小干扰RNA沉默CXCR4和CXCR7对人子宫内膜癌Ishikawa细胞裸鼠移植瘤生长的抑制作用

目的:探讨针对趋化因子受体基因CXCR4和CXCR7的小干扰RNA(siRNA)对人子宫内膜癌Ishikawa细胞裸鼠移植瘤生长的影响.方法:将子宫内膜癌Ishikawa细胞株注射于裸鼠肩胛背部皮下建立动物模型,待肿瘤最长径达5～7mm后,将所有荷瘤裸鼠随机分组,分别给予CXCR4-siRNA和CXCR7-siRNA单独或联合瘤体内注射,并以阴性对照siRNA和生理盐水作为对照组,每3d治疗一次,共6个治疗周期.观察各组裸鼠移植瘤的生长,比较各组移植瘤的体积和质量的差异,并以实时荧光定量RT-PCR、Western blotting和免疫组化技术验证CXCR4和CXCR7的基因沉默效果.结果:成功建立Ishikawa细胞裸鼠移植瘤模型,与阴性对照siRNA组和空白对照组相比,CXCR4siRNA治疗组、CXCR-siRNA治疗组和CXCR4-siRNA+ CXCR7-siRNA治疗组的肿瘤的生长均明显受到抑制(均P＜0.05);CXCR4-siRNA和/或CXCR7-siRNA瘤体内直接注射能显著下调CXCR4、CXCR7 mRNA(均P＜0.05)和蛋白(均P＜0.01)的表达.结论:siRNA干扰CXCR4和CXCR7表达能够有效抑制人子宫内膜癌Ishikawa细胞裸鼠移植瘤的生长.     

RNA干扰CXCR7基因对结肠癌SW1116细胞增殖及迁移的影响

目的:探讨趋化因子受体7(chemokine receptor 7,CXCR7)基因沉默对结肠癌细胞株SW1116增殖和迁移能力的影响。方法:采用脂质体转染的方法将CXCR7基因小干扰RNA(small interfering RNA,siRNA)转入人结肠癌SW1116细胞中,随后采用RT-PCR和蛋白质印迹法分别检测CXCR7mRNA和蛋白在SW1116细胞中的表达水平;采用CCK-8法检测对细胞增殖抑制率的影响;Transwell小室法检测干扰前后对细胞迁移能力的影响。结果:CXCR7-siRNA转染SW1116细胞24h后,CXCR7-siRNA干扰组SW1116细胞中CXCR7mRNA相对表达量为0.275±0.018,与空白对照组的0.635±0.024相比,mRNA表达抑制率为(56.6±3.8);转染48h后,CXCR7-siRNA干扰组的SW1116细胞中CXCR7蛋白的表达水平明显低于Negative-siRNA组和空白对照组(P<0.05);CXCR7-siRNA组的SW1116细胞增殖力在转染48、72、96和120h后明显低于阴性转染对照组和空白对照组(P<0.05);CXCR7-siRNA干扰组SW1116细胞穿过聚碳酸酯膜的细胞数为(22.73±2.01)个,明显少于阴性转染对照组的(49.20±3.82)个和空白对照组的(54.80±4.85)个(P<0.05)。结论:siRNA干扰下调CXCR7基因表达能抑制结肠癌细胞SW1116的增殖和迁移能力。     

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。     

小干扰RNA对骨肉瘤细胞株CXCR4基因表达和侵袭力的影响

目的：研究小干扰RNA（siRNA）对骨肉瘤细胞株中趋化因子受体CXCR4基因表达和侵袭力的影响。方法：采用单细胞克隆技术，从人骨肉瘤细胞系MG-63中分离培养出高表达CXCR4的骨肉瘤细胞株，设计合成针对CXCR4基因的siRNA，脂质体法转染骨肉瘤细胞株后用RT—PCR检测CXCR4基因表达水平的变化，荧光检测转染效率，Transwell趋化侵袭实验观察转染后细胞株侵袭能力的变化。结果：转染效率约为43．82％，CXCR4编码基因序列特异性siRNA能有效下调CXCR4基因表达水平，与空白组和阴性对照组比较，其差异有统计学意义（P〈0．05），细胞株穿过侵袭膜的细胞数也较转染前减少（P〈0．05）。结论：小分子干扰RNA技术能有效抑制骨肉瘤细胞株CXCR4基因的表达和体外侵袭能力。     

沉默CDK7对子宫内膜癌细胞顺铂敏感性的影响

  目的   研究沉默CDK7对子宫内膜癌细胞HEC-1-A顺铂化疗敏感性的影响。   方法   根据CDK7的基因序列设计合成不同的CDK7 siRNA片段并转染子宫内膜癌细胞HEC-1-A,通过实时定量PCR和Western blot验证RNA干扰的效果后,选择效果最优的CDK7 siRNA片段特异性沉默子宫内膜癌细胞中CDK7的表达,采用MTT细胞毒性实验、流式细胞仪及Hochest/PI双染色荧光显微镜技术检测转染前后该细胞系对化疗药物顺铂敏感性的变化。   结果   共选择了4个位点设计CDK7 siRNA并成功转染子宫内膜癌细胞系HEC-1-A,实时定量PCR及Western blot验证了各组的干扰作用,证实CDK7-423干扰效果最强,达70%以上。选择CDK7-423转染HEC-1-A,MTT细胞毒性实验结果发现在CDK7表达被抑制后,DDP的IC₅₀由45.122 g/mL降为3.200 g/mL,细胞毒性显著增高（P < 0.05）。流式细胞仪检测结果:CDK7低表达组细胞平均凋亡率细胞为37.57%,与高表达组细胞（11.66 %）相比,凋亡率明显增加（P < 0.05）。Hochest/PI双染色荧光显微镜下可见CDK7低表达组HEC-1-A细胞与亲代细胞比较,凋亡小体明显增多。   结论   通过CDK7 siRNA转染下调子宫内膜癌细胞中CDK7的表达以后,可以明显增强子宫内膜癌细胞对DDP的化疗敏感性,使子宫内膜癌细胞凋亡增加。CDK7可作为子宫内膜癌治疗的新靶点进行更加深入的研究。      

小干扰RNA抑制CXCR4基因表达对人肺癌细胞体外侵袭力的影响

目的：研究趋化性细胞因子受体CXCR4小干扰RNA（small interference RNA，siRNA）对人肺癌细胞体外侵袭力的影响。方法：利用T7 RNA聚合酶体外合成CXCR4特异性siRNA，用脂质体转染A549细胞。于转染后72h采用RT-PCR方法检测CXCR4 mRNA水平，用Western印迹方法检测CXCR4蛋白质水平，用Boyden小室模型检测细胞体外侵袭能力的变化，通过MTT法测定细胞增殖能力，用FCM法检测细胞周期的分布情况。结果：转染CXCR4 siRNA 72h后，CXCR4 mRNA及蛋白质水平明显下调，细胞的体外侵袭力减弱，增殖活性降低，细胞周期分布无明显改变。结论：特异的siRNA能够有效下调CXCR4基因，降低人肺癌细胞体外侵袭能力。     

MTA1影响子宫内膜癌细胞侵袭迁移的体外研究

  目的  通过siRNA(小干扰RNA)抑制MTA1(metastasis associated gene 1)在子宫内膜癌细胞系HEC-1-A中的表达, 研究MTA1对子宫内膜癌侵袭转移能力的影响, 并探索抑制子宫内膜癌侵袭转移的潜在靶点。  方法  通过脂质体介导方法, 将特异性siRNA表达载体psilencer2.0-MTA1-siRNA转染入人子宫内膜癌细胞系HEC-1-A, 以转染无关序列组psilencer2.0-neg及non-transfected组做对照, 采用RT-PCR以及Western blot检测特异性siRNA对MTA1mRNA及蛋白表达的抑制效果。应用划痕损伤实验及Transwell实验检测MTA1对子宫内膜癌细胞侵袭转移能力的影响, 以及体外实验验证应用RNA干扰技术以MTA1为靶点抑制子宫内膜癌细胞侵袭及转移的可行性。  结果  RT-PCR及Western blot结果显示, siRNA成功抑制子宫内膜癌细胞系HEC-1-A中MTA1的表达。划痕损伤实验显示转染后划痕损伤愈合明显减慢, 迁移率明显降低, Transwell体外侵袭实验结果显示, 转染后穿膜细胞百分率显著降低(P < 0.05)。  结论  体外实验显示, 应用脂质体介导的RNA干扰技术, 可有效抑制MTA1在子宫内膜癌细胞中的表达, 使之生长、侵袭及转移能力均受到抑制, 提示MTA1在子宫内膜癌的侵袭转移过程中发挥重要作用, 可能成为子宫内膜癌基因治疗的潜在靶点。      

子宫内膜癌组织中K-Cl共转运体1的表达及siRNA干扰其表达对子宫内膜癌HEC-1B细胞增殖的影响

目的:探讨K-Cl共转运体1(K-Cl cotransporter 1,KCC 1)mRNA在子宫内膜癌组织中的表达及小干扰RNA(small interfering RNA,siRNA)干扰其表达对子宫内膜癌HEC-1B细胞增殖能力和细胞周期的影响.方法:应用荧光定量PCR的方法检测正常子宫内膜和子宫内膜癌组织中KCC 1 mRNA的表达.设计并合成KCC 1特异性的siRNA,转染子宫内膜癌HEC-1B细胞后,应用RT-PCR、Western印迹法、MTT法和FCM法分别检测HEC-1B细胞中KCC 1 mRNA和蛋白的表达、细胞增殖、细胞周期和细胞凋亡的变化.结果:子宫内膜癌组织中KCC 1 mRNA的表达较正常子宫内膜组织明显增加,差异有统计学意义(P<0.05).KCC 1特异性siRNA能明显抑制HEC-1B细胞中KCC 1 mRNA和蛋白的表达(P<0.05),且HEC-1B细胞的增殖能力明显下降(P<0.05);细胞周期分析显示细胞明显阻滞于G0/G1期,与对照组比较,细胞凋亡增加(P<0.05).结论:KCC 1基因与子宫内膜癌有关,参与细胞周期的调节,具有促进细胞增殖的能力.     

小干扰RNA阻断周期蛋白依赖激酶4对子宫内膜癌细胞生物学行为的影响

背景与目的：细胞周期蛋白依赖激酶4(cyclin-dependent kinase 4,CDK4)是调控细胞周期进程的重要激酶之一,有实验报道其在子宫内膜癌中呈高表达,但是其在子宫内膜癌细胞中的生物学功能及其可能机制还不十分明确。本研究旨在通过小干扰RNA(small interfering RNA,siRNA)沉默CDK4表达,并检测其对人子宫内膜癌HEC-1B细胞生物学行为的影响及其可能机制。方法：将化学合成的CDK4-siRNA转染至HEC-1B细胞;实时荧光定量PCR法检测转染前后细胞中CDK4的mRNA表达量变化;Western blot检测转染前后细胞CDK4、视母细胞瘤基因(retinoblastoma gene,Rb)及其下游p-Rb的蛋白表达量的变化;分别采用CCK-8法、流式细胞仪、Transwell肿瘤细胞侵袭实验检测细胞增殖、周期、凋亡以及侵袭能力的变化。结果：转染后HEC-1B细胞中CDK4 mRNA及蛋白表达均明显下降(P<0.01);抑制CDK4表达后,抑制HEC-1B细胞的增殖及侵袭,转染si-CDK4组细胞发生侵袭数为(117±21)个,而转染si-control组及未处理组分别为(269±39)个和(262±35)个,差异具有统计学意义(P<0.01);细胞转染后早期凋亡率为(21.7±3.5)%,较未处理组［(12.4±2.1)%］和si-control组［(11.8±1.9)%］明显增加(P<0.01);细胞周期分布发生变化,G₁期比例增加(P<0.01),S期细胞比例降低(P<0.01);进一步的Western blot结果显示,抑制CDK4表达后,细胞内p-Rb表达下降,但是总Rb表达无明显变化。结论：针对CDK4基因的特异性小RNA干扰片段能够下调CDK4基因在子宫内膜癌细胞中的表达,抑制肿瘤生物学进程。     

CXCR4和CXCR7在肿瘤中的研究进展

以往的研究认为趋化因子受体4(chemokine receptor 4,CXCR4)是趋化因子CXCL12的唯一受体,CXCL12/CXCR4生物轴在肿瘤发展过程中起重要作用,然而最近研究发现CXCL12尚存在CXCR7这一新的受体,并且CXCL12/CXCR7生物轴同样对肿瘤的发生发展起重要作用.本文就有关趋化因子受体CXCR4和CXCR7在肿瘤中的表达、促进肿瘤增殖和转移、促进血管新生以及肿瘤治疗等方面的研究作一综述.     

The chemokine receptors CXCR4 and CXCR3 in cancer

Chemokines comprise a superfamily of at least 46 cytokines that were initially described based on their ability to bind to 18 to 22 G protein-coupled receptors to induce the directed migration of leukocytes to sites of inflammation or injury. In addition to mediating cellular migration, chemokine/chemokine receptor pairs have been shown to affect many cellular functions, including survival, adhesion, invasion, and proliferation, and to regulate circulating chemokine levels. Most malignancies also express one or more chemokine receptors. Early studies established a role for CXCR4 and CXCR7 in mediating breast cancer metastasis, but other chemokine receptors, including CXCR3, now are implicated in several malignancies as biomarkers of tumor behavior as well as potential therapeutic targets. This review summarizes our current understanding regarding the contribution of CXCR4 and CXCR3 to tumor behavior and how receptor expression is regulated, transduces intracellular signals, and contributes at the molecular level to tumor behavior. It also describes recent therapeutic approaches that target these receptors or their ligands.     

Clinical significance of CXCR3 and CXCR4 expression in primary melanoma

Tumor cell migration involved in metastases is a tightly regulated, nonrandom process. Chemokines have been identified as critical molecules guiding cell migration. We performed a prospective study to analyze a possible association between the expression of chemokine receptors CXCR3 and CXCR4 by primary melanoma and clinical outcome. Forty primary melanomas were available for analysis; 57% of the tumors expressed CXCR3 and 35% expressed CXCR4 by melanoma cells. At initial diagnosis, 5 patients had subclinical lymph node involvement and after a median follow-up time of 32 months, 2 additional patients developed regional lymph node metastases and 5 patients developed distant metastases. The expression of CXCR4, but not CXCR3, by melanoma cells in primary lesions was significantly associated with the presence of ulceration, increased tumor thickness, a greater risk of developing regional and distant metastases and a higher mortality rate. Our study underscores the value of CXCR4 expression as a useful marker for predicting outcome in patients with localized melanoma. In addition, our findings support that, among chemokine receptors, CXCR4 might be an appropriate therapeutic target for adjuvant therapy in patients at risk for metastatic disease.     

Opposing roles of CXCR4 and CXCR7 in breast cancer metastasis

Introduction  

CXCL12-CXCR4 signaling has been shown to play a role in breast cancer progression by enhancing tumor growth, angiogenesis, triggering cancer cell invasion in vitro, and guiding cancer cells to their sites of metastasis. However, CXCR7 also binds to CXCL12 and has been recently found to enhance lung and breast primary tumor growth, as well as metastasis formation. Our goal was to dissect the contributions of CXCR4 and CXCR7 to the different steps of metastasis - in vivo invasion, intravasation and metastasis formation.     

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.     

CXCR1、CXCR2在口腔鳞癌中的表达及意义

目的　探讨CXCR1、CXCR2的表达与口腔鳞癌生物学行为的关系及意义.方法　应用免疫组织化学技术检测57例口腔鳞癌手术切除标本和10例正常口腔黏膜标本中CXCR1、CXCR2的表达情况,分析其与病变类型及临床病理特征的关系.结果　口腔鳞癌标本中41例(71.9％) CXCR1呈阳性表达,35例(61.4％) CXCR2呈阳性表达.CXCR1与口腔鳞癌的病理分级、临床分期及淋巴结转移关系密切,CXCR2与口腔鳞癌的临床分期及淋巴结转移关系密切.结论　CXCR1、CXCR2在口腔鳞癌的发生、发展中起重要作用,是口腔鳞癌早期诊断和预后的潜在生物学参考指标.     

The dynamic yin-yang interaction of CXCR4 and CXCR7 in breast cancer metastasis

The contribution of CXCR7 to the tumor microenvironment has introduced a new level of complexity to CXCL12 signaling in breast cancer. In the previous issue of Breast Cancer Research, Hernandez and colleagues delineate the roles of CXCR4 and CXCR7 in tumor invasion and metastasis. The authors demonstrate that co-expression of CXCR7 and CXCR4 results in inhibition of CXCL12-mediated invasion, reduced intravasation of tumor cells into the vasculature, and fewer lung metastases compared with parental tumors. The results of this study suggest the combination of small molecule inhibitors of CXCR4 and CXCR7 could dramatically reduce invasion, intravasation, and metastasis and could be highly beneficial for the treatment of invasive breast cancer.     

CXCL12 / CXCR4 / CXCR7 chemokine axis and cancer progression

Chemokines, small pro-inflammatory chemoattractant cytokines that bind to specific G-protein-coupled seven-span transmembrane receptors, are major regulators of cell trafficking and adhesion. The chemokine CXCL12 (also called stromal-derived factor-1) is an important α-chemokine that binds primarily to its cognate receptor CXCR4 and thus regulates the trafficking of normal and malignant cells. For many years, it was believed that CXCR4 was the only receptor for CXCL12. Yet, recent work has demonstrated that CXCL12 also binds to another seven-transmembrane span receptor called CXCR7. Our group and others have established critical roles for CXCR4 and CXCR7 on mediating tumor metastasis in several types of cancers, in addition to their contributions as biomarkers of tumor behavior as well as potential therapeutic targets. Here, we review the current concepts regarding the role of CXCL12 / CXCR4 / CXCR7 axis activation, which regulates the pattern of tumor growth and metastatic spread to organs expressing high levels of CXCL12 to develop secondary tumors. We also summarize recent therapeutic approaches to target these receptors and/or their ligands.     

CXCR1 and CXCR2 enhances human melanoma tumourigenesis, growth and invasion

The aggressiveness of malignant melanoma is associated with differential expression of CXCL-8 and its receptors, CXCR1 and CXCR2. However, the precise functional role of these receptors in melanoma progression remains unclear. In this study, we investigate the precise functional role of CXCR1 and CXCR2 in melanoma progression. CXCR1 or CXCR2 were stably overexpressed in human melanoma cell lines, SBC-2 (non-tumourigenic) and A375P (low-tumourigenic) exhibiting low endogenous expression of receptors. Functional assays were performed to study the resulting changes in cell proliferation, motility and invasion, and in vivo tumour growth using a mouse xenograft model. Our data demonstrated that CXCR1- or CXCR2-overexpressing SBC-2 and A375P melanoma cells had enhanced proliferation, chemotaxis and invasiveness in vitro. Interestingly, CXCR1 or CXCR2 overexpression in SBC-2 cells induced tumourigenicity, and A375P cells significantly enhanced tumour growth as examined in vivo. Immunohistochemical analyses showed significantly increased tumour cell proliferation and microvessel density and reduced apoptosis in tumours generated from CXCR1- or CXCR2-overexpressing melanoma cells. CXCR1- or CXCR2-induced modulation of melanoma cell proliferation and migration was observed to be mediated through the activation of ERK1/2 phosphorylation. Together, these studies demonstrate that CXCR1 and CXCR2 play essential role in growth, survival, motility and invasion of human melanoma.