KR101389072B1 - siRNA for inhibiting BHRF1 expression and composition comprising the same - Google Patents

Abstract

The present invention relates to siRNA that inhibits BHRF1 expression of EBV (Epstein-Bar virus) and anticancer drug resistance inhibition using the same. More specifically, the present invention relates to a siRNA that inhibits the expression of BHRF1 and a composition for inhibiting anticancer drug resistance to EBV-positive gastric cancer comprising the same. The present invention also relates to a composition for treating EBV-positive gastric cancer comprising the siRNA and an anticancer agent.

Description

SiRNA for inhibiting expression of GHHF1 and composition comprising same {siRNA for inhibiting BHRF1 expression and composition comprising the same}

The present invention relates to siRNA for inhibiting BHRF1 expression of EBV (Epstein-Bar virus) and anticancer drug resistance inhibition using the same. More specifically, the present invention relates to a siRNA for inhibiting BHRF1 expression and a composition for inhibiting anticancer drug resistance to EBV-positive gastric cancer comprising the same. The present invention also relates to a composition for treating EBV-positive gastric cancer comprising the siRNA and an anticancer agent.

Epstein-Bar virus (EBV) is the first gamma herpes virus found in Africa associated with Burkitt's lymphoma.

The virus is associated with a variety of tumors, including Burkitt's lymphoma or nasopharyngeal tumors. Recently, it has been shown that EBV genes are expressed in some gastric cancers, and about 4-18% of gastric cancer patients worldwide are known as EBV-positive gastric cancers. However, the pathological role of EBV in gastric cancer has not been elucidated. In addition, EBV has several latent genes and lytic genes. Generally, EBV has been thought to express latent genes in EBV-related tumors, but its relationship with lytic genes has not been studied. .

The present invention is based on the discovery that EBV-positive gastric cancer exhibits high anticancer drug resistance compared to EBV-negative gastric cancer. The present inventors also found that when EBV-positive gastric cancer is treated with an anticancer agent, EBV lytic genes including BHRF1 begin to be expressed, and thus, the BHRF1 expressed in this manner may act as a factor indicating anticancer drug resistance. Therefore, in order to increase the anticancer drug sensitivity of EBV-positive gastric cancer cells showing anticancer drug resistance by reducing the BHRF1 of EBV, the study was focused on providing siRNA against BHRF1.

siRNA (small interference RNA) is a substance that induces RNA interference (RNAi) refers to a short double-chain RNA consisting of dozens to dozens of nucleotides. RNAi introduces double stranded RNA composed of sense RNA having a sequence homologous to mRNA of a target gene and antisense RNA having a complementary sequence to a cell to selectively induce degradation of the mRNA of the target gene. Or a phenomenon that can inhibit the expression of a target gene. As described above, since RNAi can selectively inhibit the expression of target genes, it has attracted considerable attention as a simple gene knock-down method that replaces conventional methods of gene destruction by inefficient homologous recombination. The siRNA is injected into cells to inhibit the gene expression of mRNA of the target gene complementary to the siRNA.

However, contrary to the original expectation that RNAi technology would be able to suppress selective expression by applying RNA to all genes, it has recently been found that some genes are not applied in the course of large-scale RNAi search due to unknown reasons. Krueger et al., Oligonucleotides, 17: 237-250, 2007). In addition, algorithms for predicting siRNA sequences through statistical analysis based on the database of the past several years have been developed, but since the computer algorithms are incomplete, all siRNAs determined by in silico method are used in real cells and in vivo. Among them, it is known that the most effective siRNA can be determined only through experimental verification, not effectively inhibiting the target mRNA (Derek et al., Annu. Rev. Biomed. Eng., 8: 377-402, 2006).

As such, the theoretically predicted siRNA does not necessarily inhibit the ability of the target mRNA effectively, and a high efficiency siRNA base sequence can be used to achieve therapeutic goals, so that specific target sites with high inhibitory efficiency on the target mRNA (hyperactive) Finding a target is very important. In addition, it is necessary to prepare siRNAs by selecting a large number of target sites per mRNA of one gene and to identify their effect at least directly at the cellular level, thereby finding a sequence having an optimum position with excellent expression inhibiting effect.

In this regard, few studies have been conducted on the development of siRNA capable of effectively inhibiting the expression of BHRF1 and on the anticancer treatment using siRNA against BHRF1.

Thus, the present inventors have tried to provide a siRNA capable of effectively inhibiting the expression of BHRF1 and to provide it for the purpose of inhibiting the anticancer resistance, it was possible to identify a target site that can effectively suppress the expression of BHRF1, Treatment of EBV-positive gastric cancer cells with an anticancer agent with siRNA complementary to the target site could reduce cell viability and promote cell death. Accordingly, the present invention has been completed by confirming that the siRNA may contribute to effective EBV-positive gastric cancer treatment by inhibiting anticancer drug resistance in EBV-positive gastric cancer cells.

An object of the present invention is to identify a target site of siRNA that can effectively inhibit the expression of BHRF1 and to provide an siRNA that binds to the target site. It is also to provide the siRNA for the purpose of inhibiting anticancer drug resistance.

Specifically, one object of the present invention is the 18-36 nucleotide sequence, 165-184 nucleotide sequence, 243-262 nucleotide sequence, 487-506 nucleotide sequence of BHRF1 mRNA having the nucleotide sequence of SEQ ID NO: 1, and To provide an siRNA that binds to a nucleotide sequence selected from the group consisting of 519 to 539 nucleotide sequences and inhibits the expression of the BHRF1 gene.

It is another object of the present invention to provide a composition for inhibiting anticancer drug resistance to EBV-positive gastric cancer comprising the siRNA.

Another object of the present invention to provide a composition for treating EBV-positive gastric cancer comprising the siRNA and an anticancer agent.

As one aspect for solving the said subject, this invention relates to the siRNA which binds to the specific target site which can suppress the expression of BHRF1 effectively.

In a preferred embodiment, the present invention provides the 18-36 nucleotide sequence, the 165-184 nucleotide sequence, the 243-262 nucleotide sequence, the 487-506 nucleotide sequence, and the 519-539 of the BHRF1 mRNA having the nucleotide sequence of SEQ ID NO: 1 The present invention relates to an siRNA that binds to a nucleotide sequence selected from the group consisting of the first nucleotide sequence and inhibits the expression of the BHRF1 gene.

Preferably, the siRNA may be a double stranded siRNA having a length of 19 to 21 nucleotides.

Also preferably, the siRNA is a nucleotide pair of SEQ ID NO: 14 and SEQ ID NO: 15, a nucleotide pair of SEQ ID NO: 16 and SEQ ID NO: 17, a nucleotide pair of SEQ ID NO: 6 and SEQ ID NO: 7, a nucleotide pair of SEQ ID NO: 28, and SEQ ID NO: 29 , Nucleotide pair of SEQ ID NO: 38 and SEQ ID NO: 39, nucleotide pair of SEQ ID NO: 40 and SEQ ID NO: 41, nucleotide pair of SEQ ID NO: 44 and SEQ ID NO: 45, nucleotide pair of SEQ ID NO: 46 and SEQ ID NO: 47, and SEQ ID NO: 62 and It may be an siRNA consisting of nucleotide pairs selected from the group consisting of nucleotide pairs of SEQ ID NO: 63.

As another aspect, the present invention relates to a composition for inhibiting anticancer drug resistance to EBV-positive gastric cancer comprising the BHRF1 siRNA.

As another aspect, the present invention relates to a composition for treating EBV-positive gastric cancer comprising BHRF1 siRNA and an anticancer agent.

Hereinafter, the present invention will be described in more detail.

As used herein, the term "siRNA" refers to a short double-chain RNA capable of inducing degradation of a target mRNA or inhibiting expression of a target gene through cleavage of a target mRNA.

The siRNA of the present invention is not limited to being completely complementary to the target mRNA and is not paired by mismatches (corresponding bases are not complementary) or bulges (no bases corresponding to one chain). This may include. The nucleotide length constituting the siRNA of the present invention may be 10 to 40 nucleotides, preferably 15 to 30 nucleotides, more preferably 19 to 21 nucleotides in length, which may interfere with the activity and expression of BHRF1. As used herein, the length of an siRNA refers to the length of a nucleotide double stranded (pairing).

The siRNA terminal structure of the present invention can be blunt or cohesive at both ends, as long as it can inhibit the expression of the target gene by the RNAi effect. The cohesive end structure can be both a structure overhanging the 3 'end and a structure overhanging the 5' end. The number of protruding nucleotides is not limited, but may be, for example, 1 to 8 nucleotides, preferably 1 to 4 nucleotides. In addition, siRNA is a sequence consistent with or complementary to the BHRF1 mRNA in the range capable of maintaining the expression inhibitory effect of the target gene, for example, dTdT, UU, or double-stranded formation at one end of the protruding portion. It may include. The siRNA terminal structure does not need to have a cleavage structure on both sides, and a stem loop structure in which one terminal region of double-stranded RNA is connected by linker RNA may be used. The length of the linker is not particularly limited as long as it does not interfere with the pairing of the stem portions.

The siRNA provided by the present invention is the BHRF1 mRNA represented by SEQ ID NO: 1 18-36 nucleotide sequence, 165-184 nucleotide sequence, 243-262 nucleotide sequence, 487-506 nucleotide sequence, and 519-539 nucleotide It is siRNA which binds to the nucleotide sequence selected from the group which consists of sequences, and suppresses the expression of BHRF1 gene.

Preferably, the siRNA that binds to the 18th to 36th nucleotide sequence of the BHRF1 mRNA may be an siRNA consisting of nucleotide pairs of SEQ ID NO: 62 and SEQ ID NO: 63. In the present specification, the siRNA is named siRNA # 26.

Preferably, the siRNA binding to the 165th to 184th nucleotide sequence of the BHRF1 mRNA may be an siRNA consisting of nucleotide pairs of SEQ ID NO: 14 and SEQ ID NO: 15, or an siRNA consisting of nucleotide pairs of SEQ ID NO: 16 and SEQ ID NO: 17. In the present specification, the siRNA is named siRNA # 2 and # 3, respectively.

Also preferably, the siRNA binding to the nucleotides 243 to 262 of the BHRF1 mRNA may be an siRNA composed of nucleotide pairs of SEQ ID NO: 6 and SEQ ID NO: 7, or an siRNA composed of nucleotide pairs of SEQ ID NO: 28 and SEQ ID NO: 29 . In the present specification, the siRNA is named siRNA #C and # 9, respectively.

Also preferably, the siRNA binding to the nucleotides 487 to 506 of the BHRF1 mRNA may be an siRNA consisting of nucleotide pairs of SEQ ID NO: 38 and SEQ ID NO: 39, or an siRNA consisting of nucleotide pairs of SEQ ID NO: 40 and SEQ ID NO: 41 . In the present specification, the siRNA is named siRNA # 14 and # 15, respectively.

Also preferably, the siRNA binding to the 519th to 539th nucleotide sequence of the BHRF1 mRNA may be an siRNA consisting of nucleotide pairs of SEQ ID NO: 44 and SEQ ID NO: 45, or an siRNA consisting of nucleotide pairs of SEQ ID NO: 46 and SEQ ID NO: 47 . In the present specification, the siRNA is named siRNA # 17 and # 18, respectively.

In addition, the siRNA of the present invention can be chemically modified in a general manner known in the art in order to prevent rapid degradation by nucleic acid enzymes in vivo and to increase in vivo stability. For example, the hydroxyl group at the 2'-position of the rib ring of at least one nucleotide contained in the siRNA is a hydrogen atom, a halogen atom, an -O-alkyl group, an -O-acyl group or an amino group, specifically H, OR, R, R'OR, SH, SR, NH ₂ , NHR, NR ₂ , N ₃ , CN, F, Cl, Br, I, etc., wherein R is alkyl or aryl, preferably 1 carbon An alkyl group of ˜6, R ′ may be alkylene, preferably alkylene having 1 to 6 carbon atoms), the phosphoric acid backbone being phosphorothioate, phosphorodithioate, alkylphosphonate, phosphoramidate, or It can be modified with boranophosphate.

In addition, the chemical modification, at least one of the nucleotides included in the siRNA of the present invention is a nucleic acid of any one of LNA (unlocked nucleic acid), UNA (unlocked nucleic acid), morpholino, peptide nucleic acid (PNA) And may be substituted to have an analog form. Preparation of these LNA, UNA, PNA, morpholino and the like can be prepared by knowledge known in the art based on the siRNA sequence information provided in the present invention, reference can be made to existing literature (Veedu RN, Wengel J. Chem Biodivers. 7 (3): 536-42, 2010; Demidov VV.Trends Biotechnol.21 (1): 4-7, 2003; Shantanu Karkare, et al., Appl Microbiol Biotechnol, 71: 575-586, 2006) .

In addition, siRNAs of the invention may include variants having one or more substitutions, insertions, deletions and combinations thereof, which are functional equivalents with changes that do not degrade their activity. For example, the siRNA of the present invention may exhibit at least 80% homology with the siRNA of each corresponding SEQ ID NO, and may preferably include 90%, more preferably at least 95% homology. Such homology can readily be determined by comparing the sequence of the nucleotide with the corresponding portion of the target gene using computer algorithms well known in the art, for example Align or BLAST algorithms.

The siRNA of the present invention is basically a form in which two strands of RNA are paired to form a double strand, that is, a form in which siRNA is directly synthesized in vitro and then introduced into a cell through transfection, or a plasmid system. It may be a modified form with a structure having a short hairpin to be used for transfection by shRNA vectors and PCR-induced siRNA expression cassettes.

As a method of preparing the siRNA, a direct chemical synthesis method (Sui G et al., Proc Natl Acad Sci USA 99: 5515-5520, 2002), a method using synthetic in vitro transcription (Brummelkamp TR et al. , Science 296: 550-553, 2002), Method for cleaving long double-stranded RNA synthesized by in vitro transcription using RNaseIII family enzymes (Paul CP et al., Nature Biotechnology 20: 505-508, 2002) And the like can be synthesized by various methods known in the art.

In a specific embodiment of the present invention, when docetaxel is treated in the EBV-negative gastric cancer cell line and the EBV-positive gastric cancer cell line, the cell survival rate is higher in the EBV-positive gastric cancer cell line than in the EBV-negative gastric cancer cell line, but the cell death rate is low. , EBV-positive gastric cancer cells were confirmed to have high anticancer drug resistance (see Fig. 1). In addition, as a result of confirming the expression and expression level of EBV lytic genes when docetaxel was treated in each of the two cell lines, EBV lysates including BHRF1 in EBV-positive gastric cancer cells at concentrations at which the two cells showed different resistance to anticancer drugs. Gene expression was confirmed (see Figure 2). This suggests that EBV lytic genes such as BHRF1 are involved in anticancer drug resistance in EBV-positive gastric cancer.

Thus, the present inventors examined whether siRNA of EBV can increase anticancer drug sensitivity of EBV-positive gastric cancer cells showing anticancer drug resistance by reducing BHRF1 expression. As a result, after treatment with BHRF1 siRNA on the EBV-positive gastric cancer cells, the anticancer drug treatment showed a 60% reduction in cell viability and induced cell death compared to the scramble siRNA treatment (FIG. 4 and 5). Furthermore, it could be confirmed that genes related to cell death and cell cycle control were involved in the process of inhibiting anticancer drug resistance (see FIG. 6). Therefore, siRNA against BHRF1 was found to be effective in inhibiting the resistance to anticancer drugs in AGS-EBV cells.

Furthermore, the present inventors have provided siRNAs against BHRF1 for the purpose of enhancing anticancer drug sensitivity by reducing BHRF1 of EBV in EBV-positive gastric cancer. It was clarified.

To this end, we synthesized 40 double-stranded siRNAs (named siRNAs # A- # E and # 1- # 35) that bind to various sites on BHRF1 mRNA (see Figure 3), and they actually expressed BHRF1 gene expression. Inhibition at the protein and mRNA levels, respectively.

As a result, siRNA # 26, which binds to the 18th to 36th nucleotide sequence of BHRF1 mRNA, siRNAs # 2 and # 3 that bind to the 165th to 184th nucleotide sequence of 40 siRNAs, and 243 to 262th nucleotide sequences of BHRF1 mRNA were identified. SiRNAs #C and # 9 that bind to, siRNAs # 14 and # 15 that bind to the 487-506th nucleotide sequence of BHRF1 mRNA, and siRNAs # 17 and # 18 that bind to the 519-539th nucleotide sequence of BHRF1 mRNA are each BHRF1 It has been shown to reduce gene expression by 80-90% (see FIGS. 7-11), and this effect was maintained in SNU-719, a naturally EBV infected gastric cancer cell line, as well as the EBV-positive gastric cancer cell line AGS-EBV (FIG. 12).

On the other hand, if any deviation from the mRNA sequence, siRNA significantly inhibited the inhibitory effect of BHRF1 expression, for example, siRNA #A, # 4 ~ # 6 in the case of siRNA # 2 and # 3 and the target sequence is a significant overlap even Almost no gene expression inhibitory effect was observed, and siRNA # 10-13 showed little gene expression inhibitory effect despite the substantial overlap of siRNA #C and # 9 with the target sequence. In addition, siRNA # 16 showed almost no gene expression inhibitory effect despite the substantial overlap of siRNA # 14 and # 15 target sequences, and siRNA # D ~ E, # 19-25 and siRNA # 17 and # 18 Despite overlapping or adjacent target sequences, the gene expression inhibitory effect was remarkably decreased. In addition, siRNA # 27 ~ 28, although adjacent to siRNA 26, gene expression inhibitory effect was significantly reduced.

Therefore, it was confirmed that the 18th to 36th, 165 to 184th, 243 to 262th, 487 to 506th, and 519 to 539th sites of the BHRF1 mRNA were hotspots that would be the main targets in siRNA production. SiRNA that binds to BHRF1 expression can be found to be effective in inducing anticancer drug inhibitory effect in EBV-positive gastric cancer.

13 illustrates a target site provided by the present invention.

Therefore, the siRNA provided by the present invention can be provided as a composition that inhibits anticancer drug resistance to EBV-positive gastric cancer by inhibiting BHRF1 expression of EBV.

As used herein, the term "anticancer drug resistance" refers to a drug that is administered or not enough to kill cancer cells when the anticancer drug is administered, and the anticancer drug resistance is the most frequent cause of anticancer drug resistance. Generally, "tolerance to anticancer drugs" means that when treating cancer patients with anticancer drugs, there is no therapeutic effect from the beginning of the treatment or early cancer treatment effect, but the cancer treatment effect is lost in the continuous treatment process. As such, it is well known that when an anticancer agent is administered to a cancer patient, the effect decreases with administration. This is due to the appearance of cancer cells resistant to anticancer agents in vivo, making the chemotherapy of cancer difficult.

As used herein, the term "inhibition of anticancer agent resistance" refers to the enhanced or increased toxicity of the anticancer agent to cancer cells when the cancer is treated with the anticancer agent than when the siRNA of the present invention is absent. In the present invention, BHRF1 siRNA can reduce the resistance of cancer cells to anticancer drugs by sensitizing EBV-positive gastric cancer cells to anticancer drugs, thereby increasing the effect of anticancer drugs. Preferably, the anticancer agent is docetaxel.

The composition of the present invention may be used alone as described above BHRF1 siRNA, or a mixture of two or more siRNA.

In the composition of the present invention siRNA may be included in complex form with various nucleic acid carriers known in the art in order to increase the efficiency of delivery in vivo. For example, siRNA can be used as a recombinant plasmid or viral vector that expresses siRNA. In addition, delivery reagents include G-fectin, Mirus TrasIT-TKO lipid affinity reagents, lipofectin, lipofectamine, cellfectin, cationic phospholipid nanoparticles, cationic polymers, cationic micelles, cationic emulsions or liposomes. It may be included as a naked siRNA in combination with a delivery reagent comprising a and administered to the subject. It may also be formulated to use common siRNA intracellular delivery techniques known in the art, such as conjugating biocompatible polymers, such as polyethyleneglycol, to increase cellular uptake in order to enhance the in vivo stability of siRNA.

The composition for inhibiting anticancer drug resistance of the present invention may be formulated with a pharmaceutically acceptable carrier in addition to siRNA or a complex of siRNA with a nucleic acid carrier. Such compositions may be formulated to provide fast release, or sustained or delayed release of the active ingredient after administration.

In the case of oral administration, binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments, flavorings, and the like can be used. It may be prepared in the form of. In the case of injections, buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, etc. may be mixed and used, and for topical administration, bases, excipients, lubricants, preservatives, etc. may be used, and unit dosage ampoules or It may be prepared in multiple dosage forms. Other inventive compositions can be prepared according to techniques commonly used in the form of various formulations.

The siRNA of the present invention may be provided in an anticancer composition with one or more anticancer agents.

Anticancer drugs are all drugs used to kill cancer cells, and most drugs block the replication, transcription, and translation of cancer cell DNA. The anticancer agent which can be used in the composition of the present invention is preferably docetaxel.

The anticancer drug resistance inhibiting composition or the anticancer composition of the present invention may be administered through any general route as long as it can reach the target tissue. Oral administration, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, intraluminal administration, intradural administration, but is not limited thereto. The BHRF1 siRNA according to the present invention may be administered together with the anticancer agent or separately from the anticancer agent at a certain time before or after administration of the anticancer agent.

The dosage of the composition may vary depending on various factors such as the age, sex, weight, pathological condition, route of administration, rate of excretion, and reaction sensitivity of the patient, and can be easily determined by those skilled in the art.

The present invention provides an siRNA that inhibits BHRF1 expression with high efficiency, and the siRNA inhibits anticancer drug resistance to EBV-positive gastric cancer, and thus can be usefully used to treat EBV-positive gastric cancer when used with an anticancer agent.

FIG. 1 (a) shows cell viability when docetaxel was treated to EBV-negative gastric cancer cells (AGS) and EBV-positive gastric cancer cells (AGS-EBV), which were repeated three times with Triplicate. The mean and SD values of the results are plotted (*, p <0.05; **, p <0.01). (b) shows the apoptosis rate when AGS cells and AGS-EBV cells were treated with docetaxel, and the graph shows the mean and SD values of the results of three repeated experiments (**, p < Means 0.01).
2 shows Western blot results confirming whether EBV lytic genes (BZLF1, BRLF1, BMRF1, BHRF1) are expressed when docetaxel is treated in AGS-EBV cells.
Figure 3 shows the mRNA sequence of the BHRF1 gene and binding sites of 40 BHRF1 siRNAs #A to #E and # 1 to # 35 produced in the present invention.
Figure 4 (A) shows the change in the number of cells when treated with docetaxel after treatment of BHRF1 siRNA #C, # 2 and # 32 to AGS-EBV cells, respectively. Scramble siRNA was used as a negative control. (B) shows the results in AGS cells.
Figure 5 shows the change in the rate of cell death when treated with docetaxel after treatment with BHRF1 siRNA #C in AGS-EBV cells. Scramble siRNA was used as a negative control.
6 shows Western blot results confirming cell death and cell cycle control-related gene expression when AGS-EBV cells and AGS cells were treated with siRNA #C and / or docetaxel, respectively. Scramble siRNA was used as a negative control.
FIG. 7A shows Western blot results confirming whether or not BHRF1 siRNA five species (#A to #E) are treated with AGS-EBV cells and then docetaxel is used to inhibit the expression of BHRF1 gene. Scramble siRNA was used as a negative control. (B) is the result of confirming once again about four BHRF1 siRNAs (# A- # D).
FIG. 8 (A) is a Western blot result confirming whether or not BHRF1 siRNA 35 species (# 1 to # 35) are treated with AGS-EBV cells and then docetaxel is used to inhibit the expression of BHRF1 gene. Scramble siRNA was used as a negative control. (B) is the result of confirmation once again. (C) is a graph showing the results of normalization of the expression of BHRF1 for β-actin used as a loading control.
9 shows BHRF1 when nine siRNAs (# 2, # 3, # 9, # 14, # 15, # 17, # 18, # 26, and # 32) were treated with AGS-EBV cells and then docetaxel. Real-time RT-PCR results confirming whether or not the expression of the gene is shown. Scramble siRNA was used as a negative control.
10 shows Western blot results confirming whether or not BHRF1 gene expression is suppressed when BHRF1 siRNAs are treated in the state of expressing BHRF1. Scramble siRNA was used as a negative control.
FIG. 11 shows Western blot results confirming whether AHs-EBV cells inhibit the expression of BHRF1 gene when BHRF1 siRNAs are treated in various concentration ranges (1, 5, 10 nM) and docetaxel is treated. Scramble siRNA was used as a negative control.
Figure 12 shows the Western blot results confirming whether or not to inhibit the expression of the BHRF1 gene when BHRF1 siRNAs treated with SNU-719 cells. Scramble siRNA was used as a negative control.
Figure 13 shows siRNA target sites on the BHRF1 mRNA provided by the present invention and siRNA binding thereto.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited by the following examples.

Example  1. Preparation of experimental material

AGS cells were used as EBV-negative gastric carcinoma (GC) cells and recombinant Akata virus (Shimizu et al., J. Virol) was used for AB cells as EBV-positive gastric carcinoma cells. 70, 7260-7263, 1996) were used to infect AGS-EBV cells. AGS is a RPMI-1640 medium (Gibco BRL, Grand Island) containing 10% fetal bovine serum (FBS; Hyclone, Logan, Utah, USA) and antibiotics (penicillin 100 Units / ml and streptomycin 100 μg / ml; Gibco BRL) , NY, USA). AGS-EBV was also cultured in medium containing 400 μg / ml Geneticin (Gibco BRL). As an anticancer agent, docetaxel (Aventis, Paris, France), a taxol-based anticancer agent, was used.

Example  2. BHRF1 For siRNA  Produce

Forty double-stranded siRNAs selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 81 were chemically synthesized (Table 1 and Table 2). The BHRF1 mRNA target site of each prepared siRNA is shown in FIG. 3. The siRNAs were custom made by Genolus (Seoul, Korea).

siRNA
No. Sense / antisense order order
number #A sense 5'-CGAAAUUCAGAGACAUUUA-3 ' 2 Antisense 5'-UAAAUGUCUCUGAAUUUCG-3 ' 3 #B sense 5'-GAAACUUGGAACAGAUUUA-3 ' 4 Antisense 5'-UAAAUCUGUUCCAAGUUUC-3 ' 5 #C sense 5'-GGAUUUUAACUCAGUAUUU-3 ' 6 Antisense 5'-AAAUACUGAGUUAAAAUCC-3 ' 7 #D sense 5'-UGACUUUGAGUCUGUUAGU-3 ' 8 Antisense 5'-ACUAACAGACUCAAAGUCA-3 ' 9 #E sense 5'-GUUAUAUGUAGUUAUUUAU-3 ' 10 Antisense 5'-AUAAAUAACUACAUAUAAC-3 ' 11

siRNA
No. Sense / antisense order order
number #One sense 5'-CUGUAGUUCUGCGUUAUCA-3 ' 12 Antisense 5'-UGAUAACGCAGAACUACAG-3 ' 13 #2 sense 5'-GGAGAUAAUUGAACGAAAU-3 ' 14 Antisense 5'-AUUUCGUUCAAUUAUCUCC-3 ' 15 # 3 sense 5'-GAGAUAAUUGAACGAAAUU-3 ' 16 Antisense 5'-AAUUUCGUUCAAUUAUCUC-3 ' 17 #4 sense 5'-GAUAAUUGAACGAAAUUCA-3 ' 18 Antisense 5'-UGAAUUUCGUUCAAUUAUC-3 ' 19 # 5 sense 5'-ACGAAAUUCAGAGACAUUU ' 20 Antisense 5'-AAAUGUCUCUGAAUUUCGU-3 ' 21 # 6 sense 5'-AAAUUCAGAGACAUUUACA-3 ' 22 Antisense 5'-UGUAAAUGUCUCUGAAUUU-3 ' 23 # 7 sense 5'-GAGACAUUUACAGAAACUU-3 ' 24 Antisense 5'-AAGUUUCUGUAAAUGUCUC-3 ' 25 #8 sense 5'-CAGAAACUUGGAACAGAUU-3 ' 26 Antisense 5'-AAUCUGUUCCAAGUUUCUG-3 ' 27 # 9 sense 5'-GAUUUUAACUCAGUAUUUU-3 ' 28 Antisense 5'-AAAAUACUGAGUUAAAAUC-3 ' 29 # 10 sense 5'-UUUUAACUCAGUAUUUUUA-3 ' 30 Antisense 5'-UAAAAAUACUGAGUUAAAA-3 ' 31 # 11 sense 5'-UUAACUCAGUAUUUUUAGA-3 ' 32 Antisense 5'-UCUAAAAAUACUGAGUUAA-3 ' 33 # 12 sense 5'-CUCAGUAUUUUUAGAGAUA-3 ' 34 Antisense 5'-UAUCUCUAAAAAUACUGAG-3 ' 35 # 13 sense 5'-CAGUAUUUUUAGAGAUAUU-3 ' 36 Antisense 5'-AAUAUCUCUAAAAAUACUG-3 ' 37 # 14 sense 5'-AGGUUUAGCUGGACUUUGU-3 ' 38 Antisense 5'-ACAAAGUCCAGCUAAACCU-3 ' 39 # 15 sense 5'-GGUUUAGCUGGACUUUGUU-3 ' 40 Antisense 5'-AACAAAGUCCAGCUAAACC-3 ' 41 # 16 sense 5'-GUUUAGCUGGACUUUGUUU-3 ' 42 Antisense 5'-AAACAAAGUCCAGCUAAAC-3 ' 43 # 17 sense 5'-GACUUUGAGUCUGUUAGUU-3 ' 44 Antisense 5'-AACUAACAGACUCAAAGUC-3 ' 45 # 18 sense 5'-CUUUGAGUCUGUUAGUUAU-3 ' 46 Antisense 5'-AUAACUAACAGACUCAAAG-3 ' 47 # 19 sense 5'-CUGUUAGUUAUAUGUAGUU-3 ' 48 Antisense 5'-AACUACAUAUAACUAACAG-3 ' 49 # 20 sense 5'-GUUAGUUAUAUGUAGUUAU-3 ' 50 Antisense 5'-AUAACUACAUAUAACUAAC-3 ' 51 # 21 sense 5'-UUAGUUAUAUGUAGUUAUU-3 ' 52 Antisense 5'-AAUAACUACAUAUAACUAA-3 ' 53 # 22 sense 5'-AGUUAUAUGUAGUUAUUUA-3 ' 54 Antisense 5'-UAAAUAACUACAUAUAACU-3 ' 55 # 23 sense 5'-UUAUAUGUAGUUAUUUAUU-3 ' 56 Antisense 5'-AAUAAAUAACUACAUAUAA-3 ' 57 # 24 sense 5'-UAUAUGUAGUUAUUUAUUU-3 ' 58 Antisense 5'-AAAUAAAUAACUACAUAUA-3 ' 59 # 25 sense 5'-AUAUGUAGUUAUUUAUUUA-3 ' 60 Antisense 5'-UAAAUAAAUAACUACAUAU-3 ' 61 # 26 sense 5'-GGAGAUACUGUUAGCCCUG-3 ' 62 Antisense 5'-CAGGGCUAACAGUAUCUCC-3 ' 63 # 27 sense 5'-AUACGGGACAGUCGUGUGC-3 ' 64 Antisense 5'-GCACACGACUGUCCCGUAU-3 ' 65 # 28 sense 5'-CUGCAUCCUGUGUUGGAGC-3 ' 66 Antisense 5'-GCUCCAACACAGGAUGCAG-3 ' 67 # 29 sense 5'-GAGACCCAAGCCUUGGGCG-3 ' 68 Antisense 5'-CGCCCAAGGCUUGGGUCUC-3 ' 69 # 30 sense 5'-GUUGGCCUGGAUGGCCUGG-3 ' 70 Antisense 5'-CCAGGCCAUCCAGGCCAAC-3 ' 71 # 31 sense 5'-AUGCAUGCCUGCAGGACAU-3 ' 72 Antisense 5'-AUGUCCUGCAGGCAUGCAU-3 ' 73 # 32 sense 5'-GUAACCAGUCUACUCCUUA-3 ' 74 Antisense 5'-UAAGGAGUAGACUGGUUAC-3 ' 75 # 33 sense 5'-GACCUGUCAGUUCGUGGGA-3 ' 76 Antisense 5'-UCCCACGAACUGACAGGUC-3 ' 77 # 34 sense 5'-AGCGAAGGCCUGGAUGGUU-3 ' 78 Antisense 5'-AACCAUCCAGGCCUUCGCU-3 ' 79 # 35 sense 5'-GCUGGUCUACAUUAAUUGA-3 ' 80 Antisense 5'-UCAAUUAAUGUAGACCAGC-3 ' 81

Experimental Example  One. EBV -Anticancer drug resistance of benign gastric cancer cells

After treatment with docetaxel in EBV-negative gastric cancer cell line (AGS) and EBV-positive gastric cancer cell line (AGS-EBV), cell viability and apoptosis rate were compared, respectively.

Specifically, to measure cell viability, AGS cells and AGS-EBV cells were each 100 μl divided into 96 well plates at a density of 5 × 10 ⁴ cells / ml and cultured for 24 hours. Next, docetaxel was treated by concentration (0-100 nM) and incubated for 72 hours, followed by dispensing 10 μl of CCK-8 (Cell Counting Kit-8; Dojindo Molecular Technologies, Tokyo, Japan) solution for each well for 3 hours. After incubation, the absorbance was measured at 450 nm using a SoftMax instrument (Molecular Devices, Sunnyvale, Calif., USA).

In addition, in order to measure the cell death rate, AGS cells and AGS-EBV cells were inoculated in a 100 mm culture dish at a density of 1 × 10 ⁶ cells / ml, respectively, and then cultured for 24 hours. Next, after treatment with 30 nM docetaxel and incubated for 72 hours, the cells were harvested, washed twice with cold saline, and 70% ethanol was added and fixed at -20 ° C overnight. The next day, after culturing for 1 hour while vortexing at room temperature every 15 minutes, the supernatant was discarded by centrifugation and washed twice with physiological saline. Next, a solution of 100 μg / ml RNase A (Invitrogen, Carlsbad, CA, USA) and 50 μg / ml PI (propidium iodide; Sigma-Aldrich, St. Louis, MO, USA) in physiological saline was added to each cell. 500 μl of the cells were released, stained and incubated for 20 minutes in a 37 ° C. water bath. The cell cycle was analyzed using a FACScaliber instrument (Becton Dickinson, San Jose, CA, USA).

As a result, as shown in Figure 1, compared to the EBV-negative gastric cancer cell line (AGS) EBV-positive gastric cancer cell line (AGS-EBV) when treated with a docetaxel concentration of 10 nM or more, while cell death is less It was confirmed that it appeared. Therefore, it was confirmed that AGS-EBV cells have higher resistance to docetaxel than AGS cells.

Experimental Example  2. Doclex  When processing EBV  Expression of lytic genes

It was confirmed whether EBV lytic genes were expressed by docetaxel treatment in AGS-EBV cells.

Specifically, AGS cells were treated with docetaxel 30 nM, and AGS-EBV cells were treated with docetaxel by concentration (0-100 nM), and cultured for 72 hours. After harvesting the cells, the proteins were extracted to obtain EBV lytic genes through Western blot. Expression was confirmed. Anti-BZLF1 (Dako, Glostrup, Denmark, 1: 500), anti-BRLF1 (Argene, Varilhes, France; 1: 500), anti-BMRF1 (Novocastra, Newcastle-upon-Tyne, UK) for the identification of EBV lytic genes 1: 500) and anti-BHRF1 (3E8, 1: 250) (Chou et al., 2004) were used as primary antibodies, respectively. Also anti-β-actin (Sigma-Aldrich, 1: 2000) was used as a loading control.

As a result, as shown in Figure 2, it was confirmed that the EBV lytic genes including BHRF1 in AGS-EBV cells when docetaxel at a concentration of 10 nM or more. That is, it was confirmed that AGS-EBV cells express EBV lytic genes at concentrations at which the two cells differ in resistance to anticancer drugs. Therefore, EBV lytic genes such as BHRF1 may be involved in anticancer drug resistance.

Experimental Example  3. BHRF1 siRNA  end EBV Cells of benign gastric cancer cells Survival rate  Identify the impact

To determine the effect of BHRF1 siRNA on the survival rate of EBV-positive gastric cancer cells against anticancer agents, siRNA #C, # 2 and # 32 were treated with AGS-EBV cells, respectively, and then treated with docetaxel and confirmed the change in cell number. It was.

Specifically, AGS cells and AGS-EBV cells were inoculated into 96 well plates at a density of 8 × 10 ³ cells / 100 μl, respectively, and 10 nM of scramble siRNA and BHRF1 siRNA #C, # 2 and # 32 were added, respectively, followed by G-fectin. Was added to transfect and incubated for 24 hours. Next, docetaxel was treated with 30 nM and further incubated for 72 hours, followed by incubation of 10 μl of CCK-8 solution for each well for 3 hours, followed by 450 nm using a SoftMax device (Molecular Devices, Sunnyvale, CA, USA). Absorbance was measured at.

As a result, as shown in Figure 4, the treatment of BHRF1 siRNA #C, # 2 and # 32 and docetaxel treatment all showed a 60% cell survival reduction effect compared to the scramble siRNA treatment. Therefore, siRNA against BHRF1 was found to be effective in inhibiting the resistance to docetaxel in AGS-EBV cells (A). However, in case of # 32, AGS cells, which are EBV-negative cells, also showed the effect of decreasing cell survival (B), which is effective in suppressing BHRF1 expression but may also affect off-target.

Experimental Example  4. BHRF1 siRNA end EBV -Identify the effects on the cell death of benign gastric cancer cells

4-1. BHRF1 siRNA  The effects of cell death on cell death

In order to confirm the effect of BHRF1 siRNA on the death of EBV-positive gastric cancer cells against anticancer agents, BHRF1 siRNA #C was treated with AGS-EBV cells, followed by docetaxel treatment, and the percentage change of apoptotic cells was confirmed. .

Specifically, AGS-EBV cells were inoculated into 6 well plates at a density of 1 × 10 ⁵ cells / ml, scramble siRNA and BHRF1 siRNA #C were added at 20 nM, and then transfected with G-fectin and incubated for 24 hours. . Next, the docetaxel was treated with 30 nM and further incubated for 72 hours, followed by annexin V staining, and the ratio of dead cells was confirmed using a FACScaliber instrument (Becton Dickinson).

As a result, as shown in FIG. 5, when a docetaxel was treated after transfection of BHRF1 siRNA #C, apoptosis occurred in more cells than when transfected with scramble siRNA. . Therefore, siRNA against BHRF1 was found to be effective in inhibiting the resistance to docetaxel in AGS-EBV cells.

4-2. BHRF1 siRNA Effect of cell death on cell death and expression of cell cycle control genes

To investigate the change in the expression of genes related to cell death and cell cycle control after treatment of docetaxel and BHRF1 siRNA #C in AGS-EBV cells at the molecular level to confirm that anti-cancer drug resistance can be reduced by using siRNA against BHRF1. It was.

20 nM of each of the scramble siRNA and BHRF1 siRNA #C used as a negative control were added, and then transfected with G-fectin and incubated for 24 hours. Next, 30 nM of docetaxel was incubated for 72 hours, and then cells were harvested and protein extracted to confirm cell death and cell cycle control-related gene expression through Western blot.

As a result, as shown in Figure 6, the treatment of docetaxel with scramble siRNA increased the expression of p53 in AGS and AGS-EBV cells to a similar degree, but in the AGS-EBV cells treated with docetaxel with BHRF1 siRNA #C It was confirmed that the expression of is increased more.

In addition, the amount of active Caspase-3 and p21 was higher in AGS cells than in AGS-EBV cells, and when docetaxel and BHRF1 siRNA #C were co-treated with AGS-EBV cells, both docetaxel and scramble siRNA were treated together. It was confirmed that the amount of activated Caspase-3 and p21 were increased than in one case.

On the other hand, Bcl-2 expression was higher in AGS-EBV cells compared to AGS cells at basal level, and Bcl-2 expression was slightly decreased when BHRF1 siRNA was treated in AGS-EBV cells. Treatment with docetaxel resulted in little Bcl-2 expression in AGS cells but increased Bcl-2 expression in AGS-EBV cells. In addition, when BHRF1 siRNA #C was treated with AGS-EBV cells like docetaxel, the expression of Bcl-2 was slightly decreased.

Therefore, it was confirmed that genes related to apoptosis and cell cycle control may be involved in the process of inhibiting the anticancer drug resistance of BHRF1 siRNA. It could be used.

Experimental Example  5. BHRF1 mRNA  top siRNA target  Identification of the site

To provide siRNAs against BHRF1 for the purpose of enhancing anticancer susceptibility in EBV-positive gastric cancer showing anticancer drug resistance, the present inventors have experimented to identify siRNA target sequences capable of inducing high anticancer susceptibility in the mRNA sequences of BHRF1. Proceeded.

5-1. BHRF1 siRNA At the protein level by BHRF1  Confirm gene silencing effect

40 double-stranded siRNAs that bind to various sites on the BHRF1 mRNA were prepared (see Example 2) and treated with each of these siRNAs in AGS-EBV cells to determine whether the gene silencing of the BHRF1 gene was expressed. Confirmation at the protein level.

Specifically, AGS-EBV cells were dispensed at a density of 1 × 10 ⁶ cells / ml in a 100 mm culture dish, and scramble siRNA used as a negative control and 40 BHRF1 siRNAs prepared in Example 3 were each added 20 nM, and then G. -fectin was added and transfected and incubated for 24 hours. Next, after treatment with docetaxel 30nM and incubated for 72 more hours, the cells were harvested and the proteins were extracted to confirm the expression change of the BHRF1 gene through Western blot.

7 shows the results of two experiments on siRNA # A ~ # E, and FIG. 8 shows the results of two experiments on siRNA # 1 ~ # 35 and normalization of BHRF1 expression on β-actin as a loading control. This is shown as a graph. As a result, among 40 kinds of siRNA, #C, # 2, # 3, # 9, # 14, # 15, # 17, # 18, # 26, and # 32 reduced BHRF1 expression by 80 ~ 90%. Confirmed. However, siRNA #A, #E, # 4-6, # 8, # 10-13, # 16, # 19- # 25, # 27-31, and # 33-35 showed almost no gene expression inhibitory effect. Appeared or weak.

5-2. BHRF1 siRNA On by mRNA  At the level BHRF1  Confirm gene silencing effect

When the BHRF1 siRNA was treated in AGS-EBV cells, the inhibitory effect of the BHRF1 gene was also examined at the mRNA level.

Specifically, AGS-EBV cells were dispensed at a density of 1 × 10 ⁶ cells / ml in a 100 mm culture dish, and scramble siRNA used as a negative control and BHRF1 siRNA 9 showing an inhibitory effect of BHRF1 expression of 80% or more in Experimental Example 5-1. Species (# 2, # 3, # 9, # 14, # 15, # 17, # 18, # 26 and # 32) were added 20 nM each, and then transfected with G-fectin and incubated for 24 hours. . Next, after treatment with docetaxel 30nM and incubated for 72 hours, cells were harvested and total RNA was extracted to confirm mRNA expression change of BHRF1 by real-time PCR. For comparison, three siRNAs (# 25, # 28 and # 29), which had little gene expression inhibitory effect in Western blot of Experimental Example 5-1, were randomly selected and examined for their effects on BHRF1 mRNA levels.

As a result, as shown in Figure 9, nine siRNA (# 2, # 3, # 9, # 14, # 15, # 17, # 18, # 26 and # 32) Western blot of Experimental Example 5-1 Similarly, the amount of mRNA was effectively suppressed.

5-3. BHRF1  In the state siRNA On by BHRF1  Confirm gene silencing effect

In Experimental Examples 5-1 and 5-2, an efficient siRNA was selected for transfection of AGS-EBV cells with BHRF1 siRNA and treatment of docetaxel to inhibit BHRF1 expression induced by docetaxel, but in this experiment AGS-EBV cells Docetaxel was first treated to transfect siRNA in the state of inducing BHRF1 gene expression, and the effect of BHRF1 gene silencing was confirmed at the protein level.

Specifically, AGS-EBV cells were dispensed at a density of 1 × 10 ⁶ cells / ml in a 100 mm culture dish, treated with docetaxel 30 nM, and cultured for 24 hours to induce BHRF1 gene expression, followed by scramble siRNA and Experimental Examples 5-1 and Among siRNAs selected in 5-2, # 2, # 3, # 9, # 14, # 17, and # 32 were each added 20 nM, and then transfected with G-fectin. After 72 hours of incubation, the cells were harvested and the proteins were extracted to confirm the expression changes of the BHRF1 gene through Western blot.

As a result, as shown in FIG. 10, even when BHRF1 siRNAs were treated in the state of expressing BHRF1, it was confirmed that all BHRF1 expressions were reduced in six siRNAs, in particular, effectively reduced by BHRF1 siRNA # 2 and # 3. It confirmed that it became.

5-4. BHRF1 siRNA  Depending on treatment concentration BHRF1 Knockdown  Check the effect

AGS-EBV cells were dispensed at a density of 1 × 10 ⁶ cells / ml in a 100 mm culture dish, and scramble siRNAs used as negative controls and siCs selected from experiments 5-1 and 5-2 were #C, # 2, # 3, # 9, # 14, # 17 and # 32 were added at 1, 5 and 10 nM, respectively, and then transfected with G-fectin and incubated for 24 hours. Next, after treatment with docetaxel 30nM and incubated for 72 more hours, the cells were harvested and the proteins were extracted to confirm the expression change of the BHRF1 gene through Western blot.

As a result, as shown in Figure 11, even when using a low concentration of BHRF1 siRNA was confirmed that the effect is good when compared to scramble siRNA.

5-5. SNU In -719 cell lines siRNA On by BHRF1  Confirm gene silencing effect

SNU-719, a natural EBV infected gastric cancer cell line, was transfected with BHRF1 siRNA to confirm the BHRF1 gene silencing effect.

G-fectin was added to SNU-719 cells by adding 20 nM of scramble siRNA and siRNA selected in Experimental Examples 5-1 and 5-2, respectively, to # 2, # 3, # 9, # 14, # 17 and # 32. Was added to transfect and incubated for 24 hours. Next, after treatment with docetaxel 30nM and incubated for 72 more hours, the cells were harvested and the proteins were extracted to confirm the expression change of the BHRF1 gene through Western blot.

As a result, as shown in FIG. 12, SNU-719, a natural EBV infected gastric cancer cell line, was able to confirm the inhibitory effect of BHRF1 gene expression by BHRF1 siRNA, and thus the effect of BHRF1 siRNA was universal in EBV-positive gastric cancer tumors. The possibility of application was confirmed.

Based on the above results, siRNA #C, # 2, # 3, # 9, # 14, # 15, # 17, # 18, # 26, and # 32 bind to each other, that is, 18 to 18 of the BHRF1 mRNA. The 36th, 165th-184th, 243-262th, 487-506th, and 519-539th sites were identified as hotspots that would be the main targets for siRNA production, and siRNA binding to these sites expressed BHRF1. Inhibition of high efficiency was found to be effective in inducing anticancer drug inhibitory effect in EBV-positive gastric cancer. 13 illustrates a target site provided by the present invention.

Attach an electronic file to a sequence list

Claims (13)

SiRNAs that inhibit expression of the BHRF1 gene, selected from the group consisting of:
SiRNA binding to the 18-36th nucleotide sequence of BHRF1 mRNA and consisting of nucleotide pairs of SEQ ID NO: 62 and SEQ ID NO: 63;
SiRNA binding to the 165th to 184th nucleotide sequence of BHRF1 mRNA and consisting of a nucleotide pair of SEQ ID NO: 14 and SEQ ID NO: 15, or a nucleotide pair of SEQ ID NO: 16 and SEQ ID NO: 17;
SiRNA that binds to the 243-262 nucleotide sequence of BHRF1 mRNA and consists of a nucleotide pair of SEQ ID NO: 6 and SEQ ID NO: 7, or a nucleotide pair of SEQ ID NO: 28 and SEQ ID NO: 29;
An siRNA that binds to the 487-506th nucleotide sequence of BHRF1 mRNA and consists of a nucleotide pair of SEQ ID NO: 38 and SEQ ID NO: 39, or a nucleotide pair of SEQ ID NO: 40 and SEQ ID NO: 41; And
An siRNA that binds to the 519 th-539 th nucleotide sequence of BHRF1 mRNA and consists of a nucleotide pair of SEQ ID NO: 44 and SEQ ID NO: 45, or a nucleotide pair of SEQ ID NO: 46 and SEQ ID NO: 47.
delete delete delete delete delete delete The siRNA of claim 1, wherein the siRNA has a locked nucleic acid (LNA), unlocked nucleic acid (UNA), morpholinos (morpholinos), or peptide nucleic acid (PNA) form.
According to claim 1, wherein the siRNA is a hydroxyl group of the 2'-position of the sugar of at least one nucleotide contained therein is substituted with any one of a hydrogen atom, a halogen atom, -O-alkyl group, -O-acyl group and amino group, or phosphoric acid An siRNA that inhibits expression of the BHRF1 gene, wherein the backbone is substituted with any one of phosphorothioate, phosphorodithioate, alkylphosphonate, phosphoramidate and boranophosphate.
A composition for inhibiting anticancer drug resistance to Epstein-Barr virus-positive gastric cancer comprising the siRNA of claim 1, 8 or 9.
The composition of claim 10, wherein the anticancer agent is docetaxel.
Epstein-Barr virus-positive gastric cancer composition comprising the siRNA of claim 1, 8 or 9 and an anticancer agent.
The composition of claim 12, wherein the anticancer agent is docetaxel.

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