KR100527372B1 - Novel Ring-type E2F decoy oligodeoxynucleotides and inhibition method of mesangial cell proliferation using the same - Google Patents

Abstract

The present invention relates to a novel circular structure E2F decoy oligodioxynucleotide and a method for inhibiting glomerular hepatic stem cell proliferation using the same. The present invention provides an excellent effect of providing a stable circular structure E2F decoy oligodioxynucleotide to nuclease. have. In addition, the present invention by transfecting the expression vector of E2F decoy oligodioxynucleotide and cell cycle regulator E2F of the circular structure in the glomerular vascular hepatocytes to inhibit the expression and cell proliferation of cell cycle regulatory genes to proliferate glomerular vascular stem cells There is an excellent effect of providing a way to suppress. Therefore, the E2F decoy oligodioxynucleotide of the circular structure of the present invention can be effectively applied to prevent and treat diabetic nephropathy even in hyperglycemic state by effectively inhibiting the proliferation of glomerular hepatic stem cells.

Description

Novel ring-type E2F decoy oligodeoxynucleotides and inhibition method of mesangial cell proliferation using the same

The present invention relates to a novel circular structure E2F decoy oligodioxynucleotide and a method for inhibiting glomerular hepatic stem cell proliferation using the same. More specifically, the present invention is characterized in that the E2F decoy oligodeoxynucleotides of circular structure designed to be stable to nucleases are cotransfected with the transcription factor E2F / DP in glomerular hepatic stem cells to inhibit the transcriptional activity of E2F. It relates to inhibiting proliferation of hepatocytes.

Proliferation of glomerular vascular hepatocytes is one of the earliest phenomena in diabetic kidney disease. Proliferation of glomerular hepatic stem cells and expansion of glomerular matrix are known to be important features of glomerulonephritis. Glomerulonephritis ultimately leads to glomerulosclerosis and kidney disease, and the suppression of proliferation of glomerular vascular hepatocytes is reported to be important to prevent the progression of kidney disease. In general, glomerular hepatic stem cells in the kidneys of adults are present in G0, which is stationary in the cell cycle, which normally stops or proliferates to low levels. As soon as they are stimulated by growth factors or hyperglycemia, they progress to G1 and G1 / S at the G0 stage of the cell cycle. An important step in the cell cycle is to go from G1 to S to lead to meiosis or cell division. The Cyclin / CDK complex phosphorylates pRB to break down several proteins, including the transcription factor E2F. E2F is an important transcription factor for cell proliferation and is known to activate genes dhfr , c-myc, DNA polymerase, cdc2, and PCNA that are involved in the early and late cell cycles. Several studies have reported that overexpression of E2F protein induces cell cycle progression and that E2F deficiency inhibits cell growth. E2F regulates the transcription of intracellular genes required for G1 to S phase progression, including DHFR, c-myc, DNA polymerase, cdc2, and PCNA, and many proteins involved in cell cycle progression, including several cdk, including the Rb gene. And physiologically important complexes are known. In addition, when cells enter the S phase, Rb protein is phosphorylated by G1 cyclin-cdk complex to separate from E2F. E2F, which activates the transcription of genes required for cell cycle entry. E2F transcription factors consist of six types of E2F proteins (E2F1-E2F6) and two DP proteins (DP1, DP2). The transcriptionally active form of E2F 1-5 complexes with the DP protein to bind DNA and rely on pRB protein to activate or inhibit the transcription of cell cycle and cell proliferation related genes. In addition, E2F1 to E2F5 activate genes dependent on E2F, and most of the products of these genes are well known as factors essential for cell cycle and cell proliferation. In contrast, E2F6 acts as a transcription inhibitor, unlike the five E2F factors discovered so far due to the deletion of the C-terminal domain to which the pRB protein binds. However, these E2F / DP complexes can regulate the transcription of reporter genes from consensus E2F binding sites, but the exact transcriptional control function of each E2F / DP complex for these genes is not fully explained.

Recently, new methods for regulating and inhibiting expression of specific genes have been developed, and these techniques have been developed as a means of treating various diseases. Numerous studies have been published to regulate mRNA, such as antisense oligonucleotides, ribozymes and RNA interference, but targeting new proteins that regulate the expression of specific genes is emerging. Among them, recent studies have been actively conducted to control gene expression in the gene transcription and transcription processes and to apply it to the treatment of diseases by using the transcription factor decoy.

The transcription factor inhibitor decoy (decoy) technology has been developed to inhibit the expression of genes whose expression is regulated by transcription factors by binding to unique transcription factors such as E2F to provide competitors. Synthetic double-stranded ODN that inhibits transcriptional activity has activity as a disruptor that inhibits the activity of transcription factors when introduced into cultured cells. It also possesses the function of inhibiting the expression of various genes by decoy characteristic. Despite the unique activity of decoy ODNs that inhibit gene expression, the main limitation of conventional decoy ODNs is that they are rapidly degraded by nucleases present in a large number of serum, cells and tissues. In this regard, ODN is chemically modified with sulfur ions, methyl groups or other foreign substances to increase the stability to nuclease activity. The stability of ODN is complemented by these chemical modifications, but due to other problems it is recognized as foreign foreign material in the cells. That is, some problems such as problems of the array specific binding effect of gene therapy and immune activation due to modification by foreign substances have been raised.

Accordingly, an object of the present invention is to provide a ring-type E2F decoy oligodeoxynucleotide (Ring type-E2F decoy ODN) newly designed to be stable to nucleases.

Another object of the present invention is to provide a method of inhibiting the proliferation of glomerular vascular stem cells by inhibiting the transcriptional activity of cell cycle regulators using the circular E2F decoy ODN.

The object of the present invention is to prepare expression vectors of transcription factor (E2F) and transcriptional activity regulator (DP) and to investigate the effects on cell proliferation by transfecting glomerular vascular hepatocytes and designed to be stable to nuclease A round-type E2F decoy ODN (R-type E2F decoy ODN) was constructed and its stability was investigated, and the decoy ODN and E2F / DP expression vectors were co-transfected into glomerular vascular stem cells. This was achieved by comparing the effect on promoter activity, the effect on cellular gene expression and the effect on cell proliferation.

EMBODIMENT OF THE INVENTION Hereinafter, the structure of invention is demonstrated concretely.

The present invention comprises the steps of preparing expression vectors of transcription factor (E2F) and transcriptional activity regulator (DP), transfection into glomerular vascular hepatocytes and effect on cell proliferation; Preparing a ring-type E2F decoy ODN and a stability step thereof; Simultaneous transfection of the decoy ODN and the E2F / DP expression vector into glomerular hepatic stem cells; The decoy ODN consists of a step of investigating the effects on the promoter activity, the effect on the expression of cell genes and on the cell proliferation of the E2F dependent gene.

The present invention provides a nucleotide sequence of E2F decoy oligodioxynucleotides of circular structure stable to the nuclease having a glomerular hepatic stem cell proliferation inhibitory ability.

The nucleotide sequence of the E2F decoy oligodioxynucleotide of the circular structure is characterized in that 5'-ATGCGCGAAACGCGTTTTCGCGTTTCGCGCATAGTTTTCT-3 '.

The present invention is a transcription factor E2F / DP involved in the expression and cell proliferation of cell cycle regulatory genes by transfecting glomerular vascular hepatocytes with co-transfected E2F decoy oligodeoxynucleotides and expression vectors of cell cycle regulators E2F and DP. It provides a method of inhibiting the proliferation of glomerular hepatic stem cells by inhibiting the transcriptional activity of the complex.

In the embodiment of the present invention, the statistical processing for all experimental results was performed by variance analysis to verify whether there is a standard difference for each experimental group, and the diversity analysis was analyzed by Duncan's test. When the P value was less than 0.05 after statistical processing, it was determined that there was statistical significance.

Hereinafter, the specific configuration of the present invention will be described through Examples, but the scope of the present invention is not limited to these Examples.

EXAMPLE

Example 1: Investigation of transcriptional activity of glomerular vascular hepatocytes of transcription factor E2F

Before presenting the E2F decoy ODN model of the present invention, the role of the transcriptional factors E2F and transcriptional activity regulator DP on the proliferation of glomerular vascular hepatocytes was investigated.

The glomerular vascular hepatocytes used in the present invention were isolated from Sprague-Dawely rats. First, rats were anesthetized with pentobarbital (50 mg / kg), and then opened, washed both kidneys with physiological saline, and extracted kidneys in phosphate buffered saline (PBS). Film was removed. Take the cortex and separate glomeruli using sieves 210, 150 and 75 μm, centrifuge at 1,500 rpm for 5 minutes, remove supernatant, resuspend the precipitate with 0.2% collagenase and shake at 37 ° C. Incubated for 30 minutes in a shaking water bath. After shaking with vigorous addition of the same amount of trypsin-EDTA, centrifuged, the supernatant was removed, and the pellet was incubated with RPMI1640 culture medium containing 20% fetal bovine serum (FBS), penicillin and streptomycin. . The culture medium was changed every 2-3 days, and 6-10 passages of glomerular vascular stem cells were used in this experiment.

In order to measure the transcriptional activity of E2F, E2F1, DHFR and PCNA promoter regions including E2F binding sites of glomerular hepatic stem cells were amplified by PCR and cloned into luciferase reporter plasmid (pGL3-Basic). Thereafter, the E2F decoy and the reporter plasmid were co-transfected by the lipofectamine plus method.

The pGL3-DHFR-WT-luciferase (DHFR-WT-luc) reporter contains 230 bp upstream of the dhfr promoter, which contains the transcriptional initiation site, the binding site of E2F and the SP1 binding site (GC boxes I-IV). Doing. The pGL3-DHFR-Sp1-Luciferase (DHFR- (ΔE2F) -luc) reporter contained IV in GC box I but the E2F binding site was removed. In addition, the [E2F] X4 luciferase reporter containing the E2F site consisting of TTTCGCGC and the TATA box of the adenovirus E1B gene was made by subcloning the promoter of chloramphenicol acetyltransferase to the reporter plasmid pGL3-Basic. E2F1-luc and E2F1- (ΔE2F) -luc reporters in the E2F1 promoter were made in the same way, and the [E2F] X4-luc, DHFR, and E2F1 reporter gene plasmids were used to investigate the transcriptional inhibitory effects of E2F decoy ODN.

Luciferase expression was measured using a luminometer using 200 μl of 1 × reporter lysis buffer (Promega, USA).

In addition, 48 hours after transfection with the pCMV expression vector, the cells were observed for the distribution of proteins in cells by immunofluorescence microscopy using antibodies of E2F1 and E2F4.

Immunostaining of glomerular hepatic stem cells was performed according to the following procedure. The isolated glomerular glomerular stem cells were washed with PBS, fixed with 4% paraformaldehyde for 15 minutes, and reacted with 0.2% Triton-X / PBS for 15 minutes. It was then blocked for 60 minutes with 0.1% azide + 0.1% Tween-20 + 2% FBS / PBS. Rabbit polyclonal antibody (1: 100) diluted with E2F1 and E2F4 (Santa Cruz Biotechnology Co.) primary antibody for 1 hour and 3 times in Tris-buffer for 10 minutes After washing with water, the anti-rabbit antibody of rabbit FITC bound as a secondary antibody was diluted 1: 100 and reacted for 1 hour. Next, washed three times with TBS-T, and then reacted with TBS-T containing 1 μg / mL propidium iodide (PI) for 1 hour at 50 ° C., washed with TBS-T, and then It observed and the result is shown in FIG. At this time, the green FITC signal represents the E2F or DP protein, and the red signal represents the DNA content (PI) of the nucleus.

As shown in FIG. 1, it was observed that the fluorescence staining of E2F1 mainly occurred in transfected cell nuclei at about 50-70% or more. E2F1 was not found in the cytoplasm without nuclear staining. On the other hand, E2F4 was evenly distributed throughout the cytoplasm in transfected MC cells and nuclear staining of E2F4 was reduced by 10% in transfected cells. E2F4 shifted to the nucleus when the nuclear localization signal was cotransfected with DP2 having the N-terminus.

In general, E2F proteins with transcriptional activity are divided into two groups, structurally and functionally. Expression of E2F1-3 is increased in the presence of cells and growth promoters, but E2F4 and 5 accumulate intracellularly during differentiation. E2F1-3 has a cyclin A binding domain at the N-terminus and is present in the nucleus of transfected cells, but E2F4 and 5 are known to have no cyclin A binding domain and therefore are present in the cytoplasm.

In the above results, it can be seen that E2F1 and E2F4 transfected to MC cells show similar intracellular positions as in other cells.

In addition, the effect of the transcription factor E2F on the proliferation of glomerular vascular hepatocytes was examined. Figure 2a is a schematic of the E2F or E2F / DP heterodimer complex to observe the promoter structure and activity for E2F. In addition, glomerular hemangioblasts of rats were infected with pCMV-E2F alone or with pCMV-DP. The reporter gene and E2F or DP protein expression plasmids were co-transfected and SV-β-gal (Promega, USA) was co-transfected for transfection. Figure 2b shows a graph of the activity of the promoter luciferase in the glomerular hepatic cells of rats infected with CMV expression plasmid.

As shown in FIG. 2B, the E2F protein elevated the activity of E2F-related promoters (p [E2F4] -luc, pE2F1-luc and pDHFR-luc; p <0.01), and DP protein compared to the E2F plasmid. Promoted activity. However, it was observed that the reporter plasmids without the E2F binding site (pE2F1 (E2F) -luc and pDHFR (E2F) -luc) were not affected by overexpression of E2F or E2F / DP protein.

Example 2 Preparation and Function of the Novel Circular Decoy Oligodioxynucleotide of the Present Invention

According to the results of Example 1, it is conceivable that E2F decoy ODN will inhibit transcriptional activation of E2F-dependent genes through competitive binding to E2F / DP complex in the E2F / DP complex activating gene expression in glomerular hepatic stem cells. Can be. Therefore, the present inventors prepared a new circular decoy ODN including the same binding sequence (TTTCGCGC) as the site to which the E2F transcription factor binds.

Prototype E2F decoy oligodioxynucleotides, phosphothioate E2F decoy oligodioxynucleotides and mutated circular E2F decoy ODNs used in the present invention. The base sequence of the decoy (M-E2F decoy) oligodioxynucleotide is as follows:

R-E2F decoy ODN: 5'-ATGCGCGAAACGCGTTTTCGCG TTTCGCGC ATAGTTTTCT-3 ';

PS-E2F decoy ODN: 5'-ATsTTAAG TTTCGCGC CCTTTsCs-3 ';

M-E2F decoy ODN: 5'-ATAATCTAAACGCGTTTTCGCG TTTAGATT ATAGTTTTCT-3 '.

The R-E2F decoy ODN formed a stem-loop structure, and the underlined portions show consensus sequences. After adding T4 DNA ligase (1 unit) and reacting at 16 ° C. for 24 hours, covalently bound circular dumbbell decoy molecules were obtained.

48 hours prior to transfection of glomerular hepatic stem cells obtained above, the cells were incubated for 48 hours, washed with Opti-MEM (Gibco BRL), and then the mass ratio was Lipofectamine Plus ^TM (Lipofectamine Plus) with DNA: lipid = 1: 3. ^TM , Gibco BRL) were transfected with various concentrations of decoy ODN (1,10,100,300 nm) bound to Gibbco BRL. E2F and DP pCMV expression vectors were used for transient expression of E2F protein and DP protein. 5 × 10 ⁵ cells / mL of cells were transfected into 6-wells with E2F decoy, respectively, mixed with Lipofectamine Plus ^™ . After incubation for 5 hours, the cells were exchanged with RPMI1640 medium containing 17% FBS for 24 hours.

Experimental Example 1: Stability test of the E2F decoy oligodioxynucleotide of the circular structure of the present invention

In order to confirm the stability of the E2F decoy of the circular structure produced in the above experiment was carried out as follows. Human serum, FBS, exonuclease III and S1 nucleases were reacted with PS-E2F decoy ODN and R-E2F decoy ODN at 1 μg each. Human serum and FBS were diluted with the addition of 50% ODN and reacted at 37 ° C. for 24 hours. In addition, exonuclease III (Takara, Japan) added 160 units / μg oligodioxynucleotide and reacted for 2 hours at 37 ° C. For S1 nuclease, 10 units / μg added oligodioxynucleotide and It was made to react at 30 degreeC. Each reaction was treated with phenol chloroform and then identified on a 15% polyacrylamide gel. Figure 3a shows the structure of the E2F decoy ODN comprising the E2F binding site in the double-stranded DNA, Figure 3b shows the structure of E2F decoy ODN in S1 nuclease, exonuclease III, human serum or fetal bovine serum The results of experimenting with stability are shown. In the gel photograph of FIG. 3, C is circular E2F decoy ODN, R is circular dumbbell E2F decoy ODN, and P is phosphorothioated E2F decoy ODN.

As shown in FIG. 3B, R-E2F and PS-E2F decoy ODN were resistant to S1 nuclease, while R-E2F decoy ODN was resistant to exonuclease but PS-E2F decoy ODN was exonuclease It was observed to dissolve completely at. In addition, PS-E2F decoy ODN was significantly degraded when incubated for 24 hours in human serum and FBS experiments, but R-E2F decoy ODN was more stable than PS-E2F decoy ODN when incubated with serum for 24 hours.

Experimental Example 2 Effect of the R-E2F Decoy ODN of the Invention on the Promoter Activity of the E2F-dependent Gene

In order to confirm E2F-dependent gene expression regulation through the E2F / DP complex, the reporter gene containing E2F binding site and E2F decoy ODN were transformed in glomerular vascular hepatocytes according to the method of Examples 1 and 2, and then E2F in the promoter. The inhibitory effect of E2F decoy ODN was observed on the promoter activity of [E2F] * 4-Luc, a reporter gene plasmid containing binding sites.

As shown in FIG. 4, simultaneous transfection of E2F decoy ODN reduced gene expression of luciferase by overexpression of E2F1 and DP1. It was also observed that R-E2F decoy ODN was more effective than PS-E2F decoy ODN in inhibiting gene expression ( p <0.05).

In order to observe the dependent E2F decoy ODN on E2F1 / DP1 of E2F1 and dhfr promoter, having a mutant E2F E2F1 and dhfr promoter luciferase plasmid results, E2F1 and dhfr promoter plasmid for reporter gene experiments, the transfected There was a significant decrease in luciferase activity by E2F decoy ODN. The mutated decoy ODN had little effect. Similarly, R-E2F decoy ODN is more effective than PS-E2F decoy ODN in the inhibition of the activity of the [E2F * 4] promoter luciferase plasmid. That is, the new circular E2F decoy more effectively inhibited the promoter activity of the E2F transcription factor than the conventional E2F decoy.

Experimental Example 3: Effect of R-E2F Decoy ODN of the Invention on Cell Gene Expression

E2F decoy ODN inhibits the activity of the E2F / DP complex in the reporter gene, indicating that decoy will increase or decrease the expression of cell cycle regulatory genes in cells. Therefore, Northern blot analysis was performed for the expression of dhfr and PCNA genes, which are used as markers for cell differentiation while being involved in cell cycle progression in response to E2F instead of E2F1. To this end, the probe was labeled with [α- ³² P] dCTP and purified on a NAP-5 column, and RNA was extracted using RNAzol ^B (Tel-Test, Inc.). 10 μg of the extracted RNA was subjected to electrophoresis on a 1% formaldehyde-agarose gel with MOPS buffer, and then, 20 × SSC was used, and then placed on a Hybond-N nylon membrane (Amersham Co., Ltd.). Transfer was carried out for 16 hours using the capillary transfer method. After washing the membrane with 6 × SSC for several minutes, RNA samples were fixed to the membrane in a UV cross-linker (120 mJ / cm ² ) and subjected to prehybridization. The prehybridized membrane was transferred to a solution containing ³² P labeled cDNA probe, reacted with PCNA and DHFR cDNA probe at 65 ° C. for 2 hours, and washed with 2 × SSC / 0.1% SDS solution. The washed membrane was dried at room temperature and then exposed to an X-ray film at -70 ° C for 24-48 hours. The band density on the X-ray was measured for each RNA band density per unit area of 18S rRNA band, which is a standard, and shows the difference in signal intensity with respect to the expression level of each group. At this time, in Figure 5 C is a pCMV-vector control, M is 100 nM M-E2F decoy ODN + pCMV-E2F1 / pCMV-DP1 or pCMV-E2F4 / pCMV-DP2 infected cells, R is 100 nM R Cells infected with E2F decoy ODN + pCMV-E2F1 / pCMV-DP1 or pCMV-E2F4 / pCMV-DP2.

As shown in Figure 5, E2F1 / DP and E2F4 / DP2 can be seen to increase the expression of the PCNA and dhfr genes and M-E2F in the group transfected E2F decoy of the circular structure to inhibit cell cycle progression Compared to the decoy, the expression of PCNA and dhfr E2F / DP complex was significantly suppressed. In contrast, 18S rRNA expression was not affected by E2F decoy ODN.

Experimental Example 4 Effect of R-E2F Decoy ODN of the Present Invention on Glomerular Vascular Hepatocellular Proliferation

The signs of hyperglycemia in renal disease generally resulted in the proliferation of glomerular vascular hepatocytes. Therefore, the proliferation of glomerular vascular hepatocytes was observed to examine the effect of E2F decoy ODN on cell proliferation. To this end, the glomerular vascular hepatocytes isolated in Example 1 were cultured in 96-well so that the cells were in the G0 state. Lipofectamine Plus: Decoy ODN (100 nM) was added to the wells and the cells were incubated at 37 ° C. for 5 hours. After 24-36 hours, cell proliferation between the experimental groups was compared using the WST cell counting kit (Wako, Japan).

As shown in Figure 6, E2F / DP1 and E2F / DP2 stimulated the growth of glomerular hepatic stem cells compared to the control. Transfection of R-E2F decoy ODN over M-E2F decoy ODN significantly inhibited cell proliferation stimulated by the E2F / DP complex.

From the above results, the novel circular structured E2F decoy ODN of the present invention showed a markedly increased stability and an effective inhibitory effect compared to the conventional linear decoy ODN. In addition, inhibition of transactivation activity of E2F protein by using R-E2F decoy ODN significantly inhibited cell cycle regulatory gene expression and cell proliferation. Therefore, R-E2F decoy ODN, which inhibits the transcriptional activity of the transcription factor E2F / DP complex, which plays an important role in the proliferation of glomerulonephritis in the pathogenesis of glomerulonephritis progression, effectively inhibits the proliferation of glomerular hepatic stem cells, thereby effectively diabetic It may be used to prevent and treat sexual nephropathy.

As described through the above Examples and Experimental Examples, the present invention relates to a novel circular structure E2F decoy oligodeoxynucleotide and a method for inhibiting glomerular vascular hepatocyte proliferation using the same, the circular structure E2F decoy oligo stable to the nuclease There is an excellent effect of providing deoxynucleotides (R-E2F decoy ODN). In addition, the present invention inhibits the proliferation of glomerular hepatocytes by inhibiting the expression and cell proliferation of cell cycle regulatory genes by simultaneously transfecting the expression vector of the novel R-E2F decoy ODN and cell cycle regulator E2F into glomerular vascular hepatocyte It provides an excellent way of doing this. Therefore, R-E2F decoy ODN of the present invention can be applied to prevent and treat diabetic nephropathy even in hyperglycemic state by effectively inhibiting the proliferation of glomerular vascular stem cells, which is a very useful invention in the medical industry.

1 is a fluorescence micrograph showing the intracellular location of E2F and DP groups in glomerular vascular hepatocytes of rats into which E2F and DP expression vectors are introduced.

FIG. 2 is a schematic diagram of E2F and DP expression vectors and promoter luciferase activity between E2F and DP proteins in glomerular hemangioblasts of rats to which they were introduced.

Figure 3 shows the structure and stability of the present invention E2F decoy ODN and conventional E2F decoy ODN.

4 is a graph showing the effect of E2F decoy ODN on promoter activity induced by E2F / DP.

Figure 5 shows the effect on E2F dependent gene expression of E2F decoy ODN of the circular structure of the present invention in glomerular hemangioblasts of rats infected with CMV expressing plasmids of E2F-1 / DP-1 or E2F-4 / DP-2. Figure 5a shows the results of Northern blot analysis for PCNA and DHFR, Figure 5b shows the results of quantifying the gene expression of PCNA and DHFR in rat glomerular vascular hepatocytes.

Figure 6 is a graph showing the effect of R-E2F decoy ODN of the present invention on the proliferation of rat glomerular vascular hepatocytes.

<110> Keimyung university CHANG, Youngchae LEE, Inseon <120> Novel Ring-type E2F decoy oligodeoxynucleotides and inhibition method of mesangial cell proliferation using the same <160> 3 <170> KopatentIn 1.71 <210> 1 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Ring-type E2F decoy oligodeoxynucleotide <400> 1 atgcgcgaaa cgcgttttcg cgtttcgcgc atagttttct 40 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Phosphorothioated E2F decoy oligodeoxynucleotide <400> 2 atsttaagtt tcgcgccctt tscs 24 <210> 3 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Mutated ring-type E2F decoy oligodeoxynucleotide <400> 3 ataatctaaa cgcgttttcg cgtttagatt atagttttct 40

Claims (3)

E2F decoy oligodioxynucleotide of circular structure set forth in SEQ ID NO: 1 having glomerular hepatic stem cell proliferation inhibitory ability. Transcription factor E2F involved in cell cycle regulatory gene expression and cell proliferation by simultaneously transfecting glomerular vascular hepatocytes with the expression vector of circular structure E2F decoy oligodioxynucleotide and cell cycle regulator E2F and DP of claim 1 A method of inhibiting proliferation of glomerular hepatic stem cells in mammals other than humans by inhibiting transcriptional activity of the / DP complex. The composition for treating glomerulonephritis according to claim 1 comprising E2F decoy oligodioxynucleotide of circular structure as an active ingredient.