Bmi-1在食管癌组织中的表达及其与浸润转移的关系

目的检测食管鳞癌组织中Bmi-1蛋白的表达,并探讨其与食管鳞癌发生及浸润转移的关系。方法应用免疫组化SP法检测50例食管鳞癌组织及其相应的28例癌旁非典型增生组织和15例正常食管黏膜组织中Bmi-1蛋白的表达。结果食管鳞癌组织中Bmi-1蛋白阳性表达率高于癌旁非典型增生组织和正常食管黏膜组织（P〈0.05）;淋巴结转移组食管鳞癌组织中Bmi-1蛋白阳性表达率低于无淋巴结转移组（P〈0.05）;浸润至外膜食管鳞癌组织中Bmi-1蛋白阳性表达率高于深肌层及浅肌层（P〈0.05）。结论 Bmi-1蛋白过表达可能参与食管鳞癌的发生及浸润转移过程。     

OVCAR细胞转染miR-130b下调多梳基因-1蛋白表达增加对卡铂敏感性

目的观察miR-130b对卡铂抗卵巢癌的增敏作用,探讨其相关的作用机制。方法 OVCAR细胞Lipofectiamine 2000转染miR-130b模拟物和无miR-130b基因功能的miR-130b阴性对照,荧光定量PCR检测转染效果。转染细胞给予对倍稀释卡铂100,50,25,12.5,6.25,3.12,1.56和0.78μmo.lL-1处理68 h。MTT法检测OVCAR细胞存活;Western印迹法检测多梳基因-1(Bmi-1)蛋白表达。结果 荧光定量PCR结果显示,正常对照组、miR-130b模拟物转染组和阴性对照组miR-130b表达的相对值分别为0.22±0.08,0.46±0.12和0.23±0.10,miR-130b模拟物转染组显著高于正常对照组和阴性对照组(P<0.01),正常对照组和阴性对照组之间无统计学差异。卡铂对正常对照组、miR-130b模拟物转染组和阴性对照组卵巢癌OVCAR细胞的IC50值分别为32.4±4.6,14.7±2.1和(31.4±4.2)μmo.lL-1,miR-130b模拟物转染组IC50值显著低于正常对照组和阴性对照组(P<0.01)。正常对照组、miR-130b模拟物转染组和阴性对照组细胞多梳基因-1蛋白的相对表达量分别为0.75±0.16,0.21±0.12和0.77±0.18,miR-130b模拟物转染组显著降低(P<0.01)。结论 OVCAR细胞转染miR-130b可以显著增加卡铂对其敏感性,下调多梳基因-1蛋白表达可能是miR-130b增敏的作用机制之一。     

结肠癌转移相关基因1在食管癌细胞中的作用

目的 探讨结肠癌转移相关基因1 (MACC1)在食管癌细胞株Eca-109中的作用及其可能机制.方法 体外培养Eca-109细胞,并分为MACC1 shRNA转染重组质粒组(A组)、转染空质粒阴性对照组(B组)和Eca-109细胞空白组(C组).采用RT-PCR和Western blot法分别检测MACC1、c-MET mRNA和蛋白表达,Transwell和划痕实验分别观察转染前后Eca-109细胞侵袭和迁移能力的变化.结果 与B、C组相比,A组MACC1、c-MET mRNA和蛋白表达下调,Eca-109细胞侵袭和迁移能力降低(P＜0.05).结论 下调MACC1表达能明显抑制食管癌细胞的侵袭和转移能力,可能是通过调节下游基因c-MET表达实现的.     

DNA修复基因XRCC1在胃癌中的表达及意义

目的探讨DNA修复基因XRCC1在胃癌中的表达及意义。方法用免疫组化方法检测胃癌组织、远癌组织及肠上皮化生组织中XRCC1的蛋白表达。结果 3组XRCC1在胃癌组织中的表达水平间,差异均有统计学意义(P<0.05)。结论 XRCC1在胃癌组织中表达异常,提示其在胃癌发生过程中可能起重要作用。     

顺铂降低铜离子转运蛋白1表达诱导人食管鳞癌细胞耐药

目的诱导对顺铂耐药的人食管鳞癌细胞株,并进一步研究细胞耐药性产生的机制。方法逐渐增加顺铂浓度处理人食管鳞癌细胞HKESC-1,诱导顺铂耐药细胞株HKESC-1/cis。MTT法观察顺铂的细胞毒作用以及细胞的生长曲线。电感耦合等离子体质谱检测细胞内顺铂的蓄积和Pt-DNA加合物的形成。Western blot检测铜离子转运蛋白(copper transporter 1,CTR1)的表达。结果顺铂耐药细胞株HKESC-1/cis较其亲代细胞HKESC-1对顺铂引起的细胞毒作用敏感性下降,耐药指数为2.9。顺铂耐药细胞株HKESC-1/cis与其亲代细胞HKESC-1的生长速度相似。然而,HKESC-1/cis细胞内顺铂蓄积和Pt-DNA加合物的形成较HKESC-1细胞减少,并且CTR1蛋白表达水平降低。结论顺铂通过降低细胞膜CTR1蛋白的表达,抑制顺铂进入细胞,导致细胞耐药性的产生。     

食管鳞癌IGF-1R、EGFR蛋白的表达及其临床意义

目的：研究IGF-1R、EGFR蛋白在食管鳞癌患者中的表达情况及其临床意义。方法应用免疫组织化学SP法检测50例食管鳞癌患者IGF-1R、EGFR蛋白的表达情况,取20例手术标本正常组织作为对照组。结果食管鳞癌中IGF-1R蛋白阳性表达率明显升高（χ2=28.29, P〈0.05）, EGFR蛋白阳性表达率明显升高（χ2=4.56, P〈0.05）。IGF-1R的阳性表达与食管癌的病理分级有关,分级越高阳性率越高,与浸润深度有关,侵及深肌层及外膜IGF-1R的表达明显高于侵及浅肌层及黏膜的表达,与TNM分期有关,分期越晚阳性率越高,差异均有统计学意义。淋巴结转移组IGF-1R或EGFR的表达明显高于非转移组。结论初步结果分析显示IGF-1R可能与食管鳞癌的病理分级及侵袭、转移有关, EGFR可能与食管鳞癌的转移有关。     

食管鳞癌组织中Netrin-1蛋白及mRNA的表达

目的 检测食管鳞癌组织中神经轴突导向因子(Netrin-1)及mRNA表达.方法 应用免疫组化SP法和原位杂交方法 检测50例食管鳞癌组织及其相应的19例癌旁不典型增生组织和20例正常食管黏膜组织中Netrin-1、mRNA的表达.结果 ①食管鳞癌组织中Netrin-1、mRNA阳性表达率均高于癌旁不典型增生组织和正常食管黏膜组织(P均<0.05).②有淋巴结转移组食管鳞癌组织中Netrin-1、mRNA阳性表达率均高于无淋巴结转移组(P均<0.05).结论 人食管鳞癌组织中Netrin-1蛋白及mRNA均呈高表达,可能参与食管癌的发生、发展.     

Chk1表达与食管鳞癌浸润转移的关系

目的 探讨Chk1表达与食管鳞癌浸润转移的关系。方法 采用Western Blot、RT-PCR技术分别检测62例食管鳞癌组织、31例癌旁不典型增生组织及62例正常食管黏膜组织中Chk1蛋白及mRNA的表达,探讨Chk1表达与食管鳞状细胞癌浸润转移之间的关系。结果 正常食管黏膜组织、癌旁不典型增生组织以及食管鳞癌组织中,Chk1蛋白、mRNA的表达水平依次升高,三者间两两比较差异均有统计学意义（P〈0.05）;深层浸润组食管鳞癌组织中Chk1蛋白、mRNA的表达水平均显著高于浅层浸润组,两组比较差异有统计学意义（P〈0.05）;有淋巴结转移组的食管鳞癌组织中Chk1蛋白、mRNA的表达水平显著高于无淋巴结转移组,两组比较差异有统计学意义（P〈0.05）。结论 Chk1高表达与食管鳞癌浸润转移有关。     

Cripto-1蛋白和mRNA在食管鳞癌中的表达及其生物学意义

目的研究Cripto-1在食管鳞癌组织中的表达情况及其与食管鳞癌发生、发展的关系。方法采用免疫组化SP法和原位杂交方法对60例食管鳞癌及60例正常食管黏膜组织进行Cripto-1蛋白和mRNA的检测。结果（1）原位杂交结果显示,Cripto-1mRNA在食管鳞癌组织中表达的阳性率为63．33％,显著高于正常食管黏膜组织中Cripto-1mRNA的表达的阳性率（8-33％）,两组之间Cripto-1mRNA表达的差异具有统计学意义（P〈0．05）。（2）免疫组织化学结果表明,Cripto-1蛋白在食管鳞癌组织中表达的阳性率为58．33％,显著高于正常食管黏膜组织中Cripto-1蛋白的表达的阳性率（6．67％）,两组之间Cripto-1蛋白表达的差异具有统计学意义（P〈0．05）。（3）对60例食管鳞癌患者的临床病理学参数进行统计分析,结果显示Cripto-1mRNA和蛋白的表达均与食管鳞癌患者的年龄和性别无关（P〉0．05）,但与食管鳞癌的组织学分级、浸润深度、淋巴结转移和TNM分期具有显著的相关性（P〈0．05）。（4）相关性分析结果表明,在60例食管鳞癌标本中,Cripto-1 mRNA阳性表达的38例患者中,Cripto-1蛋白阳性表达患者占31例。而Cripto-1mRNA阴性表达的22例患者中,Cripto-1蛋白阴性表达占18例。Cripto-1 mRNA和蛋白在食管鳞癌组织中的表达呈正相关关系（7=0．600,P=0．000）。结论食管鳞癌组织中Cripto-1 mRNA和蛋白均呈现高表达,表明Cripto-1高表达可能与食管鳞癌的发生和进展密切相关。     

HMGB1和MMP-9在食管鳞癌组织中的表达及临床意义

目的 探讨高迁移率族蛋白B1(HMGB1)和基质金属蛋白酶-9(MMP-9)在食管鳞癌组织中的表达及临床意义.方法 采用免疫组化EnVision法检测食管鳞癌组织(A组,87例)和癌旁正常黏膜组织(B组,40例)中HMGB1和MMP-9的表达.结果 A组HMGB1和MMP-9阳性表达率均高于B组(69.0％ vs.22.5％和62.1％ vs.12.5％)(P＜0.05).A组HMGB1和MMP-9阳性表达与肿瘤浸润深度、TNM分期有关(P＜0.05).A组HMGB1和MMP-9表达呈正相关(rs =0.295,P＜0.01).结论 联合检测食管鳞癌组织中HMGB1和MMP-9蛋白表达可能有助于评估食管鳞癌的恶性程度.     

NES1基因在结肠癌细胞中表达及意义

目的：探讨NES1基因在结肠癌细胞中表达及意义。方法用RT-PCR方法检测NES1mRNA在正常结肠细胞、结肠癌细胞株HT29、HCT116表达情况。结果 NES1在结肠癌细胞株中表达较正常结肠黏膜细胞明显增高。结论 NES1基因在结肠癌的发生发展中起重要作用,这为选用NES1作为结肠癌治疗靶点提供了实验依据及理论基础。     

MicroRNA-129-2靶向调控SOX4抑制食管癌细胞增殖与侵袭转移的研究

目的 探讨MicroRNA-129-2(miR-129-2)在体内外抑制食管癌细胞增殖和侵袭转移的作用及可能机制,为食管癌的预后评估及靶向治疗提供新的思路.方法 将miR-129-2 mimics及miR-129-2 mimics NC基因序列分别转染到食管癌NMC109细胞中,并设为miR-129-2 mimics高表达组和miR-129-2 mimics阴性对照组(miR-129-2 mimics NC组),将非转染的NMC109细胞设为空白对照组.体外实验:运用MTT法检测3组细胞的增殖能力、Transwell法检测细胞侵袭能力、蛋白印迹法检测SOX4蛋白表达.采用裸鼠体内移植瘤形成实验检测3组细胞在体内环境下的增殖能力.结果 miR-129-2 mimics组的食管癌细胞增殖活性及侵袭能力较miR-129-2 mimics NC组及空白对照组明显减弱(P<0.001),而空白对照组与miR-129-2 mimics NC组食管癌细胞增殖性及侵袭性无明显差异(P>0.05);miR-129-2 mimics组的食管癌细胞中,SOX4的表达下调(P<0.001);miR-129-2 mimics组裸鼠移植瘤体积及重量明显减小(P<0.001),而miR-129-2mimics NC组及空白对照组无明显差异(P>0.05).结论 miR-129-2具有抑制食管癌细胞NMC109增殖和侵袭转移的作用,其作用机制可能与靶向下调SOX4基因表达有关.     

Involvement of miR-133a and miR-326 in ADM resistance of HepG2 through modulating expression of ABCC1

Recent studies have shown that a class of small, functional RNAs, named microRNAs, may regulate multidrug resistance-associated protein 1 (ABCC1). Since ABCC1 is an important efflux transporter responsible for cellular drug disposition, the discovery of microRNAs (miRNA) brings an idea that there may be some other unknown multidrug resistance (MDR) mechanisms exist. Using computational programs, we predicted that the 3′untranslated region (3′UTR) of ABCC1 contains a potential miRNA binding site for miR-133a and also two other for miR-326. These binding sites were confirmed by luciferase reporter assay. ABCC1 mRNA degradation was accelerated dramatically in cells transfected with miR-133a or miR-326 mimics using qRT-PCR, Furthermore, western blot analysis indicated that ABCC1 protein expression was significantly down-regulated in hepatocellular carcinoma cells line HepG2 after transfection with miR-133a or miR-326 mimics, suggesting the involvement of mRNA degradation and protein expression mechanism. The effects of the two miRNAs on adriamycin (ADM) sensitivity to HepG2 cells were determined by MTT assay. Compared with mock transfection, miR-133a or miR-326 mimics transfection sensitized these cells to ADM. These findings for the first time demonstrated that the involvement of miR-133a and miR-326 in MDR is mediated by ABCC1 in hepatocellular carcinoma cell line HepG2 and suggested that miR-133a and miR-326 may be efficient agents for preventing and reversing ADM resistance in cancer cells.     

PCB126 enhanced the genotoxicity of BaP in HepG2 cells by modulating metabolic enzyme and DNA repair activities

Both of benzo(a)pyrene (BaP) and 3,3′,4,4′,5-pentachlorobiphenyl (PCB126) are ubiquitous and persistent environmental pollutants. These two chemicals coexist in various environmental media and human samples and thus may have combined effects on human health. However, the toxic effects and related mechanism of co-exposure to BaP and PCB126 remain unknown. In a series of experiments using the HepG2 cells exposed to BaP (50 μM) or/and PCB126 (0.01, 0.1, 1 and 10 nM), we measured the rate of micronucleus (MN) formation, CYP1A1 activity and expression of nucleotide excision repair (NER) proteins (XPA and XPC). We found that the exposure to BaP or PCB126 alone could effectively increase the CYP1A1 activity and the XPA expression. BaP alone had a profound enhancement of MN formation. Compared with BaP alone, co-exposure to both BaP and PCB126 significantly enhanced the CYP1A1 activity and the formation of MN but reduced the expression of both XPA and XPC. The synergistic effect of PCB126 on BaP-induced MN formation was inhibited by alpha-naphthoflavone (ANF), an inhibitor of CYP1A1. Our findings suggest that PCB126 may enhance BaP-induced DNA damage and genotoxicity by increasing cytochrome P450 1A activity and decreasing the NER capacity.     

Adenovirus-mediated expression of UHRF1 reduces the radio- sensitivity of cervical cancer HeLa cells to γ-irradiation

Aim： An in vitro study was carried out to determine the effect of UHRF1 overexpression on radiosensitivity in human cervical cancer HeLa cells using adenovirus-mediated UHRF1 gene transfer （Ad5-UHRF1）.
Methods： Cell survival was evaluated using the clonogenic survival assay and the MTT assay; apoptosis and cell cycle distribution were monitored by flow cytometry. Protein levels were measured by Western blotting. Silencing XRCC4 expression was performed by transfection of small interfering RNA （siRNA）.
Results： Increased expression of UHRF1 by Ad5-UHRF1 significantly reduced the radiosensitivity of HeLa cells. The UHRF1-mediated radioresistance was correlated with increased DNA repair capability and increased expression of the DNA damage repair protein, XRCC4. Knocking down XRCC4 expression in the cells using XRCC4 siRNA markedly reduced the UHRF1-mediated radioresistance.
Conclusion： These results provide the first evidence for revealing a functional role of UHRF1 in human cervical cancer cells as a negative regulator of radiosensitivity.     

Coptisine inhibits the malignancy of bladder carcinoma cells and regulates XPO1 expression

This work is performed to investigate the effect of coptisine (COP) on the malignant biological behaviors of bladder carcinoma cells and its underlying mechanism. Bladder carcinoma cell lines were treated with different concentrations of COP in vitro. Cell counting kit-8 (CCK-8), scratch healing assay, Transwell assay, and flow cytometry were used to detect cell growth, migration, invasion, and cell cycle progression. Bioinformatics analysis was performed to predict the molecular targets of COP. Quantitative real-time PCR and western blot were adopted to determine the expression levels of exportin 1 (XPO1) mRNA and protein, respectively. Gene set enrichment analysis was applied to predict the signaling pathways related to XPO1. This study showed that COP treatment markedly suppressed the malignant biological behaviors of bladder carcinoma cells. XPO1 was identified as a downstream molecular target of COP in bladder carcinoma, and COP treatment inhibited the expression of XPO1 in bladder carcinoma cell lines. Overexpression of XPO1 reversed the impacts of COP on the malignant biological behaviors of bladder carcinoma cells. COP treatment modulated the expression level of cyclin D1 and CYP450 via XPO1. In summary, COP represses the malignant biological behaviors of bladder carcinoma cells and regulates XPO1 expression, which is promising to be a complementary drug for bladder carcinoma treatment.     

Regulation of lysophosphatidic acid-induced COX-2 expression by ERK1/2 activation in cultured feline esophageal epithelial Cells

Lysophosphatidic acid (LPA), a potent bioactive phospholipid, mediates diverse cellular responses by binding to specific G protein-coupled receptors (GPCRs). We investigated the signaling mechanisms underlying LPA-induced COX-2 expression in primary cultures of feline esophageal epithelial cells. The identity of the cultures was confirmed by immunocytochemistry using a cytokeratin antibody. Western blot analysis revealed a concentration-and time-dependent induction of COX-2 in response to LPA. Of the three major MAPKs, only ERK1/2 was activated by LPA in a time-dependent manner. LPA-induced COX-2 expression was significantly attenuated by the MEK inhibitor, PD98059, but not by the JNK inhibitor, SP600125, or the p38 MAPK inhibitor, SB212090. LPA-induced COX-2 expression was repressed by pertussis toxin, GF109204X, and Ki16425, indicating the involvements of PTX-sensitive G_i/o protein, PKC, and the LPA_1/3 receptor, respectively. Our data suggest that in esophageal epithelial cells, LPA-induced COX-2 expression requires activation of PKC and ERK1/2 downstream of the LPA_1/3 receptor, Understanding the regulation of COX-2 expression induced by LPA in esophageal epithelial cells might provide a new therapeutic strategy for esophageal inflammatory diseases.     

Notch1 signaling inhibits growth of EC109 esophageal carcinoma cells through downmodulation of HPV18 E6/E7 gene expression

Aim： To investigate the role of the Notch1 signaling pathway in growth arrest of an esophageal carcinoma cell line （EC109） in vitro and the mechanism involved. Methods： An intracellular domain of Notch1 （ICN） was transfected into cultured EC109 cells by lipofectamine transfection. Subsequently, the proliferation of the transfected cells was measured by an MTT assay. Cell cycle distribution was analyzed by flow cytometry. Human papillomavirus type 18 （HPV18） E6/E7 mRNA expression was detected by RT-PCR, and p53 protein expression was detected by Western blot. Results： Activation of Notch1 signaling resulted in inhibition of EC109 cell proliferation with the induction of G2/M arrest, downmodulation of HPV18 E6/E7 gene expression, and upregulation of p53 expression. Conclusion：Repression of HPV18 E6/E7 expression by Notch1 signaling results in the activation of p53-mediated pathways with concomitant growth suppression of HPV18-positive EC109 cells.     

Gene expression profiles in t24 human bladder carcinoma cells by inhibiting an l-type amino acid transporter,lat1

Inhibition of LAT1 (L-type amino acid transporter 1) activity in tumor cells could be effective in the inhibition of tumor cell growth by depriving tumor cells of essential amino acids. Because of the high level of expression of LAT1 in tumor cells, LAT1 inhibitors would be useful for anticancer therapy in suppressing tumor growth without affecting normal tissues. In recent years, cDNA microarray technique is useful technology for anticancer drug development. It allows identifying and characterizing new targets for developments in cancer drug therapy through the understanding genes involved in drug action. The present study was designed to investigate gene expression profile induced by LAT1 inhibitor using gene chip technology. Human bladder carcinoma cells (T24 cells) were treated with classical system L inhibitor 2-aminobicyclo-(2, 2, 1)-heptane-2-carboxylic acid (BCH). Gene chip experiment was applied for treated and untreated cells after 3 and 12 h. Two independent experiments with a high degree of concordance identified the altered expression of 151 and 200 genes after 3 and 12 h BCH treatment. Among these genes, 132 and 13 were up-regulated and 19 and 187 were down-regulated by 3 and 12 h BCH treatment respectively. We found that BCH affected the expression of a large number of genes that are related to the control of cell survival and physiologic behaviors. These data are useful for understanding of intracellular signaling of cell growth inhibition induced by LAT1 inhibitors as candidate for anticancer drug therapy.     

姜黄素对人肝癌细胞BEL-7402中HIF-1α表达的影响

目的探讨姜黄素对人肝癌细胞BEL-7402中H IF-1α表达的影响以及蛋白酶体在其中的作用。方法以0、2.5、5、10、15、20μmol.L-1的姜黄素处理人肝癌细胞BEL-7402缺氧环境中培养6 h,W ST-8法和台盼蓝染色法分析细胞增殖和活力,采用RT-PCR和W estern B lot方法检测肝癌细胞中H IF-1α的表达;以0、10μmol.L-1姜黄素、10μmol.L-1MG-132、10μmol.L-1姜黄素+10μmol.L-1MG-132处理人肝癌细胞BEL-7402缺氧环境中培养6 h,采用W esternB lot方法检测肝癌细胞中H IF-1α的蛋白表达。结果①不同剂量的姜黄素对肝癌细胞活力和增殖率的影响与对照组相比差异无显著性;②随着姜黄素浓度的增加,H IF-1α蛋白表达逐渐降低;③不同剂量的姜黄素对H IF-1αmRNA表达的影响与对照组相比差异无显著性;④MG-132能够逆转姜黄素对H IF-1α蛋白的减少。结论姜黄素抑制人肝癌细胞BEL-7402中H IF-1α蛋白表达是通过转录后机制和蛋白酶体途径。