逆转录病毒载体介导的RNA干扰稳定抑制肌肉生长抑制素GDF-8的表达

为将肌肉生长抑制素的干扰序列表达盒hU6-siGDF-8有效导入肌成纤维细胞C2C12中, 以pAV-hU6 + 27载体为基础构建逆转录病毒载体pXSN-hU6-siGDF-8, 并使之与pVSV-G质粒共转染GP-293细胞, 用包装出的病毒粒子感染宿主细胞C2C12, G418筛选稳定整合逆转录病毒的抗性细胞库。2周后, Western Blotting和Real-Time PCR分析结果显示, 细胞内源性的GDF-8基因的表达得到了有效的抑制; MTT法和细胞流式仪分析表明, G418抗性细胞得到了更有效的增殖, 并且G0/G1期细胞数量减少了13.7%, S期细胞数量增加了14.9%。因此, 逆转录病毒载体的RNA干扰系统可以稳定抑制 GDF-8基因表达, 它将成为治疗肌肉萎缩疾病的一个强有力的工具。     

小分子G蛋白Rapl shRNA表达载体的构建和鉴定

应用RNA干扰(RNA interference,RNAi)技术抑制Rapl基因的表达,构建Rap1 shRNA(small hairpin RNA,shRNA)表达载体,观察其对小鼠肝脏细胞中Rap1 mRNA和蛋白表达的干扰作用.根据小鼠Rap1 mRNA的全序列.设计了3种Rap1 siRNA序列(Rap1 siRNA1、Rap1 siRNA2、Rap1 siRNA3)和阴性对照序列(HK);采用克隆技术,将其插入带有报告基因绿色荧光(ECFP)的pGenesil-3栽体,构建Rap1 shRNA表达载体:经双酶切和测序证实Rap1 siRNA表达载体克隆构建成功,插入片段测序结果与合成的siRNA结果一致;昆明小鼠40只,体重18～20 g,随机分成4组: Ⅰ组(转染HK组)、Ⅱ组(转染Rapl shRNAl组)、Ⅲ组(转染Rap1 shRNA2组)、Ⅳ组(转染Rapl shRNA3组).于0、16、24 h腹腔内注射Rapl shRNA 2.0～2.5 mg/kg(用PBS稀释至1 mL);48 h后收集小鼠肝脏.用显微荧光、定量RT-PCT、免疫组化检测小鼠肝细胞中Rap1shRNA的转染率、Rap1基因表达以及蛋白质表达水平.Ⅰ组、Ⅱ组、Ⅲ组、Ⅳ组小鼠肝脏细胞体内转染率均大于60%,Ⅱ组、Ⅲ组、Ⅳ组的Rap1 mRNA表达、Rap1蛋白表达均降低,其中Rap1 shRNA1干扰效果最佳.     

携带人NFBD1基因shRNA重组逆转录病毒载体的构建及鉴定

建立逆转录病毒介导的NFBD1基因RNA干扰表达体系,并观察其在宫颈癌SiHa细胞中对NFBD1表达的影响.将人NFBD1基因RNA干扰双链DNA片段重组到pSUPER Retro质粒中,构建携带人NFBD1基因RNA干扰的逆转录病毒载体pSUPER-shRNA-NFBD1,经PT67细胞包装后,产生的重组逆转录病毒感染宫颈癌细胞株SiHa细胞,并用嘌呤霉素筛选产生稳定的细胞克隆,用实时荧光定量PCR和Westernblotting检测细胞中NFBD1 mRNA和蛋白表达的变化.重组逆转录病毒质粒经测序鉴定正确;逆转录病毒感染SiHa细胞后用嘌呤霉素筛选出稳定的细胞克隆;实时荧光定量PCR和Westernblotting检测人NFBD1 mRNA和蛋白表达水平明显低于阴性对照组和未干扰组.携带人NFBD1基因RNA干扰双链DNA片段的逆转录病毒感染SiHa细胞后能明显抑制NFBD1 mRNA和蛋白表达,为进一步研究NFBD1在宫颈癌中的作用奠定了基础.     

Fluorescence-based evaluation of shRNA efficacy

RNA interference is a cellular mechanism regulating levels of mRNAs. It has been widely exploited to knock down specific protein targets. The selected interfering RNA sequence greatly influences its ability to knock down the target. Here we present a method for constructing multiple testing plasmids which express small hairpin RNAs (shRNA) targeting different regions of an mRNA. A simple fluorescence test in cultured cells allows convenient evaluation of mRNA knockdown by many different shRNAs on 96-well plates. We show that software predicted shRNAs have varying efficacies and only 2 of the 7 tested shRNAs significantly knocked down their targets.     

Erythropoietin for oncology supportive care

Recombinant human erythropoietin (rhEPO), the prototype erythropoiesis-stimulating agent developed in the 1980s, was among the first recombinant human proteins to be marketed for clinical use in the oncology setting. Anemia is a frequent concern in patients with cancer receiving myelosuppressive chemotherapy and the availability of rhEPO as an alternative to red blood cell transfusions to treat symptomatic anemia created excitement among clinicians, particularly during an era of mounting concern for transfusion-transmissible infections. Early studies of rhEPO for chemotherapy-induced anemia in patients with non-myeloid malignancies showed these agents improved hemoglobin levels and reduced transfusion rates. rhEPO therapy was reported to decrease fatigue and improve quality of life, although the magnitude and clinical meaningfulness of these effects have been debated. More recent clinical trials since 2003 linking rhEPO therapy to increased risk of tumor progression, thrombo-vascular events and mortality prompted implementation of use restrictions to minimize potential for harm. Scientific research to understand the basic mechanisms of the biologic effects of erythropoietin at the cellular receptor and signaling level has revealed pleiotropic cytokine effects extending beyond erythropoiesis regulation. The importance of erythropoietin receptor signaling in normal, non-erythroid tissues and in pre-clinical tumor models has been under intense investigation and scrutiny, as potential mechanisms of the adverse outcomes associated with rhEPO therapy have been debated. Further research will be required to clarify the complex interplay between the diverse hematopoietic and non-hematopoietic effects of erythropoietin in normal and malignant tissues and to optimize the clinical use of rhEPO in the supportive care of cancer patients.     

Synergistic interaction between leptin and cholecystokinin in the rat nodose ganglia is mediated by PI3K and STAT3 signaling pathways: implications for leptin as a regulator of short term satiety

Research has shown that the synergistic interaction between vagal cholecystokinin-A receptors (CCKARs) and leptin receptors (LRbs) mediates short term satiety. We hypothesize that this synergistic interaction is mediated by cross-talk between signaling cascades used by CCKARs and LRbs, which, in turn, activates closure of K(+) channels, leading to membrane depolarization and neuronal firing. Whole cell patch clamp recordings were performed on isolated rat nodose ganglia neurons. Western immunoblots elucidated the intracellular signaling pathways that modulate leptin/CCK synergism. In addition, STAT3, PI3K, Src, and MAPK genes were silenced by lentiviral infection and transient Lipofectamine transfection of cultured rat nodose ganglia to determine the effect of these molecules on leptin/CCK synergism. Patch clamp studies showed that a combination of leptin and CCK-8 caused a significant increase in membrane input resistance compared with leptin or CCK-8 alone. Silencing the STAT3 gene abolished the synergistic action of leptin/CCK-8 on neuronal firing. Leptin/CCK-8 synergistically stimulated a 7.7-fold increase in phosphorylated STAT3 (pSTAT3), which was inhibited by AG490, C3 transferase, PP2, LY294002, and wortmannin, but not PD98059. Silencing the Src and PI3K genes resulted in a loss of leptin/CCK-stimulated pSTAT3. We conclude that the synergistic interaction between vagal CCKARs and LRbs is mediated by the phosphorylation of STAT3, which, in turn, activates closure of K(+) channels, leading to membrane depolarization and neuronal firing. This involves the interaction between CCK/Src/PI3K cascades and leptin/JAK2/PI3K/STAT3 signaling pathways. Malfunctioning of these signaling molecules may result in eating disorders.     

Ca(V)1.2 channel N-terminal splice variants modulate functional surface expression in resistance size artery smooth muscle cells

Voltage-dependent Ca(2+) (Ca(V)1.2) channels are the primary Ca(2+) influx pathway in arterial smooth muscle cells and are essential for contractility regulation by a variety of stimuli, including intravascular pressure. Arterial smooth muscle cell Ca(V)1.2 mRNA is alternatively spliced at exon 1 (e1), generating e1b or e1c variants, with e1c exhibiting relatively smooth muscle-specific expression in the cardiovascular system. Here, we examined physiological functions of Ca(V)1.2e1 variants and tested the hypothesis that targeting Ca(V)1.2e1 modulates resistance size cerebral artery contractility. Custom antibodies that selectively recognize Ca(V)1.2 channel proteins containing sequences encoded by either e1b (Ca(V)1.2e1b) or e1c (Ca(V)1.2e1c) both detected Ca(V)1.2 in rat and human cerebral arteries. shRNA targeting e1b or e1c reduced expression of that Ca(V)1.2 variant, induced compensatory up-regulation of the other variant, decreased total Ca(V)1.2, and reduced intravascular pressure- and depolarization-induced vasoconstriction. Ca(V)1.2e1b and Ca(V)1.2e1c knockdown reduced whole cell Ca(V)1.2 currents, with Ca(V)1.2e1c knockdown most effectively reducing total Ca(V)1.2 and inducing the largest vasodilation. Knockdown of α(2)δ-1, a Ca(V)1.2 auxiliary subunit, reduced surface expression of both Ca(V)1.2e1 variants, inhibiting Ca(V)1.2e1c more than Ca(V)1.2e1b. e1b or e1c overexpression reduced Ca(V)1.2 surface expression and whole cell currents, leading to vasodilation, with e1c overexpression inducing the largest effect. In summary, data indicate that arterial smooth muscle cells express Ca(V)1.2 channels containing e1b or e1c-encoded N termini that contribute to Ca(V)1.2 surface expression, α(2)δ-1 preferentially traffics the Ca(V)1.2e1c variant to the plasma membrane, and targeting of Ca(V)1.2e1 message or the Ca(V)1.2 channel proximal N terminus induces vasodilation.     

Down-regulation of GPR137 expression inhibits proliferation of colon cancer cells

G protein-coupled receptors （GPRs） are highly related to oncogenesis and cancer metastasis. G protein-coupled re- ceptor 137 （GPR137） was initially reported as a novel orphan GPR about 10 years ago. Some orphan GPRs have been implicated in human cancers. The aim of this study is to investigate the role of GPR137 in human colon cancer. Expression levels of GRP137 were analyzed in different colon cancer cell lines by quantitative polymerase chain re- action and western blot analysis. Lentivirus-mediated short hairpin RNA was specifically designed to knock down GPR137 expression in colon cancer cells. Cell viability was measured by methylthiazoletetrazolium and colony forma- tion assays. In addition, cell cycle characteristic was investi- gated by flow cytometry. GRP137 expression was observed in aH seven colon cancer cell lines at different levels. The mRNA and protein levels of GPR137 were down-regulated in both HCTll6 and RKO cells after lentivirus infection. Lentivirus-mediated silencing of GPR137 reduced the proliferation rate and colonies numbers. Knockdown of GPR137 in both cell lines led to cell cycle arrest in the G0/G1 phase. These results indicated that GPR137 plays an important role in colon cancer cell proliferation. A better understanding of GPR137＇s effects on signal transduction pathways in colon cancer cells may provide insights into the novel gene therapy of colon cancer.