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Showing 4 results for Sirna
S. Mohebbi , M. Behmanesh , M. Nikkhah , T. Tohidi Moghadam ,
Volume 9, Issue 1 (1-2018)
Abstract
Aims: HIF-1 transcription factor is a key determinant of oxygen-dependent gene regulation, which its role has been demonstrated for the survival and progress of cancer tumors. The effect of suppression of HIF-1α on the evaluation of HIF-1 dependent processes and interference with pathophysiological events caused by hypoxia is important. The aim of this study was the apoptosis induction in glioma cells by downregulation of Hif-1α gene.
Materials and Methods: In this experimental study, a specific siRNA against the HIF1α gene was developed using OligoWalk and Mit (siRNA.wi.mit.edu) servers and the online design department of Invivogene and Qiagene companies and the efficacy of its silencing in the U87 glioma cell line was quantitatively investigated by the Real-time PCR technique. In order to find out the effect of reduction of expression in the process of cell cycle and apoptosis, staining with PI and Annexin-PI was performed and the number of cells in each phase and the rate of cell mortality with control were compared by flow cytometry.
Findings: The designed HIF-1a-siRNA was able to reduce HIF1α expression by 40%. The treatment of U87 cells after 24 hours increased the cells by 6% and after 48 hours, increased them by 12% in the sub G1 stage. Confirming the cell cycle changes, 48-hour treatment induced apoptosis in 58% of cells; regarding the 1.5% rate of apoptosis in the control cells, this cell death rate was very significant and showed the ability of the designed siRNA to induce apoptosis.
Conclusion: The apoptosis induction of specific siRNA designed against HIF1α gene has a significant effect on the reduction of HIF-1α gene expression, cell growth, and apoptosis.
Volume 11, Issue 0 (10-2009)
Abstract
Objective: CREB1 is an important downstream protein for many signaling pathways. By designing efficient siRNAs against CREB1, it may be possible to assess the role of molecules involved in signaling pathways in different cell types. In this research the efficiency of CREB1 knockdown by two different siRNAs in K562 cells has been studied.
Materials and Methods: siRNAs have been designed according to the criteria suggested by Reynolds et al. K562 cells were transfected by siRNA using Lipofectamine 2000. The efficiency of CREB knockdown has been assessed by quantitative relative Real-time PCR.
Results: Our results have shown that only one of the siRNAs has a high level of inhibitory effect on CREB1 gene expression. The expression of CREB1 by this siRNA was knocked-down by 87% in K562 cells.
Conclusion: In this research, although two siRNAs were designed according to the Reynolds et al. criteria, only one showed an inhibitory effect. Reasons other than the aforementioned criteria may be involved in effectiveness of siRNAs.
Volume 14, Issue 1 (1-2011)
Abstract
Objective: Nowadays, cord blood Hematopoietic stem cells (HSCs) are known as a valuable source for bone marrow transplantation but unfortunately their insufficient number is a limiting factor for using them in adult bone marrow transplantation. Cord blood HCSs expansion is an approach to overcome this problem, by inducing their self-renewal. TGF-b signaling pathway is a key inhibitory agent for HSCs self-renewal. In this study, we tried to enhance self-renewal of long term culture initiating cell by inhibiting TGFbR2 expression.
Materials and Methods: CD34+ HSCs were isolated from cord blood units with MACS column. SiRNA against TGFbR2 was transfected by Lipofectamine™ RNAiMAX as transfection reagent. HSCs were cultured in IMDM medium containing 10% FBS and early acting cytokines (Flt3L, SCF, Tpo) for 8 days. Then we evaluated TGFbR2 expression by QRT-PCR. The CD34+ subpopulation of cultured cells were examined by flow cytometry on the 8th day. Finally the expanded cells were evaluated for the presence of early hematopoietic stem cells by LT-CIC and clonogenic assays.
Results: According to our results, TGFbR2 down regulation increases CD34+ subpopulation of HSCs. In addition, LT-CIC assay showed an enhancement in primitive hematopoietic stem cell capable of self-renewal.
Conclusion: All in all, it seems that positive regulators have attracted more attention in the field of HSCs expansion while negative regulators have same importance in self-renewal process of HSCs and their inhibition can be a beneficial tool for enhancement of HSCs self-renewa.
Volume 23, Issue 2 (3-2020)
Abstract
Aims: Despite the efficacy of current therapies against HIV-1 infection, these methods are not a permanent treatment because they cannot prevent the return of viremia from latent cell reservoirs. On the other hand, the virus may become resistant to these drugs. Therefore, providing safer and more effective therapeutic strategies, such as inhibition of genes by siRNA, is essential. The successful therapeutic application of siRNAs requires an efficient delivery system to target cells.
Materials & Methods: In this study, a specific siRNA was designed against the HIV-1 nef gene. Then a stable HEK293 cell line expressing HIV-1 nef was developed and after fabrication and evaluation of superparamagnetic iron oxide nanoparticles (SPION) coated with trimethyl chitosan, the efficiency of nanoparticles for delivering siRNA into the cells and inhibition of nef gene was investigated.
Findings: Iron oxide nanoparticles (spherical-shaped with an average size of 85nm and the average zeta potential of +29mV) were significantly effective in transporting siRNA into HEK293 cells compared to control groups and at the same time had low toxicity to the cells. In addition, SPION-TMC containing anti-nef siRNA inhibited about 85% of the expression of this gene in stable cells (compared to control cells).
Conclusion: The optimized SPION-TMC nanocarriers can be used as a promising approach in HIV-1 infection therapy. However, pre-clinical in vivo evaluation of the drug/siRNA delivery system efficiency remains to be conducted.
