干扰ELK-3抑制人肝癌细胞的上皮-间质转换

目的研究转录因子ELK-3与人肝癌细胞系Hep G2和Hu H7上皮-间质转换(EMT)的关系及其作用机制。方法将Hep G2和Hu H7细胞分为小干扰RNA转染组和Ras-ELK-3信号通路抑制剂(XRP44X)处理组。用Western blot检测ELK-3、ELK-3靶基因EGR-1以及EMT相关分子钙黏附素E(E-cadherin)、波形蛋白(vimentin)的表达和MAPK信号通路蛋白p38的表达。结果 ELK-3干扰或Ras-ELK-3信号通路抑制剂(XRP44X)处理肝癌细胞后其ELK-3和ELK-3的靶基因EGR-1的蛋白表达均显著降低(P0.01),E-cadherin表达水平升高(P0.01),vimentin的表达及p38磷酸化水平均降低(P0.01)。结论通过干扰ELK-3下调p38抑制EMT。     

原发性肝细胞癌中干细胞标志物的表达与上皮细胞-间质转化的关系

目的 探讨原发性肝细胞癌(hepatocellular carcinoma,HCC)中肝干细胞标志物的表达及其与上皮细胞-间质转化(epithelial-mesenchymal transition,EMT)的关系.方法 采用免疫组化法检测72例HCC中肝干细胞标志物CK19、EpCAM、CD133、CD90的表达及其与EMT相关蛋白E-cadherin、vimentin和Snail的关系.结果 HCC组织中CK19、EpCAM、CD133、CD90阳性率分别为31.9％(23/72)、40.3％ (29/72)、51.4％ (37/72)、41.7％(30/72),有镜下癌栓和门静脉癌栓组中CK19、EpCAM、CD133、CD90阳性率均高于无镜下癌栓和无门静脉癌栓组,Ⅲ+Ⅳ期、肿瘤数目多发的HCC中EpCAM、CD133、CD90阳性率高于Ⅰ+Ⅱ期和单发的HCC;＜46岁、组织学Ⅲ+Ⅳ级、血清AFP≥350 ng/ml的HCC中CK19阳性率高于其相应对照组.E-cadherin与其余两种EMT标志物均呈负相关(vimentin:r=-0.572,P=0.004;Snail:r=-0.597,P=0.003),CK19、Ep-CAM、CD90阳性HCC组中vimentin阳性强度均高于阴性组,CD133阳性HCC组中E-cadherin阳性强度低于CD133阴性组,vimentin、Snail阳性强度均高于CD133阴性组.结论 与CK19、EpCAM、CD90相比,CD133阳性的肝癌干细胞与EMT的关系更加密切,CD133可作为同时针对HCC肝干细胞和EMT的治疗靶点.     

MicroRNA-9通过NRP1抑制胃癌SGC-7901细胞上皮-间充质转化功能

目的:研究微小RNA-9(microRNA-9,miR-9)对胃癌SGC-7901细胞上皮-间充质转化(EMT)功能的影响及其相关机制。方法:SGC-7901胃癌细胞株分别转染miR-9 mimics和阴性对照序列(negative control mimic,NCM),作为miR-9组和NCM组,并设立未转染对照(control)组,采用RT-qPCR法检测各组细胞miR-9的含量,Transwell实验检测3组细胞迁移能力和侵袭能力,Western blot法检测3组细胞的N-cadherin、E-cadherin、α-catenin和神经纤毛蛋白1(NRP1)表达水平。采用Western blot法检测NRP1过表达对miR-9抑制EMT的拮抗作用。双萤光素酶实验检测miR-9与NRP1的关系。结果:miR-9组的miR-9表达水平明显上调,为control组的538倍(P0.05)。miR-9组的迁移细胞数量明显低于control组(P0.05)。miR-9组的侵袭细胞数量明显低于control组(P0.05)。miR-9组细胞的N-cadherin和NRP1蛋白表达量明显降低,E-cadherin及α-catenin蛋白表达量明显升高。而NRP1及miR-9均过表达组胃癌细胞中N-cadherin蛋白表达量明显升高,E-cadherin及α-catenin蛋白表达量明显降低。双萤光素酶检验结果显示NRP1为miR-9的下游靶基因(P0.05)。结论:miR-9可能通过降低下游靶基因NRP1水平影响EMT相关蛋白表达,抑制胃癌SGC-7901细胞的EMT功能。     

Notch信号通路与Slug在调控宫颈鳞状细胞癌上皮-间充质转变中的意义

目的探讨Notch信号通路与Slug在宫颈鳞状细胞癌上皮-间充质转变(EMT)中的作用机制。方法收集临床慢性宫颈炎及宫颈鳞状细胞癌标本,分别用免疫组化及Western blot检测Notch1、NICD1、Slug、E-cadherin、α-SMA蛋白表达情况,Real-Time PCR检测Notch1、SlugmRNA表达情况。结果与慢性宫颈炎比较,Notch1、NICD1、Slug和α-SMA在宫颈鳞状细胞癌中表达增加(P<0.01),E-cadherin蛋白在癌组织中的表达降(P<0.01)。结论 Notch信号通路和Slug可能通过调控EMT在宫颈鳞状细胞癌的侵袭转移过程中发挥作用     

牛磺酸联合SN50协同抑制人肝癌细胞系HepG2的增殖

目的探讨牛磺酸联合NF-κB信号通路抑制剂SN50对肝癌细胞系HepG2增殖的影响及其机制。方法将HepG2细胞分为对照组、牛磺酸组(给予150 mmol/L牛磺酸处理24 h)、抑制剂组(给予36μmol/L SN50处理24 h)和牛磺酸+抑制剂组;用MTT法、克隆形成实验和流式细胞仪分别检测细胞的增殖和凋亡;Western blot和RT-qPCR检测细胞中cyclin D1、Bcl-2蛋白和mRNA的表达情况。结果与对照组相比,经150 mmol/L牛磺酸或36μmol/L SN50处理24 h后,HepG2细胞的生存率和集落形成率均明显降低,凋亡率明显升高,细胞中cyclin D1和Bcl-2蛋白和mRNA表达均明显受到抑制(P<0. 05)。同时,SN50增强了牛磺酸对HepG2细胞的增殖和cyclin D1、Bcl-2表达的抑制作用以及对细胞凋亡的促进作用。结论牛磺酸联合NF-κB信号通路抑制剂协同抑制肝癌细胞系HepG2的增殖。     

MYH9在人肝癌组织中的表达及其对肝细胞系增殖和凋亡的影响

目的探讨非肌肉肌球蛋白重链(MYH9)在肝癌组织中的表达及通过沉默MYH9基因对肝癌细胞系SMMC-7721的增殖及凋亡的影响。方法收集50组人肝癌组织及癌旁组织,选用人肝癌细胞系SMMC-7721和Hep G2及人正常肝细胞系LO2,免疫组织化学方法及Western blot检测肝癌组织及癌旁组织中MYH9蛋白的表达,Western blot检测SMMC-7721、Hep G2及LO2中MYH9蛋白的表达;将MYH9 siRNA转染SMMC-7721,CKK8法及流式细胞术检测沉默MYH9对肝癌细胞增殖及细胞凋亡的影响。结果 MYH9蛋白在肝癌组织中的表达明显高于癌旁(P0.05);MYH9蛋白在SMMC-7721及Hep G2中的表达均明显高于LO2(P0.05);沉默MYH9基因可抑制细胞增殖(P0.001),促进细胞凋亡(P0.05)。结论 MYH9蛋白在肝癌组织的表达显著高于癌旁组织;MYH9低表达能有效抑制肝癌细胞的增殖,促进其凋亡。     

肝星状细胞通过SDF-1/CXCR4轴诱导肝癌细胞上皮间质转分化并促进其侵袭

目的 探讨肝星状细胞是否通过SDF-1/CXCR4轴促进肝癌细胞侵袭的作用和可能机制.方法 通过Westernblot和real time RT-PCR,检测肝星状细胞LX02和肝癌细胞系SDF-1、CXCR4表达.Transwell实验检测星状细胞LX02或外源性SDF-1干预对肝癌细胞HepG2以及CXCR4基因沉默后的HepG2侵袭的影响,Westem blot检测上皮标志E-cadherin和间质标志vimentin的表达变化.结果 肝星状细胞LX02中趋化因子SDF-1高表达,4株人肝细胞癌细胞系均有CXCR4高表达,其中HepG2细胞表达最强.星状细胞或SDF-1均能诱导肝癌细胞上皮-间质分化并促进其侵袭.通过RNA干扰技术靶向沉默肝癌细胞CXCR4基因,星状细胞或SDF-1均不能增强其细胞侵袭能力,不能诱导肝癌细胞HepG2发生上皮-间质分化.结论 肝星状细胞通过趋化因子SDF-1/CXCR4轴促进肝癌细胞侵袭,其机制可能与诱导肝癌上皮-间质转化有关.     

MiR-26b inhibits hepatocellular carcinoma cell proliferation,migration, and invasion by targeting EphA2

Deregulated microRNAs (miRNAs) have been shown to play important roles in cancer progression as a result of changes in expression of their target genes. In this study, we investigated the expression of miR-16b in eight hepatocellular carcinoma (HCC) cell lines, revealed the roles of miR-26b on hepatocellular carcinoma (HCC) cell proliferation, migration, and invasion, and confirmed that EphA2 is a direct target of miR-26b. The miR-26b expression was decreased and EphA2 expression was evaluated in HCC cell lines. Luciferase assays revealed that miR-26b inhibited EphA2 expression by targeting the 3’-untranslated region of EphA2 mRNA. Overexpression of miR-26b dramatically inhibited the proliferation, invasion, and migration of HCC cells by targeting EphA2. Moreover, miR-26b down-regulated c-Myc and CyclinD1 expression, which was reversed by overexpressed EphA2. Taken together, our data demonstrated the mechanism of miR-26b contributed to HCC progression and implicated that miR-26b’s potential in HCC therapy.     

MicroRNA-194 acts as a prognostic marker and inhibits proliferation in hepatocellular carcinoma by targeting MAP4K4

MicroRNAs (miRNAs) play a crucial role in cancer development and progression of hepatocellular carcinoma (HCC). In this study, we aimed to analyze the role of microRNA-194 (miR-194) in HCC. We found that miR-194 expression was significantly reduced in HCC and its expression was an independent poor prognostic factor for HCC patient overall and disease-free survival rate. A significant correlation was observed between miR-194 reduction and unfavourable variables including tumor size (P = 0.0315), histologic grade (P = 0.0038), TNM stage (P = 0.0083), intrahepatic metastasis (P = 0.0184). Overexpression of miR-194 in HCC cell lines HepG2 and Hep3B inhibited cell proliferation by blocking G1-S transition and inducing apoptosis. Mitogen-activated protein kinase 4 (MAP4K4), a potential target gene of miR-194, was inversely correlated with miR-194 expression in HCC tissues and cell lines. Further studies demonstrated that miR-194 regulated the progression of HCC through directly inhibiting the expression of MAP4K4 and the restoration of MAP4K4 expression reversed the inhibitory effects of miR-194 on HCC cell proliferation. Together, our findings indicate that miR-194 may serve as a valuable prognostic marker and promising interventional therapeutic target for HCC.     

RAC1 overexpression promotes the proliferation,migration and epithelial-mesenchymal transition of lens epithelial cells

Cataract is a main cause of blindness worldwide. RAC1 has been reported to have a close relationship with the proliferation and migration of cells. However, the relationship between RAC1 and cataract is not yet clear. The proliferation and migration of lens epithelial cells are key factors in the formation of cataract as well as in the complication of cataract surgery. In this study, the effect of RAC1 overexpression on the proliferation and migration of lens epithelial cells was explored. Results showed that RAC1 overexpression promoted the proliferation of lens epithelial cells and increased the protein level of proliferating cell nuclear antigen. RAC1 overexpression also promoted migration and invasion of lens epithelial cells and had an influence on the epithelial-mesenchymal transition process. These results indicate that RAC1 may become a therapeutic target of cataract and inhibition of RAC1 may become a promising way for the therapy of cataract.     

Control of differentiation in human colorectal carcinoma cell lines: epithelial-mesenchymal interactions

Mesenchymal elements have been investigated for their effects on the growth and differentiation of seven human colorectal carcinoma-derived cell lines. Epithelial cells were cultured as monolayers on plastic; they were also grown on fibroblast lawns and in collagen matrices, with and without fibroblasts. In each case, differentiation was assessed morphologically with monoclonal antibodies directed against components of normal goblet and columnar cells. The results were compared with those obtained when the cell lines were grown in vivo as xenografts in athymic mice. The xenografts allowed the greatest potential for differentiation, although two cell lines showed little or no response to mesenchyme either in vivo or in vitro. The presence of fibroblasts induced branched structures in all the remaining lines when these were cultured in collagen matrices. The collage matrix alone induced the formation of well-defined glandular structures in SW1222 cells, reminiscent of those seen in SW1222 xenografts and normal colonic crypts. Epithelial response to mesenchymal factors may require specific receptors, the expression of which dictates phenotype. Isolation and analysis of such receptors and factors could lead to clarification of the mechanisms underlying normal tissue morphogenesis and the growth and spread of neoplastic cells.     

lncRNA PCAT1表达下调抑制口腔鳞状细胞癌细胞的增殖、生长、侵袭、迁移及上皮-间充质转化

目的:探讨长链非编码RNA(long non-coding RNA,lncRNA) PCAT1对口腔鳞状细胞癌(oral squamous cell carcinoma,OSCC)细胞的增殖、生长、侵袭及迁移的影响,并探讨其可能机制。方法:采用Lipofectamine 200将PCAT1 siRNA转染入OSCC细胞分别利用RT-qPCR和Western blot检测相关基因的mRNA及蛋白表达;分别利用CCK-8实验和集落形成实验检测OSCC细胞的增殖及生长能力;利用细胞侵袭实验和细胞迁移实验检测OSCC细胞的侵袭及迁移能力。结果:PCAT1在OSCC组织和细胞中的表达与癌旁正常组织和正常口腔细胞黏膜角质细胞相比显著上调(P 0. 05)。转染PCAT1 siRNA可以显著降低PCAT1在Tca8113和TSCCa细胞中的表达(P 0. 05)。PCAT1的低表达可以显著抑制Tca8133和TSCCa细胞的增殖、生长、侵袭及迁移(P 0. 05)。PCAT1在Tca8113和TSCCa细胞中的低表达可以抑制ZEB1、N-cadherin和vimentin的mRNA及蛋白表达,同时增加E-cadherin的mRNA及蛋白表达(P 0. 05)。结论:沉默PCAT1能够抑制OSCC细胞的增殖、生长及转移,该作用可能与调控上皮-间充质转化有关。     

CRNDE过表达促进人肝癌细胞系HepG2增殖和迁移

目的探讨长链非编码RNA(lncRNA)结直肠差异性表达基因(CRNDE)对HepG2细胞增殖和迁移能力的影响及作用机制。方法构建CRNDE过表达质粒,进行慢病毒包装后感染HepG2细胞。实验分别设置阴性对照组(LV5/NC)和CRNDE过表达组(LV5/CRNDE)。应用2.5μg/mL嘌呤霉素干预4~5周,筛选出CRNDE过表达HepG2细胞系;CCK8和细胞划痕实验检测细胞的增殖及迁移能力变化;real-time PCR和Western blot检测两组细胞E-cadherin、N-cadherin、Bax和Bcl-2 mRNA及蛋白表达变化。结果与LV5/NC组相比,LV5/CRNDE组CRNDE表达显著升高(P<0.01),且LV5/CRNDE组细胞的增殖和迁移能力均显著提高(P<0.01);同时,LV5/CRNDE组细胞E-cadherin和Bax表达均明显降低(P<0.01),而N-cadherin和Bcl-2表达则明显升高(P<0.01)。结论CRNDE可通过促进N-cadherin和Bcl-2表达,抑制E-cadherin和Bax表达,进而增强HepG2细胞的增殖和迁移能力。     

曲古霉素A抑制肝癌细胞的糖酵解

目的研究表观遗传药物组蛋白去乙酰化抑制剂曲古霉素A(TSA)对miR-34家族(miR-34a/b/c)在肝癌细胞中表达的影响及其抑制肝癌细胞糖酵解的分子机制。方法在肝癌细胞系中分别转染miR-34模拟物或相应阴性对照,用real-time PCR法检测miR-34a/b/c的表达以及糖酵解途径关键酶的表达;在肝癌细胞中分别转染miR-34b模拟物、相应阴性对照,或用TSA、DMSO处理肝癌细胞,用乳酸检测试剂盒、葡萄糖检测试剂盒分析miR-34b及TSA对肝癌细胞Hep G2、PLC/PRF/5糖酵解途径的影响;设计拯救实验在Hep G2细胞中研究TSA、miR-34b与肝癌细胞糖酵解调控的关系。结果 TSA可以诱导Hep G2、PLC/PRF/5肝癌细胞内源性miR-34b的表达上调(P0.01);过表达miR-34b可以抑制糖酵解关键酶LDH-A的表达(P0.05);miR-34 b可以抑制含有LDH-A 3'非翻译区的报告基因活性;敲低miR-34 b的表达可以降低TSA对肝癌细胞Hep G2糖酵解途径的抑制作用。结论 TSA通过诱导miR-34 b表达上调发挥抑制肝癌细胞的代谢转换。     

ITIH4基因沉默对人肝癌细胞HepG2增殖、迁移及侵袭的影响

目的:探讨沉默间-α-胰蛋白酶抑制剂重链H4(inter-alpha-trypsin inhibitor heavy chain H4,ITIH4)基因对人肝癌细胞系HepG2增殖、迁移及侵袭的影响.方法:将4个ITIH4基因沉默载体分别转染HepG2细胞,用2 mg/L嘌呤霉素筛选获得稳定表达细胞株.Western印迹评价沉默效率,并选择效率最高的细胞株(si-ITIH4)用于后续实验.转染无意义序列的细胞株(si-Control)作为对照组.分别用细胞计数及CCK-8细胞计数试剂盒评价两组细胞增殖.用Transwell小室评价两组细胞的迁移及侵袭.结果:成功获得ITIH4基因沉默的HepG2细胞株si-ITIH4,与si-Control组相比,si-ITIH4组沉默效率达到86%.与si-Control组相比,si-ITIH4组细胞增殖显著降低、细胞迁移及侵袭均显著减少,差异有统计学意义(P<0.05).结论:ITIH4基因沉默可显著抑制HepG2的增殖、迁移及侵袭,提示其可能是参与肝细胞癌发生发展的重要基因之一.     

miR-497与肾癌预后的关系及其对肾癌786-0细胞增殖、凋亡和侵袭的影响

目的:探讨微小RNA-497(miR-497)与肾癌预后的关系及其对肾癌786-0细胞增殖、凋亡和侵袭的影响。方法:收集2011年~2015年80例肾细胞癌患者手术切除的癌组织和癌旁组织并回顾性分析患者随访资料。采用实时定量PCR法检测癌组织及配对的癌旁组织中miR-497的表达。在肾癌细胞系786-0中转染miR-497模拟物,应用MTT法、台盼兰拒染法活细胞计数、流式细胞术和Transwell小室实验检测miR-497对786-0细胞增殖、凋亡和侵袭能力的影响。通过生物信息学预测miR-497在786-0细胞中作用的靶基因。Western blotting法检测miR-497对靶基因蛋白表达影响。结果:肾癌组织中miR-497的表达量明显低于癌旁组织(P0.05)。786-0中miR-497表达量明显低于正常肾上皮细胞中miR-497表达量(P0.05)。随访时间2~48个月,中位随访时间29个月,失访6例,随访率92.5%。76例患者3年无复发生存率(recurrence-free survival,RFS)为55.2%。高miR-497表达组和低miR-497表达组肾癌患者的RFS分别为71.2%和40.1%,差异有统计学显著性(P0.05)。过表达miR-497可显著抑制786-0细胞的增殖和侵袭能力,显著促进细胞凋亡(P0.05)。过表达miR-497可显著抑制786-0细胞中cyclin D1蛋白的表达(P0.05)。结论:miR-497与肾癌预后相关,miR-497能明显抑制肾癌786-0细胞的增殖和凋亡,其机制可能与其靶向抑制cyclin D1有关。     

Bufalin对食管癌ECA109细胞中FAK活化和上皮-间质转化的影响

目的探讨Bufalin对食管鳞状细胞癌ECA109细胞中FAK活化和上皮-间质转化(epithelial-mesenehymal transition,EMT)的影响。方法采用RT-PCR法检测不同浓度Bufalin对食管鳞状细胞癌ECA109细胞中FAK、E-cadherin、vimentin mRNA表达的影响。Western blot法检测不同浓度Bufalin对ECA109细胞中FAK活化水平及FAK、E-cadherin、vimentin蛋白表达的影响。采用Transwell小室实验检测不同浓度Bufalin对ECA109细胞迁移与侵袭能力的影响。结果 RT-PCR结果表明Bufalin抑制FAK、E-cadherin、vimentin的mRNA表达。Western blot结果表明Bufalin抑制E-cadherin、vimentin的表达和FAK的活化。Transwell实验结果表明Bufalin可以抑制ECA109细胞的迁移及侵袭。药物干预组(20、40、60、80、100 nmol/L Bufalin及PF562271组)与阳性对照组相比,Transwell迁移实验结果显示穿过基膜的细胞个数由107.00±8.19下降至78.67±3.06、61.67±3.06、42.67±3.512、24.33±2.517、10.33±3.215、9.00±2.65;Transwell侵袭实验结果显示穿过基膜的细胞个数由127.67±8.02下降至102.33±4.51、87.33±7.10、73.00±4.58、57.33±2.52、39.00±3.61、37.33±2.52,差异均有统计学意义(P0.05)。结论 Bufalin可以抑制食管鳞状细胞癌中FAK的活化,提示Bufalin可能是通过下调FAK来抑制食管鳞状细胞癌EMT过程及食管癌的迁移及侵袭。     

Knockdown of long noncoding RNA SPRY4-IT1 suppresses glioma cell proliferation,metastasis and epithelial-mesenchymal transition

Long noncoding RNAs (lncRNAs), a class of ribonucleic molecules, participate in various cellular processes. They are highly expressed in several types of cancer and their expression was related to pathophysiological characteristics of tumor growth, therefore, they can be considered as a promising diagnostic tool and a convenient prognostic biomarker. SPRY4-IT1, belonging to a group of intron-retained lncRNAs, was reported to affect tumor development of many types of cancer. However, the expression and the role of SPRY4-IT1 in glioma are still unclear. Therefore, in this study, we examined for the first time the expression and role of SPRY4-IT1 in glioma cells. The results of our study showed that SPRY4-IT1 was up-regulated in human glioma tissues and cell lines. Knockdown of SPRY4-IT1 could inhibit glioma cell growth and migration. Moreover, knockdown of SPRY4-IT1 could inhibit epithelial-mesenchymal transition (EMT) phenotype in glioma cells. Based on these findings, SPRY4-IT1 may be used as a new target for diagnosis and treatment of glioma.