microRNAs are post-transcriptional regulators of gene expression that recruit RNA silencing complexes to target transcripts to prevent translation and promote their degradation. Experimental studies suggest that micro...microRNAs are post-transcriptional regulators of gene expression that recruit RNA silencing complexes to target transcripts to prevent translation and promote their degradation. Experimental studies suggest that microRNA binding to target transcripts can result in as much as a 90% decrease in gene expression. Because of this feature, the microRNA pathway has been utilized as a vehicle for potent RNA interference (RNAi). In recent years, significant advances have been made in engineering artificial microRNA vectors for RNAi in a number of biological systems, with the most progress in plants but also some success in mouse and human cell lines. In this mini-review, we provide a brief discussion of the potential of this technology in comparison with other RNAi strategies, and the current challenges in the design of microRNA-based RNAi vectors, particularly for animal systems.展开更多
CRABS ClAW （CRC） is a member of the YABBYA transcription factor gene family that plays an important role in floral organ development of plants. This study aimed to further investigate the regulatory function of CRC ...CRABS ClAW （CRC） is a member of the YABBYA transcription factor gene family that plays an important role in floral organ development of plants. This study aimed to further investigate the regulatory function of CRC transcription factor in the development of floral organs of rape （Brassica napus L. ）. A 580 bp fragment of CRC gene was cloned by RT-PCR from total RNA of buds of rape cultivar Ningyou No. 10 to construct an inverted repeated expression cassette of CRC gene using intermediate vector pHturieane. Firstly, CRC gene fragment was positively inserted into the 5＇ end of a spliceable intron and negatively inserted into the 3＇ end of the intron. Subsequently, CaMV35S promoter sequence and inverted repeated expression cassette of CRC gene were transferred into pUC18 multiple clone site of binary expression vector pCAMBIAI1390. The constructed interference expression vector was named pA6-CRCi, which was further confirmed by restriction enzyme digestion and sequencing.展开更多
AIM： To observe the inhibition of hepatitis B virus replication and expression by transfecting vector-based small interference RNA （siRNA） pGenesiI-HBV X targeting HBV X gene region into HepG2.2.15 cells. METHODS：...AIM： To observe the inhibition of hepatitis B virus replication and expression by transfecting vector-based small interference RNA （siRNA） pGenesiI-HBV X targeting HBV X gene region into HepG2.2.15 cells. METHODS：pGenesil-HBV X was constructed and transfected into HepG2.2.15 cells via lipofection. HBV antigen secretion was determined 24, 48, and 72 h after transfection by time-resolved immunofluorometric assays （TRFIA）. HBV replication was examined by fluorescence quantitative PCR, and the expression of cytoplasmic viral proteins was determined by immunohistochemistry. RESULTS： The secretion of HBsAg and HBeAg into the supernatant was found to be inhibited by 28.5% and 32.2% （P 〈 0.01）, and by 38.67% （P 〈 0.05） and 42.86% （P 〈 0.01） at 48 h and 72 h after pGenesil-HBV X transfection, respectively. Immunohistochemical staining for cytoplasmic HBsAg showed a similar decline in HepG2.2.15 cells 48 h after transfection. The number of HBV genomes within culture supernatants was also significantly decreased 48 h and 72 h post-transfection as quantified by fluorescence PCR （P 〈 0.05）. CONCLUSION： In HepG2.2.15 cells, HBV replication and expression is inhibited by vector-based siRNA pGenesil- HBV X targeting the HBV X coding region.展开更多
AIM： To investigate RNA interference targeting signal transducer and activator of transcription-3 （STAT3） on invasion of human pancreatic cancer cells.METHODS： We constructed three plasmids of RNA interference tar...AIM： To investigate RNA interference targeting signal transducer and activator of transcription-3 （STAT3） on invasion of human pancreatic cancer cells.METHODS： We constructed three plasmids of RNA interference targeting the STAT3 gene. After LV （lentivirus）-STAT3siRNA （STAT3 small interfering RNA） the vector was transfected into the human pancreatic cell line, SW1990 and cell proliferation was measured by the MTT assay. Flow cytometry was used to assess cell cycle. Vascular endothelial growth favor （VEGF） and matrix metalloproteinase-2 （MMP-2） mRNA and protein expression were examined by quantitative PCR and western blotting, respectively. The invasion ability of SW1990 cells was determined by cell invasion assay.RESULTS： We successfully constructed the LVSTAT3siRNA lentivirus vector and proved that it can suppress expression of STAT3 gene in SW1990 cells. RNA interference of STAT3 by the LV-STAT3siRNA construct significantly inhibited the growth of SW1990 cells, in addition to significantly decreasing both VEGF and MMP-2 mRNA and protein expression. Moreover, suppression of STAT3 by LV-STAT3siRNA decreased the invasion ability of SW1990 cells.CONCLUSION： The STAT3 signaling pathway may provide a novel therapeutic target for the treatment of pancreatic cancer since it inhibits the invasion ability of pancreatic cancer cells.展开更多
Primary liver cancer is the fifth most common malignan- cy in the world and is a leading cause of cancer-related mortality.Available treatment for hepatocellular carcino- ma(HCC),the commonest primary liver cancer,is ...Primary liver cancer is the fifth most common malignan- cy in the world and is a leading cause of cancer-related mortality.Available treatment for hepatocellular carcino- ma(HCC),the commonest primary liver cancer,is rarely curative and there is a need to develop therapy that is more effective.Specific and powerful gene silencing that can be achieved by activating RNA interference(RNAi) has generated enthusiasm for exploiting this pathway for HCC therapy.Many studies have been carried out with the aim of silencing HCC-related cellular oncogenes or the hepatocarcinogenic hepatitis B virus(HBV)and hepatitis C virus(HCV).Proof of principle studies have demonstrated promising results,and an early clinical trial assessing RNAi-based HBV therapy is currently in progress.Although the data augur well,there are several significant hurdles that need to be overcome before the goal of RNAi-based therapy for HCC is realized.Particu- larly important are the efficient and safe delivery of RNAi effecters to target malignant tissue and the limitation of unintended harmful non-specific effects.展开更多
Interleukin (IL)-8 is a potent neutrophil chemotactic factor and a crucial mediator in neutrophil-dependent inflammation.Various cell types produce IL-8, either in response to external stimuli such as cytokines or bac...Interleukin (IL)-8 is a potent neutrophil chemotactic factor and a crucial mediator in neutrophil-dependent inflammation.Various cell types produce IL-8, either in response to external stimuli such as cytokines or bacterial infection, or aftermalignant transformation. Anti-IL-8 strategies have been considered for anti-inflammatory therapy. In this paper wedemonstrate that the RNA interference technique can be used to efficiently down-regulate IL-8 protein expression inairway epithelial cells. We used a helper-dependent adenoviral vector to express a small hairpin (sh)RNA targetinghuman IL-8 in cultured airway epithelial cells (IB3-1, Cftr-/-; C38, Cftr-corrected) stimulated with TNF-α, IL-1β orheat-inactivated Burkholderia cenocepacia. Stimulated IL-8 expression in IB3-1 and C38 cells was significantly reducedby shRNA expression. The shRNA targeting IL-8 had no effect on the activation of NF-κB, or on the protein levels ofIκB or IL-6, suggesting that this anti-IL-8 strategy was highly specific, and therefore may offer potential for thetreatment of inflammatory diseases.展开更多
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) replica...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.展开更多
Colorado potato beetle(CPB),Leptinotarsa decemlineata,is a notorious destructive pest that mainly feeds on the leaves of potato and several other solanaceous plants.CPB is widely recognized for its adaptation to a rem...Colorado potato beetle(CPB),Leptinotarsa decemlineata,is a notorious destructive pest that mainly feeds on the leaves of potato and several other solanaceous plants.CPB is widely recognized for its adaptation to a remarkable variety of host plants and diverse climates,and its high resistance to insecticides and Bacillus thuringiensis toxins.RNA interference(RNAi)is a sequence-specific,endogenous gene silencing mechanism evoked by small RNA molecules that is used as a robust tool for virus and pest control.RNAi has been extensively tested for CPB management by employing various target genes and delivery methods.This article reviews the screening of RNAi target genes,efficient RNAi delivery systems,and factors affecting RNAi efficiency in CPB,which may help understand the mechanisms of RNAi and its application in CPB control strategy.展开更多
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...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.展开更多
Objective：To construct and identify the recombinant plasmids PPARγ-pSUPER-EGFP for RNA interference. Methods： The pSUPER-EGFP vectors were used to transcribe functional short interfering RNA （siRNA）. Four pairs o...Objective：To construct and identify the recombinant plasmids PPARγ-pSUPER-EGFP for RNA interference. Methods： The pSUPER-EGFP vectors were used to transcribe functional short interfering RNA （siRNA）. Four pairs of 64 nt PPARγ siRNA encoding sequences were inserted into the downstream of the H1 promoter. The recombinant plasmids were confirmed by double digestion with the enzymes and sequencing. Western blotting was used to examine the silencing effect of PPARγ gene in RAW264.7 cells. Following procedures were used to optimize the experiments： the oligonucleotides were incubated 5 min at 95 C and cooled automatically in boiled water bath to anneal, and then phosphorylated oligonucleotides, pSUPER-EGFP plasmids was digested with Bgl Ⅱ and Hind Ⅲ , and the product was ligated into digested pSUPER-EGFP plasmids, and transforming the ligation products followed by screening and identifying positive clones. Results ：Four kinds of positive clones producing 285 bp fragments were selected. Sequencing further proved their correctness. Four recombinant plasmids containing corresponding PPARγ gene-specific target sequences induced the silencing of its target gene more or less. Conclusion： The optimizing method in constructing these recombinant plasmids serves other plasmid-based RNA interference research. The final plasmids PPARγ-pSUPER-EGFP established the basis for research on the function of PPARγ gene.展开更多
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 sh...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.展开更多
文摘microRNAs are post-transcriptional regulators of gene expression that recruit RNA silencing complexes to target transcripts to prevent translation and promote their degradation. Experimental studies suggest that microRNA binding to target transcripts can result in as much as a 90% decrease in gene expression. Because of this feature, the microRNA pathway has been utilized as a vehicle for potent RNA interference (RNAi). In recent years, significant advances have been made in engineering artificial microRNA vectors for RNAi in a number of biological systems, with the most progress in plants but also some success in mouse and human cell lines. In this mini-review, we provide a brief discussion of the potential of this technology in comparison with other RNAi strategies, and the current challenges in the design of microRNA-based RNAi vectors, particularly for animal systems.
基金Supported by National Natural Science Foundation of China(31571710)National 948 Program of China(2011-G23)
文摘CRABS ClAW （CRC） is a member of the YABBYA transcription factor gene family that plays an important role in floral organ development of plants. This study aimed to further investigate the regulatory function of CRC transcription factor in the development of floral organs of rape （Brassica napus L. ）. A 580 bp fragment of CRC gene was cloned by RT-PCR from total RNA of buds of rape cultivar Ningyou No. 10 to construct an inverted repeated expression cassette of CRC gene using intermediate vector pHturieane. Firstly, CRC gene fragment was positively inserted into the 5＇ end of a spliceable intron and negatively inserted into the 3＇ end of the intron. Subsequently, CaMV35S promoter sequence and inverted repeated expression cassette of CRC gene were transferred into pUC18 multiple clone site of binary expression vector pCAMBIAI1390. The constructed interference expression vector was named pA6-CRCi, which was further confirmed by restriction enzyme digestion and sequencing.
基金Supported by Natural Science Foundation of Shanxi Province, China, No.20051114
文摘AIM： To observe the inhibition of hepatitis B virus replication and expression by transfecting vector-based small interference RNA （siRNA） pGenesiI-HBV X targeting HBV X gene region into HepG2.2.15 cells. METHODS：pGenesil-HBV X was constructed and transfected into HepG2.2.15 cells via lipofection. HBV antigen secretion was determined 24, 48, and 72 h after transfection by time-resolved immunofluorometric assays （TRFIA）. HBV replication was examined by fluorescence quantitative PCR, and the expression of cytoplasmic viral proteins was determined by immunohistochemistry. RESULTS： The secretion of HBsAg and HBeAg into the supernatant was found to be inhibited by 28.5% and 32.2% （P 〈 0.01）, and by 38.67% （P 〈 0.05） and 42.86% （P 〈 0.01） at 48 h and 72 h after pGenesil-HBV X transfection, respectively. Immunohistochemical staining for cytoplasmic HBsAg showed a similar decline in HepG2.2.15 cells 48 h after transfection. The number of HBV genomes within culture supernatants was also significantly decreased 48 h and 72 h post-transfection as quantified by fluorescence PCR （P 〈 0.05）. CONCLUSION： In HepG2.2.15 cells, HBV replication and expression is inhibited by vector-based siRNA pGenesil- HBV X targeting the HBV X coding region.
基金Supported by The Affiliated First People’s Hospital, Shanghai Jiao Tong University and the Board of Education Fund for Scientific Research of Shanghai, China, No. 06BE067
文摘AIM： To investigate RNA interference targeting signal transducer and activator of transcription-3 （STAT3） on invasion of human pancreatic cancer cells.METHODS： We constructed three plasmids of RNA interference targeting the STAT3 gene. After LV （lentivirus）-STAT3siRNA （STAT3 small interfering RNA） the vector was transfected into the human pancreatic cell line, SW1990 and cell proliferation was measured by the MTT assay. Flow cytometry was used to assess cell cycle. Vascular endothelial growth favor （VEGF） and matrix metalloproteinase-2 （MMP-2） mRNA and protein expression were examined by quantitative PCR and western blotting, respectively. The invasion ability of SW1990 cells was determined by cell invasion assay.RESULTS： We successfully constructed the LVSTAT3siRNA lentivirus vector and proved that it can suppress expression of STAT3 gene in SW1990 cells. RNA interference of STAT3 by the LV-STAT3siRNA construct significantly inhibited the growth of SW1990 cells, in addition to significantly decreasing both VEGF and MMP-2 mRNA and protein expression. Moreover, suppression of STAT3 by LV-STAT3siRNA decreased the invasion ability of SW1990 cells.CONCLUSION： The STAT3 signaling pathway may provide a novel therapeutic target for the treatment of pancreatic cancer since it inhibits the invasion ability of pancreatic cancer cells.
基金The Cancer Association of South Africa,theSixth Research Framework Programme of the European UnionProject RIGHT,LSHB-CT-2004-005276South African NationalResearch Foundation and Poliomyelitis Research Foundationsupport research
文摘Primary liver cancer is the fifth most common malignan- cy in the world and is a leading cause of cancer-related mortality.Available treatment for hepatocellular carcino- ma(HCC),the commonest primary liver cancer,is rarely curative and there is a need to develop therapy that is more effective.Specific and powerful gene silencing that can be achieved by activating RNA interference(RNAi) has generated enthusiasm for exploiting this pathway for HCC therapy.Many studies have been carried out with the aim of silencing HCC-related cellular oncogenes or the hepatocarcinogenic hepatitis B virus(HBV)and hepatitis C virus(HCV).Proof of principle studies have demonstrated promising results,and an early clinical trial assessing RNAi-based HBV therapy is currently in progress.Although the data augur well,there are several significant hurdles that need to be overcome before the goal of RNAi-based therapy for HCC is realized.Particu- larly important are the efficient and safe delivery of RNAi effecters to target malignant tissue and the limitation of unintended harmful non-specific effects.
文摘Interleukin (IL)-8 is a potent neutrophil chemotactic factor and a crucial mediator in neutrophil-dependent inflammation.Various cell types produce IL-8, either in response to external stimuli such as cytokines or bacterial infection, or aftermalignant transformation. Anti-IL-8 strategies have been considered for anti-inflammatory therapy. In this paper wedemonstrate that the RNA interference technique can be used to efficiently down-regulate IL-8 protein expression inairway epithelial cells. We used a helper-dependent adenoviral vector to express a small hairpin (sh)RNA targetinghuman IL-8 in cultured airway epithelial cells (IB3-1, Cftr-/-; C38, Cftr-corrected) stimulated with TNF-α, IL-1β orheat-inactivated Burkholderia cenocepacia. Stimulated IL-8 expression in IB3-1 and C38 cells was significantly reducedby shRNA expression. The shRNA targeting IL-8 had no effect on the activation of NF-κB, or on the protein levels ofIκB or IL-6, suggesting that this anti-IL-8 strategy was highly specific, and therefore may offer potential for thetreatment of inflammatory diseases.
基金The Shanghai committee of Science and Technology(Grant No.10PJ1404800)the National Natural Science Foundation of China(Grant No.31072244)
文摘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.
基金funded by the National Natural Science Foundation of China (31572071)
文摘Colorado potato beetle(CPB),Leptinotarsa decemlineata,is a notorious destructive pest that mainly feeds on the leaves of potato and several other solanaceous plants.CPB is widely recognized for its adaptation to a remarkable variety of host plants and diverse climates,and its high resistance to insecticides and Bacillus thuringiensis toxins.RNA interference(RNAi)is a sequence-specific,endogenous gene silencing mechanism evoked by small RNA molecules that is used as a robust tool for virus and pest control.RNAi has been extensively tested for CPB management by employing various target genes and delivery methods.This article reviews the screening of RNAi target genes,efficient RNAi delivery systems,and factors affecting RNAi efficiency in CPB,which may help understand the mechanisms of RNAi and its application in CPB control strategy.
基金This work was supported by the NationalPostdoctoral Science Foundation of China(No.2003034300)
文摘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.
基金Supported by Open Fund of State Key Laboratory of Trauma, Burns and Combined Injury of Third Military Medical University and the National Funds for Outstanding Youth Scientists (30325040)
文摘Objective：To construct and identify the recombinant plasmids PPARγ-pSUPER-EGFP for RNA interference. Methods： The pSUPER-EGFP vectors were used to transcribe functional short interfering RNA （siRNA）. Four pairs of 64 nt PPARγ siRNA encoding sequences were inserted into the downstream of the H1 promoter. The recombinant plasmids were confirmed by double digestion with the enzymes and sequencing. Western blotting was used to examine the silencing effect of PPARγ gene in RAW264.7 cells. Following procedures were used to optimize the experiments： the oligonucleotides were incubated 5 min at 95 C and cooled automatically in boiled water bath to anneal, and then phosphorylated oligonucleotides, pSUPER-EGFP plasmids was digested with Bgl Ⅱ and Hind Ⅲ , and the product was ligated into digested pSUPER-EGFP plasmids, and transforming the ligation products followed by screening and identifying positive clones. Results ：Four kinds of positive clones producing 285 bp fragments were selected. Sequencing further proved their correctness. Four recombinant plasmids containing corresponding PPARγ gene-specific target sequences induced the silencing of its target gene more or less. Conclusion： The optimizing method in constructing these recombinant plasmids serves other plasmid-based RNA interference research. The final plasmids PPARγ-pSUPER-EGFP established the basis for research on the function of PPARγ gene.
基金Supported by a grant from the Natural Science Foundation of Shanghai （No. 03ZR14004）.
文摘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.
