KR101620623B1 - THE shRNA FOR INHIBITING EXPRESSION OF CYCLIN D1 - Google Patents

Abstract

The present invention relates to shRNAs that inhibit cyclin D1 expression, and more particularly to shRNAs for preventing or treating cancer caused by cyclin D1 overexpression.
According to the present invention, shRNA inhibiting cyclin D1 expression and a recombinant vector expressing the shRNA are provided to prevent cancer such as gastric cancer, thyroid cancer, skin cancer, uterine cancer and ovarian cancer caused by overexpression of cyclin D1 Or treatment.

Description

The shRNA inhibiting Cyclin D1 expression (the shRNA expression inhibition expression of CYCLIN D1)

The present invention relates to shRNAs that inhibit cyclin D1 expression, and more particularly to shRNAs for preventing or treating cancer caused by cyclin D1 overexpression.

Gastric cancer is one of the common causes of cancer-related deaths worldwide. The survival rate of patients with gastric cancer has increased with advances in diagnostic and therapeutic technologies, but there is still a problem with prognosis and long-term survival due to the evolutionary stage of diagnosis and high relapse. In addition, the biological heterogeneity of gastric cancer is a major cause of failure in cancer treatment and therefore requires treatment of new gastric cancer.

In normal cells, Cyclin D1 is a cell cycle-regulating kinase that regulates the progression from the G1 phase to the S phase in the cell cycle. However, cyclin D1 overexpressed in cancer cells is known to be closely related to cell cycle disturbance, apoptosis inhibition, poor prognosis, and increased resistance to anticancer drugs. Cyclin D1 regulatory disorders cause tumor progression in animal models, demonstrating a relationship between cyclin D1 and carcinogenesis. Therefore, inhibition of cyclin D1 will be an innovative approach to new cancer therapies.

Target therapy has become an important cancer treatment. Gene targets have emerged as an important treatment for a variety of malignancies in the past decade, but there is little research on target therapy for gastric cancer in comparison to other cancers. RNAi is one of the most potent target therapies that inhibit specific gene expression at the post-transcriptional stage, with two artificial RNAi derivatives of siRNA and shRNA.

siRNA has disadvantages such as inadequate introduction into cells, instability, rapid elimination and ineffective delivery in vivo and clinically, while viral vector-mediated shRNAs have long-term stability and persistent gene silencing It is possible, and most cells can be transfected. The hairpin structure of the shRNA is cleaved by other substances in the cell to become an siRNA. This siRNA is attached to the RNA-induced silencing complex (RISC), and the complex is attached to the mRNA having a region that matches the siRNA cleaves mRNA and inhibits gene expression.

Korean Patent No. 10-1099705 (inhibition of cancer cell growth through cancer protein regulation and anticancer drug sensitization enhancer) is known as a related art for suppressing cancer cell growth through protein regulation, but target treatment using shRNA that inhibits cyclin D1 expression The method has not been disclosed.

It is an object of the present invention to prevent or treat cancer caused by overexpression of cyclin D1 by providing an shRNA that inhibits cyclin D1 expression and a recombinant vector that expresses the shRNA.

In order to accomplish the above object, the present invention provides a short-hairpin RNA (shRNA) which inhibits the expression of cyclin D1 with the nucleotide sequence of SEQ ID NO: 1 as a target sequence.

The present invention also provides an anticancer composition comprising the shRNA as an active ingredient.

The anticancer composition is characterized by inhibiting cancer cell proliferation induced by cyclin D1 overexpression and inducing apoptosis.

The anticancer composition is characterized by preventing or treating gastric cancer, thyroid cancer, skin cancer, uterine cancer or ovarian cancer caused by overexpression of cyclin D1.

In addition, the present invention provides a recombinant vector expressing the shRNA.

Wherein the recombinant vector is a plasmid vector or a viral vector.

At this time, the viral vector is characterized in that it is derived from retrovirus.

The present invention also provides an anticancer composition comprising the recombinant vector as an active ingredient.

The anticancer composition is characterized by inhibiting cancer cell proliferation induced by cyclin D1 overexpression and inducing apoptosis.

The anticancer composition is characterized by preventing or treating gastric cancer, thyroid cancer, skin cancer, uterine cancer or ovarian cancer caused by overexpression of cyclin D1.

According to the present invention, shRNA inhibiting cyclin D1 expression and a recombinant vector expressing the shRNA are provided to prevent cancer such as gastric cancer, thyroid cancer, skin cancer, uterine cancer and ovarian cancer caused by overexpression of cyclin D1 Or treatment.

FIG. 1 shows the effect of ShCCND1 on cyclin D1 expression and cell proliferation inhibition in NCI-N87 cells. (A) is the result of Western blotting in which ShCCND1_3 decreased cyclin D1 protein expression level, and (B) ShKCCND1 is the result of CCK-8 test in which cell proliferation is inhibited. Experimental data are mean ± standard error (** P <0.01 versus the ShScramble-transduced cells).
Figure 2 shows the results of colony formation (A) and scratch-wound healing test (B). Experimental data were mean ± standard error (*** P <0.001 versus the ShScramble-transduced cells).
Figure 3 shows the effect of ShCCND1 on NCI-N87 cell cycle and apoptosis. (A) shows a cell cycle distribution graph in which ShCCND1 has an increased G1 group as compared to ShScramble, and (B) shows a significant increase in ShCCND1 cell death as compared to ShScramble. Experimental data were mean ± standard error (** P <0.01 and *** P <0.001 versus the ShScramble-transduced cells).
Figure 4 shows the effect of ShCCND1 on tumor growth in a xenograft mouse model. (A) is a comparative photograph of ShCCND1 xenografts showing smaller tumor volume than ShScramble xenotransplantation, (B) is a graph showing that tumor volume is reduced compared to ShScramble xenotransplantation during ShCcNDb xenotransplantation, (C) ShCCND1 xenotransplantation shows a decrease in tumor weight compared to ShScramble xenotransplantation. Experimental data were mean ± standard error (** P <0.01 and *** P <0.001 versus the ShScramble-transduced cells).
Figure 5 shows the effect of intratumoral injection of Saline, Virus_Scramble and Virus_CCND1. (A) is a comparative photograph of Virus_CCND1 showing a smaller tumor volume compared to Saline or Virus_Scramble, (B) is a graph showing that tumor volume is reduced compared to Saline or Virus_Scramble during the course of the experiment in case of Virus_CCND1, In the case of Virus_CCND1, the tumor weight is decreased compared to Saline or Virus_Scramble. Experimental data were mean ± standard error (* P <0.05 and ** P <0.01 versus Saline; #P <0.05 versus Virus_Scramble).
Figure 6 shows the effect of tumor injected Saline, Virus_Scramble and Virus_CCND1 on tumor proliferation and apoptosis. In H & E staining, the tumor tissues infected with Virus_CCND1 showed a reduced cleavage pattern compared to Saline and Virus_Scramble. In the TUNEL test, the number of apoptotic cells in Virus_CCND1 treated group was increased compared to Saline and Virus_Scramble. The photograph (A) is 400 magnification and the experimental data are mean ± standard error (*** P <0.001 versus Saline and ### P <0.001 versus Virus_Scramble).

Hereinafter, the present invention will be described in detail.

The present invention provides a short-hairpin RNA (shRNA) that inhibits the expression of cyclin D1 with the nucleotide sequence of SEQ ID NO: 1 as a target sequence.

[SEQ ID NO: 1] 5'-GAAGTTCATTTCCAATCCGCCCT-3 '

The present invention also provides an anticancer composition comprising the shRNA as an active ingredient.

The anticancer composition inhibits cancer cell proliferation induced by overexpression of cyclin D1, induces apoptosis, and has an effect of preventing or treating gastric cancer, thyroid cancer, skin cancer, uterine cancer or ovarian cancer caused by overexpression of cyclin D1 have.

In addition, the present invention provides a recombinant vector expressing the shRNA.

The recombinant vector may be a plasmid vector or a viral vector. Preferably, the viral vector is derived from retrovirus, genus Lentivirus, or human immunodeficiency virus (HIV), which is capable of expressing shRNA in mammalian cells Do.

The present invention also provides an anticancer composition comprising the recombinant vector as an active ingredient.

The anticancer composition inhibits cancer cell proliferation induced by overexpression of cyclin D1, induces apoptosis, and has an effect of preventing or treating gastric cancer, thyroid cancer, skin cancer, uterine cancer or ovarian cancer caused by overexpression of cyclin D1 have.

The present inventors confirmed that the growth rate, motility and colony forming ability of NCI-N87 cancer cells inhibited the expression of cyclin D1 by shRNA in in vitro experiments, and that shRNA and 5-FU, which are cyclin D1- In the mixed treatment, the survival rate of stomach cancer cells was significantly lower than that of single treatment, suggesting that inhibition of cyclin D1 expression increased sensitivity to 5-FU treatment. Decreased expression of pAKT and pNFκB, decreased G1 arrest, and increased apoptosis support this finding.

In vivo anticancer effects of cyclin D1 - targeted lentivirus - mediated shRNAs in vitro were also examined. First, NCI-N87 gastric cancer cells that continuously express shRNA were xenotransplanted into nude mice to observe an effective gastric cancer growth inhibitory effect, and cyclin D1-target shRNAs proved in vivo anti-cancer effect were administered directly into the tumor by lentivirus The inhibition of growth was significantly inhibited in the lentivirus treated group expressing shRNA compared to the control group. This is consistent with the in vitro results and is also supported by the results of apoptosis staining of the tumor.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not construed as being limited by these embodiments.

Experimental Example 1. Cell line and cell culture

NCI-N87 cancer cells were purchased from Korean Cell Line Bank (KCLB, Seoul, Korea) and stored in RPMI 1640 containing 10% FBS and 1% penicillin / streptomycin in a 5% carbon dioxide chamber at 37 ° C.

293TN human kidney-producing cell lines were purchased from System Biosciences (SBI, Mountain View, Calif., USA) and cultured in DMEM high glucose (DMEM) supplemented with 10% FBS, glutamax and 1% penicillin / streptomycin in a 5% high-glucose medium.

Experimental Example 2. Plasmid DNA expressing ShRNA

Three different target sequences corresponding to cyclin D1 were designed using a lentiviral expression vector (pGreenPuro ^™ Vector, SBI). The target region of the human gene encoding cyclin D1 is as follows.

ShCCND1_1 sense strand, 5'-GCCCTCGGTGTCCTACTTCAAAT-3 ', ShCCND1_1 antisense strand, 5'-ATTTGAAGTAGGACACCGAGGGC-3';

ShCCND1_2 sense strand, 5'-GCACGATTTCATTGAACACTTCC-3 ', ShCCND1_2 antisense strand, 5'-GGAAGTGTTCAATGAAATCGTGC-3';

ShCCND1_3 sense strand, 5'-GAAGTTCATTTCCAATCCGCCCT-3 ', ShCCND1_3 antisense strand, 5'-AGGGCGGATTGGAAATGAACTTC-3'.

The plasmid DNA has the circular sequence 5'-CTTCCTGTCAGA-3 'and is transcribed into shRNA.

Negative control sequences that did not correspond to human proteins were designed in the same way.

ShScramble sense strand, 5'-GACTTCATAAGGCGCATGC-3 ', ShScramble antisense strand, 5'-GCATGCGCCTTATGAAGTC-3'.

E. coli DH5α was transformed with each of the above DNAs, and the ligation product was isolated by PCR purification and sequencing using a plasmid purification kit (Qiagen, Valencia, USA).

Experimental Example 3: Lenti virus and NCI-N87 cell line stably expressing shRNA

293TN cells were inoculated into a 10 cm culture plate at a density of 3 × 10 ⁶ cells. After 24 hours, the plasmid having ShCCND1 or ShScramble was transfected with the lentiviral vector expression construct using lipofectamine. After 48 hours, collect the supernatant it PEG- ^TM virus was added to the solution to precipitate the supernatant / PEG- it The mixture was centrifuged for 30 minutes at 4 ℃ to 1,500 × g and resuspended in RPMI medium at a 1/100 volume of the original (resuspend).

One day before transfection, NCI-N87 cells were plated in 24-wells at 5 × 10 ⁴ cells. After 24 hours, NCI-N87 cells were infected with lentivirus containing ShCCND1 or ShScramble and cultured in RPMI 1640 medium containing 1 ㎍ / ml of puromycin the following day. Transfection efficiency of NCI-N87 cells with GFP signal was analyzed by FACS (flow cytometry).

Experimental Example 4. Western blotting

Stable cancer cells (NCI-N87, ShScramble and ShCCND1) a protease inhibitor (Sigma-Aldrich, St. Louis, MO, USA) is eluted in a buffer solution containing RIPA ^{BCA TM protein assay kit (Thermo Scientific} , Rockford, IL, USA) was used to measure the protein concentration. The total amount of protein was electrophoresed on 10% SDS-PAGE and transferred to nitrocellulose membrane. Cells were treated with cyclin D1 (1: 500, Santa Cruz Biotechnology, Santa Cruz, Calif. Cells were incubated at 4 ° C with antibody to β-actin (1: 500, Santa-Cruz Biotechnology). The membranes were then incubated with HRP-conjugated anti-rabbit or anti-mouse secondary antibodies (1: 1000, Santa-Cruz Biotechnology) and signals were measured with ECL Test Kit (KPL, Gaithersburg, MD, USA) . Β-actin was used as an internal standard, and concentration analysis was performed with ImageJ software.

Experimental Example 5. Cell proliferation and colony formation

In vitro cell viability was analyzed by CCK-8 assay (Dojindo Laboratories, Kumamoto, Japan). NCI-N87 cells, ShScramble-introduced cells, and ShCCND1-introduced cells were inoculated into 96-well plates at a density of 10 ⁴ cells / well. After 24 hours, 10 의 of CCK-8 was added to each well and incubated for 2 hours. Absorbance (Naitoh, # 25) was observed on days 1, 2, 3 and 4 through ELISA (Tecan Sunrise, Sunnyville, CA, USA)

To measure colony forming ability, shRNA-containing NCI-N87 cells were inoculated on a 6-well plate at a density of 500 cells / well. After 3 weeks, the cells were stained with 1% crystal violet and the number of colonies (? 25 cells) was measured under a microscope. Image analysis was performed with Metamorph version 7.5.6.0 software (Molecular Devices, Sunnyvale, CA, USA).

Experimental Example 6: Scratch-wound healing assay

Scratch wound healing tests were performed to investigate the effect of cyclin D1 on cell mobility. Is an NCI-N87 cells at a back plate inoculated with 5 × 10 ⁴ cells in 60mm plates were then scratch the pipette tip when it almost covered. After 48 hours, the images of the cells migrated to the injured area were observed through a Zeiss Axiovert 200 inverted microscope (Carl Zeiss MicroImaging, Thornwood, NY, USA) with a 10 × objective and the wound area was analyzed using Metamorph version 7.5.6.0 software Respectively.

Experimental Example 7. Cell cycle and apoptosis

To measure cell cycle distribution, 10 ⁶ NCI-N87 cells were washed twice with cold PBS and resuspended in PBS containing RNase A (Sigma-Aldrich) and PI (propidium iodide, Sigma-Aldrich). The cells were incubated at 37 ° C for 1 hour with blocking of light. The percentage of cells per cell cycle was measured using FACSCalibur (Becton-Dickinson, Rutherford, NJ, USA) and analyzed with CELLQUEST software (Becton-Dickinson).

100 μl aliquots of 10 ⁶ cells / ml were incubated with Annexin-V-APC (allophycocyanin, BD Biosciences Pharmingen, San Diego, CA, USA) and PI (BD Biosciences Pharmingen) And cultured. Then, 400 μl of binding buffer solution was added, and data obtained after FACS (flow cytometry) analysis was analyzed by CELLQUEST software.

Experimental Example 8. Tumor xenotransplantation in nude mice

Five-week-old male athymic BALB / c nude mice were purchased from Nara Bio animal center (NARA Biotech, Seoul, Korea). Then, NCI-N87 cell line containing ShScramble or ShCCND1 was cultured in RPMI 1640 medium, and cells (8 x 10 ⁶ ) contained in 100 μl saline were subcutaneously injected into the right thigh. Tumors were measured by tumor diameter three times a week with digital vernier caliper from day 9 to day 35 after tumor injection. Tumor volume was calculated as d ² × D / 2 (d and D mean the minimum and maximum diameters, respectively).

EXPERIMENTAL EXAMPLE 9 Intratumoral injection of viral load and lentivirus mediated ShCCND1.

NCI-N87 cells were inoculated on a 6-well plate at a density of 4 × 10 ⁵ cells / well. After 24 hours, the cell culture medium was replaced with RPMI 1640 medium containing hexadimethrine bromide. Then, serial diluted lentivirus stocks were added to pre-plated NCI-N87 cells and cultured for 48 hours and analyzed by FACS (flow cytometer). GFP-positive NCI-N87 cells were detected by FACS. The virus titration was calculated as IU (infectious unit) / ml. IU / ml = {(cell number at starting time) x (dilution factor) x (percent infection)} / (added virus volume expressed in mL)

To measure the inhibitory effect of ShCCND1-containing virus particles on tumors, 100 μl of saline containing 4 × 10 ⁶ NCI-N87 cells in 5-week-old male athymic BALB / c nude mice (NARA Biotech, N = 20) Was subcutaneously injected into the right thigh. When tumor volume reaches 80 to 100 mm ³ (usually 6 to 8 days after injection), 50 μl (4 × 10 ⁷ IU / ml) of saline (Saline, N = 6) and a virus containing ShScramble (Virus_Scramble, N = 7) or ShCCND1 (Virus_CCND1, N = 7). After one week, Saline, Virus_Scramble or Virus_CCND1 were reinjected into the tumor mass, respectively.

Experimental Example 10: H & E and TUNEL staining

After 13 days of first intra-tumor injection, subcutaneous tumors were fixed with 10% neutralized formalin at the expense of mice, and normal paraffin sections and H & E staining were performed for histopathological experiments with fixed tumors. For quantification of mitosis, the cleavage phase was measured in five random fields (400x) per slide and expressed as the mean number of cleavage per field.

Cell apoptosis was determined by the manufacturer (ApopTag, Chemicon International Inc., Temecula, Calif., USA) and the number of dead cells was measured in 5 fields per slide (400 ×) using an image analyzer (Metamorph version 7.5. 6.0 software) and the data was expressed as an average number per field.

Experiment result.

(1) Cyclin D1 expression and cell proliferation inhibitory effect of ShCCND1 in NCI-N87 cells.

NCI-N87 cells infected with lentivirus were successfully isolated in a medium containing 1 ug / ml of puromycin. The transfection efficiency of each cell line analyzed by flow cytometry was 97.41% (ShScramble) and 95.8% (ShCCND1), and Western blotting was performed to analyze the effect of ShCCND1 on cyclin D1 expression level Western blotting was performed.

As a result, there was no difference between NCI-N87 cells and ShScramble cells at the level of cyclin D1 expression. ShCCND1_1 and ShCCND1_2 did not decrease the cyclin D1 protein compared to the control, whereas ShCCND1_3 decreased the cyclin D1 protein appear. A significant reduction (P < 0.01) was confirmed by Western blot densitometric analysis (see Figure 1A). ShCCND1_3 transfected cells were used for subsequent experiments and are referred to as ShCCND1.

The expression level of pRB, a downstream molecule of cyclin D1, in ShCCND1 was significantly inhibited. These data indicate that lentiviral mediated ShCCND1 effectively inhibits endogenous cyclin D1 expression in NCI-N87 cells. To test the silencing effect of cyclin D1 in cell proliferation, cell viability was analyzed using the CCK-8 test. As a result, as shown in FIG. 1B, ShCCND1 inhibited cell proliferation significantly in NCI-N87 cells compared with ShScramble, and inhibition of proliferation by ShCCND1 was more significant (P <0.01) at the 4th day of culture. The survival rate of cells transfected with ShCCND1 was lower than that of cells transfected with ShScramble during the experiment, indicating that ShCCND1 inhibits NCI-N87 human gastric cancer cell proliferation.

(2) Colony formation and cell mobility inhibition effect of ShCCND1 in NCI-N87 cells.

As shown in FIG. 2A, ShCCND1 (N = 21.7) showed ShScramble (N = 33.0, P < 0.05) as a result of colony formation test and examined whether ShCCND1 influenced colony formation which is an important feature of tumor growth in vivo. ). &Lt; / RTI &gt; These results suggest that cyclin D1 silencing inhibits cell proliferation and reduces the ability of cells to colonize in cancer formation in nude mice.

In addition, AGS cell mobility was analyzed by a scratch wound healing test, since the reduced colony forming ability of the cells is usually related to the ability to penetrate cancer cells. As a result, as shown in FIG. 2B, ShCCND1 (30.1%) was found to have a wider area than ShScramble (21.9%). The wound area of ShCCND1 was significantly lower than that of ShScramble, indicating that ShCCND1 transfected cells had inhibited cell migration ability.

(3) Effect of ShCCND1 on cell cycle and apoptosis.

To determine the mechanism of ShCCND1 inhibition of cell proliferation, cell cycle distribution was analyzed. As a result, ShCCND1 showed no significant change in the number of G2 cells, while it showed an increase in G1 phase (P < 0.001) compared to ShScramble by decreasing the number of S-phase cells (P = 0.058). The G1 group of ShCCND1 was distributed in 67.2% and the G1 group of ShScramble was distributed in 50.17%. These results indicate that ShCCND1 causes G1 arrest and inhibits cell proliferation.

As shown in FIG. 3B, ShCCND1-introduced cells showed a significant increase in apoptosis in comparison with NCI-N87 cells. The cell suicide rate of ShScramble was 61.7% while that of ShCCND1 was 88.4%. Therefore, it was found that knockdown of cyclin D1 increased suicidal cell death in NCI-N87 cells.

(4) Effect of ShCCND1 on tumor growth in xenotransplantation mouse model.

ShCCND1 inhibited cyclin D1 expression and tumor cell growth in vitro. In addition, a tumor xenograft model was used to test the inhibitory effect of ShCCND1 on tumor cell growth in vivo. As shown in FIG. 4, the tumor was first observed on day 9, and the tumor growth inhibitory effect of ShCCND1 was observed as the date passed. At the end of the experiment, the volume of the tumor was 2,348.2 ± 318.9 mm ³ for ShScramble, 1,115.0 ± 457.7 mm ^{3 for} ShCCND1 (P <0/001), and the mean tumor weight was 2.3 ± 0.5 g for ShScramble Whereas for ShCCND1, it was 0.6 ± 0.6 g. This result implies that lentiviral mediated ShCCND1 inhibits tumor growth of NCI-N87 cells in nude mice.

(5) tumor growth inhibitory effect of injected lentiviral mediated ShCCND1 in tumor.

NCI-N87-derived cancers were infected with Virus_Scramble and Virus_CCND1, respectively, in order to confirm that lentivirus effectively infected the tumor and inhibited their growth. Specifically, when the tumor reached 80-100 mm ³ volume, 50 μl of Saline, Virus_Scramble and Virus_CCND1 were infected, respectively. The virus titre of Virus_Scramble and Virus_CCND1 was 4 × 10 ⁷ IU / ml.

The volume of NCI-N87-derived cancers infected with Virus_CCND1 decreased by 45.6% (P <0.01) and 37.5% (P <0.05) compared to Saline and Virus_Scramble, respectively 5). Weights were also reduced by 44.7% (P <0.05) and 34.9% (P <0.05) compared to Saline and Virus_Scramble, respectively. Therefore, it was found that the growth of the infected tumor was significantly inhibited by Virus_CCND1.

(6) Effect of injected lentiviral mediated ShCCND1 on tumor proliferation and apoptosis in tumor.

To confirm the biological effect of Virus_CCND1, the xenograft was stained with H & E or TUNEL. The virus_CCND1-treated group showed decreased mitotic figures and increased TUNEL-positive cell numbers compared to Saline and Virus_Scramble. The cyclin D1 target treatment using lentiviral mediated ShCCDN1 inhibited cell proliferation in human gastric cancer in vivo and induced cell suicide Respectively.

Having described specific portions of the present invention in detail, it will be apparent to those skilled in the art that this specific description is only a preferred embodiment and that the scope of the present invention is not limited thereby. It will be obvious. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> Konkuk University Industrial Cooperation Corp <120> THE shRNA FOR INHIBITING EXPRESSION OF CYCLIN D1 <130> P13-E356-01 <140> 10-2013-0127574 <141> 2013-10-25 <160> 1 <170> KoPatentin 3.0 <210> 1 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> This artificial sequence is the shRNA for inhibiting expression          of cyclin D1 <400> 1 gaagttcatt tccaatccgc cct 23

Claims (10)

A short-hairpin RNA (shRNA) that inhibits the expression of cyclin D1 using the nucleotide sequence shown in SEQ ID NO: 1 as a target sequence.
An anticancer composition comprising the shRNA of claim 1 as an active ingredient.
3. The method of claim 2,
Wherein said anticancer composition inhibits cancer cell proliferation induced by cyclin D1 overexpression and induces apoptosis.
3. The method of claim 2,
Wherein said anticancer composition is for preventing or treating gastric cancer, thyroid cancer, skin cancer, uterine cancer or ovarian cancer caused by overexpression of cyclin D1.
A recombinant vector expressing the shRNA of claim 1.
6. The method of claim 5,
Wherein the recombinant vector is a plasmid vector or a viral vector.
The method according to claim 6,
Wherein said viral vector is derived from a retrovirus.
An anticancer composition comprising the recombinant vector of claim 5 as an active ingredient.
9. The method of claim 8,
Wherein said anticancer composition inhibits cancer cell proliferation induced by cyclin D1 overexpression and induces apoptosis.
9. The method of claim 8,
Wherein said anticancer composition is for preventing or treating gastric cancer, thyroid cancer, skin cancer, uterine cancer or ovarian cancer caused by overexpression of cyclin D1.

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