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BioMed Research International
Volume 2013 (2013), Article ID 310406, 10 pages
http://dx.doi.org/10.1155/2013/310406
Research Article

Transfected Early Growth Response Gene-1 DNA Enzyme Prevents Stenosis and Occlusion of Autogenous Vein Graft In Vivo

1First Department of General Surgery, The First Affiliated Hospital of Jiamusi University, Jiamusi 154002, China
2Department of Vascular Surgery, The First Hospital of China Medical University, Shenyang 110001, China

Received 12 July 2012; Revised 20 October 2012; Accepted 2 November 2012

Academic Editor: Joseph Fomusi Ndisang

Copyright © 2013 Chengwei Liu et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

The aim of this study was to detect the inhibitory action of the early growth response gene-1 DNA enzyme (EDRz) as a carrying agent by liposomes on vascular smooth muscle cell proliferation and intimal hyperplasia. An autogenous vein graft model was established. EDRz was transfected to the graft vein. The vein graft samples were obtained on each time point after surgery. The expression of the EDRz transfected in the vein graft was detected using a fluorescent microscope. Early growth response gene-1 (Egr-1) mRNA was measured using reverse transcription-PCR and in situ hybridization. And the protein expression of Egr-1 was detected by using western blot and immunohistochemistry analyses. EDRz was located at the media of the vein graft from 2 to 24 h, 7 h after grafting. The Egr-1 protein was mainly located in the medial VSMCs, monocytes, and endothelium cells during the early phase of the vein graft. The degree of VSMC proliferation and thickness of intima were obviously relieved compared with the no-gene therapy group. EDRz can reduce Egr-1 expression in autogenous vein grafts, effectively restrain VSMC proliferation and intimal hyperplasia, and prevent vascular stenosis and occlusion after vein graft.

1. Introduction

In 1977, Paterson et al. [1] first inhibited gene transcription using a complementary combination of single-stranded DNA and RNA in a cell-free system. Later, Stephenson and Zamecnik [2] reversely inhibited the replication of the Rous sarcoma virus using a 13 oligodeoxynucleotide and pioneered the direction of gene-based drugs by inhibiting gene expression. A variety of catalytic DNA, called DNA enzymes, was one of the important breakthroughs in life science history since the discovery of catalytic RNA (ribozyme, Rz) [37].

In 1994, Breaker and Joyce [8] found that a single-stranded DNA molecule (catalytic DNA) can catalyze the hydrolysis of RNA phosphodiester bonds. This single-stranded DNA molecule was also called DNA enzyme (DRz). The enzyme activity center was the “10–23 motif” [915] composed of 15 deoxyribonucleotides (5′-GGC TAG CTA CA A CGA-3′). Its mutation or reverse mutation variants had no activities. Both ends of the active center were substrate-binding regions that can specifically combine with the target RNA through the Watson-Crick base pairing.

Early growth response gene-1 (Egr-1) is a Cys2-His2-type zinc-finger transcription factor. A broad range of extracellular stimuli are capable of activating Egr-1, thus mediating growth, proliferation, differentiation, or apoptosis, therefore, participating in the progression of a variety of diseases such as atherosclerosis [1619]. Previous studies have demonstrated that Egr-1 can activate the restenosis process and intimal hyperplasia and inhibit vascular smooth muscle cell apoptosis in vein grafts [20]. The DNA enzyme is an oligonucleotide that bound to and interfered with translation of the Egr-1 mRNA and it could inhibit the expression of Egr-1. In the present study, an Egr-1 DNA enzyme (EDRz) was designed for Egr-1 mRNA, used a liposome as a carrying agent, and investigated the inhibitory action of the Egr-1 DNA enzyme on vascular smooth muscle cell (VSMC) proliferation and intimal hyperplasia.

2. Materials and Methods

2.1. Construction of Early Growth Response Gene-1 DNA Enzyme

The primer sequences were as follows: 5′-CC GCT GCC AGG CTA GCT ACA ACG ACC CGG ACG T-3′. The 3′ end was phosphorothioate-modified, the 5′ end was labeled with carboxyl fluorescein (FAM), and a total of 15 OD260 (495 μg) of the Egr-1 DNA enzyme was synthesized (Figure 1). Approximately 80 μL of DEPC was added to the solution (1 : 1000), mixed, centrifuged, and then added with 120 μL of the liposome Lipofectamine 2000 (Invitrogen,USA). After 10 min, 32 μL of 1 mmol/L MgCl2 and 568 μL of Pluronic gel 30% F-127 (Sigma, USA) were added to a final volume of 800 μL. The solution was oscillated and homogenized at 4°C and stored until use.

310406.fig.001
Figure 1: The construction conceptual diagram of Egr-1 DNA enzyme shear and its substrate.
2.2. Establishment of Animal Model and Sample Collection

This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The animal use protocol has been reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the First Hospital Affiliated to Jiamusi University. Ninety Wistar rats of either sex (200 g to 250 g) were used. The rats were anesthetized with an intraperitoneal injection of 10% chloral hydrate solution (300 mg/kg) and underwent a sterile microsurgery under the SXP-1B microscope (10 times). The procedure was as follows: about 5 mm of the rat’s right jugular vein was cut; the vein was flushed with heparin saline; and the vein was anastomosed to the infrarenal abdominal aorta using an 11-0 vascular suture line in an end-to-end manner. Up to 8 μL of EDRz was evenly used around the graft vein (including anastomotic) when no signs of active bleeding were found. The retroperitoneum was closed without the use of anticoagulants either before or after the surgery. The animals were randomly divided into 9 groups (10/group), namely, 1, 2, 6, and 24 h and 3, 7, 14, 28, and 42 days after the graft surgery, respectively. The graft vein specimens were cut on each time point. The no-gene therapy group was taken as control group (Figure 2).

fig2
Figure 2: (a) The picture of animal model after graft vein: (b) 28 d after graft in transfection group, the picture of animal model and (c) 28 d after graft in control group, the picture of animal model.

The specimens were fixed with 4% paraformaldehyde in 0.1% diethylpyrocarbonate (DEPC) for 2 h. The gradient sucrose was dehydrated. Then, they were frozen-embedded and cut into 5 μm thick sections. EDRz transfection on the vein graft was observed under a fluorescence microscope. The localization of EDRz was determined by confocal microscopy. The fluorescence gray value was detected using a fluorescence image analyzer and was replicated and verified in multiple samples.

2.3. Histomorphology Staining

Vein grafts were fixed in 10% neutral formalin for 24 h. Conventional dehydration was then performed. The grafts were transparent and wax-dipped. The wax block was embedded and the middle section of the vein graft was cut into 5 μm thick sections. HE staining was conducted and images of the computer image analysis system were collected. Finally, intimal hyperplasia thickness was measured. The data collection and analysis of intimal hyperplasia were performed in a blinded manner.

2.4. In Situ Hybridization

Specimens were processed with fixation. The sucrose gradient was dehydrated, and frozen-embedded, and then a constant cold slicer was used to slice them into 5 μm thick sections. Digoxigenin-labeled oligonucleotides were used as probes, and the in situ hybridization was performed according to the manufacturer’s instructions (Wuhan Boster Corporation, Wuhan, China). The specimens were dyed with DAB or AEC. The percentage of positive cells in the total cell in eight-unit perspective was randomly counted and performed in a blinded manner.

2.5. Reverse Transcription-Polymerase Chain Reaction

Total RNA was extracted from cell lines according to instructions of the kit (Wuhan Boster Corporation, Wuhan, China). Primers for Egr-1 were designed using the Jellyfish software according to the sequence in GenBank and synthesized by Shanghai Sangon. For Egr-1, the primers were 5′-CAG TCG TAG TGA CCA CCT TAC CA-3′ (Fwd) and 5′-AGG TTG CTG TCA TGT CTG AAA GAC-3′ (Rev), 448-bp long. For β-actin, the primers were 5′-TTG TAA CCA ACT GGG ACG ATA-3′ (Fwd) and 5′-GAT CTT GAT CTT CAT GGT GCT-3′ (Rev), 668-bp long. The PCR program involved the following procedures: predenaturation for 2 min at 94°C; denaturation for 30 s at 94°C; annealing for 30 s at 58°C; extension for 1 min at 72°C; and final elongation at 72°C for 10 min. Thirty cycles of PCR were performed. PCR products were analyzed by electrofluorescence on 2% agarose gel in a 1x TAE buffer at a voltage of 100 V for 1 h and EB-stained for 20 min. Band intensity was photographed and analyzed on the Gel Imaging System. The gene expression value = Egr-1mRNA/β-actin mRNA.

2.6. Immunohistochemical Staining

Conventional SABC staining was performed according to the kit’s instructions (Wuhan Boster Company, Wuhan, China). PBS was used in place of primary antibodies as the negative control. The nucleus or cytoplasm had positive brown-yellow (DAB) or red (AEC) particles at 400 times magnification under the light microscope; it was considered positive regardless of dyeing intensity as long as there was a color display. The percentage of positive cells in the total cell in the eight-unit perspective was randomly counted and was performed in a blinded manner.

2.7. Western Blot Analysis

The specimens were lysed with a cell lysis solution. The vessel tissues were cut into pieces. The specimens were ultrasound-homogenized. Proteins (100 μg/sample) were separated using 10% SDS-PAGE. The proteins were electrotransferred to nitrocellulose membranes using a semidry system. Then, the membrane was blocked in 5% skimmed milk diluted in TBST for 1 h at room temperature. Thereafter, the membranes were incubated with a primary antibody for 2 h at room temperature. Next, the membranes were further incubated with a horseradish peroxidase-labeled goat anti-mouse IgG antibody at a 1 : 500 dilution. The specimens were washed with TBST three times. Then, 12.5 mg of β-Naphthyl acid phosphate and 12.5 mg of O-Dianisidine tetrazotized (Sigma Corporation) were added to color the specimens. The NC membrane was photographed and analyzed on the Gel Imaging System.

2.8. Statistical Methods

Data were shown as mean ± SD and analyzed using the SPSS10 statistical software. The significance of the differences between the group means was determined using ANOVA and post hoc test.

3. Results

3.1. Egr-1 DNA Enzyme (EDRz) Transfection

The early growth response gene-1 DNA enzyme was mainly located in the tunica media, adventitia, and partial endothelial cells of the vein graft 1 h after the grafting in transfection group (fluorescence expression value of ) (Table 1, Figure 3(a)). The early growth response gene-1 DNA enzyme was located in the tunica media of the vein graft from 2 h to 24 h after-grafting. There was a small amount of EDRz in the tunica media of the vein graft 3 d after the grafting. It was mainly located in the intima of the vein graft 7 d after grafting (Table 1, Figure 3(b)). There were no traces of the early growth response gene-1 DNA enzyme in the vein grafts at 14, 28, and 42 d and control group (Table 1, Figure 3(c)).

tab1
Table 1: The result of thickness of intimal hyperplasia in vein graft and EDRz transfection after transfection and no-transfection EDRz ( ).
fig3
Figure 3: (a) 1 h after graft in transfection group, EDRz located in adventitia, tunica media, and partial endothelial cells by confocal microscopy (×400). (b) 7 d after graft in transfection group, EDRz located in tunica intima by confocal microscopy (×400). (c) 1 h after graft in control group, there was no EDRz in adventitia, tunica media, and endothelial cells by confocal microscopy (×400).
3.2. Changes in Histomorphology

There was no expression of PCNA protein in normal vein. There was still a small amount of slightly disordered VSMCs in the media 2 h to 6 h after the vein graft compared with the control group. Slightly positive expression of PCNA at 6 h, positive cell rate of ( )% in transfection group, ( )% in control group. Moreover, VSMCs were also found partly in the thin layer of a thrombus formation in the cavity surface of the intima. The intima was partly damaged at 24 h to 3 d after grafting. The expression of PCNA protein was increased from 24 h to 3 d. In addition, endothelial cells were shed and there was a small amount of thrombosis in the local area. The intima thickened and VSMC proliferation was visible at 7 d. Intimal hyperplasia reached a peak ( .7 μm) at 14 d. The expression of PCNA protein reached peak at 14 d, ( )% in transfection group, ( )% in control group. The vascellum basically completed endothelialization and disordered VSMC were still visible compared with the control group whose degree of VSMC proliferation and thickness of intima were obviously relieved at the same time. The difference was statistically significant (F = 3.42, ). Intimal hyperplasia thickness decreased at 28 and 42 d compared with 14 d and the expression of PCNA protein was decrease (Tables 1 and 2 and Figure 4).

tab2
Table 2: Contrast of  PCNA protein by immunohistochemistry ( , %).
fig4
Figure 4: (a) Normal vein (HE × 100). (b) 14 d after graft in transfection group, intimal hyperplasia reached a peak (HE × 100). (c) 14 d after graft in control group, intimal hyperplasia reached a peak (HE × 100).
3.3. Reverse Transcription-Polymerase Chain Reaction (RT-PCR)

Egr-1 mRNA expression reached a peak (gene expression value of ) 1 h after the EDRz was transfected. The expression decreased ( , , ) from 2 h to 24 h after grafting. The expression was weak ( ) 3 d after-grafting. There were no more Egr-1 mRNA expressions at 7, 14, 28, and 42 d after grafting (Figure 5(a)). Egr-1 mRNA expression had biphasic changes in control group. Egr-1 mRNA rapid rise at 1 h after graft, a spontaneous decline at 6 h to 3 d, increase at 7 d after graft operation, a peak at 28 days (Figure 5(b)).

fig5
Figure 5: (a) The RT-PCR results of Egr-1 mRNA in transfection group. (b) The RT-PCR results of Egr-1 mRNA in control group. M: Gene Ruler 100 bp DNA Ladder Marker; 1: normal vein; 2–10: transplantation vein at different time after operation, 2: 1 h; 3: 2 h; 4: 6 h; 5: 24 h; 6: 3 d; 7: 7 d; 8: 14 d; 9: 28 d; 10: 42 d.
3.4. In Situ Hybridization

Partial VSMC showed an Egr-1 mRNA-positive expression in the media of the vein graft 1 h after EDRz transfection. The strongest positive cell expression was ( )%. The difference was statistically significant (F = 3.25, ) compared with the rest of the time points. Its expression decreased from 2 h to 3 d after grafting. There was no Egr-1 mRNA positive expression of neointimal VSMC 7 d after grafting. The trend of positive cells was consistent with the RT-PCR results (Table 3, Figure 6(a)) in control group, and the positive expression of Egr-1 mRNA was found in the part of VSMCs of the media at 1 h after graft. A peak at 28 d, the positive rate of Egr-1 mRNA was ( )%, Egr-1 mRNA major located in the vascular smooth muscle cells of neointimal (Table 3, Figure 6(b)).

tab3
Table 3: Contrast of Egr-1 mRNA and protein by in situ hybridization and immunohistochemistry ( , %).
fig6
Figure 6: (a) 1 h after graft in transfection group, the positive cells of Egr-1 mRNA were located in cytoplasm of VSMC of neointima by ISH (×400). (b) 1 h after graft in control group, the positive cells of Egr-1 mRNA were located in cytoplasm of VSMC of neointima by ISH (×400).
3.5. Western Blot Analysis

Egr-1-positive cells were not detected in the normal vein. Egr-1 protein expression appeared 2 h after EDRz transfection. The optical density value was ( ) × 103. Its expression decreased from 6 h to 3 d after grafting, with optical density values of ( ) × 103, ( ) × 103, and ( ) × 103. Egr-1 positive cells were no longer present 7 d after grafting (Figure 7(a)). In control group, we found that Egr-1 protein was expressed at the early phase of 2 h, and continuing to 6 h, the expression of Egr-1 protein was decline from 24 h to 3 d, reincreased at 7 d, and reached peak at 28 d (Figure 7(b)).

fig7
Figure 7: (a) The results of western blot of Egr-1 protein in transfection group. (b) The results of western blot of Egr-1 protein in control group 1: normal vein; 2–10: transplantation vein at different times after operation, 2: 1 h; 3: 2 h; 4: 6 h; 5: 24 h; 6: 3 d; 7: 7 d; 8: 14 d; 9: 28 d; 10: 42 d.
3.6. Immunohistochemistry

The Egr-1 protein was mainly located in the medial VSMCs, monocytes, and endothelium cells during the early phase of the vein graft. However, there were no Egr-1 proteins in the medial and neointimal VSMCs after 7 d. The positive expression rates were as follows: positive cell rate of ( )% at 2h; positive cell rate of ( )% at 6 h; positive cell rate of ( )% at 24 h; and positive cell rate of ( )% at 3d (Figure 8(a)). In control group, the positive expression of Egr-1 protein reached peak at 28 days ( )% (Figure 8(b)).

fig8
Figure 8: (a) 2 h after graft in transfection group, the positive cells of Egr-1 protein were located in cell nucleus of VSMC of neointima by immunohistochemistry (×200). (b) 2 h after graft in control group, the positive cells of Egr-1 protein were located in cell nucleus of the VSMC of neointima by immunohistochemistry (×200).

4. Discussion

AUG (816 to 818 sequence) is a selected target of the Egr-1 mRNA. The splice site was located between 816 and 817, adding T GCA GGC CC to the 3′ end of DNA enzyme for the 807–815 sequence (A CGU CCG GG) of Egr-1 mRNA and ACC GTC GCC [2124] to the 5′ end of DNA enzyme for the 817–825 sequence (UGG CAG CGG). A phosphorothioate modification was made in the 3′ end to resist nuclease degradation, and the 5′ end was labeled with carboxy fluorescein (FAM) for detection purposes. The constructed DNA enzyme was called Egr-1 DNA enzyme (EDRz) (Figure 1). The 816 base (A) of the Egr-1 mRNA did not undergo base pairing with EDRz. Meanwhile, the rest of the EDRz sites formed the combination of base pairing with Egr-1 mRNA. Then, the latter underwent conformational changes. The 2′ end at the point of the OH proton was cut with the help of divalent metal cations, such as Mg2. Moreover, a nucleophilic attack occurred on the adjacent phosphate. The Egr-1mRNA molecular structure was dissociated by two transesterification reactions [2530].

The substrate-binding site can be applied to shear the RNA of a variety of pathogens and mRNAs of disease-related genes after changing its sequence composition in the 10–23 DNA enzyme [31, 32]. In gene therapy, 10–23 DNA enzymes have the advantages of both the ribozyme (Rz) and antisense oligodeoxynucleotide (ASODN) [33, 34]. The 10–23 DNA enzyme has the following features compared with ASODN: it not only has a substrate RNA antisense inhibitory effect by virtue of the two substrate-binding sites, but also kills virus RNA through the “shear” mechanism [3538]. Furthermore, DNA enzyme molecules can be used repeatedly, which means that they can shear a number of RNA molecules. The 10–23 DNA enzyme has the following characteristics compared with a variety of Rz: the identified splice site of 10–23 DNA enzyme is present in a range of RNA molecules, including the RNA translation initiation codon AUG of viruses. It is a good shear target and has more shearing targets to choose from compared with Rz. Its nature is relatively stable. The stability of DNA is about 100, 000 times that of RNA in the conditions of physiological pH, temperature, ionic strength, and so on. Its resistance to hydrolysis is about 100 times or more than that of a protein enzyme [3941]. The sequence of the active center is short. The molecular weight is relatively small with relatively good elasticity. Therefore, it is less affected by the secondary structure of the target sequence. The trend to the substrate is better. Thus, the specificity of the target RNA, combing stability and shear activity, is expressed better than Rz in general [4244]. It is easier to dissociate the DNA-RNA hybrid molecule than the RNA-RNA hybrid molecule. Therefore, the shear rate of the shear product DRz dissociation process is relatively small [45, 46]. The RNA of the DNA-RNA hybrid molecules can be degraded by the RNA enzyme H. Hence, the DNA enzyme can not only directly kill the target RNA such as Rz, but also cause the hydrolysis of the RNA enzyme H to target RNAs, such as ASODN [47, 48].

The results of this experiment combined with those of previous studies [8, 49, 50] indicated that the early growth response gene-1 DNA enzyme was mainly located in the media and adventitia of the vein graft 1 h after grafting and then gradually shifted to the media. There was a small amount of EDRz in the media of the vein graft 3 d after grafting and was mainly located in the media. It was mainly located in the intima of the vein graft 7 d after grafting. In addition, the Egr-1 DNA enzyme can also be found in some small newborn blood vessels. However, Egr-1 mRNA and protein expressions in the vein graft were not detected 14 d after grafting. There was no EDRz in the vein grafts, suggesting that the EDRz pathway is adventitia → medial → intima and perhaps degraded by a deoxyribonuclease in the end. Egr-1 mRNA and protein expressions decreased at the same time point. Egr-1 mRNA expression decreased obviously 1 h after grafting. This finding indicated that the Egr-1 DNA enzyme rapidly transferred from the adventitia to the media to combine with the Egr-1 mRNA under a short period of time. Hence, the role of the carrier liposome Lipofectamine 2000 was confirmed. Egr-1 proteins were mainly located in the medial VSMCs, monocytes, and endothelium cells during the early phase of the vein graft. However, there were no Egr-1 proteins in medial and neointimal VSMCs 7 d after grafting, indicating that the early growth response gene-1 DNA enzyme can reduce Egr-1 expression in an autogenous vein graft. VSMC proliferation and intimal hyperplasia reached a peak 7 and 14 d after grafting. The degree of VSMC proliferation and thickness of intima were obviously relieved at the same time compared with the no-gene therapy group. Therefore, Egr-1 DNA enzyme transfection of vein grafts with the liposome Lipofectamine 2000 as a carrier can effectively restrain VSMC proliferation and intimal hyperplasia and prevent vascular stenosis and occlusion after vein grafting.

Conflict of Interest

The authors declare that there is no conflict of interests.

Authors’ Contribution

C. Liu and X. Zhang contributed equally to this paper.

Acknowledgments

This study was supported by the National Natural Science Foundation of China (no: 30801123) and Reserve Talents of Universities Overseas Research Program of Heilongjiang, China.

References

  1. B. M. Paterson, B. E. Roberts, and E. L. Kuff, “Structural gene identification and mapping by DNA.mRNA hybrid-arrested cell-free translation,” Proceedings of the National Academy of Sciences of the United States of America, vol. 74, no. 10, pp. 4370–4374, 1977. View at Scopus
  2. M. L. Stephenson and P. C. Zamecnik, “Inhibition of Rous sarcoma viral RNA translation by a specific oligodeoxyribonucleotide,” Proceedings of the National Academy of Sciences of the United States of America, vol. 75, no. 1, pp. 285–288, 1978. View at Scopus
  3. R. Pavri, A. Gazumyan, M. Jankovic et al., “Activation-induced cytidine deaminase targets DNA at sites of RNA polymerase II stalling by interaction with Spt5,” Cell, vol. 143, no. 1, pp. 122–133, 2010. View at Publisher · View at Google Scholar · View at Scopus
  4. S. S. Roy, P. Chakraborty, P. Ghosh, J. Biswas, and S. Bhattacharya, “Influence of novel naphthalimide-based organoselenium on genotoxicity induced by an alkylating agent: the role of reactive oxygen species and selenoenzymes,” Redox Report, vol. 17, no. 4, pp. 157–166, 2012.
  5. N. Kota, V. V. Panpatil, R. Kaleb, B. Varanasi, and K. Polasa, “Dose-dependent effect in the inhibition of oxidative stress and anticlastogenic potential of ginger in STZ induced diabetic rats,” Food Chemistry, vol. 135, no. 4, pp. 2954–2959, 2012.
  6. B. O. Ajayi and F. D. Otajevwo, “Extrachromosomal DNA length and antibiograms of Staphylococcus aureus and Pseudomonas aeruginosa isolated from tears of HIV/AIDS Patients after curing with sodium dodecyl sulphate,” Global Journal of Health Science, vol. 4, no. 1, pp. 229–236, 2012.
  7. S. Collani and G. Barcaccia, “Development of a rapid and inexpensive method to reveal natural antisense transcripts,” Plant Methods, vol. 8, no. 1, article 37, 2012. View at Publisher · View at Google Scholar
  8. R. R. Breaker and G. F. Joyce, “A DNA enzyme that cleaves RNA,” Chemistry and Biology, vol. 1, no. 4, pp. 223–229, 1994. View at Publisher · View at Google Scholar · View at Scopus
  9. G. Tell, D. M. Wilson, and C. H. Lee, “Intrusion of a DNA repair protein in the RNome world: is this the beginning of a new era?” Molecular and Cellular Biology, vol. 30, no. 2, pp. 366–371, 2010. View at Publisher · View at Google Scholar · View at Scopus
  10. C. H. Lam and D. M. Perrin, “Introduction of guanidinium-modified deoxyuridine into the substrate binding regions of DNAzyme 10–23 to enhance target affinity: implications for DNAzyme design,” Bioorganic and Medicinal Chemistry Letters, vol. 20, no. 17, pp. 5119–5122, 2010. View at Publisher · View at Google Scholar · View at Scopus
  11. J. Li, N. Wang, Q. Luo, and L. Wan, “The 10–23 DNA enzyme generated by a novel expression vector mediate inhibition of taco expression in macrophage,” Oligonucleotides, vol. 20, no. 2, pp. 61–68, 2010. View at Publisher · View at Google Scholar · View at Scopus
  12. L. Robaldo, F. Izzo, M. Dellafiore et al., “Influence of conformationally restricted pyrimidines on the activity of 10–23 DNAzymes,” Bioorganic & Medicinal Chemistry, vol. 20, no. 8, pp. 2581–2586, 2012.
  13. A. A. Fokina, M. I. Meschaninova, T. Durfort, A. G. Venyaminova, and J. C. François, “Targeting insulin-like growth factor I with 10–23 DNAzymes: 2'-O-methyl modifications in the catalytic core enhance mRNA cleavage,” Biochemistry, vol. 51, no. 11, pp. 2181–2191, 2012.
  14. J. He, D. Zhang, Q. Wang, X. Wei, M. Cheng, and K. Liu, “A novel strategy of chemical modification for rate enhancement of 10–23 DNAzyme: a combination of A9 position and 8-aza-7-deaza-2′-deoxyadenosine analogs,” Organic and Biomolecular Chemistry, vol. 9, no. 16, pp. 5728–5736, 2011. View at Publisher · View at Google Scholar · View at Scopus
  15. B. Wang, L. Cao, W. Chiuman, Y. Li, and Z. Xi, “Probing the function of nucleotides in the catalytic cores of the 8-17 and 10–23 DNAzymes by abasic nucleotide and C3 spacer substitutions,” Biochemistry, vol. 49, no. 35, pp. 7553–7562, 2010. View at Publisher · View at Google Scholar · View at Scopus
  16. G. Kapakos, V. Youreva, and A. K. Srivastava, “Attenuation of endothelin-1-induced PKB and ERK1/2 signaling, as well as Egr-1 expression, by curcumin in A-10 vascular smooth muscle cells,” Canadian Journal of Physiology and Pharmacology, vol. 90, no. 9, pp. 1277–1285, 2012.
  17. A. Skorokhod, J. Bachmann, N. Giese, M. E. Martignoni, and H. Krakowski-Roosen, “Real-imaging cDNA-AFLP transcript profiling of pancreatic cancer patients: Egr-1 as a potential key regulator of muscle cachexia,” BMC Cancer, vol. 12, no. 1, article 265, 2012. View at Publisher · View at Google Scholar
  18. W. Windischhofer, E. Huber, C. Rossmann et al., “LPA-induced suppression of periostin in human osteosarcoma cells is mediated by the LPA(1)/Egr-1 axis,” Biochimie, vol. 94, no. 9, pp. 1997–2005, 2012.
  19. S. Y. Shin, J. H. Kim, J. H. Lee, Y. Lim, and Y. H. Lee, “2'-Hydroxyflavanone induces apoptosis through Egr-1 involving expression of Bax, p21, and NAG-1 in colon cancer cells,” Molecular Nutrition & Food Research, vol. 56, no. 5, pp. 761–774, 2012.
  20. C. W. Liu, X. H. Hu, X. S. Zhang, Y. W. Luo, X. W. Wang, and Q. Zhang, “Expression and significance of early growth response gene-1 in autogenons vein graft in rats,” Chinese Journal of Bases and Clinics In General Surgery, vol. 13, no. 1, pp. 23–27, 2006.
  21. J. Maeda, M. Nishida, H. Takikawa et al., “Inhibitory effects of sulfobacin B on DNA polymerase and inflammation,” International Journal of Molecular Medicine, vol. 26, no. 5, pp. 751–758, 2010. View at Publisher · View at Google Scholar · View at Scopus
  22. N. Patel and V. K. Kalra, “Placenta growth factor-induced early growth response 1 (Egr-1) regulates hypoxia-inducible factor-1α (HIF-1α) in endothelial cells,” Journal of Biological Chemistry, vol. 285, no. 27, pp. 20570–20579, 2010. View at Publisher · View at Google Scholar · View at Scopus
  23. T. Iyoda, F. Zhang, L. Sun et al., “Lysophosphatidic acid induces early growth response-1 (Egr-1) protein expression via protein kinase Cδ-regulated extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) activation in vascular smooth muscle cells,” The Journal of Biological Chemistry, vol. 287, no. 27, pp. 22635–22642, 2012.
  24. J. I. Pagel, T. Ziegelhoeffer, M. Heil et al., “Role of early growth response 1 in arteriogenesis: impact on vascular cell proliferation and leukocyte recruitment in vivo,” Thrombosis and Haemostasis, vol. 107, no. 3, pp. 562–574, 2012.
  25. T. M. Jr. Donohue, N. A. Osna, C. S. Trambly et al., “Early growth response-1 contributes to steatosis development after acute ethanol administration,” Alcoholism: Clinical and Experimental Research, vol. 36, no. 5, pp. 759–767, 2012.
  26. A. C. Jones, K. A. Trujillo, G. K. Phillips et al., “Early growth response 1 and fatty acid synthase expression is altered in tumor adjacent prostate tissue and indicates field cancerization,” Prostate, vol. 72, no. 11, pp. 1159–1170, 2012.
  27. B. P. Sullivan, W. Cui, B. L. Copple, and J. P. Luyendyk, “Early growth response factor-1 limits biliary fibrosis in a model of xenobiotic-induced cholestasis in mice,” Society of Toxicology, vol. 126, no. 1, pp. 267–274, 2012.
  28. M. G. Dickinson, B. Bartelds, G. Molema et al., “Egr-1 expression during neointimal development in flow-associated pulmonary hypertension,” American Journal of Pathology, vol. 179, no. 5, pp. 2199–2209, 2011.
  29. S. Y. Shin, C. G. Kim, Y. Lim, and Y. H. Lee, “The ETS family transcription factor ELK-1 regulates induction of the cell cycle-regulatory gene p21Waf1/Cip1and the BAX gene in sodium arsenite-exposed human keratinocyte HaCaT cells,” Journal of Biological Chemistry, vol. 286, no. 30, pp. 26860–26872, 2011. View at Publisher · View at Google Scholar · View at Scopus
  30. S. Bakalash, M. Pham, Y. Koronyo et al., “Egr1 expression is induced following glatiramer acetate immunotherapy in rodent models of glaucoma and Alzheimer's disease,” Investigative Ophthalmology & Visual Science, vol. 52, no. 12, pp. 9033–9046, 2011.
  31. K. A. McKinney, N. Al-Rawi, P. C. Maciag, D. A. Banyard, and D. A. Sewell, “Effect of a novel DNA vaccine on angiogenesis and tumor growth in vivo,” Archives of Otolaryngology, vol. 136, no. 9, pp. 859–864, 2010. View at Publisher · View at Google Scholar · View at Scopus
  32. D. Lockney, S. Franzen, and S. Lommel, “Viruses as nanomaterials for drug delivery,” Methods in Molecular Biology, vol. 726, pp. 207–221, 2011.
  33. Y. Y. Xu, Y. Y. Bao, S. H. Zhou, and J. Fan, “Effect on the expression of MMP-2, MT-MMP in laryngeal carcinoma Hep-2 cell line by antisense glucose transporter-1,” Archives of Medical Research, vol. 43, no. 5, pp. 395–401, 2012.
  34. A. Ganesh, W. Bogdanowicz, M. Haupt, G. Marimuthu, and K. E. Rajan, “Egr-1 antisense oligodeoxynucleotide administration into the olfactory bulb impairs olfactory learning in the greater short-nosed fruit bat Cynopterus sphinx,” Brain Research, vol. 1471, pp. 33–45, 2012. View at Publisher · View at Google Scholar
  35. N. El-Murr, M. C. Maurel, M. Rihova et al., “Behavior of a hammerhead ribozyme in aqueous solution at medium to high temperatures,” Naturwissenschaften, vol. 99, no. 9, pp. 931–938, 2012.
  36. P. Guo, F. Haque, B. Hallahan, R. Reif, and H. Li, “Uniqueness, advantages, challenges, solutions, and perspectives in therapeutics applying RNA nanotechnology,” Nucleic Acid Therapeutics, vol. 22, no. 4, pp. 226–245, 2012.
  37. N. Sankaran, “How the discovery of ribozymes cast RNA in the roles of both chicken and egg in origin-of-life theories,” Studies in History and Philosophy of Biological and Biomedical Sciences, vol. 43, no. 4, pp. 741–750, 2012.
  38. E. D. Egan and K. Collins, “Biogenesis of telomerase ribonucleoproteins,” RNA, vol. 18, no. 10, pp. 1747–1759, 2012.
  39. H. W. Yu, Q. F. Liu, and G. N. Liu, “Positive regulation of the Egr-1/osteopontin positive feedback loop in rat vascular smooth muscle cells by TGF-β, ERK, JNK, and p38 MAPK signaling,” Biochemical and Biophysical Research Communications, vol. 396, no. 2, pp. 451–456, 2010. View at Publisher · View at Google Scholar · View at Scopus
  40. B. Wang, L. Cao, W. Chiuman, Y. Li, and Z. Xi, “Probing the function of nucleotides in the catalytic cores of the 8-17 and 10–23 DNAzymes by abasic nucleotide and C3 spacer substitutions,” Biochemistry, vol. 49, no. 35, pp. 7553–7562, 2010. View at Publisher · View at Google Scholar · View at Scopus
  41. X. Zhong, C. J. Hale, J. A. Law et al., “DDR complex facilitates global association of RNA polymerase V to promoters and evolutionarily young transposons,” Nature Structural & Molecular Biology, vol. 19, no. 9, pp. 870–875, 2012.
  42. E. Deindl, S. Fischer, and K. T. Preissner, “New directions in inflammation and immunity: the multi-functional role of the extracellular RNA/RNase system,” Indian Journal of Biochemistry and Biophysics, vol. 46, no. 6, pp. 461–466, 2009. View at Scopus
  43. S. A. Weeks, C. A. Lee, Y. Zhao et al., “A Polymerase mechanism-based strategy for viral attenuation and vaccine development,” The Journal of Biological Chemistry, vol. 287, no. 38, pp. 31618–31622, 2012.
  44. P. A. Del Rizzo, S. Krishnan, and R. C. Trievel, “Crystal structure and functional analysis of JMJD5 indicate an alternate specificity and function,” Molecular and Cellular Biology, vol. 32, no. 19, pp. 4044–4052, 2012.
  45. J. Ni, A. Waldman, and L. M. Khachigian, “c-Jun regulates shear- and injury-inducible Egr-1 expression, vein graft stenosis after autologous end-to-side transplantation in rabbits, and intimal hyperplasia in human saphenous veins,” Journal of Biological Chemistry, vol. 285, no. 6, pp. 4038–4048, 2010. View at Publisher · View at Google Scholar · View at Scopus
  46. L. Robaldo, J. M. Montserrat, and A. M. Iribarren, “10–23 DNAzyme modified with (2′R)- and (2′S)-2′-deoxy-2′-C-methyluridine in the catalytic core,” Bioorganic and Medicinal Chemistry Letters, vol. 20, no. 15, pp. 4367–4370, 2010. View at Publisher · View at Google Scholar · View at Scopus
  47. O. F. Dyson, C. M. Traylen, and S. M. Akula, “Cell membrane-bound Kaposi's sarcoma-associated herpesvirus-encoded glycoprotein B promotes virus latency by regulating expression of cellular Egr-1,” Journal of Biological Chemistry, vol. 285, no. 48, pp. 37491–37502, 2010. View at Publisher · View at Google Scholar · View at Scopus
  48. G. N. Liu, Y. X. Teng, and W. Yan, “Transfected synthetic DNA Enzyme Gene specifically inhibits Egr-1 gene expression and reduces Neointimal Hyperplasia following balloon injury in rats,” International Journal of Cardiology, vol. 129, no. 1, pp. 118–124, 2008. View at Publisher · View at Google Scholar · View at Scopus
  49. J. Yang, X. H. Hu, C. W. Liu, Z. S. Zhang, and Q. Zhang, “Effect of local transfection of survivin antisense oligodeoxyribouocleotides on intimal hyperplasia in vein graft,” Chinese Journal of Bases and Clinics in General Surgery, vol. 13, no. 1, pp. 28–33, 2006.
  50. G. Xin, X. Zhao, X. Duan et al., “Antitumor effect of a generation 4 polyamidoamine dendrimer/cyclooxygenase-2antisense oligodeoxynucleotide complex on breast cancer in vitro and in vivo,” Cancer Biotherapy and Radiopharmaceuticals, vol. 27, no. 1, pp. 77–87, 2012.