Suppression of RNA Interference Pathway in vitro by Grass Carp Reovirus

The means of survival of genomic dsRNA of reoviruses from dsRNA-triggered and Dicer-initiated RNAi pathway remains to be defined. The present study aimed to investigate the effect of Grass carp reovirus (GCRV) replication on the RNAi pathway of grass carp kidney cells (CIK). The dsRNA-triggered RNAi pathway was demonstrated unimpaired in CIK cells through RNAi assay. GCRV-specific siRNA was generated in CIK cells transfected with purified GCRV genomic dsRNA in Northern blot analysis; while in GCRV-infected CIK cells, no GCRV-specific siRNA could be detected. Infection and transfection experiments further indicated that replication of GCRV correlated with the increased transcription level of the Dicer gene and functional inhibition of in vitro synthesized egfp-siRNA in silencing the EGFP reporter gene. These data demonstrated that although only the genomic dsRNA of GCRV was sensitive to the cellular RNAi pathway, unidentified RNAi suppressor protein(s) might contribute to the survival of the viral genome and efficient viral replication.

INFLUENCE OF p53 SMALL DOUBLE STRANDED RNA INTERFERENCE ON HEPATOMA CELL LINE SK-HEP-1

Objective: The effects on cell-cycle and p53 expression in hepatoma cell line SK-Hep-1 were explored by transfecting exogenous p53 small double stranded RNA (dsRNA) into the SK-Hep-1 cells. Methods: p53 dsRNA and EGFP dsRNA were synthesized. SK-Hep-1 (wtp53) cell line was transfected with 200 ng and 400 ng p53 dsRNA or EGFP and EGFP+EGFP dsRNA (as positive control) or 9% NaCl (as blank control) by liposome transfection technique. Flow cytometry was adopted to measure the effects of p53 dsRNA on cell cycle. Expression of p53 protein was detected by Western-Blotting at 48 h after transfecting p53 dsRNA. Results: The number of G0-G1 phase SK-Hep-1 cells, which were transfected with 200 ng p53 dsRNA, was decreased by 52.53% comparing with the control, and decreased by 50.29% (P<0.05) comparing with the positive control cells transfected with same dosage of EGFP+EGFP dsRNA. The number of S phase cells, which were transfected with 200 ng p53 dsRNA, was increased by 146.8% comparing with the control, and increased by 128.62% (P<0.05) comparing with the positive control cells transfected with same dosage of EGFP+EGFP dsRNA. The number of G2-M phase cells, which were transfected with 200 ng p53 dsRNA, was increased by 30.56% (P<0.05) comparing with the control, and increased by 21.63% (P>0.05) comparing with the positive control cells transfected with same dosage of EGFP+EGFP dsRNA. After 48 h, p53 protein expression was not detected in the SK-Hep-1 cells transfected with p53 dsRNA. Conclusion: p53 dsRNA can obviously improve the proliferation of SK-Hep-1 cells, and suppress p53 protein expression of SK-Hep-1 cells, the former may be related to of the latter.

Antitumor effect of RNA interference on non-small- cell lung cancer in vivo

Objective： Lung cancer has emerged as a leading cause of cancer death in the world. Current therapies are ineffective, thus new approaches are needed to improve the therapeutic ratio. RNA interference （RNAi） has shown promise in gene silencing in vitro, the potential of which in developing new methods for the therapy of non-small-cell lung cancer （NSCLC） needs to be further tested in vivo. In this study, chemically synthesized double-stranded RNA （dsRNA） targeting epidermal growth factor receptor （EGFR） was transfected into NSCLC cell line SPC-A1 cells and established the tumor burdened athymic nude mice model to investigate whether dsRNA could induce gene silencing in NSCLC cells in vivo. Methods： SPC-A1 was transfected with EGFR sequence-specific dsRNA formulated with Lipofectamine 2000. SPC-A1 cells （1 × 107/ mL） in 200 pL were injected s.c. into the left flank area of the mice to establish the tumor burdened athymic nude mice model. Calculate the tumor growth inhibition rate by measuring the diameter and the weight of the tumor. Immunohistochemistry and Westem blot were used to monitor the reduction in the production of the EGFR protein. Realtime RT-PCR was used to detect the silencing of the EGFR mRNA level. Results： It displayed that EGFR sequence specific dsRNA （dsRNA-EGFR） significantly inhibited the tumor growth in vivo. The tumor growth inhibition rate was 75.03%. The dsRNA-EGFR sequence specifically silenced EGFR with 53.6% of down-regulation of EGFR protein production and 32.3% of silencing of EGFR mRNA level. Conclusion： DsRNA-EGFR showed a blockbuster effect in downregulation of EGFR mRNA level and protein production, and inhibition of tumor growth in vivo.

RNA干扰技术与植物病毒病害防治

RNA干扰是生物进化的结果,是生物体对病毒基因等外源核酸侵入的一种保护性反应。它普遍存在于各种生物中,具有抗病毒、稳定转座子及监控异常表达mRNA的生物学功能。应用RNAi技术,将病毒在复制中起关键作用的基因作为目标,设计dsRNA导入植株培育抗病毒植株。

RNA-Based Biocontrols--A New Paradigm in Crop Protection

Modern agribusiness plays a vital role in safeguarding and improving the production,quality,and quantity of food,feed,fiber,and fuel.Growing concerns over the impact of chemical pesticides on health and the environment have stimulated the industry to search for alternative and greener solutions.Over the last years,the RNA interference(RNAi)process has been identified as a very promising new approach to complement the arsenal of foliar spray,soil,or seed treatments applied as chemical and biological pest control agents,and of plant-incorporated protectants(PIPs).RNA-based active ingredients(AIs)possess a unique mode of action and can be implemented via both genetic modification(GM)and biocontrol approaches.RNA-based AIs promise to deliver the selectivity and sustainability desired in future crop protection agents.This is due to their utilization of a natural process to exert control and their high level of selectivity,which leads to reduced risk for non-target organisms(NTOs).This review discusses the advantages and limitations of RNA-based solutions in crop protection and recent research progress toward RNA-based biocontrols against the Colorado potato beetle(CPB),corn rootworm(CRW),and soy stink bug(SSB).Many challenges still exist on the road to the implementation of a broad range of RNA-based products and their widespread use and application.Despite these challenges,it can be expected that RNA-based AIs will become valuable new tools complementing the current arsenal of crop-protection solutions.

Gecko crude peptides inhibit migration and lymphangiogenesis by down regulating the expression of VEGF-C in human hepatocellular carcinoma cells and human lymphatic endothelial cells

OBJECTIVE To explore the role of gecko crude peptides(GCPs)in the proliferation,apoptosis,migration and lymphangiogenesis of human hepatocellular carcinoma cells(Hep G2)and human lymphaticendothelial cells(HLECs)in vitro.METHODS The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay was used to evaluate the anti-proliferative effect of GCPs and si RNA-VEGF-C on Hep G2 cells,Hoechst 33258 staining and flow cytometry were performed to analyze cycle and apoptosis.The migration and invasion ability of cells were assayed by transwell chamber experiment and wound-healing assay.The protein and m RNA expressions of vascular endothelial growth factor-C(VEGF-C)and CXC chemokine receptor-4(CXCR4)were detected by q-PCR,immunofluorescence,Western blot.The protein expressions of the extracellular signal regulated kinase(ERKI/2),c-Jun N-terminal kinase(JNK),p38-mitogen activated protein kinases(p38 MAPK),serine/threonine kinase(Akt)and phosphatidylinositol-3-kinase(PI3K)were detected by western blot.The anti-lymphangiogenesis effect of GCPs on the HLECs was analyzed using an in vitro tube-formation assay.The protein and m RNA expressions of vascular endothelial growth factor receptor-3(VEGFR-3)and stromal cell-derived factor-1(SDF-1)were detected by q-PCR,Western blot.RESULTS GCPs and si RNA-VEGF-C inhibited Hep G2 proliferation,invasion and migration,and the most obvious inhibitory effect was both synergistic effects.Thus,GCPs suppressed HLECs proliferation,migration and tubelike structure formationin a dose-dependent manner,and had inhibitory effect of tumor-induced lymphangiogenesis in vitro.Additionally,we found that GCPs and si RNA-VEGF-C decreased the expressions of MMP-2,MMP-9,VEGF-C,CXCR4,phospho-ERK1/2,phospho-P38,phospho-JNK and PI3K in Hep G2 cells.Moreover,GCPs had a dose-dependent depressive effecton the expressions of VEGFR-3,SDF-1 in HLECs.CONCLUSION The low expression of VEGF-C mediated by si RNA-VEGF-C and GCPs inhibit tumor proliferation,invasion and migrationby suppressing the MAPK signaling pathway through reduced levels of VEGF-C,and GCPs inhibit tumor lymphangiogenesis by suppressing the CXCR4/SDF-1 signaling pathway through suppressed VEGF-C/VEGFR-3.

The inhibitory effects of siRNA expression vector on CXCR4 expression in prostate carcinoma cell lines

Objective： To investigate the inhibitory effects of RNAi （ RNA interference, RNAi） expression vector on CXCR4 expression in prostate carcinoma cell lines. Methods： Small interference RNA （siRNA） expression vectors for CXCR4 gene were constructed and transfected into prostate carcinoma cell lines（PC-3m and LNCaP）with liposomes. T expression of CXCR4 was detected by RT-PCR and western blot. Results： T expression of CXCR4 mRNA and protein in the PC-3m and LNCaP cells was reduced by RNAi expression vectors. The inhibitory rate of CXCR4 mRNA expression in the PC-3m cells was 87.81% ± 10.20% ,56.10% ± 9.32% at the 24th hour and the 48th hour, compared with 56.93% ±8.78% ,49.24% ± 11.23% in LNCaP cells. The inhibitory rate of the expression of CXCR4 protein was 64.71% ± 6.68% ,58.66% ± 11.56% respectively. Conclusion： The expression of CXCR4 gene can effectively be inhibited by RNAi expression vectors.

Effect of HMGA2 shRNA on the Cell Proliferation and Invasion of Human Colorectal Cancer SW480 Cells In vitro

High mobility group A2（HMGA2） protein is a small nonhistone chromosomal protein that can modulate transcription of an ample number of genes.Many previous studies demonstrate that up-regulation of HMGA2 expression occurrs in many kinds of cancers including colorectal cancer,suggesting that HMGA2 might play a critical role in the progression of various tumors.However,the exact role of HMGA2 in colorectal cancer has not been determined.To verify the essential role of HMGA2 in the growth and invasiveness of colorectal cancer,HMGA2 expression was down-regulated by RNA interference（RNAi） in SW480 cells.We observed that the knockdown of HMGA2 led to the significant inhibition of proliferation and invasion of SW480 cells in vitro.These results suggest that HMGA2 might play a crucial role in the progression of colorectal cancer,and be a potential therapeutic target for human colorectal cancer.

Recombinant adenovirus-mediated shRNA silencing of midkine gene in BxPC-3 cells

Objective：To investigate the silencing effects of recombinant adenovirus Ad-shRNA-MK on midkine（MK） gene in pancreatic cancer cells. Methods：Ad-shRNA-MK was used to infect pancreatic cancer BxPC-3 cells. Assays were conducted for knockdown of the MK gene on the day of infection and on the 1 ^st, 3^rd, 5^th, 7^th, and 9^th days post-infection by using immunocytochemistry, real-time RT-PCR, and Western blot analysis. Results：The adenoviral Ad-shRNA-PTN was constructed successfully, and infection was confirmed by electron microscopic observation. By using real-time RT-PCR, the inhibition rates of MK mRNA expression in the BxPC-3 cells were 20%, 80%, 55%, and 23% on the 1st, 3^th, 5^th, and 7^th days post-infection. Immunocytochemistry and Western blot analysis confirmed this effect at the gene product level. Conclusion：Efficient and specific knockdown of MK in pancreatic cancer cells by adenoviral Ad-shRNA-PTN is a potentially powerful tool for the study of gene therapy of pancreatic cancer nerve infiltration.

Survivin:an overexpression protein with notable cellular localization and multiple roles in colon cancer

Objective:Survivin has attracted abundant interest in tumor research since it was discovered in 1997.But several studies indicated that the relationship between survivin expression and tumor behavior is still not fully understood.So our main aim was to observe the localization of survivin in colon cancer and its influence on the colon cancer biocharacteristics.Methods:We screened the expression and localization of survivin in SW480 by immunofluorescence and immunocytochem-istry.Then, we constructed the recombinant adenovirus (Ad-survivin/shRNA), which contained the short hairpin RNA (shRNA) of survivin and transfected it into SW480 cells.We detected survivin gene expression after shRNA interference by RT-PCR and Western blot, and its influence on the proliferation, apoptosis and cell cycle was analyzed by MTT, colony-formation, and flow cytometry.Results:Survivin was expressed at a high level in SW480 cells and the sub-cellular localization was in the cytoplasm.Recombinant adenovirus could suppress survivin expression efficiency and inhibit proliferation, induce apoptosis, and affected the mitosis in vitro.Conclusion:Survivin plays an important role in controlling tumor growth by a variety of molecular regulatory mechanisms.Cytoplasmic survivin silence could induce apoptosis, effect the mitotic cycle and inhibit cell proliferation.

Effects of transfecting Bcl-2 shRNA and celecoxb on the SGC-7901 cell line

Objective:The aim of this study was to study the changes of SGC-7901 cells transfecting small hairpin RNA(shRNA) targeted Bcl-2 and celecoxib in vitro.Methods:To use the effective siRNA targeted to Bcl-2 by Lipofectamine 2000,the rate of cell growth inhibition of all groups was detected by multiply-table tournament(MTT) method,apoptosis rate was examined by flow cytometry and the expression of Bcl-2 was assayed by Elisa.Results:After siRNA was transfected to SGC-7901 cells,the rate of cell growth inhibition was increased,the joint group was higher by flow cytometry and the expression of Bcl-2 was lower by Elisa.Conclusion:The growth of SGC-7901 cells which was transfected siRNA has been inhibited,and the sensitivity to celecoxib has been increased.

Mechanism Analyses for Elucidating the Role of LOXL2 Silencing in Hepatocellular Carcinoma

The paper aimed to study the effect of lysyl oxidase-like 2 （LOXL2） on hepatocellular carcinoma （HCC） and explore the biological mechanisms of tumorigenicity and progression in HCC. The authors used four HCC cell lines to identify LOXL2. A lentiviral vector containing LOXL2-siRNA was constructed to silence the LOXL2 gene in SMMC-7721 cell line, and mRNA of the target gene was detected by real-time polymerase chain reaction （RT-PCR）. The effect of LOXL2 silencing on the growth of SMMC-7721 cells was explored with flow cytometry profiling and BrdU labeling. Downstream genes of LOXL2 were selected by microarray and verified by Western Blotting. In the results, LOXL2 expression was significantly up-regulated in four types of HCC cell lines, therefore, SMMC-7721 cell line was selected for further exploration. When SMMC-7721 cell line was infected with LOXL2-siRNA, the expression of LOXL2 mRNA decreased. The silencing of LOXL2 resulted in the cell cycle arrest at the G 1-phase, the increased apoptosis and the decreased growth of SMMC-7721 cells on the indicated days by BrdU. Moreover, the MDM2, BIRC3, CDC42, FOS and TGFBR2 genes were selected and verified to be the downstream genes of LOXL2. In conclusion, LOXL2 contributes to the genesis and progression of HCC cells and works by regulating downstream genes of LOXL2 in certain pathways. Therefore, LOXL2 may play an important role in the progression and prognosis of HCC.

Applications of RNA interference highthroughput screening technology in cancer biology and virology

RNA interference （RNAi） is an ancient intra-cellular mechanism that regulates gene expression and cell function. Large-scale gene silencing using RNAi highthroughput screening （HTS） has opened an exciting frontier to systematically study gene function in mammalian cells. This approach enables researchers to identify gene function in a given biological context and will provide considerable novel insight. Here, we review RNAi HTS strategies and applications using case studies in cancer biology and virology.

Development of a RNAi-based release of insects carrying a dominant lethal (RIDL) system in Drosophila melanogaster

Since the first report of the release of insects carrying a dominant lethal （RIDL） strategy, RIDL strains have been constructed in species including fruit flies and mosquitoes. However, in many insects, identification of sterile and lethal genes needed to generate a RIDL strain is limited by the lack of molecular and genetic information. Here, we created RIDL strains of Drosophila melanogaster using RNA interference （RNAi） of the Pygopus （Pygo） gene, a key component of the Wingless/Wnt signaling pathway. In two transgenic lines, XDll and XD15, we verified lethality in the absence of tetracycline, but we were unable to demonstrate sex-specific lethality. We found that male XD15 adults maintained on medium without tetracycline had a longer lifespan than wild type. This RNAibased RIDL strain may therefore offer the advantages of a transgene that promotes the expression of two contrary actions at different life stages： lethality in larvae and prolonged lifespan in adults, actions that could work together to provide prolonged delivery of lethality by the RIDL system. Use of RNAi can facilitate the development and application of RIDL strategies in a wide range of species.

RNAi technique, how far is it from pediatrics?

The new technology of ribonucleic acid interference(RNAi)or small/short interfering RNA(siRNA)can be used to reduce expression of genes in a sequence specific manner,and thereby can treat various diseases caused by expression or overexpression of genes.Phase 1 and phase 2 clinical studies on application of this technology to treat diseases have demonstrated efficacy and safety of this approach in a few specialties/subspecialties.However,no clinical trials have been reported in the fields of pediatrics.This article aimed to describe very briefly what the RNAi technique is,examples of demonstration of the efficacy and safety of RNAi techniques in a few different fields of clinical medicine,and to encourage pediatricians and pediatric researchers to actively participate in studies on this new therapeutic approach for treatment of various pediatric diseases.

Effect of interrupted endogenous BMP/Smad signaling on growth and steroidogenesis of porcine granulosa cells

Bone morphogenetic proteins(BMPs) play a critical role in the growth and steroidogenesis of granulosa cells(GCs).BMP signals act through membrane-bound heteromeric serine/threonine kinase receptors.Upon ligand binding,BMPs activate intracellular Smad proteins and regulate growth and apoptosis in various cell types.The objective of this study was to demonstrate the effects of BMP/Smad signal on growth and steroidogenesis of porcine GCs.A strategy of RNA interference(RNAi)-mediated 'gene silencing' of Smad4,a core molecule mediating the intracellular BMP/Smad signal transduction pathways,was used to interrupt endogenous BMP/Smad signaling.Results indicate that Smad4-small interfering RNA(siRNA) caused specific inhibition of Smad4 mRNA and protein expression after transfection.Interrupted endogenous BMP/Smad signaling significantly inhibited growth,and induced apoptosis of porcine GCs,while decreasing estradiol production.In addition,interrupted BMP/Smad signaling significantly(P<0.05) changed the expression of Cyclin D2,CDK4,Bcl-2,and Cyp19a1.These findings provide new insights into how BMP/Smad signaling regulates the growth and steroidogenesis of porcine GCs.

The Isogene 1-Deoxy-D-Xylulose 5-Phosphate Synthase 2 Controls Isoprenoid Profiles, Precursor Pathway Allocation, and Density of Tomato Trichomes

Plant isoprenoids are formed from precursors synthesized by the mevalonate （MVA） pathway in the cytosol or by the methyl-D-erythritol 4-phosphate （MEP） pathway in plastids. Although some exchange of precursors occurs, cytosolic sesquiterpenes are assumed to derive mainly from MVA, while plastidial monoterpenes are produced preferentially from MEP precursors. Additional complexity arises in the first step of the MEP pathway, which is typically catalyzed by two divergent 1-deoxy-D-xylulose 5-phosphate synthase isoforms （DXS1, DXS2）. In tomato （Solanum lycopersicum）, the SIDXS1 gene is ubiquitously expressed with highest levels during fruit ripening, whereas SIDXS2 transcripts are abundant in only few tissues, including young leaves, petals, and isolated trichomes. Specific down-regulation of SIDXS2 expression was performed by RNA interference in transgenic plants to investigate feedback mechanisms. SIDXS2 down-regulation led to a decrease in the monoterpene β-phellandrene and an increase in two sesquiterpenes in trichomes. Moreover, incorporation of MVA-derived precursors into residual monoterpenes and into sesquiterpenes was elevated as determined by comparison of ^13C to ^12C natural isotope ratios. A compensatory up-regulation of SIDXS1 was not observed. Down-regulated lines also exhibited increased trichome density and showed less damage by leaf-feeding Spodoptera littoralis caterpillars. The results reveal novel, non-redundant roles of DXS2 in modulating isoprenoid metabolism and a pronounced plasticity in isoprenoid precursor allocation.

Effective suppression of fibronectin synthesis by retrovirus delivered shRNA in rat cardiac fibroblasts

Extracellular matrix overexpression is a common final pathway that leads to ventricular remodeling. Fibronectin plays a pivotal role in this progress. In the work presented here, we explored the possibility of direct inhibition of fibronectin synthesis in rat cardiac fibroblasts by small interference RNA (siRNA). We found that siRNA could successfully suppress the fibronectin overexpression stimulated by angiotensin II. To overcome the limitations of plasmid-based siRNA, we subcloned the H1 promoter into pLXIN, a commercially available retroviral vector. We found that H1 promoter worked very well to form the small hairpin RNA (shRNA) on the retroviral vector, and the fibronectin expression was dramatically down regulated by shRNA. We think the retroviral shRNA delivery system that we have constructed may have potential roles in treating ventricular remodeling. Keywords RNA interference (RNAi) - Angtiotensin II (ATII) - extracellular matrix - fibronectin

Silencing suppressors:viral weapons for countering host cell defenses

RNA silencing is a conserved eukaryotic pathway involved in the suppression of gene expression via sequence-specific interactions that are mediated by 21–23 nt RNA molecules.During infection,RNAi can act as an innate immune system to defend against viruses.As a counter-defensive strategy,silencing suppressors are encoded by viruses to inhibit various stages of the silencing process.These suppressors are diverse in sequence and structure and act via different mechanisms.In this review,we discuss whether RNAi is a defensive strategy in mammalian host cells and whether silencing suppressors can be encoded by mammalian viruses.We also review the modes of action proposed for some silencing suppressors.

Transgenic cotton expressing C YP392A4 double-stranded RNA decreases the reproductive ability of Tetranychus cinnabarinus

As a polyphagous pest, Tetranychus cinnabarinus has the ability to overcome the defense of various hosts, and causes severe losses to various economically important crops. Since the interaction between pest and host plants is a valuable clue to investigate potential ways for pest management, we intend to identify the key genes of T. cinnabarinus for its adaption on cotton, then, with RNA interference （RNAi） and transgenic technology, construct a transgenic cotton strain to interfere with this process, and evaluate the effect of this method on the management of the mites. The difference of gene expression of T. cinnabarinus was analyzed when it was transferred to a new host （from cowpea to cotton） through high-throughput sequencing, and a number of differentially expressed genes involved in detoxification, digestion and specific processes during the development were classified. From them, a P450 gene CYP392A4 with high abundance and prominent over-expression on the cotton was selected as a candidate. With transgenic technology, cotton plants expressing double-stranded RNA of CYP392A4 were constructed. Feeding experiments showed that it can decrease the expression of the target gene, result in the reduction of reproductive ability of the mites, and the population of T. cinnabarinus showed an apparent fitness cost on the transgenic cotton. These results provide a new approach to restrict the development of mite population on the host. It is also a useful attempt to control piercing sucking pests through RNAi and transgenic technology.