RNA interference

摘要： The brown planthopper(BPH)(Nilaparvata lugens St?l)is a highly destructive pest that seriously damages rice(Oryza sativa L.)and causes severe yield losses.To better understand the physiological and metabolic mechanisms through which BPHs respond to resistant rice,we combined mass-spectrometry-based lipidomics with transcriptomic analysis and gene knockdown techniques to compare the lipidomes of BPHs feeding on either of the two resistant(NIL-Bph6 and NIL-Bph9)plants or a wild-type,BPH susceptible(9311)plant.Insects that were fed on resistant rice transformed triglyceride(TG)to phosphatidylcholine(PC)and digalactosyldiacylglycerol(DGDG),with these lipid classes showing significant alterations in fatty acid composition.Moreover,the insects that were fed on resistant rice were characterized by prominent expression changes in genes involved in lipid metabolism processes.Knockdown of the NlBmm gene,which encodes a lipase that regulates the mobilization of lipid reserves,significantly increased TG content and feeding performance of BPHs on resistant plants relative to dsGFP-injected BPHs.Our study provides the first detailed description of lipid changes in BPHs fed on resistant and susceptible rice genotypes.Results from BPHs fed on resistant rice plants reveal that these insects can accelerate TG mobilization to provide energy for cell proliferation,body maintenance,growth and oviposition.

摘要： RNA interference,widely regarded as a key mechanism for cells to regulate gene expression,is a natural gene silencing phenomenon.It can be used as the gene knockdown to reverse the multidrug resistance of tumor cells and has been applied in the field of biomedicine,exhibiting huge potential in drug target identification,optimization of drug targets,multidrug resistance,etc.This paper first introduces the mechanism of RNA interference and the formation mechanism of multidrug resistance of tumor cells,on the basis of which it reviews the application and challenges of RNA interference technology in reversing multidrug resistance.Additionally,the development of the siRNA delivery system is illustrated.

摘要： OBJECTIVE Phosphodiesterase 4(PDE4),specific for cyclicAMP(cAMP)-hydrolyzing,has four isoforms(PDE4A-D) with at least 25 splice variants. PDE4 inhibitors produce definite antidepressant-like and cognitive-enhancing effects. However,none of PDE4 inhibitors has yet been approved for clinical utility so far due to the concomitant side effects. The present research is to explore the splice variants of PDE4 D responsible for antidepressant-like and cognitive-enhancing effects of PDE4 inhibitors but not side effects. METHODS Long-form PDE4 Ds were silenced by the bilateral microinfusion of lentiviral vector containing mi RNAs(4Dmi R) into the prefrontal cortex(PFC),PDE4D4 or D5 was overexpressed by the bilateral microinfusion of lentiviral vector containing full c DNA into hippocampus. Antidepressant-like behaviors were measured by tail-suspension test(TST),forced swimming test(FST)and chronic unpredictable stress model. Cognitive behaviors were measured by the novel object recognition test(NOR) and Morris water maze test(MWM) in both normal mice and the mice with chronic unpredictable stress-induced memory deficits. The emetic potential was evaluated by the assessment of the anaesthetic reversal effect,a surrogate of the emesis test in non-vomiting species. The expressions of PDE4 isoforms/splice variants and cAMP level were examined by Western-blot and ELISA analysis. The dendritic complexity and spine density were assessed by Golgi staining. RESULTS(1)High and specific expression of EGFP(green,indicator of 4Dmi R expression) in PFC was observed under fluorescence microscopy.(2) 4Dmi R significantly down-regulated PDE4D4/5 splice variants,but not PDE4 A,PDE4 B or PDE4D1/2/3.(3) 4Dmi R treatments significantly increased cAMP signaling and dendritic complexity in PFC.(4) Rolipram and/or 4Dmi R treatments significantly decreased immobility in TST and FST.(5) Rolipram and/or 4Dmi R treatments reversed the depressive-like behaviors in chronically stressed mice,including the reduced sucrose preference,prolonged latency to novelty-suppressed feeding and increased immobility in FST.(6) Rolipram and/or 4Dmi R treatments significantly increased the recognition index in NOR task and both the entries and durations in MWM task.(7) Rolipram and/or 4Dmi R treatments reversed the memory deficits in chronically stressed mice,including the reduced the recognition index in NOR task and the decreased durations in MWM task.(8) Rolipram and/or 4DmiR treatments reversed the decreased cA MP signaling,dendritic complexity and spine density.(9) Rolipram or plus 4Dmi R treatment significantly decreased the duration of anaesthesia in the alpha2 adrenergic receptor-mediated anesthesia,but not 4Dmi R treatment alone.(10)Hippocampal overexpression of PDE4D5,but not PDE4D4,produced depressive-like and cognitive defect behaviors,which were reversed by rolipram.The measurements including cAMP signaling,dendritic complexity and in vivo hippocampal LTP,showed the same changes. CONCLUSION Long-form PDE4 Ds,especially the PDE4D5,are the major isoforms responsible for antidepressant-like and cognitive-enhancing effects with little side effects. The critical roles of long-form PDE4 Ds are mediated by their regulation of cAMP signaling pathway and neuroplasticity.

摘要： Safe and effective gene therapy approaches require targeted tissue-specific transfer of a therapeutic transgene.Besides traditional approaches, such as transcriptional and transductional targeting, micro RNA-dependent posttranscriptional suppression of transgene expression has been emerging as powerful new technology to increase the specificity of vector-mediated transgene expression. Micro RNAs are small non-coding RNAs and often expressed in a tissue-, lineage-, activation- or differentiation-specific pattern. They typically regulate gene expression by binding to imperfectly complementary sequences in the 3' untranslated region(UTR) of the m RNA. To control exogenous transgene expression, tandem repeats of artificial micro RNA target sites are usually incorporated into the 3' UTR of the transgene expression cassette, leading to subsequent degradation of transgene m RNA in cel s expressing the corresponding micro RNA. This targeting strategy, first shown for lentiviral vectors in antigen presenting cells, has now been used for tissue-specific expression of vector-encoded therapeutic transgenes, to reduce immune response against the transgene, to control virus tropism for oncolytic virotherapy, to increase safety of live attenuated virus vaccines and to identify and select cell subsets for pluripotent stem cell therapies, respectively. This review provides an introduction into the technical mechanism underlying micro RNA-regulation, highlights new developments in this field and gives an overview of applications of micro RNA-regulated viral vectors for cardiac, suicide gene cancer and hematopoietic stem cell therapy, as well as for treatment of neurological and eye diseases.

摘要： Acute and chronic hepatitis B virus(HBV) infections remain to present a major global health problem. The infection can be associated with acute symptomatic or asymptomatic hepatitis which can cause chronic inflammation of the liver and over years this can lead to cirrhosis and the development of hepatocellularcarcinomas. Currently available therapeutics for chronically infected individuals aim at reducing viral replication and to slow down or stop the progression of the disease. Therefore, novel treatment options are needed to efficiently combat and eradicate this disease. Here we provide a state of the art overview of gene therapeutic approaches to inhibit HBV replication. We discuss non-viral and viral approaches which were explored to deliver therapeutic nucleic acids aiming at reducing HBV replication. Types of delivered therapeutic nucleic acids which were studied since many years include antisense oligodeoxynucleotides and antisense RNA, ribozymes and DNAzymes, RNA interference, and external guide sequences. More recently designer nucleases gained increased attention and were exploited to destroy the HBV genome. In addition we mention other strategies to reduce HBV replication based on delivery of DNA encoding dominant negative mutants and DNA vaccination. In combination with available cell culture and animal models for HBV infection, in vitro and in vivo studies can be performed to test efficacy of gene therapeutic approaches. Recent progress but also challenges will be specified and future perspectives will be discussed. This is an exciting time to explore such approaches because recent successes of gene therapeutic strategies in the clinic to treat genetic diseases raise hope to find alternative treatment options for patients chronically infected with HBV.

摘要： AIM: To investigate a therapeutic method for gastrointestinal stromal tumor (GIST) based on KIT RNA interference (RNAi) with AdMax adenovirus. METHODS: KIT short hairpin RNA (shRNA), whose lateral sides were decorated with restriction endonuclease sequences, was designed. T 4 DNA ligase catalyzed the joint of the KIT shRNA and the green fluorescent protein-containing PDC316-EGFP-U6 to form PDC316EGFP-U6-KIT. Homologous recombination of AdEGFPU6-KIT was performed with the AdMax system. Heterotopically transplanted GISTs were established in nude mice. AdEGFP-U6-KIT was intratumorally injected. The volume, inhibition ratio of tumor and CD117 expression of GIST graft tumor in nude mice were compared between test and control groups. RESULTS: The length of KIT shRNA was determined to be about 50bp by agarose electrophoresis. Gene se-quencing detected the designed KIT RNAi sequence in PDC316-EGFP-U6-KIT. After transfection with AdEGFPU6-KIT, 293 cells displayed green fluorescence. The physical and infective titers of AdEGFP-U6-KIT were 5 × 10 11 viral particles/mL and 5.67 × 10 7 plaque forming units/mL, respectively. The mean volume of the grafted tumor was significantly smaller in test mice than in control mice (75.3 ± 22.9 mm 3 vs 988.6 ± 30.5 mm 3 , t = -18.132, P < 0.05). The inhibition ratio of the tumors was 59.6% in the test group. CD117 positive expression was evident in two cases (20%) in the test group and 10 cases (100%) in the control group (χ 2 = 10.2083, P < 0.005). CONCLUSION: AdEGFP-U6-KIT is successfully constructed, and KIT RNAi mediated with Admax vector system can effectively inhibit the expression of the KIT gene and the growth of GIST in nude mice.

摘要： AIM:To investigate whether silencing Fas-associated phosphatase 1(FAP-1)expression enhances the efficiency of chemotherapy for colon carcinoma with oxaliplatin.METHODS:Expression of FAP-1 in mRNA and protein was detected by reverse transcription polymerase chain reaction(RT-PCR)and flow cytometry.Small interfering RNA(siRNA)was designed according to the FAP-1 mRNA sequence.Cell proliferation was evaluated by methyl thiazolyl tetrazolium(MTT)assay.Anenxin V-and propidine iodine(PI)were assayed by flow cytometry for the detection of apoptosis. RESULTS:The expression of FAP-1 was increased in SW480 cells after chemotherapy with oxaliplatin. Transfection of FAP-1 siRNA into SW480 cells silenced the expression of FAP-1 and consequently abolished the inhibitory function of Fas/FasL-mediated apoptosis pathway,thus increasing the efficacy of chemotherapy for colon carcinoma with oxaliplatin. CONCLUSION:RNA interference combined with conventional chemotherapy is more effective against colon cancer.

摘要： AIM:To investigate the effects and mechanism of disruption of focal adhesion kinase(FAK) expression on collagen metabolism in rat hepatic stellate cells(HSC).METHODS:The plasmids expressing FAK short hairpin RNA(shRNA) were transfected into HSC-T6 cells,and the level of FAK expression was determined by both real-time quantitative polymerase chain reaction(QPCR) and Western blotting analysis.The production of type collagen and type collagen in FAK-disrupted cells was analyzed by real-time Q-PCR.The level of collagen metabolism proteins,including matrix metalloproteinases-13(MMP-13) and tissue inhibitors of metalloproteinases-1(TIMP-1) was also determined by both real-time Q-PCR and Western blotting analysis.RESULTS:The transfection of FAK shRNA plasmids into HSC resulted in disrupted FAK expression.Compared with the HK group,the levels of type collagen and type collagen mRNA transcripts in FAK shRNA plas-mid group were signif icantly decreased(0.69 ± 0.03 vs 1.96 ± 0.15,P = 0.000;0.59 ± 0.07 vs 1.62 ± 0.12,P = 0.020).The production of TIMP-1 in this cell type was also signif icantly reduced at both mRNA and protein levels(0.49 ± 0.02 vs 1.72 ± 0.10,P = 0.005;0.76 ± 0.08 vs 2.31 ± 0.24,P = 0.000).However,the expression of MMP-13 mRNA could be significantly up-regulated by the transfection of FAK shRNA plasmids into HSC(1.74 ± 0.20 vs 1.09 ± 0.09,P = 0.000).CONCLUSION:These data support the hypothesis that shRNA-mediated disruption of FAK expression could attenuate extracellular matrix(ECM) synthesis and promote ECM degradation,making FAK a potential target for novel anti-f ibrosis therapies.

摘要： This study aims to demonstrate that blocking the receptor-interacting protein2(Rip2)expression can decrease inflammatory cytokine production by macrophage and protect mice from endotoxin lethality.Murine Rip2 small interfering RNA(siRNA)plasmids were constructed and transfected into macrophage and Rip2 expression was detected with reverse transcription-polymerase chain reaction(RT-PCR)and western blot.Cell proliferation was assayed with MTT.TNF-α concentration was assayed with ELISA and high-mobility group box 1 protein(HMGB1)level with semi-quantitative western blot after lipopolysaccharide(LPS)stimulation.LPS challenge was given after the plasmids were injected into mice and the survival rate was calculated.Rip2 siRNA plasmid could block the mRNA and protein expression of Rip2 and promote cell proliferation.Blocking Rip2 could attenuate LPS-induced TNF-~ and HMGB1 production.The HMGB1 expression in the liver decreased to(40.21±11.03)pg/g,and serum TNF-α level decreased to(300.43±59.26)ng/L(P＜0.05).The survival rate of mice from endotoxemia was also improved(P＜0.05).The results demonstrate that Rip2 siRNA plasmid can block the expression of Rip2,decrease the production of TNF-α and HMGB1 and protect mice from fatal endotoxemia.

摘要： Three plasmids (pGenesil-P1, pGenesil-P2, pGenesil-P3) with different p27Kipl-shRNA sequences were designed and synthesized. Their effects on the proliferation of bovine corneal endo- thelial cells (bCEC) were investigated. Plasmid expressing irrelevant shRNA with a random combi- nation was used as negative control (pGenesil-HK). The recombination of four plamids was con- firmed by restrictive enzyme digestion and sequence analysis. The expression of mRNA and protein of p27Kipl was detected by RT-PCR and Western blotting after stable transfection. The expressions of p27Kipl mRNA and p27Kipl protein of pGenesil-P1 group, pGenesil-P2 group and pGenesil-P3 group were all lower than those in the pGenesil-HK group and the blank group (non-transfected group), pGenesil-P3 had the strongest inhibitory effect and was selected for the next steps. The pro- liferation rates of the pGenesil-P3 group, the pGenesii-HK group and the blank group were assessed by MTT. The influence of shRNA-p27Kipl on bCEC cell cycle was detected by flow cytometry (FCM). Compared with the control groups, the proliferation rate of the pGenesiI-P3 group was increased significantly, and the ratio of S-phase also increased. It is concluded that shRNA-p27Kipl could down-regulate the expression of p27Kipl effectively and increase the proliferation of bCEC. RNA interference (RNAi) may be an effective means to promote the proliferation of CEC.