A-kinase anchoring protein 13 interacts with the vitamin D receptor to alter vitamin D-dependent gene activation in uterine leiomyoma cells
3
Citation
69
Reference
10
Related Paper
Citation Trend
Abstract:
To determine if A-kinase anchoring protein 13 (AKAP13) interacts with the vitamin D receptor (VDR) to alter vitamin D-dependent signaling in fibroid cells. Uterine leiomyomas (fibroids) are characterized by a fibrotic extracellular matrix and are associated with vitamin D deficiency. Treatment with vitamin D (1,25-dihydroxyvitamin D3) reduces fibroid growth and extracellular matrix gene expression. A-kinase anchoring protein 13 is overexpressed in fibroids and interacts with nuclear hormone receptors, but it is not known whether AKAP13 may interact with the VDR to affect vitamin D signaling in fibroids.Laboratory studies.Translational science laboratory.Human immortalized fibroid or myometrial cells were treated with 1,25-hydroxyvitamin D3 (1,25(OH)2D3) and transfected using expression constructs for AKAP13 or AKAP13 mutants, RhoQL, C3 transferase, or small interfering ribonucleic acids (RNAs).Messenger ribonucleic acid (mRNA) levels of AKAP13, fibromodulin, and versican as measured by quantitative real-time polymerase chain reaction. Glutathione S-transferase-binding assays. Vitamin D-dependent gene activation as measured by luciferase assays.1,25(OH)2D3 resulted in a significant reduction in mRNA levels encoding AKAP13, versican, and fibromodulin. Small interfering RNA silencing of AKAP13 decreased both fibromodulin and versican mRNA levels. Glutathione S-transferase-binding assays revealed that AKAP13 bound to the VDR through its nuclear receptor interacting region. Cotransfection of AKAP13 and VDR significantly reduced vitamin D-dependent gene activation. RhoA pathway inhibition partially relieved repression of vitamin D-dependent gene activation by AKAP13.These data suggest that AKAP13 inhibited the vitamin D receptor activation by a mechanism that required, at least in part, RhoA activation.Keywords:
Versican
Objective To construct and screen neurite outgrowth inhibitory 66-samll interfering RNA(nogo66-siRNA)eukaryotic expression vectors of effective interference,so as to lay a foundation for further reconstruction of related viral vector.Methods The nogo66-siRNA fragments were designed and cloned into pGenesil-1.1,4 plasmids of pGenesil-nogo66-siRNA-1,pGenesil-nogo66-siRNA-2,pGenesil-nogo66-siRNA-hk,and pGenesil-nogo66-siRNA-kb were obtained,sequenced and identified,then were transfected into C6 cell line.The transfection efficiency was measured by fluorescence microscope.RT-PCR and Western blot were used to detect the expression of nogo gene and select the plasmid of effective interference.Results DNA sequencing results showed interference sequences were correct.The bands of 800 bp and 4.3 kb were detected when pGenesil-nogo66-siRNAs were digested by Kpn I/Xho I.The expression of green fluorescent protein could be detected under fluorescence microscope,and the transfection efficiency was about 73%.RT-PCR and Western blot results showed that compared to non-transfected cells,the transfection of pGenesil-nogo66-siRNA-1 made the expression of nogo gene decline 22% and the expression of nogo protein decline 73%;the transfection of pGenesil-nogo66-siRNA-2 made the expression of nogo gene decline 28%and the expression of nogo protein decline 78%;the differences were significant(P0.05);and the transfection of pGenesil-nogo66-siRNA-hk and pGenesil-nogo66-siRNA-kb did not make the expressions of nogo gene and nogo protein decrease significantly(P0.05).Conclusion Nogo66-siRNA eukaryotic expression vector is successfully constructed,it lays an experimental foundation for repair of spinal cord injury.
Cite
Citations (0)
Objective To investigate the suppressive effects of siRNA on the BCSG-1 gene expression in breast cancer MCF-7 cells. Methods The four-pair designed and chemically synthesized in vitro of small interfering RNA (siRNA) sequences ( BCSG-1-siRNA-1 ,-2,-3 and 4) for BCSG-1, and one pair of non-specific siRNA sequence were transducted into plasmid vectors, following by transfection into into MCF-7 cells. Blank control group (empty vector transfection) and normal control group (no transfection) were set up. After 48 h, the mRNA and protein expression levels of BCSG-1 in transfected cells were detected by using reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry respectively. Results The expression of BCSG1 protein was 4.27 ±0. 12, 4.19 ±0.22, 4.15 ± 0.14,4.17 ±0.13, 7.92 ±0.22, 8.02 ±0.13, 8.02 ±0.13, and that of BCSG1 mRNA was 0.624 ±0.010,0.626±0.013, 0.634 ±0.008, 0.631 ±0.010, 0.976±0.076, 0.983 ±0.052, 1.014±0.034 respectively. As compared with control groups, there was significant difference in the expression of BCSG-1 mRNA and protein among the above groups (P ＜0.01 ). The expression level of BCSG-1 mRNA and protein in the groups of MFC-1 cells transfected with BCSG-1-siRNA was lower than that in control groups.Conclusion Expression of BCSG-1 gene can be effectively inhibited by siRNA in MCF7 cells.
Key words:
BCSG1; RNA-interference; Transfection; Inhibition
MCF-7
Cite
Citations (0)
Objective To evaluate the effects of small interfering RNA(siRNA) targeting alpha-globin gene on the expression level of alpha-globin chain mRNA in erythroid cells cultured in vitro.Methods siRNA for alpha-globin gene was designed and chemical synthesized,and transferred into the normal adult donor originated erythroid cells cultured in vitro with liposome-induced gene transfection.After transfection,at the time of 24,48,72 h,the transfection efficiencies were inspected by flow cytometry,the level of mRNA in alpha-globin gene was detected by real-time quantitation PCR.Results Liposomal could effectively transfect the FCM-labeled siRNA into erythroid cells cultured in vitro.The transfection efficiencies were 61.2%,44.3%,33.7% at 24,48,72 h respectively.Results of RT-PCR indicated siRNA could down-regulated alpha-chain mRNA expression.The inhibition efficiency was higher as the siRNA concentration increased.The trypan blue exclusion rates of erythroid cells were decreased as the concentration increased and time lasts.Conclusion The siRNAs targeting alpha-globin gene transferred into erythroid cells cultured in vitro with liposome-induced gene transfection can down-regulate the alpha-chain mRNA expression level,which may be the new target for gene therapy in β-thalassemia.
Trypan blue
Alpha (finance)
Cite
Citations (0)
Cite
Citations (35)
In order to study the effect of pcsk9 siRNA on THP-1 derived macrophages apoptosis induced by oxLDL, THP-1 derived macrophages were induced to differentiate into macrophages by PMA treatment for 24 h. The experiments were designed as follows: cells were incubated with oxLDL with a concentration of 0, 25, 50, 75, 100 mg/L for 48 h respectively. The apoptosis of THP-1 derived macrophages was observed by staining with Hoechst33258. The expression of pcsk9 was analyzed by reverse transcription polymerase chain reaction and Western blot. The siRNAs for pcsk9 gene were designed and synthesized then transfected into THP-1 derived macrophages by positive ion liposome Lipofectamine 2000. Transfection efficiency was assessed by fluorescence microscope assay. RT-PCR and Western blot were conducted to detect the expression of pcsk9 after 24 h, 48 h respectively. The most efficient siRNA was selected to transfect into THP-1 derived macrophages. 24 h after transfection, cells were treated with oxLDL for 48 h, and then Hoechst 33258 staining. The results showed that the number of cells with nuclear condensation induced by 75 mg/L oxLDL increased significantly. In THP-1 derived macrophages, pcsk9 was upregulated with increasing concentration of oxLDL, while 75 mg/L oxLDL increased significantly. The RNA interference experiment showed that siRNA was successfully transfected into cells and 80 nmol/L as most effective dose of siRNA was selected by RT-PCR and Western blot. Compared with control, the suppression of apoptosis in THP-1 cells transfected with 80 nmol/L of siRNA for 24 h and incubated with 75 mg/L of oxLDL for another 48 h was detected by Hoechst 33258 staining and flow cytometer. Together, these results reveal the expression of pcsk9 mRNA and protein were increased by oxLDL in a concentration-dependent manner. Expression of pcsk9 gene could be effectively suppressed by siRNA. The apoptosis of THP-1 derived macrophages induced by oxLDL could be effectively suppressed by pcsk9 siRNA.
Lipofectamine
THP1 cell line
Cite
Citations (0)
Objective To assess the expression state of mixed-lineage leukemia 1(MLL1) gene in human CD4+CD25-T cells transfected by MLL1-specific small interfering RNA(siRNAs),explore the influence on regulatory T cell(iTreg) and provide theoretical basis for curing clinical diseases associated with Treg unbalance. Methods CD4+CD25-T cells(purity85%)were isolated with magnetic cell sorting(MACS) from healthy human peripheral blood.Specific siRNAs of MLL1 SET domain,which was important for the histone(H3) lysine 4(K4) methylation,were chemically synthesized,and they contained the following groups:A green fluorescent siRNA(siRNA-FAM),3 expe-rimental groups(siRNA-1,siRNA-2,siRNA-3),and a negative control(siRNA-NC).Firstly,human CD4+CD25-T cells isolated after 24 hours were transfected by siRNA-FAM.After 6 hours,transfection efficiency of siRNA was detected by fluorescence microscopy in order to find the optimal transfection condition.Based on the above condition,siRNA-1,siRNA-2,siRNA-3,siRNA-NC were transfected to human CD4+CD25-T cells.After 48 hours,intracellular RNA was extracted from 2×106 cells of each group and reversed the cDNA.Then,the cDNA fragments were amplified using polymerase chain reaction and the amplified DNA were detected by agarose gel.It was found that siRNA-2 significantly inhibited MLL1 gene expression.The ratio of iTreg induced with transforming growth factor-β1 was measured by flow cytometry in the MLL1-silencing cells. Results When the siRNA was 100 pmol and liposome was 5×10-6 L,the transfection efficiency was highest(50.80±0.61)%.The level of MLL1 mRNA was decreased significantly in transfected with MLL1-specific siRNA-2(F=5.820,P0.05).The radio of iTreg was significantly lower in the MLL1-silencing cells than the control groups(F=4.046,P0.05). Conclusions The MLL1-specific siRNA-2 can significantly inhibite the expression of MLL1 gene on CD4+CD25-T cells,and the optimal siRNA sequence segment is identified.The inducing of iTreg is regulated by nuclear factor MLL1.
Jurkat cells
Cite
Citations (0)
Objective To investigate the inhibitory effect of RNA interference （RNAi） on Glil, Bcl-2, Box and cycin DI gene expressions in U251 cell line and the proliferation of U251 cells. Methods Small interfering RNA （siRNA, at locus of 58, 59, 60 and 61） targeted for Glil gene was designed and transfected into U251 cells. RT-PCR was emplyed to detect the mRNA expression of Glil gene to select the siRNA interference fragment （siRNA-Glil） that could most efficiently inhibit the mRNA expression of Glil gene. The mRNA and protein expressions of Glil gene at different times after siRNA-Glil transfection were detected to determine the time law of this interference. U251 cells at logarithmic phase were divided into 3 groups： siRNA-Glil group （transfection of selected siRNA-Glil fragments）, siRNA-NC （transfection of siRNA fragments） and siRNA-N group （blank controls）. The mRNA and protein expressions of Bcl-2, Box and cycin D1 gene were assessed by RT-PCR and Western blotting. Proliferation of cells was measured by MTT assay, and cell apoptosis and cell cycles were detected by flow cytometry （FCM）. Results Transfection efficiency of interference fragments （at locus of 58, 59, 60 and 61, and NC） reached 69.2%; RT-PCR indicated that no obvious Glil mRNA expression was noted at U251-60 cells 48 h after the transfection, therefore, locus 60 was the best interference fi＇agment and 48 h was the best time. The mRNA and protein expressions of Bcl-2 and cycin D1 genes were obviously suppressed by siRNA, and the rnRNA and protein expressions of Bax gene were significantly up-regulated in the siRNA-Glil group as compared with those in the siRNA-N and siRNA-NC groups 48 h after transfection （P〈0.05）. Silencing Glil by RNAi significantly inhibited the proliferation and induced the apoptosis of U251 cells as compared with siRNA-N and siRNA-NC groups 24, 48 and 72 h after transfection （P〈0.05）. Cells at GO and G1 phases were obviously increased and those at S phase were significantly decreased in the siRNA-Glil group as compared with those in the siRNA-N and siRNA-NC groups （P〈0.05）. Conclusion Expression of Glil gene can be effectively inhibited by specific siRNA targeting Gill gene in U251 cells and the proliferation of U251 cells can be significantly inhibited, which may possibly be related to that siRNA-Glil decreases the expressions of Bcl-2 and cycin D1 and alters the ratio of Bcl-2/Bax.
Key words:
Hedgehog signal way; Signal transduction; RNA interference
Cite
Citations (1)
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.
K562 cells
Cite
Citations (0)
Aim: To investigate the influence of small interfering RNA(siRNA) on the expression of cathepsin B(CB) gene in human esophageal carcinoma EC9706 cells. Methods: Specific siRNA of CB gene was obtained by in vitro transcription. EC9706 cells were transfected by specific siRNA at different concentrations (10 μmol/L, 15 μmol/L, 20 μmol/L) for 24 h, 48 h, or 72 h, respectively. The non-specific siRNA group, empty liposome group, and normal control group were used as control. The expression of CB mRNA and protein was detected using in situ hybridization and immunohistochemistry. Results: Compared with control groups, there was a significant decrease in the CB protein and mRNA level in specific siRNA transfection groups, and the inhibition effect was in a concentration dependent manner. It was observed that some EC9706 cells had morphological changes after the transfection.Conclusion: Specific siRNA can effectively inhibit the expression of CB gene in EC9706 cells.
Cite
Citations (0)