent group) and NC-GFP-RNAi-LV (negative control group) cellular plasmids. The vshRNA interference resulted in a down-regulation of the CXCR4 gene mRNA and protein expressions in Eca109 cells. CXCL12 promoted the proliferation of EsCa cell lines Eca109. The speed of EsCa cell proliferation became slower in the silencing group than in the normal control (also the control) and the negative control groups (P < 0.05). However, there was no significant difference in comparison of the proliferation speeds between the negative control and the normal control groups (P > 0.05). In the silencing group, the proportion of the cells in phase G0/G1, phase S and phase G2/M was respectively (69.9 ± 5.0)%, (17.1 ± 2.5)% and (13.0 ± 7.4)%, and the apoptotic rate achieved (7.27 ± 0.50)%. In the normal control group, the proportion of the cells in phase G0/G1, S and G2/M was respectively (55.9 ± 4.6)%, (30.2 ± 3.9)% and (13.8 ± 1.4)%, and the apoptotic rate was (3.30 ± 0.70)%. In the negative control group, the proportion of cells in phase G0/G1, S and G2/M was respectively (52.7 ± 7.8)%, (25.3 ± 2.3)% and (21.9 ± 7.4)%, with an apoptotic rate of (4.03 ± 1.37)%. Compared with the normal control and negative control groups, there was an apparent growth of cells in the phase G0/G1 (P < 0.05), and a greatly increased number of cells in phase S (P < 0.05) in the silencing group. There was no signi. cant di.erence in comparison of those between the normal control and negative control groups (P > 0.05). The apoptotic rate was obviously higher in the cells of the silencing group than in the normal control and the negative control groups (P < 0.05). There was no signi. cant di.erence in comparison of the apoptotic rate between the normal control and the negative control groups (P > 0.05). CONCLUSION CXCR4-vshRNA can specifically and effectively inhibit CXCR4 expression of Eca109 cells. CXCR4-vshRNA can inhibit the proliferation and enhance the apoptosis rate of Eca109 cells through intervening the expression of CXCR4, suggesting that CXCL12/CXCR4 might have an important role in the progression of Escc Thisslow virus-induced shRNA can effectively silence the expression of CXCR4 gene in the EsCa cells; block up the biological e.ect of CXCL12/CXCR4 axle; and e.ectively inhibit the potency of proliferation in the EsCa cell line Eca109, thus advancing apoptosis. It suggests that the CXCL12/CXCR4 plays an important role in the progression of EsCa.     

乳腺癌趋化因子受体CXCR4的表达及其意义

目的研究趋化性细胞因子受体CXCR4在乳腺癌中的表达,探讨其与淋巴结转移、远处转移及预后的关系。方法84例正常乳腺组织及84例乳腺癌组织、43例区域淋巴结转移癌组织标本（依据组织类型及临床病理特性分组）,取自福建医科大学附属第二医院2000年1月至2002年12月手术切除的病理存档蜡块,应用免疫组化方法（二步法）分别检测3组标本CXCR4的表达情况。对计数资料采用χ^2检验,对生存率采用时序检验进行统计学分析。结果正常乳腺组织、乳腺癌组织、区域淋巴结转移癌组织CXCR4阳性率分别为11．9％、53．6％、74．4％;转移组织CXCR4阳性表达率明显高于原发肿瘤。乳腺癌组织CXCR4阳性表达率与淋巴结转移、HER-2表达、临床分期密切相关（P〈0．05）,而与患者年龄、肿瘤直径、ER表达、PR表达、组织学类型及肿瘤分化程度无关（P〉0．05）。发生远处转移组CXCR4表达水平（74．1％）高于未发生远处转移组（43．9％,P〈O．01）。CXCR4阳性表达组5年生存率66．7％（30／45）明显低于阴性表达组87．2％（84／89,P〈0．05）。结论CXCR4阳性表达与乳腺癌的淋巴结转移、远处转移有关,有助于预后判断。     

趋化因子受体CXCR4和CCR7及其配体与食管癌转移相关性的研究进展

目的:总结国内外趋化因子受体CXCR4、CCR7及其配体与食管癌转移关系的研究进展.方法:应用PubMed及CNKI期刊全文数据库检索系统,以"CXCR4,CCR7,肿瘤,食管癌"等为关键词,检索2000-01-2010-01年的相关文献,共检到英文文献1 750篇,中文文献453篇.纳入标准:1)CXCR4、CCR7的结构和功能;2)CXCR4、CCR7在常见恶性肿瘤中的异常表达与预后;3)CXCR4、CCR7在食管癌中的表达情况.根据纳入标准,最后纳入分析65篇文献.结果:CXCR4、CCR7及其配体在多种恶性肿瘤中表达,与肿瘤的浸润、淋巴结转移、远处转移以及预后密切相关.CXCR4、CCR7及其配体在食管癌组织中异常表达,并与肿瘤的增殖分化、细胞凋亡、血管新生和侵袭转移等密切相关.结论:趋化因子受体CXCR4、CCR7及其配体在食管癌发生、发展中起重要作用,以CXCR4、CCR7为靶点有望为食管癌的治疗提供新的思路.