COMPOSITION FOR CANCER CELL SENSITIZATION CONTAINING AS ACTIVE INGREDIENT SUBSTANCE INHIBITING EXPRESSION OF ONCOGENE OF HPV VIRUS

Abstract

The present invention relates to a composition for cancer cell sensitization containing as an active ingredient a substance inhibiting the expression of an oncogene of a HPV virus, and more specifically, to a composition for radiosensitization which is used for the treatment of cancer induced by HPV infection and contains as an active ingredient oligonucleotide having any one nucleic acid sequence selected from the group consisting of sequence numbers 1 to 10. The oligonucleotide of the present invention inhibits the expressions of E6 and E7 which are HPV virus oncoproteins, thereby inhibiting the proliferation of tumor cells, and enhances the sensitivity of tumor cells to radiation, thereby exhibiting an excellent effect as a sensitizer that can maximize the cell death effect.

Claims

1. A sensitizing composition for treating HPV (human papilloma virus)-induced cancer with radiation therapy, comprising an oligonucleotide having any one of the nucleic acid sequences selected from the group consisting of SEQ ID NOs: 1 to 10 as an active ingredient.

2. The composition according to claim 1, wherein the oligonucleotide is a small interfering RNA (siRNA) complementarily hybridized with a sense oligonucleotide and a complementary antisense oligonucleotide.

3. The composition according to claim 1, wherein HPV (human papilloma virus)-induced cancer is selected from the group consisting of cervical cancer, vaginal cancer, vulvar cancer, anal cancer, penis cancer, tonsillar cancer, pharynx cancer, laryngeal cancer, head & neck cancer, and adenocarcinoma of lung.

4. The composition of claim 1, wherein the composition is administered sequentially or concurrently with radiation therapy.

5. The composition of claim 1, wherein the composition is for concurrent chemoradiation therapy.

6. The composition of claim 1, wherein said composition comprises all of siRNA molecules hybridized with SEQ ID NOs: 1 and 2, and siRNA molecules hybridized with SEQ ID NOs: 3 and 4.

7. The composition of claim 1, wherein said composition comprises all of siRNA molecules hybridized with SEQ ID NOs: 5 and 6, siRNA molecules hybridized with SEQ ID NOs: 7 and 8, and siRNA molecules hybridized with SEQ ID NOs: 9 and 10.

8. A sensitizing composition for treating HPV (human papilloma virus)-induced cancer with radiation therapy, comprising an oligonucleotide having any one of the nucleic acid sequences selected from the group consisting of SEQ ID NOs: 1 to 10 as an active ingredient.

9. (canceled)

10. A sensitizing method for treating HPV (human papilloma virus)-induced cancer with radiation therapy, the method comprising administering to a subject in need thereof an effective amount of a composition which comprises an oligonucleotide having any one of the nucleic acid sequences selected from the group consisting of SEQ ID NOs: 1 to 10.

11. The method according to claim 10, wherein the oligonucleotide is a small interfering RNA (siRNA) complementarily hybridized with a sense oligonucleotide and a complementary antisense oligonucleotide.

12. The method according to claim 10, wherein HPV (human papilloma virus)-induced cancer is selected from the group consisting of cervical cancer, vaginal cancer, vulvar cancer, anal cancer, penis cancer, tonsillar cancer, pharynx cancer, laryngeal cancer, head & neck cancer, and adenocarcinoma of lung.

13. The method of claim 10, wherein the composition is administered sequentially or concurrently with radiation therapy.

14. The method of claim 10, wherein the composition is for concurrent chemoradiation therapy.

15. A method of for treating HPV (human papilloma virus)-induced cancer with radiation therapy, the method comprising steps (a) administering to a subject in need thereof an effective amount of a composition which comprises an oligonucleotide having any one of the nucleic acid sequences selected from the group consisting of SEQ ID NOs: 1 to 10; and (b) performing a radiotherapy on said subject.

16. The method according to claim 15, wherein HPV (human papilloma virus)-induced cancer is selected from the group consisting of cervical cancer, vaginal cancer, vulvar cancer, anal cancer, penis cancer, tonsillar cancer, pharynx cancer, laryngeal cancer, head & neck cancer, and adenocarcinoma of lung.

17. The method of claim 15, wherein the radiation therapy is concurrent chemoradiation therapy.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0061] FIG. 1 is a graph showing the inhibitory effect on cell proliferation in a cervical cancer cell line infected with HPV type 18 or type 16 virus by siRNA treatment of the present invention;

[0062] (FIG. 1A: Effect on HPV type 18 infected-HeLa cell line, Comparative Example (1, 2, 3, 4, 6 and 7), siRNA 426 hybridized with SEQ ID NOs: 1 and 2 (5)

[0063] (FIG. 1B: Effect on HPV type 16 infected-SiHa cell line, Comparative Example (1, 3, and 4), siRNA 497 hybridized with SEQ ID NOs: 9 and 10 (2))

[0064] FIG. 2 shows the results of evaluation of expression of TP53 and E7 proteins in cervical cancer cell lines infected with HPV type 18 or type 16 virus by siRNA treatment of the present invention (FIG. 2A: effect on HPV type 18 infected-HeLa cell line, FIG. 2B: effect on HPV type 16 infected-SiHa cell line).

[0065] FIG. 3 shows the results of evaluation of expression of E6 mRNA in cervical cancer cell lines infected with HPV type 18 or type 16 virus by siRNA treatment of the present invention (FIG. 3A: effect on HPV 18 infected-HeLa cell line, FIG. 3B: effect on HPV 16 infected-SiHa cell line).

[0066] FIG. 4 shows the results of evaluation of expression of CDKN1A mRNA in a cervical cancer cell line infected with HPV type 18 or type 16 virus by siRNA treatment of the present invention (FIG. 4A: effect on HPV 18 infected-HeLa cell line, FIG. 4B: effect on HPV 16 infected-SiHa cell line).

[0067] FIG. 5 shows the results of evaluating the plasma stability of the siRNA of the present invention (FIG. 5A: 426 siRNA, FIG. 5B: 497 siRNA).

[0068] FIG. 6 is a graph showing the inhibitory effect of the combination treatment of siRNA of the present invention and radiation on cell proliferation in a cervical cancer cell line infected with HPV type 18 or type 16 virus (FIG. 6A: effect on HPV type 18 infected-HeLa cell line, FIG. 6B: effect on HPV type 16 infected-SiHa cell line).

[0069] FIG. 7 shows the results of evaluating the expression of TP53 and E7 proteins in cervical cancer cell lines infected with HPV type 18 or type 16 virus by the combination treatment of siRNA of the present invention and radiation (FIG. 7A: effect on HPV type 18 infected-HeLa cell line, FIG. 7B: effect on HPV type 16 infected-SiHa cell line).

[0070] FIG. 8 shows the results of evaluating the apoptosis-inducing effect of cervical cancer cell lines infected with HPV type 18 or type 16 virus by the combination treatment of siRNA of the present invention and radiation (FIG. 8A: effect on HPV type 18 infected-HeLa cell line, FIG. 7B: effect on HPV type 16 infected-SiHa cell line).

[0071] FIG. 9 shows the results of evaluating the effect of inducing cell senescence in cervical cancer cell lines infected with HPV type 18 or type 16 virus by the combination treatment of siRNA of the present invention and radiation (FIG. 9A: effect on HPV type 18 infected-HeLa cell line, FIG. 9B: effect on HPV type 16 infected-SiHa cell line).

DETAILED DESCRIPTION OF THE INVENTION

[0072] Hereinafter, the present invention will be described in detail.

[0073] However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Experimental Method

[0074] 1. Cell Lines and Western Blotting

[0075] HeLa cells are HPV18-positive human cervical cancer cell lines. HeLa-Luc (stably transfected bioluminescent human tumor cell line derived from HeLa cell line) was obtained from Xenogen Corp. (Alameda, Calif., USA). The HPV16-positive human cervical cancer cell line, SiHa, was obtained from American Type Culture Collection (Manassas, Va., USA) and validated through short nucleotide sequence repeat. Cells were routinely tested for mycoplasmas.

[0076] Whole-cell lysate was extracted using RIPA buffer (10 mM Tris (pH.4), 0.15 M NaCl, 5 mM EDTA, 1% Triton X-100, 0.5% deoxycholic acid sodium salt and 0.1% SDS). After centrifugation, the protein concentration of the supernatant was measured using a BCA protein assay kit (Thermo Fisher Scientific Inc). Protein samples were placed in sample buffer for complete denaturation and boiled for 5 minutes. The samples were then applied to 6%, 10%, or 15% polyacrylamide gels and transferred to a 0.4 m PVDF membrane (Milipore, Bilecica). After transfer, the membranes were blocked with 5% skim milk, incubated with appropriate concentrations of TP53, E7, -actin primary antibody and horseradish peroxidase-conjugated IgG. Finally, the expression level of the protein was detected using ECL solution.

[0077] 2. siRNA Transfection and Ray-Irradiation

[0078] siRNA for HPV18 E6/E7 (426, 450), HPV16 E6/E7 (366, 448, 497) and control siRNA were synthesized by BIONEER (Dae-joen, Korea). siRNA transfection was performed with DharmaFect (Dharmacon, Lafayette, Colo., USA) and performed according to manufacturer's instructions. Chemically modified (2-sugar modification by 2-OMe, 2-F) siRNA duplex derivatives were designed and synthesized by BIONEER (Dae-jeon, Korea). All siRNAs were diluted with DEPC treated water and the final concentration was 5 ug/uL. For irradiation, the cells were cultured at a density of 510.sup.5 cells/100 mm dish. Twenty-four hours later, cells were plunged into a gamma ray of 2-3 GY from a GC 3000 Elan irradiator (MDS Nordion, Canada). On the next day, the cells were trypsinized and re-plated at a density of 310.sup.5 cells/100 mm dish.

[0079] 3. Flow Cytometry Analysis and Detection of Senescence-Associated -Galactosidase (SA--Gal)

[0080] Apoptotic cells were stained with annexin V-fluorescence isothiocyanate (FITC) and Propidium iodide (BD PharMingen, San Diego, Calif., USA) according to manufacturer's instructions and quantitated by flow cytometry. For senesence analysis, the cells were fixed in 2% formaldehyde, 2% glutaraldehyde, and cells were stained at pH 6.0 using X-Gal (5-bromo-4-chloro-3-indolyl--D-galactopyranoside) for senesence analysis. The SA--Gal positive cells immediately underwent cell count in three representative fields.

[0081] 4. Quantitative Real-Time PCR Analysis

[0082] Total RNA was extracted from cells by Trizol RNA isolation method (Invitrogen, Carlsbad, Calif., USA) and then reverse transcribed using oligo-dT primer. Primers and probes for PCR were prepared in TIB MOLBIOL (Berlin, Germany). qRT-PCR was performed using a LightCycler Real-time PCR detection system (Roche Diagnostics, Basel, Switzerland). The expression levels of mRNA in cells and tissues were measured at 530 nm and 705 nm, respectively. The PCR conditions were as follows: 95 C. for 10 minutes, 95 C. for 10 seconds, 45 cycles repeated, 55 C. for 30 seconds, and finally 40 C. for 30 seconds.

[0083] 5. Serum Stability

[0084] The siRNA double strand (9 ug) was maintained at 90 uL of 10% human serum at 37 C. 12 ul of samples were obtained at each time point (0, 1, 2, 3, 4, 24 h), respectively, and frozen and stored at 70 C. immediately. RNA was isolated using unmodified 15% polyacrylamide-TBE, followed by EtBr staining and imaging by UV-transilluminator.

Example 1

[0085] Production of E6/E7 siRNA of HPV Types 16 and 18

[0086] The inventors of the present invention prepared siRNAs for E6/E7 oncogenic proteins of HPV types 16 and 18 as shown in the following Table. In the following Table, the siRNA sequence indicated by m represents a base substituted with a 2-O-Me modified nucleotide in which a methyl group is bound to a base residue. That is, in case of 2-0-Me modified U, it is indicated as mU, or in case of 2-O-Me modified G. it is indicated as mG

TABLE-US-00001 TABLE1 siRNAforHPVtype16and18 SEQID name NO: sequence HPVtype18 SEQID 5-CAACCmGAmGCACmGACAmGmGAA-3 NO:1 siRNA426 SEQID 5-UUCCUGUCGUGCUCGGUUG-3 NO:2 HPVtype18 SEQID 5-CCAACmGACmGCAmGAmGAAACA-3 NO:3 siRNA450 SEQID 5-UGUmUUCUCmUGCGmUCGmUUGG-3 NO:4 HPVtype16 SEQID 5-GCAAAGACAUCmUmGmGACAAA-3 NO:5 siRNA366 SEQID 5-UUUGUCCAGAUGUCUUUGC-3 NO:6 HPVtype16 SEQID 5-UCAAmGAACACmGUAmGAmGAAA-3 NO:7 siRNA448 SEQID 5-UUUCUCUACGUGUUCUUGA-3 NO:8 HPVtype16 SEQID 5-GACCGGUCGAUGUAUGUCUUG-3 NO:9 siRNA497 SEQID 5-AGACAmUACAmUCGACCGGmUCCA-3 NO:10

Comparative Example

[0087] Production of E6/E7 siRNA of HPV Types 16 and 18

[0088] The inventors of the present invention compared HPV type 18 siRNA 426 and HPV type 16 siRNA 497 prepared in Example 1 with siRNAs chemically modified in different bases as shown in Table 2 below. In the following Table, an siRNA sequence indicated by m represents a base substituted with a 2-O-Me modified nucleotide in which a methyl group is bound to a base residue, and the siRNA sequence designated f refers to a 2-fluorinated base. That is, in the case of 2-O-Me modified U, it is indicated as mU. In the case of 2-O-Me modified G, it is indicated as mG or in the case of 2-fluorinated C, it is indicated as fC.

TABLE-US-00002 TABLE2 siRNAvariantsforHPVtype16and18 SEQID name NO: Sequence HPV18 SEQID 5-CAACCGAGCACGACAGGAA-3 type NO:11 siRNA SEQID 5-UUCCUGUCGUGCUCGGUUG-3 426 NO:12 SEQID 5-fCAAfCfCGAGfCAfCGAfCAGGAA-3 NO:13 SEQID 5-UUCCUGUCGUGCUCGGUUG-3 NO:14 SEQID 5-fCAAfCfCGAGfCAfCGAfCAGGAA-3 NO:15 SEQID 5-UUCCmUGmUCGmUGCmUCGGUUG-3 NO:16 SEQID 5-fCAAfCfCGAGfCAfCGAfCAGGAA-3 NO:17 SEQID 5-mUmUCCmUGmUCGmUGCmUCGGUUG-3 NO:18 SEQID 5-CAACCmGAmGCACmGACAmGmGAA-3 NO:19 SEQID 5-UUCCmUGmUCGmUGCmUCGGUUG-3 NO:20 SEQID 5-CAACCmGAmGCACmGACAmGmGAA-3 NO:21 SEQID 5-mUmUCCmUGmUCGmUGCmUCGGUUG-3 NO:22 HPV16 SEQID 5-GACCGGUCGAUGUGUGUCUUG-3 type NO:23 siRNA SEQID 5-AGACAUACAUCGACCGGUCCA-3 497 NO:24 SEQID 5-GACCGGmUCGAmUGmUAmUGmUCmUUG-3 NO:25 SEQID 5-AGACAUACAUCGACCGGUCCA-3 NO:26 SEQID 5-GACCGGmUCGAmUGmUAmUGmUCmUUG-3 NO:27 SEQID 5-AGACAmUACAmUCGACCGGmUCCA-3 NO:28

Example 2

[0089] Cell Culture and siRNA Transfection

[0090] Cervical cancer cells, Hela cervical cancer cell lines infected with HPV type 18 virus (HeLa; ATCC CCL-2), SiHa Cervical cancer cell lines infected with HPV type 16 virus (SiHa; ATCC HTB-35) were seeded in 6-well plates at 510.sup.4 cells or 110.sup.5 cells and cultured in RPMI 1640 or DMEM medium for 24 hours at 37 C. and 5% carbon dioxide, respectively. siRNA transfection was performed using DarmaFect (Darmacon, Lafayette, Colo., USA) and performed according to manufacturer's instructions.

Example 3

[0091] Evaluation of Inhibitory Effect of siRNA on Cell Proliferation

[0092] siRNA targeting HPV type 16 or E6/E7 of Example 1 was transfected into cervical cancer cell line infected with the HPV type 18 virus-infected HeLa or HPV type 16-infected SiHa by the method of Example 2, and after an additional one day of culture, siRNA was transfected again by the method of Example 2. After the transfection, the cells were cultured for 3 days and then the number of cells was measured. GFP siRNA was used as a control.

[0093] The results are shown in FIG. 1.

[0094] As shown in FIG. 1A, in a HeLa cell line infected with HPV type 18 virus, siRNA 426 hybridized with SEQ ID NOs: 1 and 2 showed an excellent inhibitory effect on cell proliferation (FIG. 1A, No. 5). This effect was confirmed as the best one by comparing with these of siRNA hybridized with SEQ ID NOs: 11 and 12 (No. 1 in FIG. 1A), siRNA hybridized with SEQ ID NOs: 13 and 14 (No. 2 in FIG. 1A), siRNA hybridized with SEQ ID NOs: 15 and 16 (No. 3 in FIG. 1A), siRNA hybridized with SEQ ID NOs: 17 and 18 (No. 4 in FIG. 1A), siRNA hybridized with SEQ ID NOS 19 and 20 (No. 6 in FIG. 1A) and siRNA hybridized with SEQ ID NOS 21 and 22 (No. 7 in FIG. 1A).

[0095] As shown in FIG. 1B, in a SiHa cell line infected with HPV type 16 virus, siRNA 497 hybridized with SEQ ID NOs: 9 and 10 showed an excellent inhibitory effect on cell proliferation (No. 2 in FIG. 1B). This effect was confirmed as the best one by comparing with these of siRNA hybridized with SEQ ID NOs: 23 and 24 (No. 1 in FIG. 1B), SEQ ID NOs: 25 and 26 (No. 3 in FIG. 1B), and siRNA hybridized with SEQ ID NOs: 27 and 28 (No. 4 in FIG. 1B).

Example 4

[0096] The Effect on TP53 and E7 Protein Expression

[0097] E6 and E7 oncogenic proteins encoded by HPV play an important role in the development of cervical cancer. Most of HPV-associated cervical cancers, unlike other cancers, have a wild-type TP53 gene and levels of TP53 protein in cervical cancers remain significantly low. Because it is consistently degraded by the E6 virus protein as a target. Thus, the expression of TP53 and E7 protein in HPV type 18 or 16 infected cervical cancer cell lines treated with E6/E7 siRNA of the present invention was confirmed by Western blotting.

[0098] The results are shown in FIG. 2.

[0099] As shown in FIG. 2, siRNA 426 hybridized with SEQ ID NOs: 1 and 2 in a Hela cell line infected with HPV type 18 virus, and siRNA 497 hybridized with SEQ ID NOs: 9 and 10 in a SiHa cell line infected with HPV type 16 virus, induced strongly the expression of TP53 protein and inhibited the expression of E7 protein. This effect was remarkably superior to the siRNA prepared in Comparative Example.

Example 5

[0100] The Effect on E6 and CDKN1A mRNA Expression

[0101] The effect of siRNA according to the present invention on mRNA expression of E6 and CDKN1A in HPV-infected cervical cancer cell line was confirmed. The expression of CDKN1A is regulated by the p53 protein, a tumor suppressor gene, and CDKN1A is closely related to the p53-dependent G1 phase cell cycle arrest of the cells. The expression levels of E6 and CDKN1A mRNA were analyzed using TaqMan quantitative real-time PCR (qRT-PCR).

[0102] The results are shown in FIGS. 3 and 4.

[0103] As shown in FIGS. 3 and 4, siRNA 426 hybridized with SEQ ID NOs: 1 and 2 in a Hela cell line infected with HPV type 18 virus, and siRNA 497 hybridized with SEQ ID NOs: 9 and 10 in a SiHa cell line infected with HPV type 16 virus, inhibited most effectively the expression of E6 mRNA and induced most effectively the expression of CDKN1A mRNA.

Example 6

[0104] Serum Stability Evaluation of siRNA

[0105] As shown in FIG. 5, the serum stability of siRNA 426 hybridized with SEQ ID NOs: 1 and 2 and siRNA 497 hybridized with SEQ ID NOs: 9 and 10 were found to be superior to that of siRNA that was not chemically modified (SiRNA hybridized with SEQ ID NOs: 11 and 12 in FIG. 5A, No. 1 and siRNA hybridized with SEQ ID NOs: 23 and 24) in FIG. 5B, No. 1.

Example 7

[0106] The Inhibitory Effects of Combination Treatment with siRNA and Radiation on Cell Proliferation

[0107] Experiments were conducted to confirm whether siRNAs having the nucleic acid sequences of SEQ ID NOs: 1 to 10 exhibited the effect as a sensitizing agent for combination therapy with radiation. siRNAs having the nucleic acid sequences of SEQ ID NOs: 1 to 10 were transfected into HeLa cells or SiHa cells, respectively, in the same manner as in Example 3, and then radiation was applied to evaluate the effects of combination therapy.

[0108] The results are shown in FIG. 6.

[0109] As shown in FIG. 6A, in HeLa cells infected with HPV type 18 virus, the group of combination therapy of siRNA 426 (426_d5 in FIG. 6A) or siRNA 450 (450_d4 in FIG. 6A) and irradiation showed more than two-fold improved inhibitory effect on cell proliferation when compared to the group treated with siRNA 426 or siRNA 450 alone. In addition, the group of combination therapy of siRNA 426 or siRNA 450 and irradiation showed more than two-fold improved inhibitory effect on cell proliferation when compared to the mock control group that was irradiated only. Therefore, it was confirmed that siRNA 426 or siRNA 450 showed excellent sensitizing effect on radiotherapy.

[0110] As shown in FIG. 6B, in SiHa cells infected with HPV type 16 virus, the group of combination therapy of siRNA 366 (366_d2 in FIG. 6B), siRNA 448 (448_d2 in FIG. 6B) and siRNA 497 (497_d2 in FIG. 6B) and irradiation showed more than two-fold improved inhibitory effect on cell proliferation when compared to the mock control group that was irradiated only. Therefore, it was confirmed that siRNA 366, siRNA 448 and siRNA 497 exhibited excellent sensitizing effect on radiotherapy.

Example 8

[0111] Changes of TP53 and E7 Protein Expression by Combination Treatment of siRNA and Radiation

[0112] The expression of TP53 and E7 protein was evaluated by the same method as in Example 4, by combination treatment of siRNA and radiation.

[0113] The results are shown in FIG. 7.

[0114] As shown in FIG. 7, the expression of TP53 protein was significantly increased and the expression of E7 protein was significantly decreased in the group co-treated with siRNA 426, siRNA 450 siRNA 366, siRNA 448 or siRNA 497 and radiation, therefore, it was confirmed that siRNA 426, siRNA 450 siRNA 366, siRNA 448 and siRNA 497 exhibited excellent sensitizing effect on radiotherapy.

Example 9

[0115] Inducing Effect on Cell Apoptosis by Combination Treatment of siRNA and Radiation

[0116] In the same method as described above, siRNA was transfected alone into HeLa cells or SiHa cell lines, or combined with radiotherapy, and cultured for one day. Cells were stained with Annexin V and PI (propidium iodide) reagents using a cell death kit (BD, USA), and reacted at room temperature for 30 minutes. Cell death was confirmed using a flow cytometer.

[0117] The results are shown in FIG. 8.

[0118] As shown in FIG. 8, annexin-v-positive apoptotic cells were significantly increased in the group of combination therapy of siRNA 426, siRNA 450 siRNA 366, siRNA 448 or siRNA 497 and irradiation when compared to the mock control group that was irradiated only. Therefore, it was confirmed that siRNAs of the present invention were effective as a sensitizing agent for increasing the apoptotic effect of radiation therapy on cancer cells.

Example 10

[0119] Inducing Effect on Cell Senescence by Combination Treatment of siRNA and Radiation

[0120] In the same method as described above, siRNA was transfected alone into HeLa cells or SiHa cell lines, or combined with radiotherapy, and cultured for one day. For senescence analysis, cells were fixed in 2% formaldehyde/0.2% glutaraldehyde, and cells were stained at pH 6.0 using X-Gal (5-bromo-4-chloro-3-indolyl--D-galactopyranoside) as previously performed in the method. The SA--Gal positive cells immediately underwent cell count in three representative fields.

[0121] The results are shown in FIG. 9.

[0122] As shown in FIG. 9, the group of combination therapy of siRNA 426, siRNA 450 siRNA 366, siRNA 448 or siRNA 497 and irradiation showed SA- Gal activity increased by 10-20 folds when compared to the mock control group that was irradiated only. Therefore, it was confirmed that the siRNA variants of the present invention showed excellent sensitizing effect on radiotherapy.

Example 11

[0123] Evaluation of Radiation Sensitization Effect of siRNA Pool (SP)

[0124] In nature, siRNAs generated by Dicer represent a siRNA pool that silence together the expression of the gene. The siRNA pool obtained in this way is an efficient silencer and has a low off-target effect. The inventors of the present invention evaluated the apoptotic effects of HPV type 18 siRNA pool (SP) using 10 nM of siRNA described above and 7 nM and 10 nM of HPV type 16 siRNA pool (SP), respectively.

[0125] As shown in FIGS. 6 to 9, the siRNA pool (SP) showed excellent inhibitory effect of cell proliferation, induction effect of TP53 protein expression, inhibitory effect of E7 protein expression, induction effect of apoptosis, and induction effect of cell senescence, and it was confirmed that these effects were maximized by radiation therapy.

[0126] Therefore, it is considered that the siRNA pool in which each of the siRNA variants according to the present invention is mixed can exhibit an excellent radiation sensitizatizing effect while exhibiting low side effects.

INDUSTRIAL AVAILABILITY

[0127] The oligonucleotides of the present invention inhibit the expression of E6 and E7, which are oncogenic proteins of HPV virus, resulting in inhibiting the proliferation of tumor cells and enhancing the sensitivity of tumor cells to radiation, thus, they have excellent effect as a sensitizing agent capable of maximizing the apoptosis effect and are highly applicable to be used in industry.