Specific inhibition of gene expression of lung resistance-related protein by short interfering RNA.
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To investigate inhibitory effect of short interfering RNA (siRNA) on the expression of lung resistance-related protein (LRP) in leukemia cells.The eukaryotic vectors of LRP, pcDNA3.0/LRP, were constructed. The transfection protocol of K562 cells grown in standard conditions consisted of different combinations of pcDNA3.0/LRP, pEGFP-C1 expressing mammalian enhanced green fluorescent protein (GFP), and their gene-specific siRNAs. RT-PCR and flow cytometry were employed to evaluate the mRNA and protein expression of LRP and fluoroscopy was performed for assay of GFP expression in the transfected cells.Compared with untreated K562 cells, pcDNA3.0/LRP-transfected cells showed increased LRP mRNA and protein expression and the positive cell percentage reached 30%. In the cells co-transfected with LRP gene-specific siRNA and pcDNA3.0/LRP, both LRP mRNA and protein expression decreased significantly to a level defined as negative results; the GFP expression showed no significant difference between the cells transfected with pEGFP-C1 and those co-transfected with LRP gene-specific siRNA and pEGFP-C1. LRP mRNA and protein expressions were also similar between the cells transfected with pcDNA3.0/LRP and those co-transfected with GFP gene-specific siRNA and pcDNA3.0/LRP.The LRP gene-specific siRNA we designed is capable of degrading LRP mRNA and inhibiting the protein expression effectively and specifically, which shed light on the potential application of siRNA for gene-specific therapy to reverse LRP-induced multidrug resistance of leukemia cells.Keywords:
K562 cells
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To Knockdown Homeobox A5 (HOXA5) expression by HOXA5-specific siRNA and evaluate the effects on Livin and Smac proteins expression in acute T cell leukemia Jurkat cells.We designed and constructed HOXA5-specific siRNA, and using liposomes we transfected Jurkat cells with this siRNA. The experiment was designed for three groups: (i) experimental group with Jurkat cells transfected with HOXA5-specific siRNA (siRNA transfection group), (ii) negative control group (irrelevant siRNA transfection) with Jurkat cells transfected with pRNAT-U6.1-siD and (iii) normal control group (untransfected Jurkat cells, only with equivalent amounts of cells and medium). We used FQ-PCR and Western blot to detect the relative expression levels of HOXA5 mRNA and protein in each group separately. The Western blot was also used to detect Livin and Smac protein levels in Jurkat cells.Expression levels of HOXA5 mRNA and protein were significantly reduced in the group with Jurkat cells transfected with HOXA5 siRNA (p<0.05). The expression of Livin protein was significantly down-regulated (p<0.05) while the expression of Smac protein was significantly up-regulated (p <0.05).HOXA5-specific siRNA effectively silenced the HOXA5 gene expression and down-regulation of HOXA5 induced the down-regulation of Livin protein expression and up-regulation of Smac protein. We suggest the HOXA5 gene to be considered as the new target for acute leukemia gene therapy.
Jurkat cells
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To construct an expression vector of siRNA targeting human MTA1 gene and observe its gene-silencing effect in esophageal carcinoma cells.The siRNA sequences targeting MTA1 gene were designed and synthesized with two complementary oligonucleotide strands. The oligonucleotide strands were annealed and recombined into pRNAT-U6.2 vector, which was identified by sequencing following transformation and amplification. The siRNA expression vector pRNAT-U6.2-MTA1 was transfected into human esophageal carcinoma EC9706 cells via liposome. RT-PCR and Western blotting were used to detect expression levels of MTA1 mRNA and protein in the transfected EC9706 cells, respectively.The double-stranded oligonucleotide fragments of the siRNA targeting MTA1 gene were cloned into pRNAT-U6.2 vector, which was validated by sequence analysis. RT-PCR and Western blotting indicated that MTA1 mRNA and protein expressions were significantly decreased in the transfected cells, especially in those transfected with the siRNA targeting the sequence of GACCCTGCTGGCAGATAAA (481-499), which induced almost complete silencing of MTA1 protein expression.The siRNA expression vector pRNAT-U6.2-MTA1 for silencing MTA1 gene expression in the esophageal carcinoma cells has been successfully constructed, which may facilitate further study for decreasing the invasive and metastatic potentials of malignant tumors by MTA1 gene silencing.
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The siRNA off-target effects is a great concern in the applications of RNA interference.To investigate specificity of siRNAs,siRNA eukaryotic expression plasmid p-Mat01-1 targeting to c-myc oncogene and its mismatch plasmid p-Mis09-1 were constructed,and stably transfected into MCF-7 human breast cancer cells.Proteomic analyses for the off-target effects on the plasmid-mediated c-myc siRNA were performed using a pEGFP-C1 plasmid as the control.The RT-PCR and Western blot results showed that the c-MYC expressions were decreased in the p-Mat01-1 transfected MCF-7 cell pool.2-DE and LC-ESI-MS/MS analyses on p-Mat01-1 and pEGFP-C1 transfected stable clones revealed 47(about 11.1%) non-target proteins of the total 423 up-or down-regulated spots,including proteins of cytoskeleton,metabolism,proliferation,signal transduction,molecular chaperones and detoxification/redox proteins.The results showed that plasmid-mediated c-myc siRNA resulted in a substantial off-target effect in MCF-7 cells.Our findings suggested that nonspecific effects on siRNA inhibition of mammalian protein expressions should be considered in the siRNA designs and the experimental applications.
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【Objective】 Observing the effect of RNA interference(RNAi) technique to suppress the expression of G250 gene in G250 positive renal cell carcinoma cell line 786-0,in order to establish stable RNAi technique and use this technique for further gene research.【Methods】 Four small fragments of siRNA against the G250 gene and one negative control were sequenced and cloned into vector pRNAT-U6.1/Neo,The recombinant plasmids were then transfected into renal cell carcinoma cell line 786-0 cells respectively,by positive liposomal transfection assay.G250 mRNA expression was measured using real-time fluorescence quantitative RT-PCR,G250 protein expression was analyzed by Western-blot.【Results】 DNA sequencing and enzyme digestion confirmed that the siRNA expression vectors targeting G250 gene were successfully constructed.35.56%,49.27%,45.88% and 65.13% silencing of G250 mRNA expression were observed respectively.The corresponding decrease of G250 protein expression was confirmed.【Conclusions】 These results demonstrated that siRNA against G250 gene could effectively down regulate G250 gene expression in 786-0 cell line,it can be used for next experiment.
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Objective:To observe the apoptotic changes in liver cancer cell HepG-2 transfected by siRNA of inhibitory member of the ASPP family(iASPP) gene.Methods:The specific small interfere RNA(siRNA) sequence was designed according to the gene bank,and then the obtained siRNA sequence was cloned into PGCsilencerTM H1/Neo/GFP plasmid.After the recombinant plasmid was transfected into HepG-2 by using LipofectamineTM 2000,the mRNA transcription and protein expression of iASPP gene was analyzed by RT-PCR and Western blot,respectively,and then the cell apoptosis was detected by flow cytometry.Results:Both the mRNA transcription and protein expression of iASPP gene in HepG-2 cell lines decreased,but the apoptosis rate increased after the siRNA transfection.Conclusion:Inhibition of endogenous iASPP may restore the cancer suppressor function of p53 in HepG-2.
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Survivin
HeLa
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RNA interfering (RNAi) is a process of sequence-specific, posttranscriptional gene silencing in animals and plants. In mammalian cells, duplexes of 19～25 nts (nucleotides) RNAs efficiently inhibit gene expression. The pBS/H1SP vector expressing siRNA is used which inhibit specific HIV-1 gene expression. To assess the intracellular effect of this H1 promoter-driven siRNA, a reporter plasmid pEGFP-C1-HIV protein which expresses fusion protein of enhanced green fluorescent protein (EGFP) and HIV protein was used. The expression of the reporters can be easily visualized by fluorescence microscopy in living cells. siRNA-generating vectors targeted to several HIV-1 genomes were constructed and then co-transfected with respond reporter expression vectors into HEK293 cells. Cells transfected with pHIV-siRNA exhibited a significant inhibition of pEGFP-HIV expression compared with cells transfected with control vectors. By this way, it is successfully to select effective siRNA for silencing target HIV-1 genes. Then two or three siRNA transcripts targeted to different HIV genes were expressed by one plasmid, and a relative strong inhibition effect was observed.
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Activation of hTERT, the human telomerase catalytic subunit, has been implicated as the critical event in triggering telomerase activity of cancer cells. In present research, we investigated whether RNA interference (RNAi) induced by small interference RNA (siRNA) could suppress human telomerase catalytic unit (hTERT) gene expression in gastric SGC7901 cells.As a pilot study, we utilized green fluorescent protein (GFP) plasmid pCX-GFP (5 510 bp) as a reporter system and generated constructs SHi-pU6-GFP expressing small hairpin RNA (shRNA) specific for green fluorescence protein (GFP) in K562 and SGC7901 cell respectively. Furthermore, we constructed pU6-hTERT-siRNAs carried hairpin siRNA for hTERT gene and transfected in SGC7901 by using Lipofectamine trade mark 2000. The expression of hTERT gene was detected by reverse transcription polymerase chain reaction (RT-PCR) and fluorescence quantitative polymerase chain reaction (FQ-PCR) assay.Our pilot study showed the short hairpin RNA (shRNA) expression vector driven by the murine U6 small nuclear RNA promoter can specifically induce potent gene knockdown effect (i.e., inhibit GFP expression specifically) when transfected transiently into SGC7901 cell. The constructed pU6-hTERT-siRNAs carried hairpin siRNA for hTERT gene was proved to be the same as designed by restriction endonuclease analysis. pU6-hTERT-siRNAs were successfully transferred into SGC7901 cell and their stable expression were obtained. The expression of hTERT gene were specific inhibited by pU6-hTERT-siRNAs in SGC7901 cell.Short hairpin RNAs (shRNAs) could induce sequence-specific hTERT gene silencing in SGC7901 cell. Our results prove the feasibility of the U6 promoter-driven shRNA expression technique to be used to cancer gene therapy.
Lipofectamine
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AIM: To construct RNAi plasmids and synthesize siRNA in vitro, transport them into Hep-3B cells, so as to find some effective VEGF RNAi plasmids and siRNA segments. METHODS: The RNAi plasmids pcDUVEGF-1, pcDUVEGF-2 and siRNA segments TWvegf-1, TWvegf-2 were transported into Hep-3B cells, and the expressions of VEGF mRNA and protein in the transfected Hep-3B cells were checked using RT-PCR, Wes-tern Blot and ELISA. The results were compared between the transfected and the normal Hep-3B cells. RESULTS: There were prominent differences between the two groups in depressing the expression of mRNA (0.28, 0.47, 0.76, 0.58 vs 1.2, P0.01) and protein (138, 121, 249, 227 vs 389 μg/L, P0.01) of VEGF gene. CONCLUSION: The two RNAi plasmids and siRNA segments inhibit the expression of VEGF gene effectively.
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ABSTRACT RNA interference (RNAi) is a cellular process of post-transcriptional gene silencing in which a short interfering dsRNA (siRNA, 21–23 nt) targets a homologous mRNA for degradation by ribonuclease. RNAi has been used successfully to inhibit targeted gene expression and viral replication in mammalian cells. In this study we established an RNAi transfection protocol for primary porcine alveolar macrophages and evaluated potential off-target effects of siRNA introduction into these cells. Porcine alveolar macrophages were transfected using a fluorescence-labeled siRNA to compare transfection reagents from different suppliers. Under optimized transfection conditions, up to 95% of macrophages were fluorescent at 12 and 24 h post-transfection using an amine-based transfection reagent. An siRNA targeting GAPDH suppressed macrophage endogenous GAPDH transcript levels as much as 60% through 24 h. Further, we did not detect a significant interferon response following siRNA transfection. These data suggest that RNAi will be an efficient and convenient approach for studying loss of gene function in primary porcine alveolar macrophages.
RNA Silencing
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