Lentiviral-mediated miRNA against osteopontin suppresses tumor growth and metastasis of human hepatocellular carcinoma

In our previous study, osteopontin (OPN) was identified as one of the leading genes that promote the metastasis of hepatocellular carcinoma (HCC). However, the mechanism by which OPN promotes metastasis of HCC is not understood. In this study, RNA interference mediated by viral vectors-which could induce a long-lasting down-regulation in gene expression-was applied to analyze the role of OPN in metastasis of HCC. Three lentiviral vectors encoding microRNA against OPN, Lenti.OPNi-1, Lenti.OPNi-2, and Lenti.OPNi-3, were constructed and found to down-regulate the OPN level by 62%, 78%, and 95%, respectively, in HCCLM3 cells which had an overexpression of OPN and a higher metastatic potential. Consequently, both Lenti.OPNi-2 and Lenti.OPNi-3 induced a significant decrease in matrix metalloproteinase (MMP)-2 and urokinase plasminogen activator expression, and led to an obvious inhibition of both in vitro invasion and in vivo lung metastasis of HCCLM3 cells (P < 0.001). Moreover, Lenti.OPNi-3, rather than Lenti.OPNi-2, could also suppress in vitro proliferation and in vivo tumor growth of HCCLM3. Smaller detectable tumors were found in only 50% of mice after implantation of Lenti.OPNi-3-transfected HCCLM3 cells (341 +/- 502.6 mm(3) versus >3500 mm(3) in controls; P < 0.001). Lenti.OPNi-3, not Lenti.OPNi-2, significantly suppressed the MEK/ERK1/2 pathway in HCCLM3 cells. Recombinant OPN was found to induce translocation of p65 into the nucleus of HCC cells and activation of MMP-2 and MEK/ERK/1/2, which were suppressed by the nuclear factor kappaB (NF-kappaB) inhibitor pyrrolidine dithiocarbamate. Conclusion: OPN plays an important role in metastasis as well as tumor growth of HCC, in which different minimum threshold levels of OPN are needed. These effects may occur through activation of the mitogen-activated protein kinase and NF-kappaB pathways, and MMP-2. OPN could be a hopeful target for the control of HCC.     

应用蛋白质组学技术筛选胰腺癌生物标志物的研究进展

胰腺癌是一种十分凶险的恶性肿瘤.胰腺癌早期症状不明显,临床确诊的患者多已处于中晚期,大多失去手术治疗机会.蛋白质组技术的快速发展为研究胰腺癌发病机制、发现早期诊断和预后的生物标志物提供了可能.本文就蛋白质组学技术在胰腺癌细胞系、组织、血清和胰液等方面的应用作一综述.     

针对HER2/neu的siRNA对非小细胞肺癌细胞生长的抑制作用

目的:构建针对HER2/neu的RNA干涉载体,观察对HER2/neu表达抑制及抑制后对非小细胞肺癌生长的影响. 方法:设计和合成长度为64nt的脱氧寡核苷酸链,退火互补成双链,克隆至pSUPER质粒载体,转化DH5α菌株,提取质粒,转染过表达HER2/neu的肺腺癌细胞系SPC-A-1,RT-PCR检测HER2/neu的mRNA表达,Western blot和间接免疫荧光法检测HER2/neu的蛋白表达,FCM分析细胞周期变化,MTT法观察细胞生长. 结果:肺腺癌细胞系SPC-A-1存在HER2/neu过表达;成功构建了HER2/neu特异性RNA干涉载体pSUPER-siHER2;瞬时转染SPC-A-1细胞,HER2/neu的mRNA及蛋白表达均降低;G1期细胞较亲代增加9.9%;肿瘤细胞增殖速度减慢(P＜0.05). 结论:成功构建了HER2/neu RNA干涉载体,转染后可有效降低肺腺癌细胞系SPC-A-1 HER2/neu的mRNA转录及蛋白水平表达,阻滞转染细胞于G1期,造成转染细胞生长速度减慢,这有望为肺癌基因治疗提供一种选择手段.     

Anticancer effect and neurotoxicity of S-(+)-deoxytylophorinidine, a new phenanthroindolizidine alkaloid that interacts with nucleic acids

Phenanthroindolizidine alkaloids are a family of plant-derived compounds with significant antineoplastic activity as well as other effects like antiamebicidal, antiviral, and anti-inflammatory activities. The specific biomolecular targets of these compounds have not yet been clearly identified. S-(+)-Deoxytylophorinidine (CAT) is a new phenanthroindolizidine alkaloid, originally extracted from the roots of Tylophora atrofolliculata and Tylophora ovata. Potent anticancer activity was observed in vitro and in vivo. Neurotoxicity of CAT was also studied and it was far less serious than that of vinblastine. Interactions between this compound and DNA had been studied in detail in our laboratory previously, and we further studied its interactions with RNA.     

Validation and application by HPLC for simultaneous determination of vitexin-2″-O-glucoside,vitexin-2″-O-rhamnoside,rutin, vitexin,and hyperoside

A simple, precise, and rapid high-performance liquid chromatographic method was developed and validated for the simultaneous determination of vitexin-2″-O-glucoside, vitexin-2″-O-rhamnoside, rutin, vitexin, and hyperoside. The HPLC separation was performed using a Shim-pack VP-ODS C₁₈ column (250 mm×4.6 mm i.d., 5 μm) with the isocratic mobile phase consisting of tetrahydrofuran/ acetonitrile/0.05% phosphoric acid solution (20:3:77, v/v/v), and the flow rate was set at 1.0 mL/min. UV detection was carried out at a wavelength of 360 nm and the whole analysis took 25 min. The method was linear in the range of 4.12–206.00 μg/mL for vitexin-2″-O-glucoside, 4.05–202.50 μg/mL for vitexin-2″-O-rhamnoside, 1.64–82.00 μg/mL for rutin, 1.74–87.00 μg/mL for vitexin, and 1.41–70.60 μg/mL for hyperoside with the correlation coefficient for each analyte more than 0.998. The limit of detection (LOD) and limit of quantitation (LOQ) were 0.6 and 2 ng for vitexin-2″-O-glucoside, 0.6 and 2 ng for vitexin-2″-O-rhamnoside, 0.3 and 1 ng for rutin, 1 and 3 ng for vitexin, and 0.5 and 2 ng for hyperoside, respectively. Intra- and inter-day precision and accuracy (RSD) were less than 3%. The developed HPLC method was successfully applied to the analysis of five flavonoids in hawthorn leaves, hawthorn fruits, and the preparations containing hawthorn leaves or fruits.     

Protective effects of hydroxyethyl starch 130/0.4 against myocardial ischemia/reperfusion injury in rats

Background  The effects of hydroxyethyl starch 130/0.4 (HES130/0.4) on myocardial ischemia/reperfusion (I/R) injury and its mechanism are uncertain. The aim of this study was to investigate the protective effects of HES 130/0.4 on myocardial I/R injury.

Methods  Forty-eight Sprague-Dawley rats were assigned to sham-operation group (S group), ischemia-reperfusion group (I/R group), albumin-I/R group (A-I/R group) and HES130/0.4-I/R group (H-I/R group). The fluids were administered at 25 minutes after ischemia. H-I/R group was given 7.5 ml/kg of HES 130/0.4; I/R group and A-I/R group received the same volume of normal saline and 5% albumin, respectively. The rats in S group were sham operated and received the same fluid as I/R group. After 30 minutes of ischemia and 3 hours of reperfusion, blood samples were taken for cytokines assay, myocardium was excised for detection of NF-κB activity and myocardial infarction areas were taken for immunohistochemical analysis.

Results  Hemodynamic parameters of H-I/R group were better than I/R and A-I/R groups at all designated time points. The results of 2,3,5-triphenyl-tetrazolium (TTC) and HE staining were better in the H-I/R group. Myeloperoxidase (MPO), NF-κB activity and concentrations of TNF-α, IL-1β were elevated markedly in I/R groups. HES130/0.4 lessened the release of TNF-α and IL-1β consistent with the reduction of MPO activity, and HES 130/0.4 inhibited the activity of NF-κB in H-I/R group. The number of apoptotic cells in the H-I/R group was also significantly reduced compared with I/R and A-I/R group

Conclusion  HES130/0.4 has a protective effect on I/R injured myocardium, probably by inhibiting NF-κB activity, reducing the release of pro-inflammatory cytokines and interfering with the apoptosis of cardiomyocytes.

     

CCPG1 involved in corneal Aspergillus fumigatus infection

AIM: To investigate whether non-canonical autophagy transport receptor cell cycle progression 1 (CCPG1) is involved in the corneal antifungal immune response. METHODS: Human corneal epithelial cells (HCECs) and human myeloid leukemia mononuclear cells (THP-1) macrophages stimulated by Aspergillus fumigatus (A. fumigatus) were used as cell models. The expression of CCPG1 mRNA was detected by qRT-PCR. Western blot was used to determine the protein expression of CCPG1 and interleukin-1β (IL-1β). The dectin-1 neutralizing antibody was used to detect the association between dectin-1 and CCPG1. Immunofluorescence was used to observe the colocalization of CCPG1 and C-type lectin-like receptor-1 (CLEC-1) in THP-1 macrophages. RESULTS: The expression of CCPG1 started to increase at 4h after infection and increased in a time-dependent manner in HCECs and THP-1 macrophages. With dectin-1 neutralizing antibody pretreatment, the expression of IL-1β was down-regulated. CCPG1 up-regulation in response to A. fumigatus infection was independent of dectin-1. Immunofluorescence showed the colocalization of CCPG1 and CLEC-1 in THP-1 macrophages. CONCLUSION: As a specific autophagy protein of non-canonical autophagy pathway, CCPG1 is involved in corneal infection with A. fumigatus.