RhoC在肝癌细胞生长中的作用及分子机制

目的 研究RhoC在肝癌细胞生长中的作用及分子机制,为抑制肝癌细胞生长寻找分子靶点.方法 构建siRNA-RhoC载体,建立RhoC基因沉默的肝癌细胞株.采用四甲基偶氮唑蓝(MTT)法检测细胞生长,硝酸银染色检测细胞增殖,平板克隆实验检测细胞克隆形成能力,流式细胞术(FACS)检测细胞周期,逆转录聚合酶链反应(RT-PCR)检测细胞周期蛋白表达.PcDNA3-RhoC转染肝细胞,建立RhoC高表达细胞株,检测RhoC基因对正常肝细胞生长的影响.结果 siRNA-RhoC载体转染Bel7402细胞后,RhoC基因表达的抑制效率为82.3%.细胞培养第4天,RNAi组吸光度(A)值为0.41±0.10,显著低于Bel7402细胞组(0.73±0.11,P＜0.05)和阴性对照组(0.71±0.07,P＜0.05).此后,RNAi组的细胞增殖速度持续低于Bel7402细胞组和阴性对照组,直到第7天实验结束.RNAi组的细胞核平均银染颗粒数明显少于Bel7402细胞组和阴性对照组(分别为1.23±0.35、3.47±0.93和3.17±0.78,均P＜0.01);G0/G1期细胞显著升高[分别为(73.14±5.93)%、(57.05±5.97)%和(52.99±4.80)%,均P＜0.05];cyclin D1表达下降(分别为0.45±0.21、1.25±0.24和1.12±0.15,均P＜0.05);CDK4表达下降(分别为0.55±0.08、1.18±0.32和1.10±0.29,均P＜0.05);p16表达升高(分别为1.07±0.23、0.36±0.12和0.35±0.13,均P＜0.01);p21表达升高(分别为0.42±0.12、0.17±0.06和0.19±0.08,均P＜0.05).HL7702细胞转染PcDNA3-RhoC质粒后,RhoC 高表达,至转染的第3天,RhoC转染组A值为0.83±0.10,显著高于HL7702细胞组(0.54±0.11,P＜0.05)和空载体对照组(0.58±0.55,P＜0.05).其后,RhoC转染细胞生长速度持续高于HL7702细胞组和空载体对照组,直至第7天实验结束.结论 RhoC是调控肝癌细胞生长的关键分子,是抑制肝癌细胞生长的良好分子靶点.

Abstract：

Objective To clarify the role of RhoC in the growth of hepatocellular carcinoma cells and its molecular mechanism, so as to explore the molecular target of tumor cell growth.Methods siRNA-RhoC plasmid was constructed and RhoC gene silencing the cell-line of hepatocellular carcinoma was setup.Cell growth was assessed by MTT assay.AgNORs staining was applied to determine cell proliferation.Plate cell clone test was conducted to examine the capacity of cell clone formation.FACS was adopted to measure the course of cell cycle and semi-quantitative RT-PCR was used to determine the expression of cell cycle proteins.In order to further determine the effect of RhoC expression on cell growth, a RhoC over-expression human hepatocellular cell line was setup by PcDNA3-RhoC plasmid transfection.Results The inhibition rate of RhoC was 82.3%.From the fourth day of cell culture, the growth of cells in RNAi group was significantly slower than that in parental Bel7402 and negative control groups (0.41 ±0.10 vs.0.73 ±0.11and 0.71 ±0.07 respectively, P ＜0.05).AgNORs staining showed that average cell stained particles in RNAi group was significantly lower than that in parental Bel7402 and negative control(1.23 ±0.35 vs.3.47 ±0.93 and 3.17 t0.78,P ＜0.01 ).Plate clone formation test showed that clone formation efficiency in the RNAi group was notably lower than that in the control group [(20.33 ± 5.42 ) % vs.(70.58 ± 10.10) %and (69.83 ± 14.77) %, respectively, P ＜ 0.01].Cell cycle analysis by FACS showed that G0/G1 cell percentage in the RNAi group was significantly higher than that in the control group [(73.14 ±5.93)% vs.(57.05 ± 5.97 ) % and (52.99 ± 4.80) %, P ＜ 0.05].Compared with Bel7402 and negative control groups, the expression of following growth associated genes was significantly decreased: cyclin D1 (0.45 ±0.21 vs.1.25 ± 0.24 and 1.12 ± 0.15, respectively, P ＜ 0.05 ) and CDK4 (0.55 ± 0.08 vs.1.18 ± 0.32and 1.10 ± 0.29, respectively, P ＜ 0.05 ); the following genes were notably increased: p16 (1.07 ± 0.23vs.0.36±0.12 and 0.35 ±0.13, respectively, P ＜0.01)and p21 (0.42 ±0.12 vs.0.17 ±0.06 and 0.19±0.08, respectively, P ＜ 0.05).RhoC was highly expressed in PcDNA3-RhoC transfected hepatocellular cell line.From the third day on of the cell culture, cell growth in PcDNA3-RhoC group was remarkably higher than that in the HL7702 and PcDNA3 groups(0.83 ±0.10 vs.0.54 ±0.11 and 0.58 ±0.55, respectively, P ＜ 0.05 ).Conclusions RhoC is the key molecule in promoting hepatocellular cell growth, and is a promising target for tumor cell growth controlling.     

斑点型POZ蛋白对膀胱癌T24细胞增殖和迁移能力的影响

目的 探讨RNA干扰斑点型POZ蛋白（SPOP）基因表达对人膀胱癌T24细胞生物学行为的影响。 方法 采用阳离子脂质体转染试剂Lipofectamine 2000将靶向沉默SPOP基因的小干扰RNA（siRNA）序列siRNA1和siRNA2分别转染至T24细胞株（siRNA1组和siRNA2组）,设转染随机序列的T24细胞为对照组,采用实时定量逆转录聚合酶链反应（qRT-PCR）和Western blotting分别检测各组转染48 h后的SPOP mRNA及蛋白水平,细胞增殖活性检测试剂盒（CCK-8）检测细胞体外增殖活力,Transwell迁移试验及划痕试验观察各组的迁移能力。 结果 对照组、siRNA1组和siRNA2组的SPOP mRNA相对表达量为1.012±0.025、0.281±0.023和0.274±0.053,蛋白相对表达量为0.712±0.153、0.358±0.073和0.317±0.084,siRNA1组和siRNA2组的SPOP mRNA和蛋白水平均低于对照组（P＜0.05）,siRNA1组和siRNA2组SPOP mRNA和蛋白水平的差异无统计学差异（P＞0.05）;与对照组比较,siRNA1组和siRNA2组的增殖活性升高,差异有统计学意义（P＜0.05）,且siRNA1组和siRNA2组的增殖率随转染时间的延长而增加（P＜0.05）;Transwell迁移试验结果显示siRNA1组和siRNA2组的穿膜细胞数分别为（128.6±9.3）个和（134.5±8.6）个,均高于对照组的（92.5±8.4）个,差异有统计学意义（P＜0.05）;划痕试验结果显示siRNA1组和siRNA2组的划痕愈合率为（89.1±4.5）%和（93.7±5.1）%,均高于对照组的（32.6±2.8）%,差异有统计学意义（P＜0.05）。 结论SPOP与膀胱癌的恶性行为有关,可能发挥抑癌基因的作用,如抑制增殖及迁移。     

RhoC小干扰RNA抑制胃癌细胞的迁移和侵袭

目的：研究Ras homologyC（RhoC）在胃癌转移中的作用。方法：利用Vector NTI软件设计并构建RhoC的小干扰RNA（siRNA）载体，利用脂质体介导法将一组RhoCsiRNA分别转染胃癌SGC7901细胞。筛选出稳定抗性克隆；Westernblot检测RhoCsiRNA转染后的抑制效应，Cell Counting Kit-8检测RhoC siRNA转染细胞株的生长速度；MTT法检测转染细胞株对化疗药物的敏感性；损伤刮擦实验和TRANSWELL小室实验分别检测转染细胞株的迁移与侵袭能力。结果：在计算机辅助下成功地设计并构建了5个RhoC的小干扰RNA载体，Western blot显示其中一个RhoC的小干扰RNA RhloC-s362对RhoC的表达有明显的抑制作用；尽管下调RhoC的表达对胃癌细胞的生长和药物敏感性元明显影响，但可显著抑制肿瘤细胞的迁移与侵袭。结论：RhoC siRNARhoC-s362能够明显抑制胃癌SGC7901细胞中RhoC的表达，并进而抑制胃癌细胞的迁移与侵袭，提示RhoC可能在胃癌转移中发挥重要作用。     

RhoC and ROCKs regulate cancer cell interactions with endothelial cells

RhoC is a member of the Rho GTPase family that is implicated in cancer progression by stimulating cancer cell invasiveness. Here we report that RhoC regulates the interaction of cancer cells with vascular endothelial cells (ECs), a crucial step in the metastatic process. RhoC depletion by RNAi reduces PC3 prostate cancer cell adhesion to ECs, intercalation between ECs as well as transendothelial migration in vitro. Depletion of the kinases ROCK1 and ROCK2, two known RhoC downstream effectors, similarly decreases cancer interaction with ECs. RhoC also regulates the extension of protrusions made by cancer cells on vascular ECs in vivo. Transient RhoC depletion is sufficient to reduce both early PC3 cell retention in the lungs and experimental metastasis formation in vivo. Our results indicate RhoC plays a central role in cancer cell interaction with vascular ECs, which is a critical event for cancer progression.     

RhoC及其调节因子RhoGDla在非小细胞肺癌中的表达及临床意义

背景与目的RhoC在许多癌组织中过表达,而且与肿瘤的转移密切相关。RhoGDIα(Rho GDP dissociation inhibitor)被认为是Rho的负性调节因子,但目前其在肿瘤中的研究有限,而且在不同肿瘤中结果不一。本研究旨在探讨RhoC及其调节因子RhoGDIa在非小细胞肺癌的表达、二者间的关系及临床意义。方法应用免疫组织化学、Western blot、RT-PCR分别检测100例石蜡包埋肺癌组织、60例新鲜肺癌组织、正常支气管上皮细胞、肺癌细胞系中RhoC、RhoGDIa蛋白和mRNA的表达;分析其与临床病理参数间的关系。结果RhoC、RhoGDIa蛋白和mRNA在非小细胞肺癌组织和肺癌细胞系中的表达均明显高于相应的正常肺组织及正常支气管上皮细胞。在肺巨细胞癌高侵袭转移能力亚型BE1中,RhoC和RhoGDIa表达高于低侵袭转移能力亚型LH7。RhoC和RhoGDIa表达与淋巴结转移和临床分期有关。RhoC、RhoGDIα的过表达与患者的生存率低相关,在一定程度上提示患者的不良预后。RhoC的表达与RhoGDIα的表达正相关。结论RhoC、RhoGDIa可能参与肺癌的发生、发展。二者的表达与肺癌细胞的转移相关,可分别作为影响肺癌预后的独立风险因素。     

PTP1B基因对人胃癌细胞株的增殖和迁移能力的影响

背景与目的：胃癌是我国常见的消化道恶性肿瘤,术后易复发和转移。前期研究发现,蛋白酪氨酸磷酸酶1B(protein tyrosine phosphatase 1B,PTP1B)基因与胃癌肿瘤大小、淋巴结转移及TNM分期有关。本研究通过RNA干扰技术和基因克隆技术分别沉默和上调胃癌细胞中PTP1B基因的表达,观察PTP1B基因对胃癌细胞增殖和迁移能力的影响。方法：将靶向沉默PTP1B基因的短发夹RNA(short hairpin RNA,shRNA)序列和克隆人的PTP1B cDNA基因序列分别转染MKN28和MKN45细胞,采用实时定量PCR(quantitative realtime PCR,qRT-PCR)、蛋白［质］印迹法(Western blot)分别检测转染后细胞中PTP1B基因和蛋白的表达水 平,使用细胞计数试剂盒(cell counting kit-8,CCK-8)、Transwell迁移试验和划痕试验分别观察PTP1B基因对细胞增殖和迁移能力的影响。结果：转染shRNA后,MKN28细胞中PTP1B mRNA和蛋白表达量与空白对照组、阴性对照组相比抑制显著(P＜0.05)。CCK-8增殖活性实验显示,shRNA沉默PTP1B基因表达后能显著抑制胃癌MKN28细胞在48、72和96 h的增殖活性(P＜0.05)。Transwell迁移试验和划痕实验显示,PTP1B表达下调后胃癌MKN28细胞的迁移能力受到显著抑制(P＜0.05)。而提高PTP1B在MKN45细胞中表达后,细胞的增殖和迁移能力则显著提高(P＜0.05)。结论：PTP1B基因是胃癌细胞增殖和迁移的重要调控因子。     

RNAi沉默KPNA2对人膀胱癌细胞系5637转移潜能的影响

目的 探讨RNA干扰技术(RNAinterference,RNAi)沉默KPNA2(karyopherin a2)基因表达对人膀胱癌细胞系5637迁移和侵袭能力的影响.方法 实验分为siRNA敲低组和阴性对照组.siRNA敲低组设计合成靶向KPNA2序列的小分子干扰RNA(small interfering RNA,siRNA),用LipofectaminTM 2000方法瞬时转染膀胱癌细胞系5637;阴性对照组采用无关序列RNA采集.转染后48 h,应用蛋白质印迹法检测KPNA2的蛋白表达;Transwell实验检测细胞的迁移和侵袭能力.结果 蛋白质印迹法检测结果显示,膀胱癌5637细胞转染KPNA2特异性siRNA 48 h,与阴性对照组相比较,敲低组细胞KPNA2蛋白表达水平下降92.1％,差异有统计学意义,P=0.004 3.Transwell实验检测结果显示,敲低组细胞迁移能力明显受到抑制,细胞抑制率为61.7％,差异有统计学意义,P=0.038;敲低组细胞侵袭能力也明显受到抑制,细胞抑制率为58.6％,差异有统计学意义,P=0.026.结论 靶向KPNA2的特异siRNA能够下调KP-NA2在膀胱癌5637细胞中的蛋白表达水平,有效抑制膀胱癌5637细胞的迁移和侵袭能力.以KPNA2为靶点的RNA干扰技术有望成为膀胱癌基因治疗的新方法.     

利用siRNA阻抑survivin基因表达诱导乳腺癌细胞系MCF-7细胞凋亡的研究

背景与目的:survivin属IAP基因家族成员,表达于多种肿瘤组织中,能促使细胞逃避凋亡,并促进细胞的异常有丝分裂。本研究旨在探讨敲除survivin基因后,癌细胞的增殖和凋亡情况。方法:利用RNAi阻抑人乳腺癌细胞内survivin基因的表达,RT-PCR和Westernblot法分析survivin基因mRNA和蛋白的表达,MTT法检测细胞生长增殖抑制率,流式细胞术检测细胞凋亡率。结果:survivinsiRNA转染组,survivin基因表达水平与未转染组比较下调了64%。随着siRNA浓度的增加,细胞增殖抑制率逐渐增高,200nmol/L剂量组细胞增殖抑制率最高,可达60.9%。不同浓度的siRNA可不同程度地诱导细胞凋亡,200nmol/L剂量组凋亡率最高,可达29.0%。结论:survivinsiRNA有可能成为治疗乳腺癌的新药物。     

RhoC表达与体外乳腺癌细胞系侵袭行为的相关性

目的研究RhoC在不同转移能力乳腺癌细胞系中的表达及其与乳腺癌侵袭行为的相关性。方法利用逆转录-聚合酶链反应、Western blot、细胞爬片免疫组织化学和免疫荧光组织化学染色检测乳腺癌低转移细胞系MCF-7和高转移细胞系MDA-MB-231中RhoC mRNA和蛋白表达水平。构建RhoC真核表达载体,并转染乳腺癌低转移细胞系MCF-7;利用Matrigel穿膜侵袭实验检测转染后细胞的体外侵袭能力,划痕修复实验检测运动迁移能力,组织化学染色检测微丝骨架结构的变化,并利用Western blot检测Akt磷酸化水平变化。结果RhoC在不同转移能力的乳腺癌细胞系中呈不同程度表达,在高转移细胞系MDA-MB-231中高表达,在低转移细胞系MCF-7中低表达,差异有统计学意义(P<0.01)。基因转染过表达RhoC后,乳腺癌细胞的体外侵袭能力明显增强,正义转染组穿膜细胞数(56.88±4.18)与其相应空载体组(23.77±3.64)、反义组(23.12±3.22)和未转染对照组(28.44±2.48)比较,明显增多(P<0.01);运动迁移能力明显增强,正义转染组24 h划痕修复率(58.28%±2.14%)明显高于转染空载体组(28.23%±2.62%)、反义组(22.36%±2.73%)和未转染对照组(30.18%±2.86%),差异有统计学意义(P<0.01);组织化学染色显示,正义转染组细胞内肌动蛋白多聚体减少,微丝骨架重构;此外,RhoC正义转染组细胞p-Akt水平升高。结论RhoC过表达促进乳腺癌细胞的体外侵袭能力,并与乳腺癌细胞系转移潜能正相关,有望成为阻断乳腺癌转移的新靶点。     

肿瘤群体性迁移模型与机制

群体性迁移是胚胎发育过程中重要的细胞运动模式。近年研究发现,群体性迁移是恶性肿瘤细胞侵袭、转移的主要运动模式之一。肿瘤细胞能以群体的方式向间质及血管侵袭,其特征与单个肿瘤细胞侵袭运动明显不同,而其发生机制与肿瘤细胞-细胞间连接、肿瘤细胞与间质微环境的相互作用密切相关。随着群体性迁移观察模型的逐步完善,参与调控群体性迁移的物理引导及化学引导机制以及相关信号调控、表观调节等分子机制正逐步被揭示。     

RNA干扰YAP基因对人膀胱癌T24细胞株增殖和迁移能力的影响

背景与目的：膀胱尿路上皮癌是泌尿统系最常见的肿瘤,术后易复发及转移,Yes相关蛋白(Yes associated protein,YAP)基因与膀胱癌关系密切,本研究探讨通过RNA干扰技术沉默膀胱癌T24细胞中YAP基因的表达,观察YAP基因沉默后对人膀胱癌T24细胞增殖、迁移能力的影响。方法：用阳离子脂质体转染试剂LipofectamineTM2000将靶向沉默YAP基因的小干扰RNA(small interfering RNA,siRNA)序列转染至膀胱癌T24细胞株中,采用实时定量逆转录聚合酶链反应(quantitative real time-polymerase chain reaction,qRT-PCR)、蛋白质印迹法(Western blot)检测转染后T24细胞中YAP基因及蛋白的表达水平,细胞增殖活性检测试剂盒(cell counting kit-8,CCK-8)、Transwell迁移试验以及划痕实验观察siRNA在体外对人膀胱癌T24细胞增殖、迁移能力的影响。结果：转染siRNA后,YAP RNA和蛋白表达量同空白对照以及阴性对照组相比被显著抑制(RNA：F=93.91,P<0.000 1;蛋白：F=4.62,P<0.05),CCK-8增殖活性实验结果显示,RNAi干扰YAP表达可以显著抑制膀胱癌T24细胞的增殖活性(12 h：F=6.00,P=0.037;24 h：F=41.72,P=0.000 3;36 h：F=462.8,P<0.000 1;48 h：F=236.6,P<0.000 1;72 h：F=140.5,P<0.000 1),通过Transwell实验和划痕试验发现,RNAi干扰YAP表达显著抑制膀胱癌T24细胞的迁移能力(Transwell：F=43.55,P<0.05;划痕：F=43.55,P<0.05)。结论：YAP基因是膀胱肿瘤增殖及迁移的重要调控因子。     

ＲｈｏＣ和ＬｙＧＤＩ在肺癌中的表达及临床意义

目的　探讨ＲｈｏＣ和ＬｙＧＤＩ在肺鳞癌、肺腺癌组织中的表达情况及其相关性的临床意义。方法　收集肺鳞癌、腺癌及其癌旁组织标本各４８例。应用免疫组织化学法、蛋白免疫印迹法检测ＲｈｏＣ、ＬｙＧＤＩ在肺癌及癌旁组织中的表达情况,并结合临床资料分析。结果　ＲｈｏＣ、ＬｙＧＤＩ在肺癌组织中的表达量分别为２.１９７±０.２２０和１.０３５±０.１１８,均高于其癌旁组织中的表达量１.８１０±０.０９９和０.８５９±０.０９１。ＲｈｏＣ、ＬｙＧＤＩ的阳性率分别为７７.１％和６８.８％,二者均与ＴＮＭ分期和淋巴结转移有关,但二者无相关性（ｒ＝－０.０１１,Ｐ＝０.９４２）。结论　ＲｈｏＣ、ＬｙＧＤＩ都参与了肺癌的发生,ＲｈｏＣ可能促进肺癌的进展及淋巴结转移,而ＬｙＧＤＩ可能抑制肺癌的进展及淋巴结转移。     

巨噬细胞游走抑制因子在卵巢癌侵袭中的作用与意义

背景与目的:巨噬细胞游走抑制因子(maerophage migration inhibitory factor,MIF)与肿瘤的恶性进展密切相关.本研究探讨MIF基因对卵巢癌HO-8910和OVCAR-3细胞侵袭和增殖能力的影响及其在卵巢癌组织中表达的意义.方法:瞬时转染小干扰RNA(small interfering RNA,siRNA)靶向敲除MIF基因,RT-PCR和Western blot检测MIF在mRNA和蛋白水平的表达,通过体外迁移、侵袭实验和噻唑蓝比色法(MTT)分析MIF对HO-8910和OVCAR-3细胞迁移、侵袭和增殖能力的影响;免疫组织化学检测MIF蛋白在卵巢癌组织中的表达情况.结果:与阴性对照组细胞比较,瞬时转染MIF siRNA的HO-8910和OVCAR-3细胞中MIF基因表达水平明显降低.MTT结果显示,转染MIF siRNA的两株卵巢癌细胞增殖率显著低于阴性对照组(P＜0.05).转染MIF siRNA的HO-8910细胞穿膜细胞数(MIF-si1:48.0±7.3;MIF-si2:38.0±3.6)与阴性对照组(78.0±8.5)比较差异有统计学意义(P＜0.05),其穿过铺了Matrigel膜的细胞数(MIF-sil:35.0±5.0:MIF-si2:30.0±5.6)也显著少于阴性对照组(P＜0.05).同样,转染了MIF siRNA的OVCAR-3细胞穿膜细胞数(MIF-si1:40.0±4.5;MIF-si2:42.0±3.0)与阴性对照组(65±2.1)比较,差异也有统计学意义(P＜0.05),其穿过铺了Matrigel膜的细胞数(MIF-sil:25.0±3.0:MIF-si2:27.0±3.4)也明显低于阴性对照组(P＜0.05).53.6%(37/69)卵巢癌组织中有MIF蛋白表达.MIF蛋白表达与肿瘤临床分期呈显著正相关(P＜0.01).结论:MIF基因可能通过促进肿瘤细胞的增殖和侵袭能力在卵巢癌的发生发展中发挥重要作用,有可能成为分子靶向治疗卵巢癌的潜在靶点.     

RNAi干扰食管癌EC9706细胞MTA1基因表达对侵袭和迁移的影响

Objective  To observe the effect of MTA1 gene silencing by RNA interference on invasion and migration of esophageal carcinoma 9706 cells. Methods  siRNA expression vector targeting MTA1 gene was transfected into EC9706 cells by Lipofectamine method. MTA1 mRNA and protein expressions were detected through quantitative RT-PCR and Western Blot, respectively. The invasion and migration of EC9706 cells were evaluated by scrape wound healing assay and cell invasion assay in vitro. Results  MTA1 gene expression decreased significantly. The scrape wound of EC9706 cells healed more slowly and the cell population that cut through Matrigel were less in the EC9706 cells transfected with siRNA expression vector than non-transfected EC9706 cells and EC9706 cells transfected with blank vector (P < 0.05). Conclusion  MTA1 gene silencing by RNAi can inhibit the invasion and migration of esophageal carcinoma effectively. It is supposed that MTA1 gene may be a prospective molecule target in tumor therapy. Supported by a grant from the “Tenth Five-Year Plan” Research Foundation for the Key Construction Project (211 Projects) by Ministry of Education of China (2002).     

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。     

Co-ordination of cell cycle,migration and stem cell-like activity in breast cancer

Migration, proliferation and stem cell-like activity are all key cellular characteristics which aid the formation and progression of breast cancer, in addition to involvement in treatment resistance. Many current therapies aim to target tumour proliferation, and although successful, mortality rates in breast cancer remain significant. Our main objectives were to investigate the relationship between proliferation, migration and stem cell-like activity in breast cancer.We used a panel of cell lines and primary human breast cancer samples to assess the relationship between migration, proliferation and stem cells. We performed live cell sorting according to cell cycle (Hoechst-33324) and in combination with stem-cell markers (CD44/CD24/ESA) followed by assessment of migration and stem cell activity (mammosphere formation).We identified an inverse relationship between proliferation and migration/stem cell-like activity. G0/1 cells showed increased migration and mammosphere formation. Furthermore we identified a subpopulation of low proliferative stem-like cells (CD44+/24lo/ESA+) with increased migration and mammosphere formation that are specifically inhibited by Dickkopf 1 (DKK1) and Dibenzazepine (DBZ) known stem-cell inhibitors.These data show the co-ordination of migration, proliferation and stem cell activity in breast cancer, and has identified a sub-population of stem-like cells, greatly adding to our understanding of the complex nature of stem cell biology.     

壳聚糖纳米粒介导人表皮生长因子受体2小干扰RNA抑制乳腺癌细胞迁移

目的以壳聚糖纳米粒作为人类表皮生长因子受体2小干扰RNA（HER-2siRNA）的载体转染人乳腺癌细胞SK—BR-3,观察HER-2沉默对其体外迁移能力的影响。方法采用离子凝胶法制备壳聚糖．HER．2siRNA纳米粒,原子力显微镜观察纳米粒的形态。分4组进行转染,分别加入转染试剂壳聚糖-HER-2siRNA纳米粒（实验组）、Lipofectamine^TM2000和HER-2siRNA（阳性对照组）、壳聚糖-阴性对照siRNA纳米粒（阴性对照组）和空白壳聚糖纳米粒（空白组）。-步法逆转录-聚合酶链反应（RT—PCR）和Westernblot鉴定HER-2在SK—BR-3细胞中的表达变化。Transwell小室法检测HER-2siRNA对细胞体外迁移能力的影响。统计分析采用单因素方差分析、K—WH秩和检验。结果成功制备了壳聚糖-siRNA纳米粒,呈球形,大小约100nm。实验组能有效抑制HER-2mRNA和蛋白的表达以及SK—BR-3细胞的体外迁移能力,与阴性对照组和空白组相比差异有显著的统计学意义（P均=0．000）。结论壳聚糖纳米粒介导的HER-2siRNA能有效抑制人乳腺癌细胞SK—BR-3中HER-2的表达及其体外迁移能力。