反义miR-30a-5p抑制脑胶质瘤生长的体内实验研究

ObjectiveTo study the inhibitory effect of knocking down miR-30a-5p on the U87 human glioma xenograft growth and its possible mechanism. MethodsNude mice bearing subcutaneous U87 human glioblastoma were established and treated with miR-30a-5p antisense oligonucleotides (AS-miR-30a-5p) subcutaneous injection. Tumor size was measured every other day until the observation period ended. Researchers executed the animals after the treatment, stripped tumor tissues and extracted RNA and protein. Real-time PCR were conducted to detect the expression of miR-30a-5p. The histopathological characteristics and proliferation and apoptosis biological characters (including SEPT7, PCNA, cyclinD1, MMP-2, apoptosis related factor P53, bcl-2 and caspase3)were evaluated by HE and immunohistochemical staining, Western blot analysis respectively, and the cell apoptosis was detected by TUNEL method.ResultsIn AS-miR-30a-5p treated group, the tumor growth was delayed and the final tumor volume was smaller than that in the control and scr-ODN treated group (F=7.167, P＜0.05), and the expression of miR-30a-5p was knocked down. The expression of PCNA、cyclinD1 were significantly down-regulated while P53、SEPT7 and caspase3 up-regulated. Apoptotic index was increased significantly. ConclusionAs-miR-30a-5p suppresses the growth of U87 human gliomas xenografts significantly. Malignant phenotype of tumors are reversed to a considerable degree. Therefore, miR-30a-5p can be a candidate for targeted therapy of human glioma.     

Novel pharmacological agents in clinical development for solid tumours

For decades, cancer therapy has focused on DNA-directed mechanisms of cytotoxicity, utilising agents with limited efficacy and significant toxicity. Recent advances in tumour biology have elucidated the molecular pathways implicated in the pathogenesis and progression of cancers and have resulted in the discovery of a variety of novel molecular targets for therapeutic intervention. Promising novel agents targeting signal transduction pathways, cell cycle regulation, angiogenesis and apoptosis are in clinical testing and are discussed in this review.     

Use of RNA in drug design

This is a review of RNA as a target for small molecules (ribosomes, riboswitches, regulatory RNAs) and RNA-derived oligonucleotides as tools (antisense/small interfering RNA, ribozymes, aptamers/decoy RNA and microRNA). This review highlights the present state of research using RNA as a drug target or as a potential drug candidate and explains at which stage and to what extent rational design could eventually be involved. Special attention has been paid to the recent potential clinical applications of RNA either as drugs or drug targets. The review deals mainly with mechanistic approaches rather than with physicochemical or computational aspects of RNA-based drug design.     

内皮素A型受体反义基因减弱内皮细胞条件培养液刺激的血管平滑肌细胞增殖

目的 :研究内皮素 （Endothelin,ET） A型受体 （ETA）反义寡核苷酸 （Oligonucleotide,OGN）对内皮细胞 （En-dothelial cells,ECs）条件培养液 （ECCM）刺激的人大隐静脉 （hum an saphenous vein,HSV）血管平滑肌细胞 （Vas-cular sm ooth m uscle cells,VSMCs）增殖的影响。方法 :分离、培养 HSV的 ECs和 VSMCs。收集 ECCM,分别用正常的 DMEM（Dulbecco’s Mod Ified Eagle medium ）、含 ET- 1的 DMEM、ECCM和加入 Phophoramidon（ET- 1转化酶抑制剂 ）的 ECCM培养 VSMCs。以放射免疫法测定 ECs培养基中 ET- 1水平 ;分别采用放射受体结合分析法和四唑盐 （MTT）比色实验检测 ETA 蛋白表达和细胞的增殖。结果 :ETA- OGN（15 μm ol/L）以时间依赖的方式抑制ETA 表达。 12～ 4 8h HSV的 ECs培养上清液 ET- 1水平呈现上升的趋势 ,4 8h达到高峰。 ECCM显著刺激 VSMCs增殖 （P<0 .0 1） ,ET- 1能够模拟 ECCM的促增殖作用。在 ECCM中加入 Phosphorimidon,不能使 ECCM的促增殖效应消失 （P<0 .0 5 ）。ETA- OGN（15 μm ol/L）对无血清正常的 DMEM培养基培养的 VSMCs增殖没有显著影响 ,但显著抑制 ECCM和 ET- 1的促增殖作用 （P<0 .0 1vs ECCM）。结论 :ECs通过分泌包括 ET- 1在内的多种因子促进 VSMCs增殖 ,其中 ET- 1发挥主要的作用。 E     

Proto-oncogene c-mpl is involved in spontaneous megakaryocytopoiesis in myeloproliferative disorders

Spontaneous megakaryocytic colonies (CFU-MK) formation without the addition of Meg-CSA in myeloproliferative disorders (MPD) has been reported by many laboratories. The mechanism by which this occurs is still unknown. In our previous work we have found that the spontaneous colonies persisted in serum-free agar culture although the colony cells were smaller and the colony numbers fewer than in plasma clot culture and that monoclonal antibodies against IL3, IL6 and GM-CSF had no inhibitory effect on spontaneous CFU-MK in both semi-solid cultures. Recently, proto-oncogene c-mpl and c-mpl ligand, thrombopoietin (TPO), have been shown to specifically participate in the regulation of normal human megakaryocytopoiesis. In order to test the hypothesis that c-mpl c-mpl ligand pathway is involved in the spontaneous growth of megakaryocyte progenitors, we investigated mRNA expressions of c-mpl and TPO in cells grown in serum-free liquid culture using RT-PCR. The c-mpl expression was detected in the cultured cells from all nine patients (six with ET, two with PV, one with PMF) who had spontaneous CFU-MK in clonal assays. However, none of the patients expressed TPO mRNA in these cells. Pre-incubation of nonadherent mononuclear cells with thioester-modified antisense oligodeoxynucleotide to c-mpl at a concentration of 6μ M significantly decreased the cloning efficiency of spontaneous megakaryocyte growth by 42.5% ( P <0.05) in plasma clot assay (seven with ET, one with PV) and 69.6% ( P <0.05) in serum-free agar culture (six with ET, one with PV). In control experiments the introduction of a scrambled oligomer to antisense oligodeoxynucleotide had no such effect on spontaneous colony formation. These results indicate that c-mpl exerts an important effect in the growth of spontaneous megakaryocytopoiesis in MPD.     

Proto-oncogene c-mpl is involved in spontaneous megakaryocytopoiesis in myeloproliferative disorders

Spontaneous megakaryocytic colonies (CFU-MK) formation without the addition of Meg-CSA in myeloproliferative disorders (MPD) has been reported by many laboratories. The mechanism by which this occurs is still unknown. In our previous work we have found that the spontaneous colonies persisted in serum-free agar culture although the colony cells were smaller and the colony numbers fewer than in plasma clot culture and that monoclonal antibodies against IL3, IL6 and GM-CSF had no inhibitory effect on spontaneous CFU-MK in both semi-solid cultures. Recently, proto-oncogene c-mpl and c-mpl ligand, thrombopoietin (TPO), have been shown to specifically participate in the regulation of normal human megakaryocytopoiesis. In order to test the hypothesis that c-mplc-mpl ligand pathway is involved in the spontaneous growth of megakaryocyte progenitors, we investigated mRNA expressions of c-mpl and TPO in cells grown in serum-free liquid culture using RT-PCR. The c-mpl expression was detected in the cultured cells from all nine patients (six with ET, two with PV, one with PMF) who had spontaneous CFU-MK in clonal assays. However, none of the patients expressed TPO mRNA in these cells. Pre-incubation of nonadherent mononuclear cells with thioester-modified antisense oligodeoxynucleotide to c-mpl at a concentration of 6μM significantly decreased the cloning efficiency of spontaneous megakaryocyte growth by 42.5% (P<0.05) in plasma clot assay (seven with ET, one with PV) and 69.6% (P<0.05) in serum-free agar culture (six with ET, one with PV). In control experiments the introduction of a scrambled oligomer to antisense oligodeoxynucleotide had no such effect on spontaneous colony formation. These results indicate that c-mpl exerts an important effect in the growth of spontaneous megakaryocytopoiesis in MPD.     

Evidence for a critical role of DNA topoisomerase IIα in drug sensitivity revealed by inducible antisense RNA in a human leukaemia cell line

To examine the role of human DNA topoisomerase IIα (topo IIα) in drug resistance, we selectively inhibited topo IIα gene expression in U937 human monocytic leukaemia cells stably transfected with a plasmid that allowed for Zn-mediated conditional expression of a human α-topo IIα antisense sequence. Expression of topo IIα mRNA was reduced to <30%, whereas no significant alteration of topo IIβ mRNA expression was observed. Under these conditions, drug sensitivity to the topo-II-directed agents, etoposide and daunorubicin, was reduced to approximately 50%, whereas sensitivity to 4-hydroperoxy-cyclophosphamide (4-HC) was not altered. This suggests that a reduced amount of topo IIα mRNA may be sufficient for the resistance to topo II inhibitors in leukaemia cells.