抑制P18^INK4C表达对胃腺癌细胞侵袭的影响

应用基因芯片技术筛选胃腺癌转移相关基因的过程中 ,发现CDK抑制因子 (CKI)P18INK4C在人类胃腺癌转移细胞株RF 4 8中的表达 ,较其原发灶细胞株RF 1明显下调。这提示 ,P18INK4C表达差异与胃腺癌细胞的侵袭转移 ,可能有一定程度的相关性。为此 ,通过反义RNA技术抑制在RF 1中的表达 ,研究其对胃腺癌原发灶细胞体外运动、侵袭转移能力以及生长特性的影响 ,进一步明确P18INK4C与人类胃腺癌侵袭转移之间的关系。结果发现 ,抑制P18INK4C的表达 ,可以使胃腺癌原发灶细胞的体外侵袭能力明显增加 ,抑制前RF 1细胞的体外侵袭能力仅为抑制后的 4 4 %。然而 ,RF 1的细胞周期和生长增殖能力 ,并未因为P18INK4C表达的改变而受到影响。上述结果提示 ,P18INK4C参与人类胃腺癌转移过程 ;在此过程中 ,其主要的作用可能并不是调节细胞周期 ,而是与胃腺癌原发灶细胞侵袭转移能力的调节密切相关。     

M-CSF上调cyclinD1/D3和CDK2/6的表达促进HeLa细胞增殖

非分泌型巨噬细胞集落刺激因子(M-CSF)的表达在肿瘤的发生发展过程中发挥重要作用,为探讨胞质M-CSF对细胞增殖的影响,采用基因重组技术构建胞内稳定表达M-CSF的HeLa细胞系,以空载体(pCMV/myc/cyto)转染HeLa细胞和未转染HeLa细胞作为对照,MTT法及反义寡核苷酸抑制实验分析M-CSF对细胞增殖的影响,并计算细胞倍增时间,RT-PCR观察胞内M-CSF对G1期细胞周期相关蛋白的影响．结果显示,与对照组比较,转染M-CSF的HeLa细胞倍增时间明显缩短、增殖能力显著增强,M-CSF的特异性反义寡核苷酸能抑制转染M-CSF的HeLa细胞的增殖,且抑制率随着反义寡核苷酸浓度的增高而增强,转染M-CSF 的HeLa细胞的cyclinD1/D3和CDK2/6 mRNA表达显著升高(P < 0.05)．提示：M-CSF可上调cyclinD1/D3和CDK2/6的mRNA表达,促进HeLa细胞的增殖．     

增强P18~(INK4C)表达对胃腺癌细胞侵袭的影响(英文)

在应用基因芯片技术筛选胃腺癌转移相关基因的过程中 ,发现CDK抑制因子P18INK4C在人类胃腺癌转移细胞株RF 4 8中的表达较其原发灶细胞株RF 1明显下调 .这提示P18INK4C表达差异与胃腺癌细胞的侵袭转移可能有一定程度的相关性 .通过构建P18INK4C 表达质粒并将其转染入RF 4 8增强P18INK4C的表达 ,研究其对胃腺癌原发灶细胞体外运动、侵袭转移能力以及生长特性的影响 ,进一步明确P18INK4C与人类胃腺癌侵袭转移之间的关系 .结果发现 ,增强P18INK4C表达可以使胃腺癌原发灶细胞的体外侵袭能力明显下降 ,而对RF 4 8的细胞周期和生长增殖能并力未产生影响 .上述结果提示 ,P18INK4C参与人类胃腺癌转移过程 ,在此过程中其主要的作用可能并不是调节细胞周期 ,而是与胃腺癌原发灶细胞侵袭转移能力的调节密切相关 .     

靶向Survivin的反义寡核苷酸对肿瘤细胞增殖的抑制作用

 Survivin是新近克隆的一种凋亡抑制蛋白 (IAP)家族成员 ,在几乎所有肿瘤组织中特异性表达 ,而在正常成年终末分化组织中低表达甚至不表达 .采用四唑盐 (MTT)比色实验法比较 2 0条抗人survivin反义寡核苷酸对HeLa细胞增殖的抑制效果 ,并从中筛选效果显著的反义寡核苷酸 ,在体外水平进一步验证其抑制survivin表达的能力 .在用 4 0 0nmol L反义寡核苷酸转染HeLa细胞 4 8h后 ,有 4条反义寡核苷酸对细胞增殖的抑制率超过 4 0 %,其中 4 5号反义寡核苷酸的抑制率可达5 9%,而阳性对照序列ISIS2 372 2的抑制率仅达 30 %.Northern和Western印迹分析证明 :4 5号反义寡核苷酸可明显降低细胞中survivin基因的mRNA含量和蛋白水平 .4 5号反义寡核苷酸还可在较低浓度 (2 0 0nmol L)显著增强HeLa细胞对化疗药三尖杉酯碱的敏感性 .因此 ,4 5号反义寡核苷酸有望应用于survivin高表达肿瘤的辅助治疗之中     

ppGalNac-T10对人结直肠癌细胞株LoVo的生物学特性的影响

探讨多肽N-乙酰氨基半乳糖转移酶10(ppGalNAc-T10)对人结直肠癌细胞株LoVo细胞特性的影响.ppGalNAc-T10正义真核表达载体pcDNA3.1-T10(+)、反义真核表达载体pcDNA3.1-T10(-)与空载体pcDNA3.1分别转染LoVo细胞,Western blot检测ppGalNAc-T10蛋白水平表达的变化,确定转染效果.CCK8法检测转染后各实验组细胞增殖的变化,细胞划痕实验检测细胞迁移能力的变化,穿膜实验检测细胞侵袭能力的变化.Western blot证明各实验组经转染不同ppGalNAc-T10载体后,ppGalNAc-T10蛋白表达量发生变化,转染pp-GalNAc-T10正义真核表达载体的LoVo细胞,ppGalNAc-T10的蛋白表达量增加,同时细胞的增殖受到抑制,迁移能力和侵袭能力降低;而转染ppGalNAc-T10反义真核表达载体的LoVo细胞,ppGalNAc-T10的蛋白表达量降低,细胞生长加快,迁移能力和侵袭能力增强.ppGalNAc-T10可能影响人结直肠癌细胞株LoVo细胞的增殖、迁移能力和侵袭能力.     

过表达外源M-CSF促进MCF7细胞增殖

为探讨过表达外源巨噬细胞集落刺激因子(M-CSF)对细胞增殖的影响,将重组载体 pCMV/cyto/myc-M-CSF转染MCF7细胞、G418筛选,RT-PCR、Western 印迹和免疫荧光鉴定M-CSF的mRNA表达、蛋白表达及定位,通过计算细胞倍增时间、MTT法及反义寡核苷酸抑制实验分析M-CSF对细胞增殖的影响. 结果表明,转染M-CSF的MCF7细胞过表达M-CSF-mRNA及蛋白,并且定位表达于细胞质;与转染空载体及未转染M-CSF组细胞比较,转染M-CSF的MCF7细胞倍增时间明显缩短、增殖能力显著增强,M-CSF的特异性反义寡核苷酸能抑制转染M-CSF的MCF7细胞的增殖,且抑制率随着反义寡核苷酸浓度的增高而增强;以上结果提示,胞质过表达M-CSF可促进MCF7细胞的增殖.     

反义细胞周期蛋白E真核表达载体的构建及其对体外人乳腺癌细胞的抑制作用

 为了探讨细胞周期蛋白 E(cyclin E)与人乳腺癌细胞恶性特征间的相关性 ,利用反义 RNA抑制基因表达的技术 ,构建了细胞周期蛋白 E反义 RNA的真核表达载体并转入人乳腺癌细胞中 .通过 G41 8筛选出阳性克隆 ,经 PCR和 Western印迹检测 ,确定细胞中含有重组质粒 ,并且细胞周期蛋白 E蛋白的水平明显降低 ,由此获得了反义 RNA表达载体导致的细胞周期蛋白 E表达受抑制的细胞 .细胞模型建立后 ,观察分析了细胞形态 ,细胞生长的血清依赖性以及软琼脂成集落能力 ,与对照细胞相比所发生的变化 .结果显示 ,细胞周期蛋白 E受抑制后 ,乳腺癌细胞体积变大 ,细胞生长对血清依赖性增加 ,低血清培养到第 6d时 ,细胞密度约为对照细胞的五分之一 ,细胞成集落能力也显著下降 ,软琼脂中克隆形成率下降 57% .这些变化都表明乳腺癌细胞恶性程度由于细胞周期蛋白 E表达受抑制而减弱 ,可以推测 cyclin E与乳腺癌细胞的恶性增殖及非锚定依赖性生长有着明显的关系 .     

HBXIP基因对乙肝病毒X蛋白诱导细胞凋亡的影响

探讨乙型肝炎病毒X蛋白结合蛋白(hepatitisBXinteractingprotein ,HBXIP)基因在乙型肝炎病毒X蛋白(HBX)诱导肝癌细胞凋亡时对细胞周期的影响.构建HBXIP基因真核表达载体pcDNA3 hbxip ,进行瞬时基因转染,将克隆有HBx基因的pCMV X (分别为1μg、2 μg和3μg)和pcDNA3 hbxip质粒分别和共转染至人H74 0 2肝癌细胞中(总体积分别为5 0 μl) .发现瞬时转染3μgpCMV X质粒后,肝癌细胞凋亡发生率为34 4 % ,肝癌细胞的细胞周期相关蛋白p2 7表达水平发生明显上调;与对照组相比,瞬时转染1μg、2 μg和3μg时,细胞周期蛋白D和细胞周期蛋白E的表达水平均发生明显上调,但随着HBX水平的增加细胞周期蛋白D和细胞周期蛋白E的表达水平发生明显下降;在稳定转染pCMV X质粒的H74 0 2 X肝癌细胞中无明显的细胞凋亡发生,研究发现p2 7的表达水平发生了明显下调,而细胞周期蛋白D和细胞周期蛋白E的表达水平发生了明显上调;当pcDNA3 hbxip质粒与pCMV X质粒进行共瞬时转染时,细胞凋亡发生率由pcDNA3质粒与pCMV X质粒共转染时的2 9 2 %下降为13 3% ,p2 7的表达水平发生了下调,但细胞周期蛋白D和细胞周期蛋白E的表达水平无明显变化.研究结果表明,瞬时转染一定剂量的x基因可导致肝癌细胞发生凋亡,细胞周期相关蛋白p2 7、细胞周期蛋白D和     

荧光差异显示PCR克隆参与胃腺癌转移的基因wcl1

使用来源于同一个胃腺癌病人的原发灶RF 1(ATCC编号CRL 186 4 )和转移灶RF 4 8(ATCC编号CRL 186 3)作为研究肿瘤转移的模型 .通过荧光差异显示PCR(FDD PCR)技术 ,克隆了 4 5个涉及胃腺癌转移相关基因 .和原发灶CRL 186 4相比 ,发现在转移灶CRL 186 3细胞中有 38个基因被显著上调 ,7个基因被显著下调 ,包括未被发现的基因 3个 .利用生物信息学技术对其中 1个在RF 4 8中高度上调的wcl1进行克隆和鉴定 ,发现wcl1的cDNA全长为 6 6 4bp ,含有 1个 2 4 0bp的完整阅读框 .用RT PCR和Northern印迹证实 ,结果与克隆拼接的大小完全一致 (NCBI数据库收录号AF36 4 86 3) .wcl1编码肽位于胞浆内含 79个氨基酸 ,理论上的pI Mr:5 2 8 896 1 72 .氨基酸序列分析发现 ,其N端 4～ 5 5氨基酸处为未知功能区UPF0 0 16蛋白 ,5 7～ 78处有一段亮氨酸拉链结构域 ,未发现与已知的蛋白质有高度的同源性 .该基因定位于 11q14.组织分布表明 ,wcl1除在分化差的胃腺癌中的表达要高于相应的正常胃组织 ,在微血管内皮中表达也明显高于乳腺管癌、脑纤维状星形细胞瘤 ,未检测到在其它组织有分布 .研究提示 ,Wcl1可能为胃腺癌转移过程中参与核内基因转录的相关蛋白质 .     

Effects of Helicobacter pylori on biological characteristics of gastric epithelial cells

Infection with Helicobacter pylori (H. pylori) strains is linked to an increased risk of inflammation and gastric cancer. To investigate the effects of H. pylori on biological characteristics of gastric epithelial cells SGC-7901, derived from human adenocarcinoma, morphological appearances of both the pathogen and these cells, as well as features of attachment and internalization were observed by using transmission electron microscopy (TEM). We also investigated cell junctions and invasion by TEM and Transwell Invasion Assay. Cell proliferation and apoptosis were assessed by using chromogenic methylthiazol tetrazolium bromide (MTT) dye and flow cytometry. Three types of H. pylori were observed around, attaching to, or invading tumor cells. Cellular damage was characterized by vacuolar degeneration, dilated endoplasmic reticulum (ER), and reduction of organelles. Cell junctions and cell microvilli reduced or disappeared. H. pylori inhibited cell proliferation, whereas it had no effect on apoptosis. It also promoted gastric carcinoma cell invasion. H. pylori damages cell construction, destroys cell junctions, inhibits cell proliferation, promotes cell invasive ability, and, therefore, might accelerate the malignant progress and metastasis of gastric cancer.     

Cyclic AMP-Response Element Regulated Cell Cycle Arrests in Cancer Cells

Recently, we have demonstrated that trichosanthin (TCS), a promising agent for the treatment of cervical adenocarcinoma, inhibited HeLa cell proliferation through the PKC/MAPK/CREB signal pathway. Furthermore, TCS down-regulated Bcl-2 expression was abrogated by a decoy oligonucleotide (OGN) to the cyclic AMP-responsive element (CRE). The decoy OGN blocked the binding of CRE-binding protein (CREB) to Bcl-2. These results suggested that CRE-mediated gene expression may play a pivotal role in HeLa cell proliferation. However, little is known about the effect of TCS on cell cycle arrests, particularly, whether the genes involved in cell cycle were regulated by CRE. Our present study shows that the arrests of S, G1 and G2/M phases were accompanied by the significant down-regulation of cyclin A, D1 and CDK 2, 4 in HeLa cells, cyclin D1, E and CDK 2, 4 in Caski and C33a cells, and cyclin A, B1, E and CDK 2 in SW1990 cells. However, the cell cycle arrests were reversed via the significant up-regulation of cyclin A and D1, by the combined treatment of TCS and CRE. In conclusion, these data demonstrate for the first time that specific cell cycle arrests in cancer cells can be induced by TCS by inhibiting the binding of CREB to CRE on genes related to cell proliferation.     

Antibiotic tigecycline inhibits cell proliferation,migration and invasion via down-regulating CCNE2 in pancreatic ductal adenocarcinoma

Recently, many researches have reported that antibiotic tigecycline has significant effect on cancer treatment. However, biomedical functions and molecular mechanisms of tigecycline in human pancreatic ductal adenocarcinoma (PDAC) remain unclear. In the current study, we tried to assess the effect of tigecycline in PDAC cells. AsPC-1 and HPAC cells were treated with indicated concentrations of tigecycline for indicated time, and then, MTT, BrdU and soft agar assay were used to test cell proliferation. The effect of tigecycline on cell cycle and cellular apoptosis was tested by cytometry. Migration and invasion were detected by wound healing assay and transwell migration/invasion assay. Expressions of cell cycle-related and migration/invasion-related protein were determined by using Western blot. The results revealed that tigecycline observably suppressed cell proliferation by inducing cell cycle arrest at G0/G1 phase and blocked cell migration/invasion via holding back the epithelial-mesenchymal transition (EMT) process in PDAC. In addition, tigecycline also remarkably blocked tumorigenecity in vivo. Furthermore, the effects of tigecycline alone or combined with gemcitabine in vitro or on PDAC xenografts were also performed. The results showed that tigecycline enhanced the chemosensitivity of PDAC cells to gemcitabine. Interestingly, we found CCNE2 expression was declined distinctly after tigecycline treatment. Then, CCNE2 was overexpressed to rescue tigecycline-induced effect. The results showed that CCNE2 overexpression significantly rescued tigecycline-inhibited cell proliferation and migration/invasion. Collectively, we showed that tigecycline inhibits cell proliferation, migration and invasion via down-regulating CCNE2, and tigecycline might be used as a potential drug for PDAC treatment alone or combined with gemcitabine.     

RASAL1 influences the proliferation and invasion of gastric cancer cells by regulating the RAS/ERK signaling pathway

The aim of this study was to investigate the biological characteristics of the RASAL1 gene in a well-differentiated gastric cancer cell line MKN-28 and a poorly differentiated gastric cancer cell line BGC-823 cells, using RNA interference and gene transfection technology, respectively. MKN-28 cells were transfected with the shRNA of RASAL1 and BGC-823 cells were transfected with the pcDNA 3.1 plasmid vector containing RASAL1. RT-PCR and western blotting were then used to detect the expression of RASAL1 mRNA and protein. The activities of RAS and extracellular signal-regulated kinase 1/2 were analyzed by the pull-down method and western blotting. The proliferate capacity, apoptosis rate, invasive and migratory potentials of MKN-28 or BGC-823 cells were also measured by Cell Counting Kit-8 cell proliferation assay, propidium iodide/Annexin V staining coupled with flow cytometry, and transwell chamber assays, respectively. Measurement of RASAL1 mRNA and protein expression in two cells revealed successful transfection of the shRNA of RASAL1 and RASAL1-pcDNA3.1 plasmid into these two cells. Moreover, decreased expression of RASAL1 in MKN-28 cells resulted in increased expression of RAS-GTP and p-ERK1/2. Interestingly, decreased expression of RASAL1 inhibited apoptosis and facilitated cell proliferation, invasion and migration. The increased expression of RASAL1 in BGC-823 cells caused declined expression of RAS-GTP and p-ERK1/2, as well as promoted apoptosis and restrained cell proliferation, invasion and migration. The down-regulation of RASAL1 promoted the proliferation, invasion and migration of gastric cancer MKN-28 cells, and up-regulation of RASAL1 inhibited the proliferation, invasion and migration of BGC-823 gastric cancer cells by regulating the RAS/ERK signaling pathway. Thus, our results suggest that RASAL1 may play an important role as a tumor suppressor gene in gastric cancer.     

Downregulation of RPL6 by siRNA inhibits proliferation and cell cycle progression of human gastric cancer cell lines

Our previous study revealed that human ribosomal protein L6 (RPL6) was up-regulated in multidrug-resistant gastric cancer cells and over-expression of RPL6 could protect gastric cancer from drug-induced apoptosis. It was further demonstrated that up-regulation of RPL6 accelerated growth and enhanced in vitro colony forming ability of GES cells while down-regulation of RPL6 exhibited the opposite results. The present study was designed to investigate the potential role of RPL6 in therapy of gastric cancer for clinic. The expression of RPL6 and cyclin E in gastric cancer tissues and normal gastric mucosa was evaluated by immunohistochemisty. It was found that RPL6 and cyclin E were expressed at a higher level in gastric cancer tissues than that in normal gastric mucosa and the two were correlative in gastric cancer. Survival time of postoperative patients was analyzed by Kaplan- Meier analysis and it was found that patients with RPL6 positive expression showed shorter survival time than patients that with RPL6 negative expression. RPL6 was then genetically down-regulated in gastric cancer SGC7901 and AGS cell lines by siRNA. It was demonstrated that down-regulation of RPL6 reduced colony forming ability of gastric cancer cells in vitro and reduced cell growth in vivo. Moreover, down-regulation of RPL6 could suppress G1 to S phase transition in these cells. Further, we evidenced that RPL6 siRNA down-regulated cyclin E expression in SGC7901 and AGS cells. Taken together, these data suggested that RPL6 was over-expressed in human gastric tissues and caused poor prognosis. Down-regulation of RPL6 could suppress cell growth and cell cycle progression at least through down-regulating cyclin E and which might be used as a novel approach to gastric cancer therapy.     

Inhibitory effects of γ-tocotrienol on invasion and metastasis of human gastric adenocarcinoma SGC-7901 cells

Natural vitamin E is a mixture of two classes of compounds, tocopherols and tocotrienols. Recent research has revealed that tocotrienols, especially γ-tocotrienol, exhibit not only the same antioxidant ability as tocopherols, but also remarkable anticancer capacity in cancer cell lines. In this study, the invasion and metastatic capacities of gastric adenocarcinoma SGC-7901 cells and the correlation with antimetastasis mechanisms induced by γ-tocotrienol were explored. The results showed the inhibitory effects of γ-tocotrienol at doses of 15, 30, 45 and 60 μmol/L for 48 h on cell migration and cell matrigel invasion; activities of matrix metalloproteinase (MMPs) increased in SGC-7901 cells when compared to the control group (P<.05 or P<.01). An increasing trend in the chemotactic responses to fibronectin (FN) in SGC-7901 cells was found in the γ-tocotrienol treatments. SGC-7901 cell attachment decreased in the γ-tocotrienol-treated groups in comparison with the control group (P<.01). The mRNA expressions of MMP-2 and MMP-9 showed that γ-tocotrienol significantly reduced the matrigel invasion capability through down-regulation of the mRNA expressions of MMP-2 and MMP-9 (P<.01), and up-regulation of tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2 in SGC-7901 cells by treatment with γ-tocotrienol for 48 h (P<.05). γ-Tocotrienol also significantly increased the mRNA expression of nm23-H1 in SGC-7901 cells (P<.01). These findings suggest a potential mechanism of γ-tocotrienol-mediated antitumor metastasis activity and indicate the role of vitamin E as potential chemopreventative agents against gastric cancer.     

MicroRNA-221 regulates osteosarcoma cell proliferation,apoptosis, migration,and invasion by targeting CDKN1B/p27

MicroRNAs (miRNAs, miR) are of critical importance in growth and metastasis of cancer cells; however, the underlying functions of miRNAs in osteosarcoma (OS) remain largely unknown. This study was aimed to elucidate the role of miR-221 in regulating the biological behavior of OS cells. The proliferation ability was examined by cell counting kit-8 (CCK-8) and cell cycle assay. The abilities of cell migration, invasion, and apoptosis were monitored by transwell assay and flow cytometry, respectively. The effect of miR-221 on cyclin-dependent kinase inhibitor 1B (CDKN1B) expression was evaluated by luciferase assays, real-time polymerase chain reaction, and Western blot analysis. We found that miR-221 was elevated in OS cell lines compared with the normal osteoblastic cell line. Transfection of the miR-221 inhibitor into MG63 and U-2OS cell lines obviously suppressed cell proliferation, migration, and invasion, which is accompanied with cell cycle arrest in G0/G1 phase. Furthermore, luciferase reporter assays indicated that CDKN1B is directly targeted by miR-221 in OS cells. Knockdown of CDKN1B inhibited the effects of miR-221 inhibitor, along with decreased Bax and caspase-3 and increased cyclin E, cyclin D1, Bcl-2, Snail, and Twist1 expression. The results suggested that miR-221 might act as a potentially useful target for treatment of OS.