Siglec-15与CD4^(+)、CD8^(+)在肝细胞癌中的表达关系及其临床意义

目的研究在肝细胞癌(hepatocellutar carcinoma,HCC)中唾液酸免疫球蛋白型凝集素-15(Sialic acid immunoglobulin type lectin 15,Siglec-15)与CD4^(+)、CD8^(+)的表达及相关性,并分析其与临床病理特征及预后的影响。方法通过TCGA数据库分析HCC与正常组织中Siglec-15、CD4^(+)T、CD8^(+)T mRNA的表达水平。收集我院2019年1月至2022年1月行肝癌根治切除术的27例新鲜HCC组织及正常组织标本和56例HCC组织及正常组织石蜡切片标本,qRT-PCR检测Siglec-15和CD4^(+)T、CD8^(+)T mRNA表达水平,IHC检测Siglec-15和CD4^(+)、CD8^(+)蛋白表达水平,并对Siglec-15与CD4^(+)、CD8^(+)蛋白之间的关系及临床病理因素和预后进行分析。结果HCC患者中Siglec-15在HCC组织表达高于正常组织(P<0.05),CD4^(+)、CD8^(+)在HCC组织表达低于正常组织(P<0.05);在HCC组织中,Siglec-15表达与CD4^(+)、CD8^(+)蛋白表达呈负相关(P<0.05)。在HCC组织中,Siglec-15蛋白的表达与患者的TNM分期、肿瘤大小相关(P<0.05),CD4^(+)蛋白的表达与患者的肿瘤大小相关(P<0.05),CD8^(+)蛋白的表达与患者的TNM分期、远处转移相关(P<0.05)。Siglec-15及CD4^(+)的表达与患者的无进展生存期具有相关性(P<0.05)。结论HCC组织中Siglec-15高表达、CD4^(+)、CD8^(+)低表达,且Siglec-15与CD4^(+)、CD8^(+)表达呈负相关。Siglec-15及CD4^(+)的表达高低与HCC患者进展及不良预后相关,可作为预测HCC患者的独立预后因素。

喉鳞状细胞癌组织中SIGLEC-15表达及其与患者临床病理特征及预后的关系

目的分析喉鳞状细胞癌组织中SIGLEC-15表达及其与患者临床病理特征及预后的关系。方法选取106例喉鳞状细胞癌组织标本及30例癌旁正常组织标本,免疫组化染色检测SIGLEC-15蛋白表达,分析SIGLEC-15表达及其与患者临床病理特征的关系,Kaplan-Meier生存曲线分析SIGLEC-15蛋白阳性表达与患者预后的关系。结果癌组织中SIGLEC-15阳性表达率(61.32%)高于癌旁组织(P<0.05)。SIGLEC-15蛋白阳性表达与喉鳞状细胞癌的分化程度、TNM分期、淋巴结转移相关(P<0.05)。随访截止至2024年3月,106例喉鳞状细胞癌患者死亡36例、生存70例。SIGLEC-15蛋白阳性表达患者累积生存率为49.23%(32/65),低于阴性表达患者的累积生存率92.68%(38/41)(Log Rankχ^(2)=15.955,P<0.001)。结论喉鳞状细胞癌组织中SIGLEC-15的阳性表达率较高,与患者的肿瘤分化程度、TNM分期、淋巴结转移有关。SIGLEC-15的阳性表达可能预示着患者预后较差,可能成为喉鳞状细胞癌患者预后评估的一个重要标志物。

吉九里香碱体外诱导HCT-15细胞凋亡的研究

目的:探讨中药单体吉九里香碱是否通过诱导 HCT-15细胞凋亡而发挥其抑制肿瘤细胞增殖的作用。方法:采用 MTT法检测细胞增殖抑制作用;采用透射电子显微镜观察细胞凋亡的形态学变化;库尔特全自动颗粒粒度分析药物引起 HCT-15细胞体积大小分布的变化;琼脂糖凝胶电泳测定 DNA ladder 的发生;通过流式细胞仪应用 Annexin V-FITC 和 Rhodamine123检测磷脂酰丝氨酸(PS)及线粒体跨膜电位(△ψm)。结果:MTT 结果显示吉九里香碱以浓度和时间依赖方式抑制 HCT-15细胞的增殖,抑制率依赖于吉九里香碱的浓度和作用时间;50 μmol·L^(-1)吉九里香碱处理 HCT-15细胞24 h 时,细胞出现凋亡典型的形态学特征;50 μmol·L^(-1)吉九里香碱分别处理 HCT-15细胞6,12,24 h 及不同浓度吉九里香碱处理 HCT-15细胞24 h 时,能够使细胞产生明显的 DNA ladder 和体积大小变化,呈一定的量效和时效关系;细胞 PS 外翻随作用时间的延长而增加,分别为15.34%(6 h),20.20%(12 h),39.37%(24 h);50 μmol·L^(-1)吉九里香碱处理 HCT-15细胞导致线粒体△ψm以时间依赖方式下降。结论:吉九里香碱通过诱导 HCT-15细胞凋亡而发挥其抗 HCT-15细胞增殖的作用,致凋亡机理可能与线粒体△ψm快速下降有关。

17-AAG阻滞HCT-15细胞周期并诱导其凋亡

目的：观察17-AAG对人结直肠癌HCT-15细胞株凋亡和周期及相关分子机制的影响。方法：体外培养HCT-15细胞，分别给予不同剂量的17-AAG及不同作用时间，采用四甲基偶氮唑盐微量酶反应比色法（ MTT法）检测细胞的活力；Annexin V-FITC／PI双标记流式细胞术检测细胞凋亡；流式细胞术检测细胞周期的变化；RT-PCR和Western blotting法检测凋亡和周期相关因子的表达水平。结果：1．25～20 mg／L浓度的17-AAG作用24 h和48 h后对HCT-15细胞有显著抑制作用，且有明显的时间、剂量依赖性；0.425、0.85和1.7 mg／L浓度的17-AAG作用48 h后，各组细胞发生G1期阻滞及明显凋亡，STAT3 mRNA和蛋白的表达水平明显下降，凋亡和周期相关因子cyclin D1 mRNA和蛋白表达水平下调，Cyt C、caspase 9及caspase 3 mRNA和蛋白表达水平上调。结论：17-AAG对人结直肠癌细胞的活力具有明显抑制作用，使细胞周期发生G1期阻滞和细胞凋亡。17-AAG可能通过阻断STAT3通路下调cyclin D1而发生G1期阻滞；17-AAG可能通过阻断STAT3通路启动线粒体凋亡通路来诱导细胞凋亡。

鬼臼苦素对人结直肠癌HCT-15细胞增殖和周期的影响及其作用机制

目的探讨鬼臼苦素(PPP)对人结直肠癌细胞系HCT-15细胞增殖和周期的影响及其作用机制。方法不同浓度PPP处理HCT-15细胞,采用细胞计数试剂盒8(CCK-8)法检测HCT-15细胞的增殖活性;倒置显微镜下观察HCT-15细胞的形态变化;流式细胞术检测HCT-15细胞周期的改变;Western blotting检测增殖细胞核抗原(PCNA)及细胞周期蛋白D1(cyclin D1)的蛋白表达水平。结果CCK-8法结果显示,PPP能显著抑制HCT-15细胞的增殖活性,并呈剂量-时间依懒性;倒置显微镜下观察发现,细胞失去原有形态回缩为圆形,折光性降低,活细胞数量明显减少;流式细胞术结果显示,G2/M期细胞比例显著升高,G0/G1期和S期细胞比例明显降低;Western blotting结果显示,PCNA、细胞周期蛋白D1的表达水平明显降低。结论PPP可显著抑制人结直肠癌HCT-15细胞的增殖,其作用机制可能是通过下调PCNA、cyclin D1的表达水平,进而将细胞阻滞于G2/M期发挥抑制作用。

联合应用罗格列酮与维甲酸对HCT-15细胞增殖和凋亡的影响

目的:探讨联合应用罗格列酮(ROS)与维甲酸(ATRA)对人结直肠癌细胞株HCT-15细胞增殖和凋亡的影响。方法:实验分为ROS单药组(ROS浓度分别为6.25、12.5、25、50μmol/L)、ATRA单药组(ATRA浓度为2μmol/L)、ROS与ATRA联合用药组(ROS以上各浓度+2μmol/L ATRA)。在分别或联合应用不同浓度ROS和ATRA干预人结直肠癌细胞株HCT-15不同时间后,MTT法检测药物对细胞生长的抑制作用;流式细胞仪检测细胞发生凋亡和细胞生长周期的变化;免疫细胞化学法检测干预前后细胞COX-2、MMP-7和TIMP-1表达蛋白水平的变化。结果:单独应用ROS可抑制HCT-15细胞的增殖,并呈浓度-时间依赖性,与ATRA联合应用则有明显的协同作用(q>1.15)。流式细胞术结果显示:单独应用ROS和ATRA可导致G0/G1期细胞比例上升,而S期比例下降明显,诱导HCT-15细胞凋亡;而联合应用对细胞G1期的阻滞作用更强(P<0.05)。免疫细胞化学结果显示:COX-2、MMP-7和TIMP-1在对照组人结直肠癌细胞HCT-15中阳性表达率分别为66.79%、73.21%、64.08%,联合应用后3种蛋白的表达率为19.33%,20.58%,13.13%,二者差异有统计学意义(P<0.01)。结论:ROS和ATRA可降低HCT-15细胞中COX-2、MMP-7和TIMP-1的表达,使细胞周期阻滞于G1期,诱导细胞凋亡,从而发挥抑制细胞增殖的作用。

单纯疱疹病毒2型ICP27_(377-513)核酸疫苗联合IL-15核酸疫苗免疫效果的观察

目的构建表达单纯疱疹病毒2型感染细胞蛋白27(infected cells protein 27,ICP27)的重组质粒pcDNA3.1-ICP27377-513并观察白细胞介素(IL)-15核酸疫苗联合HSV-2-ICP27377-513核酸疫苗的免疫效果。方法采用分子克隆技术构建pcDNA3.1-ICP27377-513重组质粒。将BALB/c雌性小鼠随机分为pcDNA3.1-ICP27377-513联合pcDNA3.1-IL-15(pIL-15)组、pcDNA3.1-ICP27377-513组、pcDNA3.1组和pIL-15组,共免疫3次,每次间隔2周。末次免疫后28 d取血,用微量中和实验法检测血清中特异性中和抗体,ELISA法检测血清中IL-4、IL-2和干扰素-γ(IFN-γ)水平。阴道给予致死量HSV-2攻击小鼠,分别于接种后3 d、7 d和14 d收集阴道冲洗液,用荧光定量PCR法检测生殖道病毒载量。结果pcDNA3.1-ICP27377-513联合pIL-15组和pcDNA3.1-ICP27377-513组中和抗体效价分别为40.00±8.16、28.67±4.47,但两组间差异无统计学意义(P>0.05)。pcDNA3.1-ICP27377-513联合pIL-15组IFN-γ、IL-4水平明显高于pcDNA3.1-ICP27377-513组(P<0.05),但IL-2水平两组间差异无统计学意义(P>0.05)。阴道给予致死量HSV-2攻击后,pcDNA3.1-ICP27377-513联合pIL-15组和pcDNA3.1-ICP27377-513组小鼠阴道冲洗液中病毒载量随着时间延长逐渐减低,pcDNA3.1-ICP27377-513联合pIL-15组和pcDNA3.1-ICP27377-513组相比差异有统计学意义(P<0.05)。结论IL-15核酸疫苗联合HSV-2-ICP27377-513核酸疫苗对小鼠有较好的免疫保护作用。

鬼臼苦素对人结直肠癌HCT-15细胞增殖的影响

目的:研究鬼臼苦素对人结直肠癌细胞HCT-15增殖的影响。方法:体外培养人结直肠癌HCT-15细胞,采用CCK-8法检测不同浓度鬼臼苦素作用HCT-15细胞24h、48h、72h后的增殖活性,并在倒置显微镜下观察HCT-15细胞形态的变化。结果:CCK8法结果显示鬼臼苦素能显著抑制HCT-15细胞的增殖活性,鬼臼苦素浓度在0.125μmol/L～2μmol/L范围内,随着鬼臼苦素浓度的增加和作用时间的延长,HCT-15细胞的抑制率逐渐增加,呈剂量-时间依赖性。倒置显微镜下观察发现,鬼臼苦素作用后HCT-15细胞不同程度皱缩、折光性降低、活细胞数明显减少。结论:鬼臼苦素对人结直肠癌HCT-15细胞具有明显的抑制作用。

唾液酸结合免疫球蛋白样凝集素-15在食管鳞癌组织中的表达及意义

目的探讨免疫检查点唾液酸结合免疫球蛋白样凝集素-15(Siglec-15)在食管鳞癌中的表达及意义。方法免疫组化检测食管鳞癌组织、癌旁正常食管组织中Siglec-15表达情况,并比较分析其与食管鳞癌患者临床病理特征的关系。结果Siglec-15在食管鳞癌组织中表达阳性率为60.0%(48/80),高于癌旁正常食管组织中的23.75%(19/80)(χ^(2)=21.595,P<0.001)。食管鳞癌组织中Siglec-15蛋白表达与患者肿瘤直径、T分期、TNM分期、N分期及分化程度有关(χ^(2)=7.500,P=0.006;χ^(2)=10.342,P=0.001;χ^(2)=22.547,P=0.016;χ^(2)=20.508,P<0.001;χ^(2)=12.586,P=0.002)。结论Siglec-15在食管鳞癌组织中高表达,与患者的疾病进展显著相关。

Claudin-15 overexpression inhibits proliferation and promotes apoptosis of Schwann cells in vitro

Our previous experiments have discovered that Claudin-15 was up-regulated in Schwann cells of the distal nerve stumps of rat models of sciatic nerve injury.However,how Claudin-15 affects Schwann cell function is still unknown.This study aimed to identify the effects of Claudin-15 on proliferation and apoptosis of Schwann cells cultured in vitro and explore the underlying mechanisms.Primary Schwann cells were obtained from rats.Claudin-15 in Schwann cells was knocked down using siRNA(siRNA-1 group)compared with the negative control siRNA transfection group(negative control group).Claudin-15 in Schwann cells was overexpressed using pGV230-Claudin-15 plasmid(pGV230-Claudin-15 group).The pGV230 transfection group(pGV230 group)acted as the control of the pGV230-Claudin-15 group.Cell proliferation was analyzed with EdU assay.Cell apoptosis was analyzed with flow cytometric analysis.Cell migration was analyzed with Transwell inserts.The mRNA and protein expressions were analyzed with quantitative polymerase chain reaction assay and western blot assay.The results showed that compared with the negative control group,cell proliferation rate was up-regulated;p-AKT/AKT ratio,apoptotic rate,p-c-Jun/c-Jun ratio,mRNA expression of protein kinase C alpha,Bcl-2 and Bax were down-regulated;and mRNA expression of neurotrophins basic fibroblast growth factor and neurotrophin-3 were increased in the siRNA-1 group.No significant difference was found in cell migration between the negative control and siRNA-1 groups.Compared with the pGV230 group,the cell proliferation rate was down-regulated;apoptotic rate,p-c-Jun/c-Jun ratio and c-Fos protein expression increased;mRNA expression of protein kinase C alpha and Bax decreased;and mRNA expressions of neurotrophins basic fibroblast growth factor and neurotrophin-3 were up-regulated in the pGV230-Claudin-15 group.The above results demonstrated that overexpression of Claudin-15 inhibited Schwann cell proliferation and promoted Schwann cell apoptosis in vitro.Silencing of Claudin-15 had the reverse effect and provided neuroprotective effect.This study was approved by the Experimental Animal Ethics Committee of Jilin University of China(approval No.2016-nsfc001)on March 5,2016.

Cellular Adhesion Tripeptide RGD Inhibits Growth of Human Ileocecal Adenocarcinoma Cells HCT-8 and Induces Apoptosis

The tripeptide,Arg-Gly-Asp（RGD）motif is an integrin-recognition site found in adhesive proteins present in extracellular matrices（ECM）and in the blood.HCT-8 cells were treated with cellular adhesion tripeptide RGD at various concentrations.MTT assay was performed to examine the growth and proliferation of HCT-8 cells after treatment with RGD for 48 h.Haematoxylin and Eosin（HE）staining and electromicroscope were used to observe the morphology of apoptotic cells.Survivin and flow cytometry were also used to analyze the HCT-8 apoptosis.Cellular adhesion tripeptide RGD significantly inhibits the growth and proliferation of HCT-8 cells in a dose-dependent manner and induces apoptosis of HCT-8.These results indicate that cellular adhesion tripeptide RGD inhibits the growth and proli-feration of tumor HCT-8 cell,probably by the aid of inducing apoptosis of HCT-8 cell.

虎杖白藜芦醇对人结肠癌HCT-15细胞增殖及凋亡的影响

目的观察虎杖白藜芦醇对人结肠癌HCT-15细胞增殖及凋亡的影响。方法以0、25、50、100、200μmol/L白藜芦醇作用人结肠癌HCT-15细胞24h以及50μmol/L白藜芦醇处理HCT-15细胞0、12、24、48、72h。采用噻唑蓝(MTT)法检测白藜芦醇对HCT-15细胞增殖的影响,倒置相差显微镜观察细胞形态变化,流式细胞术检测细胞周期的分布以及细胞凋亡率,免疫印迹方法以及聚合酶链反应法观察主要的细胞周期蛋白以及基因的表达情况。结果虎杖白藜芦醇能显著抑制HCT-15细胞生长,并呈现时间-浓度依赖性。细胞形态学检测显示随着药物浓度升高,细胞明显皱缩。流式细胞检测显示,随着白藜芦醇浓度的增加以及作用时间的延长,G0/G1期细胞比例明显增加,其中0、50、100、200μmol/L白藜芦醇处理24h后分别为(42.81±1.21)%、(49.25±0.92)%、(54.67±1.03)%、(64.24±1.17)%,50μmol/L白藜芦醇作用0、24、48、72h后分别为(32.41±1.09)%,(48.35±1.03)%,(55.61±1.33)%,(70.24±1.43)%,各组数据差异均有统计学意义(P<0.05)。0、50、100、200μmol/L白藜芦醇组S期细胞比例随着浓度增大而减小,分别为(34.89±0.66)%、(31.07±1.75)%,(28.66±1.71)%,(23.65±0.62)%,各组数据差异均有统计学意义(P<0.05)。免疫印迹法检测显示,白藜芦醇以时间依赖方式下调周期蛋白cyclin D1表达,上调p21cip1蛋白以及细胞凋亡相关蛋白剪切行Caspase-3和PRAP表达。结论白藜芦醇能明显抑制HCT-15细胞增殖,并诱导其细胞凋亡,可引起HCT-15细胞的S期阻滞。

Sequencing of p53 mutation in established human hepatocellular carcinoma cell line of HHC4 and HHC15 in nude mice

AIM To set up cell lines of human hepatocellular carcinoma in nude mice for the research of cell biology and gene therapy. METHODS Xenotransplantation of human hepatoma into nude mice was carried out and the growth rate, histopathology and immunology of the nude mice were studied. The DNA from xenografts were analyzed by HBV gene and PCR amplification of a fragment of p 53 gene exon 7, which were identified by dot blot hybridization, restriction fragments length polymorphism and DNA sequencing. RESULTS hHCC4 and hHCC415 cell lines could be successively transplanted in nude mice and the population doubling time was 7 and 5 days respectively. These strains retained the original characteristics of histopathology, secreting AFP and heteroploid karyotypes in human hepatocellular carcinoma. The fragment of HBV gene was detected in the genomic DNA of both hHCC4 and hHCC15, however only hHCC4 secreted HBsAg. The mutation at 250 code (C→A) and 249 code (G→T) were detected respectively in the genomic DNA of hHCC4 and hHCC15. CONCLUSION The two cell lines are useful material for studying cell biology and gene therapy in human hepatocellular carcinoma and provide molecular biological trace of the relationship between high mortality of hepatoma and AFB1 severe pollution of the daily common foods in this district.

15-PGDH is reduced and induces apoptosis and cell cycle arrest in gastric carcinoma

AIM: To investigate the expression of 15-hydroxyprostaglandin dehydrogenase (15-PGDH) in human gastric cancer and it's mechanism in apoptosis and cell cycle arrest. METHODS: Expression of 15-PGDH mRNA and protein was examined by immunohistochemistry, immunocytochemistry, reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting in tissue from human gastric cancer, gastric precancerous state (gastric polyps and atrophic gastritis), normal stomach, and gastric cancer cell lines. The relationship between gastric cancer, gastric precancerous state and 15-PGDH expression was determined. The association between expression of 15-PGDH and various clinicopathological parameters in gastric cancer was evaluated. Human gastric cancer cell line SGC-7901 was transfected with 15-PGDH expression plasmids. The effect of 15-PGDH on the cell cycle was examined by flow cytometry. The effect of 15-PGDH on apoptosis was examined by transmission electron microscopy, flow cytometry and transferasemediated nick end labeling (TUNEL) assay. Expression of cell cycle (p21, p27, p16 and p53) and apoptosis (Survivin, BCL-2, BCL-XL, BAK and BAX) genes was analyzed by RT-PCR. RESULTS: Expression of 15-PGDH mRNA and protein in human gastric cancer tissues was significantly lower than in normal gastric tissues (P < 0.01). Expression in human gastric cancer cell lines MKN-28 and MKN-45 was reduced, and absent in SGC-7901 cells (P < 0.05). Reduction of 15-PGDH expression was also found in precancerous tissues, such as gastric polyps and atrophic gastritis (P < 0.01). There was a significant difference in expression of 15-PGDH among various gastric cancer pathological types (P < 0.05), with or without distant metastasis (P < 0.05) and different TNM stage (P < 0.01). Flow cytometry demonstrated a significant increase in apoptotic cells in SGC-7901 cells transfected with pcDNA3/15-PGDH plasmid for 24 and 48 h (P < 0.01), and an increased fraction of sub-G1 phase after transfection (P < 0.05). TUNEL assay showed an increased Apoptotic Index in cells overexpressing 15-PGDH (P < 0.01). After transfection, expression of proapoptotic genes, such as BAK (P < 0.05), BAX and p53 (P < 0.01), was increased. Expression of antiapoptotic genes was decreased, such as Survivin, BCL-2 and BCL-XL (P < 0.01). Expression of cyclin-dependent kinase inhibitors p21 and p16 (P < 0.01) was significantly upregulated in cells overexpressing 15-PGDH. CONCLUSION: Reduction of 15-PGDH is associated with carcinogenesis and development of gastric carcinoma. 15-PGDH induces apoptosis and cell cycle arrest in SGC-7901 cells.

Adenoviral 15-lipoxygenase-1 gene transfer inhibits hypoxia-induced proliferation of retinal microvascular endothelial cells in vitro

AIM: To investigate whether 15-Lipoxygenase-1 (15-LOX-1) plays an important role in the regulation of angiogenesis, inhibiting hypoxia-induced proliferation of retinal microvascular endothelial cells (RMVECs) and the underlying mechanism. METHODS: Primary RMVECs were isolated from the retinas of C57/BL63 mice and identified by an evaluation for FITC-marked CD31. The hypoxia models were established with the Bio-bag and evaluated with a blood-gas analyzer. Experiments were performed using RMVECs treated with and without transfer. Ad-15-LOX-1 or Ad-vector both under hypoxia and normoxia condition at 12, 24, 48, 72 hours. The efficacy of the gene transfer was assessed by immunofluorescence staining. Cells proliferation was evaluated by the CCK-8 method. RNA and protein expressions of 15-LOX-1, VEGF-A, VEGFR-2, eNOs and PPAR-r were analyzed by real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blot. RESULTS: Routine evaluation for FITC-marked CD31 showed that cells were pure. The results of blood-gas analysis showed that when the cultures were exposed to hypoxia for more than 2 hours, the Po2 was 4.5 to 5.4 Kpa. We verified RMVECs could be infected with Ad-LS-LOX-for Ad-vectorvia Fluorescence microscopy. CCK-8 analysis revealed that the proliferative capacities of RMVECs in hypoxic group were significantly higher at each time point than they were in normoxic group (P <0.05). In a hypoxic condition, the proliferative capacities of RMVECs in 15-LOX-1 group were significantly inhibited (P<0.05). Real-time RT-PCR analysis revealed that the expressions of VEGF-A, VEGF-R2 and eNOs mRNA increased in hypoxia group compared with normoxia group (P<0.01). However, the expressions of 15-LOX-1, PPAR-r mRNA decreased in hypoxia group compared with normoxia group (P<0.01). It also showed that in a hypoxic condition, the expressions of VEGF-A, VEGF-R2 and eNOs mRNA decreased significantly in 15-LOX-1 group compared with hypoxia group (P<0.01). However, 15-LOX-1 and PPAR-r mRNA increased significantly in 15-LOX-1 group compared with hypoxia group (P<0.01). There was no significant difference of the mRNA expressions between vector group and hypoxia group (P>0.05). Western blot analysis revealed that the expressions of relative proteins were also ranked in that order. CONCLUSION: Our results suggested that 15-LOX-1 and PPAR-r might act as a negative regulator of retinal angiogenesis. And the effect of 15-LOX-1 overexpression is an anti-angiogenic factor in hypoxia-induced retinal neovascularization (RNV). Overexpression 15-LOX-1 on RMVECs of hypoxia-induced RNV blocked signaling cascades by inhibiting hypoxia-induced increases in VEGF family. PPAR-r effect on VEGFR(2) could be an additional mechanism whereby 15-LOX-1 inhibited the hypoxia-induced RNV.

TATA-box-binding protein-associated factor 15 is a novel biomarker that promotes cell proliferation and migration in gastrointestinal stromal tumor

BACKGROUND Gastrointestinal stromal tumor(GIST)is a common neoplasm with high rates of recurrence and metastasis,and its therapeutic efficacy is still not ideal.There is an unmet need to find new molecular therapeutic targets for GIST.TATA-boxbinding protein-associated factor 15(TAF15)contributes to the progress of various tumors,while the role and molecular mechanism of TAF15 in GIST progression are still unknown.AIM To explore new molecular therapeutic targets for GIST and understand the biological role and underlying mechanisms of TAF15 in GIST progression.METHODS Proteomic analysis was performed to explore the differentially expressed proteins in GIST.Western blotting and immunohistochemical analysis were used to verify the expression level of TAF15 in GIST tissues and cell lines.Cell counting kit-8,colony formation,wound-healing and transwell assay were executed to detect the ability of TAF15 on cell proliferation,migration and invasion.A xenograft mouse model was applied to explore the role of TAF15 in the progression of GIST.Western blotting was used to detect the phosphorylation level and total level of RAF1,MEK and ERK1/2.RESULTS A total of 1669 proteins were identified as differentially expressed proteins with 762 upregulated and 907 downregulated in GIST.TAF15 was selected for the further study because of its important role in cell proliferation and migration.TAF15 was significantly over expressed in GIST tissues and cell lines.Overexpression of TAF15 was associated with larger tumor size and higher risk stage of GIST.TAF15 knockdown significantly inhibited the cell proliferation and migration of GIST in vitro and suppressed tumor growth in vivo.Moreover,the inhibition of TAF15 expression significantly decreased the phosphorylation level of RAF1,MEK and ERK1/2 in GIST cells and xenograft tissues,while the total RAF1,MEK and ERK1/2 had no significant change.CONCLUSION TAF15 is over expressed in GIST tissues and cell lines.Overexpression of TAF15 was associated with a poor prognosis of GIST patients.TAF15 promotes cell proliferation and migration in GIST via the activation of the RAF1/MEK/ERK signaling pathway.Thus,TAF15 is expected to be a novel latent molecular biomarker or therapeutic target of GIST.

Expression of Interleukin-15 and Its Receptor on the Surface of Stimulated Human Umbilical Vein Endothelial Cells

Summary： Human interleukin-15 （hIL-15） is an important cytokine to activate endothelial cells and can be regulated by many other cytokines. The aim of this study is to examine the ability of interferon-γ （IFN-γ）, and tumor necrosis factor-or （TNF-α to induce the production of human interleukin-15 （hIL-15） and IL-15 receptor （IL-15Rα by human umbilical vein endothelial cells （HUVECs）. The data are summarized as follows： 1. Northern blot revealed that IL-15 mRNA was up-regulated by IFN-γ and TNF-α 2. Intracellular IL-15 protein was visualized by fluorescence microscopy, whereas the expression of IL-15 on the surface of HUVECs was detected by fluorescence activated cell sorting （FACS）, and no detectable IL-15 in the medium was verified by ELISA. 3. IL-15Rα was detected on the surface of HUVECs by FACS after IFN-α and TNF-α stimulation, whereas Western blotting revealed that the elevated expression on surface IL-15Rα was not due to the increased protein expression. The conclusion demonstrated from our results is that IFN-α and TNF-α play an important role in regulating the expression of IL-15 and IL-15Rα on the surface of HUVECs.

NT4(Si)-p53(N15)-antennapedia induces cell death in a human hepatocellular carcinoma cell line

AIM： To construct the recombinant lentivirus expression plasmid, pLenti6/V5-NT4 p53（N 15）-antennapedia （Ant）, and study its effect on HepG2 cells. METHODS： Plasmid pLenti6/V5-NT4 p53（N15）-Ant was constructed incorporating the following functional regions, including signal peptide sequence and proregion of neurotrophin 4, N-terminal residues 12-26 of p53 and 17 amino acid drosophila carrier protein, Ant. Hepatocellular carcinoma （HepG2） cells were used for transfection. 3-[4,5-climethyl-thiazol-2yl]-2,5 diphenyl tetrazolium bromide （MI-I） assay, lactate dehydrogenase （LDH） release assay, transmission electron microscopy （TEM） and flow cytometric analysis （FCM） were employed to investigate the effects of LV-NT4（Si）- p53（N15）-Ant in vitro on HepG2 cells. In vivo experiment was also performed to investigate the inhibitory effect of LV-NT4（Si）-p53（N15）-Ant on tumor growth in nude mice.RESULTS： LV-NT4（Si）-p53（N15）-Ant significantly suppressed the growth of HepG2 cells. MTT assay showed that the growth of HepG2 cells was mucj more significantly inhibited by LV-NT4（Si）-p53（N15）-Ant than by LV-EGFP. The inhibition rate for HepG2 cell growth in the two groups was 46.9% and 94.5%, respectively, 48 h after infection with LV-NT4（Si）-p53（N15）-Ant, and was 33.9% and 95.8%, respectively, 72 h after infection with LV-NT4（Si）-p53（N15）-Ant （P 〈 0.01）. Light microscopy and TEM showed morphological changes in HepG2 cells infected with LV-NT4（Si）-p53（N15）-Ant, but no significant changes in HepG2 cells infected with LV-EGFR Changes were observed in ultra-structure of HepG2 cells infected with LV-NT4（Si）-p53（N15）-Ant, with degraded membranes, resulting in necrosis. LDH release from HepG2 cells was analyzed at 24, 48, 72 and 96 h after infection with LV-NT4（Si）-p53（N15）-Ant and LV-EGFP, which showed that LDH release was significantly higher in LV-NT4（Si）-p53（N15）-Ant treatment group （682 IU/L） than in control group （45 IU/L, P 〈 0.01）. The longer the time was after infection, the bigger the difference was in LDH release. FCM analysis showed that LV-NT4（Si）- p53（N15）-Ant could induce two different kinds of cell death： necrosis and apoptosis, with apoptosis being the minor type and necrosis being the main type, suggesting that LV-NT4（Si）-p53（N15）-Ant exerts its anticancer effect on HepG2 cells by inducing necrosis. The in vivo study showed that LV-NT4（Si）-p53（N15）-Ant significantly inhibited tumor growth with an inhibition rate of 66.14% in terms of tumor size and weight. CONCLUSION： LV-NT4（Si）-p53（N15）-Ant is a novel recombinant lentivirus expression plasmid and can be used in gene therapy for cancer.

Inhibition of Growth of Human Ileocecal Adenocarcinoma Cells HCT-8 and Inducing Apoptosis by Different RGD-containing Peptides

Human ileocecal adenocarcinoma cells HCT-8 were treated with RGD-containing cellular adhesion peptides including RGD, RGD（NH2）2（i.e., RGE-NH2）, RGDS, and RGDS-NH2. MTT assay was prepared to examine their inhibiting effects on HCT-8 cells after treatment. The methods including Haematoxylin and Eosin（HE） staining, transmission electron microscopy（TEM）, immunohistochemistry, flow cytometry, and Reverse TranscriptionPolymerase Chain Reaction（RT-PCR） were used to observe the morphology of the apoptotic cells and analyze the mechanism of apoptosis. The experimental results indicate that RGD-containing cellular adhesion peptides can inhibit the growth and proliferation of tumor HCT-8 cells in a dose-dependent manner and induce the apoptosis of HCT-8 cells. At the same time, the high conservative property of RGD was confirmed again.

MicroRNA-15a-cell division cycle 42 signaling pathway in pathogenesis of pediatric inflammatory bowel disease

AIM To determine whether cell division cycle(Cdc)42 is regulated by microRNA(miR)-15 a in the development of pediatric inflammatory bowel disease(IBD).METHODS We cultured 293 T cells, used plasmids and performed dual-luciferase assay to determine whether Cdc42 is a miR-15 a target gene. We cultured Caco-2 cells, and stimulated them with tumor necrosis factor(TNF)-α. We then employed lentiviruses to alter the expression of miR-15 a and Cdc42. We performed quantitative reverse transcription polymerase chain reaction(qRTPCR) and immunofluorescence to determine whether Cdc42 is regulated by miR-15 a in Caco-2 cells. Finally, we collected ileocecal tissue by endoscopy from patients and performed qRT-PCR to examine the expression of miR-15 a and Cdc42 in pediatric IBD patients.RESULTS Target Scan and dual-luciferase assay revealed thatCdc42 was a miR-15 a target gene. MiR-15 a expression increased(P = 0.0038) and Cdc42 expression decreased(P = 0.0013) in cells stimulated with TNF-α, and the expression of the epithelial junction proteins zona occludens(ZO)-1(P < 0.05) and E-cadherin(P < 0.001) decreased. Cdc42 levels decreased in miR-15 a-mimic cells(P < 0.001) and increased in miR-15 a inhibitor cells(P < 0.05). ZO-1 and E-cadherin decreased in mi R-15 a-mimic cells(P < 0.001) but not in the miR-15 a inhibitor + TNF-α cells. In Lv-Cdc42 + TNF-α cells, ZO-1 and E-cadherin expression increased compared to the Lv-Cdc42-NC + TNF-α(P < 0.05) or miR-15 a-mimic cells(P < 0.05). Fifty-four pediatric IBD patients were included in this study, 21 in the control group, 19 in the Crohn's disease(CD) active(AC) group, seven in the CD remission(RE) group, and seven in the ulcerative colitis(UC) group. MiR-15 a increased and Cdc42 decreased in the CD AC group compared to the control group(P < 0.05). miR-15 a decreased and Cdc42 increased in the CD RE group compared to the CD AC group(P < 0.05). miR-15 a was positively correlated with the Pediatric Crohn's disease Activity Index(PCDAI)(P = 0.006), while Cdc42 was negatively correlated with PCDAI(P = 0.0008). Finally, miR-15 a expression negatively correlated with Cdc42 in pediatric IBD patients(P = 0.0045).CONCLUSION Mi R-15 a negatively regulates epithelial junctions through Cdc42 in Caco-2 cells and pediatric IBD patients.