WO2016140552A1 - Biomarker composition for diagnosing sensitivity to anticancer agent in anticancer agent-resistant breast cancer - Google Patents

Abstract

The present invention relates to a biomarker composition for diagnosing the sensitivity to an anticancer agent in anticancer agent-resistant breast cancer, a diagnosis kit, a diagnosis method, or a pharmaceutical composition for promoting anticancer effects, wherein it is possible to help effective treatment of tumors by restoring the sensitivity to anticancer agents such as tamoxifen, etc. if the expression of LY6K is inhibited or miR-192-5p is suppressed in breast cancer cells. Also, it is possible to help effective treatment of tumors by restoring the sensitivity to anticancer agents such as tamoxifen, etc. if the expression of miR-500a-3p is induced or miR-500a-3p is applied in breast cancer cells.

Description

Biomarker composition for diagnosing anticancer sensitivity of anticancer agent resistant breast cancer

The present invention relates to a biomarker composition for diagnosing anticancer sensitivity of an anticancer drug-resistant breast cancer comprising LY6K or miR-192-5p, a diagnostic kit, a diagnostic method, and a pharmaceutical composition for enhancing anticancer effect comprising a LY6K expression inhibitor or a miR-192-5p inhibitor. In this regard, the mechanism of resistance to anti-hormonal drugs such as tamoxifen, a breast cancer drug, is revealed, and it was confirmed that miR-192-5p inhibition in LY6K overexpressing breast cancer cells restored tamoxifen sensitivity by ERα re-expression.

In addition, the present invention is an anti-cancer drug sensitivity diagnostic biomarker composition, diagnostic kit, diagnostic method of the anti-cancer drug-resistant breast cancer comprising miR-500a-3p and a pharmaceutical composition for enhancing anticancer effect comprising any one of miR-500a-3p or its expression promoter. In the present invention, the mechanism of resistance to anti-hormonal drugs such as tamoxifen, a breast cancer drug, is disclosed, and the activation of miR-500a-3p in LY6K overexpressing breast cancer cells has recovered tamoxifen sensitivity by ERα re-expression. And inhibited sensitivity to anticancer drugs.

Breast cancer is a common cancer in women around the world. According to practice breast cancer is divided into several subgroups depending on the expression of receptors such as estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2). Most breast cancers are ER dependent. However, about 30% of breast cancers are classified as ER negative breast cancers, especially ERα negative breast cancers. ERα negative breast cancers do not respond to anti-estrogen therapy and tamoxifen, a selective estrogen receptor modulator. Tamoxifen is clinically associated with growth inhibition and apoptosis in breast cancer cells by disturbing estrogen binding to ER in ERα positive breast cancers, but it is well known that apoptosis occurs only at high tamoxifen concentrations in ERα negative breast cancers.

Lymphocyte antigen 6 complex locus K (LY6K) is a Ly-6 / urokinase-type plasminogen activator receptor (uPAR) superfamily member. LY6K has been reported as a molecular biomarker of esophageal squamous cell carcinomas, bladder cancer and breast cancer. In breast cancer cells, LY6K expression is mediated by AP-1 transcription factors and enhances cell proliferation, invasion and metastatic capacity. However, little is known about the effect of LY6K on breast cancer.

MicroRNA (miRNA) is a small non-coding RNA consisting of 20-22 nucleotides. miRNAs recognize seed sequences of several target genes by binding to the 3'UTR and then degrade or inhibit translation of the target mRNA. In many carcinomas, various cellular activities such as metabolism, apoptosis and proliferation are regulated by miRNAs. The relevance of miRNAs for increasing tamoxifen sensitivity has been studied. For example, re-expression of miR-320a, miR-375 and miR-342 can restore tamoxifen sensitivity by inhibiting the target.

An object of the present invention is to provide a biomarker composition, a diagnostic kit, a diagnostic method, a pharmaceutical composition for enhancing anti-cancer effects, or a cancer composition for preventing or treating cancer.

In order to achieve the above object, the present invention provides a biomarker composition for diagnosing anticancer agent sensitivity of anticancer agent resistant breast cancer, including LY6K.

In order to achieve the above object, the present invention provides an anti-cancer drug sensitivity diagnostic kit of anti-cancer drug-resistant breast cancer, comprising an agent for detecting LY6K.

In order to achieve the above object, the present invention comprises the steps of analyzing the expression of LY6K in a sample isolated from the subject; And comparing the expression level with the LY6K expression level in the normal group, providing a method for diagnosing anticancer sensitivity of anticancer agent resistant breast cancer.

In order to achieve the above object, the present invention provides a pharmaceutical composition for enhancing the anticancer effect comprising a LY6K inhibitor and enhances the anticancer effect of the anticancer agent against anticancer drug resistant breast cancer.

In order to achieve the above object, the present invention includes a LY6K inhibitor and an anticancer agent, and provides a pharmaceutical composition for preventing or treating cancer disease to enhance the anticancer effect of the anticancer agent against the anticancer agent resistant breast cancer.

In order to achieve the above object, the present invention provides a biomarker composition for diagnosing anticancer agent sensitivity of anticancer drug resistant breast cancer, comprising miR-192-5p.

In order to achieve the above object, it provides an anti-cancer drug sensitivity diagnostic kit of anti-cancer drug-resistant breast cancer, comprising an agent for detecting miR-192-5p.

In order to achieve the above object, the present invention comprises the steps of analyzing miR-192-5p expression in a sample isolated from the individual; And comparing the expression level with miR-192-5p expression level in the normal group, and provides an anticancer drug sensitivity diagnosis method for anticancer drug resistant breast cancer.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating cancer disease comprising a miR-192-5p inhibitor and enhancing the anticancer effect of an anticancer agent against anticancer drug resistant breast cancer.

In addition, the present invention includes a miR-192-5p inhibitor and an anticancer agent, and provides a pharmaceutical composition for preventing or treating a cancer disease that enhances the anticancer effect of the anticancer agent against the anticancer drug resistant breast cancer.

In order to achieve the above object, the present invention provides a biomarker composition for diagnosing anticancer agent sensitivity of anticancer agent resistant breast cancer, comprising miR-500a-3p.

In order to achieve the above object, the present invention provides an anti-cancer drug sensitivity diagnostic kit of anti-cancer drug-resistant breast cancer, comprising an agent for detecting miR-500a-3p.

In order to achieve the above object, the present invention comprises the steps of analyzing miR-500a-3p expression in a sample isolated from the individual; And comparing the expression level with miR-500a-3p expression levels in the normal group, and provides an anti-cancer drug sensitivity diagnostic method for anti-cancer drug-resistant breast cancer.

In order to achieve the above object, the present invention provides a pharmaceutical composition for enhancing the anticancer effect comprising miR-500a-3p or an expression promoter thereof and enhancing the anticancer effect of the anticancer agent against anticancer drug resistant breast cancer.

In order to achieve the above object, the present invention comprises any one of miR-500a-3p or its expression promoting agent and an anticancer agent, and a pharmaceutical composition for preventing or treating cancer disease to enhance the anticancer effect of the anticancer agent against the anticancer agent resistant breast cancer to provide.

According to the present invention, inhibiting the expression of LY6K or inhibiting miR-192-5p in breast cancer cells may restore sensitivity to anticancer drugs such as tamoxifen and may help smooth tumor treatment. In addition, inducing expression of miR-500a-3p in breast cancer cells or adding miR-500a-3p may restore sensitivity to anticancer drugs such as tamoxifen, thereby helping to smooth tumor treatment.

1A and B show that LY6K expression is negatively correlated with ERα. The relationship between ERα and LY6K mRNA, protein expression was tested by RT-PCR (A) and Western blotting (B). h18S rRNA and β-actin are endogenously regulated (C and D). Potential expression of LY6K in MCF7 and T47D lowers the transcriptional activity (C) and protein activity (D) of ERα.

2 shows that ectopic expression of ERα reduces both LY6K mRNA (A) and protein (B) expression in ERα negative breast cancer cells.

3 shows that decreased Ago2 expression restores ERα expression. Simultaneously with Ago2 siENA treatment and overexpression of LY6K, gene expression levels were determined in mRNA and protein after 48 hours. mRNA and protein expression were analyzed by qRT-PCR (A) and Western blotting (B), respectively.

Figure 4 confirms the response to knockdown AGO2 following control and ERα treatment for 48 hours. Expression of LY6K mRNA (A) and protein (B), respectively, was confirmed by qRT-PCR (FIG. 1E) or western blotting.

5 confirms primary and mature miRNAs induced by LY6K. Hierarchical clustering data confirmed mature miRNAs based on miRNA microarray analysis of T47D / Mock cells compared to T47D / LY6K cells (A) and ESR1 3′UTR in miRanda (http://www.microrna.org/). MiRNA binding sites were predicted (B).

6 confirms primary and mature miRNAs induced by LY6K. Primary miRNAs (pri-miRNAs) were upregulated in T47D / LY6K cells compared to T47D / Mock cells (A) and mature miRNAs induced by LY6K in ERα positive breast cancer cell lines (B).

Figure 7 identifies primary and mature miRNAs induced by ERα. Hierarchical clustering data is based on miRNA microarray analysis between MCF7-ADR / Mock and MCF7-ADR / ERα.

Figure 8 identifies primary and mature miRNAs induced by ERα. Comparison of MCF7-ADR / Mock and MCF7-ADR / ERα by qRT-PCR confirmed up-regulation of primary-miRNAs (pri-miRA) expression (A) ERα negative breast cancer cell lines MCF7-ADR (left) and MDA-MB Mature miRNA induced by ERα at -468 (right) was identified (B).

9 shows that ERα is a direct target of miR-192-5p. ESR1 gene structure showing that the expected target site of miR-192-5p is at 3′UTR (A). Luciferase assay in MCF7 cells showed miR-192-5p independent wild type (WT) ERα 3'UTR inhibition (B), while miR-192-5p mutant (MT) did not affect the inhibition of the expected binding site (C). Luciferase assays in MCF7 cells showed that miR-29b-3p and miR-29c-5p showed little difference between the expected binding sites of wild type and mutant types (D and E).

10 shows that LY6K is a direct target of miR-500a-3p and downregulates its expression. The gene structure of LY6K shows that there are two predicted target sites of miR-500a-3p at 3'UTR (A). Dual Luciferase assay in HEK293T cells shows LY6K (left) and miR-500a-3p (right) dependent inhibition of wild type (WT) LY6K 3'UTR, whereas mutant (MT) of seed sequence Luciferase activity is restored (B). Ectopic expression of miR-500a-3p inhibits LY6K mRNA and protein expression in MCF7-ADR (left) and MDA-MB-468 (right) (C).

In FIG. 11, A represents miR-34a and miR-194-5p expected binding sites in the gene structure of LY6K 3′UTR, and miRNA binding sites of wild type (WT) and mutant (MT). B in Figure 11 shows that miR-34a (left) and miR-194-5p (right) do not affect luciferase activity in HEK293T cells.

12 shows regulation of ERα by miR-192-5p. Transfection effect of miR-192-5p mimic in MCF7 and T47D cells (top). Potential expression of miR-192-5p downregulated ERα mRNA levels (bottom) (A). Re-expression of miR-192-5p in MCF7 and T47D cells inhibited the protein levels of ERα (B). MRNA and protein expression of LY6K in cell lines stabilized to overexpress human LY6K were measured by qRT-PCR and Western blot (C). Transfection efficiency of miR-192-5p inhibitor in T47D / LY6K cells stably expressing LY6K (left). miR-192-5p inhibition increased mRNA expression (right) (D). Reduced miR-192-5p expression inhibited ERα expression (E).

13 shows that miR-192-5p reduces tamoxifen induced autologous cell death. Three hours after treatment with varying amounts of tamoxifen, cell viability of T47D cells and T47D / LY6K cells was measured (A). In T47D / LY6K cells, transfection with a miR-negative control (NC) and a miR-192-5p inhibitor was performed 48 hours after transfection and cell viability was measured (B).

Figure 14 shows that miR-500a-3p reexpression increases tamoxifen sensitivity in ERα negative breast cancer (A). Cell survival rate when treated with miR-negative control (NC) or miR-500a-3p after 24 hours incubation with different tamoxifen doses in MCF7-ADR. After 48 hours of treatment with carrier or tamoxifen caspase-3 activation was increased by miR-500a-3p (B).

In the present invention it was found that there is an inverse correlation between the expression of ERα and LY6K. The hypothesis of the present invention is that miR-192-5p is upregulated by LY6K via miRNA microarrays when comparing T47D cells with T47D cells overexpressing LY6K. In addition, the inventors confirmed that miR-192-5p directly targets 3'UTR in ERα positive breast cancer cells to inhibit ERα activity and affect endocrine resistance. Taken together, LY6K is a key factor in ERα regulation in breast cancer cells and provides the molecular mechanism of ERα deregulation.

Breast cancer can be classified into ER, PR and Her2 according to the patient's receptor status. Of these major receptors, ER, in particular ERα, has an important effect in prescribing drugs suitable for breast cancer patients. However, in some patients, tumors were resistant to hormonal treatments such as tamoxifen and fulvestrant. Among the many causes associated with hormone therapy resistance, loss of ERα is one of the most important aspects of breast cancer. In that regard, we have found that ERα negatively correlates with the human LY6K gene. In addition, ERα expression was downregulated when human LY6K gene was overexpressed in ERα positive cells. This reduction in ERα expression by LY6K can provide clues to the problem for hormone therapy resistant patients.

Many mechanisms have been proposed for reducing ERα expression. Epigenetic regulatory mechanisms are also an important part of loss of ERα expression in breast cancer. Subsequently, ERα promoter activity is inhibited and regulated by Twist, hMAPK. Regulation by miRNAs is also involved in epigenetic programs such as methylation and acetylation. The miRNA microarrays of the invention showed that miR-192-5p is particularly overexpressed upon upregulation of human LY6K gene. In addition, pri-miR-194-2 / 192 expression was upregulated in T47D cells transiently overexpressing LY6K. The manner in which LY6K modulates the transcriptional activity of miR-192-5p is still unknown because the LY6K position is unclear. However, these results indicate that the human LY6K gene can activate miR-192-5p at the transcription level and mature miR-192-5p inhibits ERα expression in ERα positive breast cancer cells.

Interestingly, various miRNAs in breast cancer have been reported to be involved in ERα signaling and expression. However, miR-192-5p has not been reported as breast cancer related miRNA in previous studies. In addition, several studies have reported on miRNAs that act as ERα signaling inhibitors and target ERα directly, suggesting that these miRNAs may be involved in endocrine resistance in breast cancer. We have confirmed that ERα is a direct target of miR-192-5p. This suggests that miR-192-5p not only affects ERα signaling but also affects treatment resistance targeting ERα.

Recent chemotherapy resistant developmental profiling studies have identified miR-192-5p as a potential target for esophageal cancer cells. Indeed, new cancer-related miRNAs (oncomiR), miR-519a, have been reported to confer tamoxifen resistance through cell proliferation and autologous cell death by targeting tumor suppressor genes. They have shown that induction of cell proliferation in breast cancer is associated with a decrease in autologous apoptosis with high doses of tamoxifen. We have observed that miR-192-5p induced by human LY6K gene in breast cancer cells is associated with tamoxifen resistance through cell proliferation. In this regard, the inhibition of miR-192-5p may be related to the apoptosis induced by tamoxifen, thus analyzing the more detailed relationship between miR-192-5p inhibition and apoptosis by tamoxifen regimen. There is a need.

In addition, the inventors found for the first time that ERα-induced miR-500a-3p in ERα-negative breast cancer cells directly regulates human LY6K expression and is effective in enhancing tamoxifen sensitivity. This logically supports the understanding that LY6K targeting or miR-500a-3p overexpression is useful for treating ERα negative breast cancer patients, as well as understanding the mechanism by which LY6K expression reduction affects apoptosis induced by tamoxifen.

It is well known that miRNAs can play an important role as tumor genes or tumor suppressor genes, depending on the particular tumor subtype. In breast cancer, some miRNAs have been reported to affect tumorigenic processes and increase response to treatments such as anti-endocrine and chemotherapy. In the present invention, miR-500a-3p was induced by ERα through miRNA microarray comparing MCF7-ADR / control with MCF7-ADR / ERα. In addition, it was confirmed that miR-500a-3p directly binds to the 3'UTR of human LY6K. miR-500a-3p is involved in cell proliferation and metastasis and reduced LY6K expression in vitro. These results suggest a new mechanism that the regulation of LY6K degradation via the miR-500a-3p axis will delay metastasis and proliferative capacity in ERα negative breast cancer.

Through the data of the present invention, we found that LY6K is expressed at very high levels in ERα negative breast cancer. In addition, ERα and LY6K have a negative correlation in breast cancer cells. LY6K expression is downregulated by ectopic expression of ERα, which is an important target receptor for breast cancer target therapy. Tamoxifen, a selective estrogen antagonist, is well known as a medicament for ERα positive breast cancer and has no effect on ERα negative breast cancer patients. Many researchers have tried to restore ERα expression to increase tamoxifen sensitivity in ERα negative breast cancer. Histone deacetylase (HDAC) inhibitors can re-express ERα transcriptional activity and improve endocrine therapy in combination with DNA damaging agents. CpG methylation of the estrogen receptor promoter by inhibiting DNA Methyltransferase (DNMT) activity can reactivate ER due to transcriptional silencing in ERα negative breast cancer cells. In addition, ER reactivated by tamoxifen-binding recruits inhibitor complexes to regulate ER-reactive genes. In a similar view, we observed that primary and mature miR-500a-3p is upregulated by ERα overexpression in ERα negative breast cancer cells. In addition, miR-500a-3p was able to increase apoptosis observed by tamoxifen-induced cell viability and caspase-3 activation. Although the 4-OHT (10 μM) concentration is high compared to the effective tamoxifen dose for ERα positive breast cancer, overexpression of miR-500a-3p helps to enhance tamoxifen sensitivity in ERα negative breast cancer. These results suggest that decreased LY6K expression through increased miR-500a-3p may be more responsive to tamoxifen treatment in patients with ERα negative breast cancer.

In conclusion, the regulatory mechanism of the LY6K gene is well known in breast cancer, but the epigenetic mechanisms associated with miRNA are not well known. We have found a negative correlation between LY6K and ERα mediated by miR-192-5p in breast cancer cells. This suggests that miR-192-5p induced by LY6K activation causes resistance to tamoxifen, an anti-hormonal therapy. These results suggest that miR-192-5p may be the target of treatment in patients with ERα-reduced breast cancer.

We also found a negative correlation between ERα and LY6K in breast cancer and found that overexpressed ERα can regulate LY6K expression through miR-500a-3p activation. In addition, the data of the present invention show that increased miR-500a-3p expression by ERα induced in ERα negative breast cancer cells can increase tamoxifen sensitivity through cell viability and apoptosis. The present invention has established a relationship between miR-500a-3p and LY6K, which can be used as an effective therapeutic agent for the treatment of ERα negative breast cancer patients.

The present invention provides a biomarker composition for diagnosing anticancer agent sensitivity of anticancer agent resistant breast cancer, including LY6K.

The present invention also provides an anti-cancer drug sensitivity diagnostic kit for anti-cancer drug-resistant breast cancer, comprising an agent for detecting LY6K.

In addition, the present invention comprises the steps of analyzing the expression of LY6K in a sample isolated from the individual; And comparing the expression level with the LY6K expression level in the normal group.

The present invention also provides a pharmaceutical composition for enhancing the anticancer effect, comprising a LY6K inhibitor and enhancing the anticancer effect of the anticancer agent against anticancer drug resistant breast cancer.

The LY6K inhibitor is one or more selected from the group consisting of a LY6K gene-specific siRNA, a LY6K gene-specific shRNA, a recombinant expression vector comprising a LY6K gene-specific siRNA, and a recombinant expression vector comprising a LY6K gene-specific shRNA. It is not limited.

The LY6K gene-specific siRNA is characterized in that represented by any one of SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17.

The LY6K gene specific shRNA is characterized in that represented by any one of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21.

The expression vector is characterized in that any one selected from the group consisting of lentiviral vector, retrovirus vector and adenovirus vector.

In addition, the present invention includes a LY6K inhibitor and an anticancer agent, and provides a pharmaceutical composition for preventing or treating cancer disease that enhances the anticancer effect of the anticancer agent against the anticancer drug resistant breast cancer.

The anticancer agent is tamoxifen, 5-Fluorouracil, doxorubicin, doxorubicin, mitomycin, cisplatin, paclitaxel, docetaxel, docetaxel, irinotecan, Irinotecan. At least one selected from the group consisting of Xeloda, Oxalopatin, and etoposide, and more preferably tamoxifen, but is not limited thereto.

In another aspect, the present invention provides a biomarker composition for diagnosing anticancer agent sensitivity of anticancer agent resistant breast cancer, comprising miR-192-5p.

The present invention also provides an anti-cancer drug sensitivity diagnostic kit for anti-cancer drug-resistant breast cancer, comprising an agent for detecting miR-192-5p.

In addition, the present invention comprises the steps of analyzing miR-192-5p expression in a sample isolated from the individual; And comparing the expression level with miR-192-5p expression level in the normal group, and provides an anticancer drug sensitivity diagnosis method for anticancer drug resistant breast cancer.

In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising a miR-192-5p inhibitor and enhancing an anticancer effect of an anticancer agent against anticancer drug resistant breast cancer.

The miR-192-5p inhibitor is any one of an anti-miR-192-5p oligonucleotide or an expression vector including the same, and the anti-miR-192-5p oligonucleotide is characterized in that represented by SEQ ID NO: 22.

The expression vector is any one selected from the group consisting of lentiviral vectors, retroviral vectors and adenovirus vectors, but is not limited thereto.

In addition, the present invention includes a miR-192-5p inhibitor and an anticancer agent, and provides a pharmaceutical composition for preventing or treating a cancer disease that enhances the anticancer effect of the anticancer agent against the anticancer drug resistant breast cancer.

The anticancer agent is tamoxifen, 5-Fluorouracil, doxorubicin, doxorubicin, mitomycin, cisplatin, paclitaxel, docetaxel, docetaxel, irinotecan, Irinotecan. At least one selected from the group consisting of Xeloda, Oxalopatin, and etoposide, and more preferably tamoxifen, but is not limited thereto.

The present invention also provides a biomarker composition for diagnosing anticancer sensitivity of anticancer agent resistant breast cancer, comprising miR-500a-3p.

The present invention also provides an anti-cancer drug sensitivity diagnostic kit for anti-cancer drug-resistant breast cancer, comprising an agent for detecting miR-500a-3p.

In addition, the present invention comprises the steps of analyzing miR-500a-3p expression in a sample isolated from the individual; And comparing the expression level with miR-500a-3p expression levels in the normal group, and provides an anti-cancer drug sensitivity diagnostic method for anti-cancer drug-resistant breast cancer.

The present invention also provides a pharmaceutical composition for enhancing anticancer effect comprising miR-500a-3p or an expression promoter thereof and enhancing the anticancer effect of the anticancer agent against anticancer drug resistant breast cancer.

The miR-500a-3p is characterized in that represented by SEQ ID NO: 23.

The expression promoter is any expression vector selected from the group consisting of lentiviral vectors, retroviral vectors and adenovirus vectors expressing miR-500a-3p, but is not limited thereto.

In addition, the present invention includes any one of miR-500a-3p or its expression promoter and an anticancer agent, and provides a pharmaceutical composition for preventing or treating cancer disease to enhance the anticancer effect of the anticancer agent against the anticancer drug resistant breast cancer.

The anticancer agent is tamoxifen, 5-Fluorouracil, doxorubicin, doxorubicin, mitomycin, cisplatin, paclitaxel, docetaxel, docetaxel, irinotecan, Irinotecan. At least one selected from the group consisting of Xeloda, Oxalopatin, and etoposide, and more preferably tamoxifen, but is not limited thereto.

In the post-genome era, small RNAs are emerging as new gene expression regulators. Small RNA is produced at sites that were considered garbage DNA sequences that do not encode proteins. Small RNAs do not produce proteins, and their molecules themselves inhibit sequence-specific expression of target genes by mechanisms known as RNAi (RNA interference) or microRNA-mediated regulation. Thus, such RNA interference or microRNA technology is one of the technologies that are attracting interest in new gene therapy.

Representative small RNAs, siRNA and miRNA, are produced by dicer enzymes belonging to the RNase III group, a dsRNA (double strandRNA) -specific endonuclease.

siRNA is a dsRNA consisting of about 21 nucleotides and consists of about 19 base pairs and two nucleotide overhangs present on the 3 'side. These 3 ′ overhangs function as intermediate mediators in the RNAi process. siRNAs are produced when long dsRNAs are made from transposons, viruses, or genes in vivo or by artificially injecting dsRNAs into cells from outside.

miRNAs are made naturally in cells, and are RNAs that do not have the genetic information to produce proteins. The formation process of miRNA is as follows. It is transcribed into primary miRNAs by genes in the nucleus and then cleaved by drosha to form precursor miRNAs in the form of short hairpins, which then migrate from the nucleus to the cytoplasm. In the cytoplasm, a small stem loop of a precursor miRNA is cleaved by a diser enzyme, a kind of ribonuclease, and matures into a single strand of miRNA.

siRNA inhibits gene function by inducing degradation of RNA through the RNA-induced silencing complex (RISC), while miRNA inhibits the translation of mRNA, which is part of the nucleotide sequence of the 3'-UTR (untranslated region) of the target mRNA. Complementary binding inhibits gene function by inhibiting the translation of mRNA into proteins in ribosomes.

Early RNAi-related studies mostly used synthetic siRNA (double stranded RNA oligonucleotide). RNAi using synthetic siRNA is a specific nucleic acid sequence in a short time by transfection of dsRNA to a target cell directly to the cell in vitro. There is an advantage that can be screened for the effect of inhibiting gene expression.

In the case of RNAi using short hairpin RNA (shRNA), instead of directly injecting synthetic dsRNA, plasmid DNA is used to transcribe single-stranded RNA of hairpin structure, and shRNA transcribed from the DNA injected into the cell undergoes a process similar to that of microRNA. Finally converted to siRNA. Specifically, in the case of RNAi using shRNA, the plasmid controlled by the RNA polymerase III promoter is transcribed into single-stranded RNA having a loop similar to pre-miRNA in the nucleus of the target cell, and then moved to the cytoplasm by Exportin-5. It is converted into siRNA during processing by Dicer.

In order to express the above-mentioned shRNA in a cell, DNA capable of artificially expressing the shRNA is recombinantly injected into the cell. Thereafter, the shRNA is expressed in the cell by the expression mechanism of the miRNA present in the cell, thereby suppressing the expression of the target gene. Such RNA is sometimes called "artificial microRNA".

The miRNA expression vector can be transformed or infected into a cell so that the miRNA of the present invention can be expressed temporarily or permanently in the cell. The recombinant expression vector of the present invention may be constructed by recombinant DNA methods known in the art. The expression vector may be selected from the group consisting of plasmids, lentiviral vectors, retroviral vectors, adenovirus vectors known in the art, used for replication and expression of mammalian cells or other target cell types. Especially preferred are viral vectors in the present invention. The reason for this is that viral vectors have high efficiency of nucleic acid delivery in vivo, and therefore, the effect is expected to be high when applied to RNAi.

In the present invention, it is particularly preferable to use a lentivirus. Lentiviruses are a type of retroviral vector that can transduce even non-dividing cells, and the injected gene can be expressed for several months or more, which is advantageous in maintaining RNAi for a long time.

The present invention also provides a transformed cell prepared by introducing the expression vector into a host cell.

The expression vector into which the gene encoding the miRNA sequence is inserted can be introduced into a host cell by methods well known to those skilled in the art. Introduction methods include, but are not limited to, electroporation and lipofection, and methods known in the art may be selected.

The shRNA or siRNA can be designed by putting the sequence of the target gene in the Internet site such as Ambion, invitrogen, RNAi codex. The Gregory Hannon lab site also designs siRNAs or shRNAs for these genes, and targets genes in other sites and programs, including http://www.dharmacon.com/designcenter/designcenterpage.aspx and Invivogen's siRNA Wizard v3.1. Inserting a sequence allows the design of shRNA or siRNA sequences. The shRNA or siRNA sequence for the target gene is not limited and can be easily designed by those skilled in the art through various sites and programs as described above.

Exemplary LY6K siRNA sequences of the present invention include 5'-GCAAAUGGACAGAGCCAUA-3 ', 5'-ACAAUAGAGUGUGGUGUCAUGUUUG-3', 5'-CAAAUGGACAGAGCCAUACUGCGUU-3 'and the like. However, the LY6K siRNA sequence of the present invention is not limited to the three sequences exemplified above.

The LY6K shRNA sequences of the present invention include 5'-UGUGGACAGACGCCAACCUGACUGCGAGA-3 ', 5'-GCAAAUGGACAGAGCCAUACUGCGUUAUA-3', 5'-GAGGAGAAGCGGUUUCUCCUGGAAGAGGCC-3 ', and 5'-UCCUGCUGCUGGCCUC shUCC-3, etc. It is not limited to the sequences exemplified above.

In addition, miR-192-5p anti-oligonucleotide sequence of the present invention is the same as 5'-GGC TGT CAA TTC ATA GGT CAG-3 '.

In addition, miR-500a-3p sequence of the present invention is the same as 5'-AUGCACCUGGGCAAGGAUUCUG-3 '.

The composition according to the present invention preferably comprises a gene carrier or cell comprising LY6K siRNA, LY6K shRNA or miR-500a-3p, but is not limited thereto. The gene carrier is preferably a vector or a recombinant virus, but is not limited thereto. The vector may be a linear DNA vector, a plasmid vector, a vector containing a viral expression vector or a recombinant retrovirus vector, a recombinant adenovirus vector, a recombinant adeno-associated virus expressed in human or animal cells. It is preferably a recombinant viral vector including a virus, AAV) vector, a recombinant herpes simplex virus vector or a recombinant lentivirus vector. The recombinant virus is preferably a retrovirus, adenovirus, adeno-associated virus, herpes simplex virus or lenti virus, but is not limited thereto.

The composition may be administered parenterally during clinical administration, and may be administered by intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intrauterine dural injection, cerebrovascular injection or intrathoracic injection during parenteral administration. And can be used in the form of general pharmaceutical formulations. The composition can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy and biological response modifiers. The daily dosage of the composition is about 0.0001 to 1000 mg / kg, specifically 0.001 to 100 mg / kg, can be administered once or several times a day, but the weight, age, sex, health, diet, administration of the patient The range varies depending on the time, the method of administration, the rate of excretion and the severity of the disease.

Vectors containing LY6K siRNA, LY6K shRNA or miR-500a-3p of the present invention specifically contain 0.01 to 500 mg, more specifically 0.1 to 300 mg, LY6K siRNA, LY6K shRNA or miR- for recombinant viruses containing 500a-3p, specifically 10 ³ ~ 10 ¹² IU contain (10 to ¹⁰ 10 PFU), one or more specifically, contains 10 ⁵ to 10 ¹⁰ IU, but not limited thereto.

In addition, the cell containing the LY6K siRNA, LY6K shRNA or miR-500a-3p of the present invention specifically contains 10 ³ to 10 ⁸ , more specifically contains 10 ⁴ to 10 ⁷ , It is not limited.

In addition, the effective dose of the composition containing a vector or cells comprising LY6K siRNA, LY6K shRNA or miR-500a-3p of the present invention as an active ingredient is 0.05 to 12.5 mg / kg for a vector per kg of body weight, recombinant virus In the case of 10 ⁷ to 10 ¹¹ virus particles (10 ⁵ to 10 ⁹ IU) / kg, 10 ³ to 10 ⁶ cells / kg for the cells, specifically 0.1 to 10 mg / kg for the vector, recombinant virus In the case of 10 ⁸ to 10 ¹⁰ particles (10 ⁶ to 10 ⁸ IU) / kg, in the case of cells 10 ² to 10 ⁵ cells / kg, it can be administered 2-3 times a day. The composition is not necessarily limited thereto, and may vary depending on the condition of the patient and the degree of onset.

The composition can be administered in various parenteral formulations during actual clinical administration, when formulated using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants and the like commonly used. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

Hereinafter, the configuration of the present invention will be described in more detail with reference to specific embodiments. However, it is apparent to those skilled in the art that the configuration of the present invention is not limited by the scope of the embodiments.

Example 1 Experimental Method

1. Cell Culture and Transfection

MCF7, MCF7 / ADR, MDA-MB-468 and T47D cells were cultured in DMEM (WelGENE, Korea) medium containing 10% fetal calf serum (FBS, Gibco). These cells were maintained at 37 ° C in humid conditions in 5% CO ₂ and 95% air. T47D cell line stably overexpressing the human LY6K gene was made with G418 selection.

MCF7 and T47D cells were transfected for 48 h with hLY6K / pCMV6-infiltrating clones purchased from Origene using FuGENE® preparation (Promega). MCF7-ADR and MDA-MB-468 were transfected with human ERα pCMV plasmid using Fugene® reagent (Promega) according to the manufacturer's instructions. MCF7 and T47D cells were transfected with miR-192-5p mimics (miRVana ™ miRNA mimic, ambion) for 48 hours using siPORT ™ NeoFX ™ transfection agent (Ambion). Controls were transfected with negative control miRNA mimics (miVana ™ miRNA mimic, Negative control # 1, ambion). MCF7 and T47D cells were transfected with 10 nM Argonatue 2 siRNA (SMARTpooled, Dharmacon) and 4 μg of hLY6K / pCMV6 clone using Lipofectamin 2000 (Invitrogen ™) according to the manufacturer's instructions.

MCF7-ADR cells were transfected with 10 nM Argonatue 2 siRNA (Dharmacon, USA) and 5 μg of human ERα clones using Lipofectamin 2000 (Invitrogen, USA) for 48 hours as directed by the manufacturer. Transfection with scrambled siRNA (Dharmacon, USA) and pCMV-Flag plasmid is a control experiment. Subsequent experiments were performed 48 hours after transfection. After 48 hours, each was separated and used for qRT-PCR and Western blotting.

2. Transfection with mimic miRNAs and inhibitors

Manufacturer's instructions using siPORT ™ NeoFX ™ Transfection agent (Ambion, USA) to final concentrations of MCF7-ADR and MDA-MB-468 cells with miRVana ™ miR-500a-3p mimic (Ambion, USA) Reverse transfection was performed. Control experiments were transfected with miRVana ™ miR-negative control mimic (Negative control # 1, Ambion, USA).

3. Total RNA Extraction and miRNA Expression Analysis

Total RNA was extracted using Trizol® (ambion) according to the manufacturer's instructions for miRNA-microarray. 500 ng of total RNA was reverse transcribed using TaqMan® MicroRNA Reverse Transcription Kit (Applied Biosystems). Quantitative RT-PCR was performed with TaqMan Universal PCR Master Mix (Applied Biosystems) using ABI Real-time PCR System 7500 or LightCycler® 96 System (Roche), and RNU48 was used as the miRNA endogenous control. 500 ng of total RNA was reverse transcribed into TaqMan® MicroRNA Reverse Transcription Kit (Applied Biosystems) to detect has-miR-500a-3p.

4. Quantitative RT-PCR (qRT-PCR)

RNA was extracted using the NucleoSpin® RNA / Protein kit (MACHEREY-NAGEL). RNAs (5 μg) were reverse transcribed using M-MLV reverse transcriptase (Promega), 100 nM oligo-dT, 2.5 nM dNTP mixture and RNase inhibitor. The primers used are shown in Table 1 below. Quantitative RT-PCR (qRT-PCR) was performed using HiFast SYBR Lo-Rox (genepole, Korea) according to the manufacturer's instructions. Human 18s rRNA was used as anti-zone gene.

Name	Primer	Number
Human ERα	Forward: 5'-GGCCCAGCTC CTCCTCATG-3 'Reverse: 5'-AGTGGCTTTGGTCCGTCTCC-3'	12
Human LY6K	Forward: 5'-AGCCCATGCCCTTCTTTTAC-3 'Reverse: 5'-CCAGCCACAGCCCACCACAG-3'	34
Human Ago2	Forward: 5'-C TAGACCCGACTTTGGGACCT-3 'Reverse: 5'-GGGCACTTCTCTGG CTTGATA-3'	56
pri-miR-29a / b1	Forward: 5'-TGCCAGGAGCTGGTGATTTCCT-3 'Reverse: 5'-ACGGGCGTACAGAGGATCCCC-3'	78
pri-miR-29b2 / c	Forward: 5'-GGCTGGGTCTTCCGATTGAATCTCC-3 'Reverse: 5'-TCCACCCTTGGCTGTGCTGCA-3'	910
pri-miR-194-2 / 192	Forward: 5'-TCACAGGTATGTTCGCCTCA-3 'Reverse: 5'-CTCTGCTGACTGCTGGACAC-3'	1112
Human 18S rRNA	Forward: 5'-GTCGGCGTCCCCCAACTTCTT-3 'Reverse: 5'-CGTGCAGCCCCGGACATCTA-3'	1314

5. Western Blotting

Proteins were extracted using NucleoSpin® RNA / Protein kit (MACHEREY-NAGEL). Proteins were analyzed using BCA (Bicinchoninic acid) solution (Sigma, B9643) and copper sulfate solution (Sigma, C2284). Proteins were SDS-PAGE (12% separation gel, 5% accumulation gel; H ₂ O, 30% acrylamide (Bio-Rad, # 161-0156), 1.5 M pH 8.8 Tris, 10% SDS, 10% and Ammonium persulfate, TEMED (Sigma, T9281) was separated and transferred to a clean membrane (Atto, AE-6667-P) The antibodies used in the experiments are shown in Table 2. The immunoreactive proteins were horseshoe. Dish peroxidase was detected with a bound secondary antibody and amplified with a chemiluminescent material called EzWestLumi plus (ATTO, JAPAN) β-actin was used as loading control.

Name	Catalog No.	Dilution
Anti-LY6K	IL-14, sc-87282	1: 1000
Anti-ERα	HC-20, sc-543	1: 2000
Anti-argonate 2	ab57113	1: 1000
Anti-βactin	A300-491A	1: 5000

6. Luciferase analysis method

3'UTR of human ERα was amplified by PCR from T47D cDNA and cloned into the XbaI site of pGL3-control vector (Promega). Seed sequences miR-29b, miR-29c and miR-192 on the 3'UTR of human ERα were mutated with a PCR based approach. T47D and MCF7 cells were simultaneously transfected with Lipofectamin 2000 (Invitrogen) with luciferase constructs (1.8 μg reporter gene / well in 6 well plates) and pCMV-LY6K vector (200 ng / well in 6 well plates). . Cells were lysed after 48 hours and measured by Dual Luciferase Assay Kit (promega). Relative luciferase activity was normalized to the Renilla luciferase activity of the phRL-CMV vector (40 ng / well in 6 well plate; promega).

Human LY6K 3'UTR containing the expected miR-500a-3p seed sequence was amplified by PCR from human genomic DNA and psiCHECK ™ -2 vector (Promega, USA) using an In-fusion® HD cloning kit (Clontech Laboratories, USA) ) The miR-500a-3p seed sequence on LY6K 3'UTR was mutated using the QuickChange II XL site-directed mutation kit (Agilent Technologies, USA). We transfected HEK293T cells using a Lipofectamin 2000 (Invitrogen, USA) with a luciferase reporter construct containing LY6K 3'UTR variant and 30 nM miR-500a-3p mimics (mimics) or negative control miRNAs. . After 48 hours, cells were manually lysed and measured with a dual luciferase assay system (Promega, USA).

7. Cell Viability Analysis

Cell viability was measured using the WST-8 assay. MCF7-ADR, MDA-MB-468 cells, T47D or T47D / LY6K cells were seeded in 1 × 10 ⁴ cells / well in 96 well plates. Cells were treated with tamoxifen (Sigma, H790) after incubation for 24 hours and incubated again for three hours. Cells were seeded and transfected with siPORTTM NeoFXTM transfection preparation (Ambion) with negative miRNA mimics or mature miR-192-5p inhibitors (30 nM, ambion). 48 hours cells were treated with tamoxifen (4-Hydroxytamoxifen; "4-OHT"; Sigma, USA) at a concentration of 1-10 μM. WST-8 (Enzo0R) labeling mixtures were added to each well and quantified using a scanning multi-well spectrophotometer according to the manufacturer's instructions.

8. Caspase-3 Activity Assay

MCF7-ADR cells were seeded in 100 mm dishes for 24 hours using siPORT ™ neoTX ™ transfection Agent (Ambion, USA) with miR-500a-3p mimics (mimics) or negative control miRNAs. Then, 4-hydroxytamoxifen (4-OHT) was treated to a final concentration of 30 nM. After 48 hours, the cells were lysed and caspase-3 activity was measured on the cell lysate according to the manufacturer's instructions with the Caspase-3 / CPP32 Colorimetric Assay Kit (Biovision, USA).

9. Statistical Analysis

Two-tailed t-tests were performed with GraphPad InStat (Graphpad software, La Jolla, Calif., USA). Values are expressed as mean ± standard deviation. P <0.05 was determined to be significant. (* P <0.05; ** P <0.01; *** P <0.001; **** P <0.0001)

In Table 3, three LY6K siRNA sequences, four LY6K shRNA sequences, and one miR-192-5p anti-oligonucleotide sequence are shown in detail. However, it is apparent to those skilled in the art that the LY6K siRNA sequence of the configuration of the present invention is not limited only to the following sequences, and may be generated using a sequence generation program as necessary.

Name	Primer	Number
LY6K siRNA (1)	5'-GCAAAUGGACAGAGCCAUA-3 '	15
LY6K siRNA (2)	5'-ACAAUAGAGUGUGGUGUCAUGUUUG-3 '	16
LY6K siRNA (3)	5'-CAAAUGGACAGAGCCAUACUGCGUU-3 '	17
LY6K shRNA (1)	5'-UGUGGACAGACGCCAACCUGACUGCGAGA-3 '	18
LY6K shRNA (2)	5'-GCAAAUGGACAGAGCCAUACUGCGUUAUA-3 '	19
LY6K shRNA (3)	5'-GAGGAGAAGCGGUUUCUCCUGGAAGAGCC-3 '	20
LY6K shRNA (4)	5'-UCCUGCUGCUGGCCUCCAUUGCAGCCGGC-3 '	21
anti-miR-192-5pnucleotide	5'- GGC TGT CAA TTC ATA GGT CAG -3 '	22
miR-500a-3p	5'-AUGCACCUGGGCAAGGAUUCUG-3 '	23

Example 2 Experimental Results

1. LY6K expression is negatively correlated with ERα.

To determine if LY6K is involved in regulating ERα expression, we tested mRNA and protein in breast cancer cell lines, MCF7, MCF7-ADR, MDA-MB-468 and T47D. We found that ERα positive breast cancer cell lines, MCF7 and T47D, did not express LY6K mRNA and protein, whereas ERα negative breast cancer cell lines, MCF7-ADR and MDA-MB-468, expressed LY6K mRNA and protein (FIG. 1A, B). In order to confirm the inverse correlation between ERα and LY6K, we observed changes in ERα expression by LY6k overexpression in ERα positive breast cancer cell lines. As a result, ERα mRNA and protein were downregulated by LY6K (FIG. 1C, D). In addition, the expression of LY6K was decreased by the ectopic expression of ERα in MCF7-ADR and MDA-MB-468, which are well known as ERα negative breast cancer cells. Ectopic expression of ERα decreased both LY6K mRNA and protein expression in ERα negative breast cancer cells (FIGS. 2A, B).

2. LY6K regulates ERα expression after transcription.

To test the mechanism of ERα regulation by LY6K overexpression in ERα positive breast cancer cell lines, we hypothesized that miRNAs upregulated by LY6K would inhibit ERα expression. To confirm this hypothesis, ERα expression levels were confirmed by simultaneously performing Ago2 (Argonate 2) knockdown and LY6K overexpression, which are involved in the processing of mature miRNAs, as members of the RISC complex protein. As a result, ERα mRNA and protein expression were reduced by transpotent expression of LY6K, while Ago2 knockdown restored ERα expression (FIG. 3A, B). Taken together, these results indicate that LY6K inhibits ERα expression through post-transcriptional mechanisms, in particular miRNA regulation.

We also hypothesized that miRNAs upregulated by ERα would regulate the expression of LY6K mRNA and protein in order to identify the molecular mechanisms involved in LY6K expression reduction by ectopic ERα. To confirm this hypothesis, we observed whether the expression of LY6K mRNA and protein is affected by knockdown of AGO2 , a key factor in miRNA biosynthesis, and simultaneously by ERα overexpression. Ectopic expression of ERα in MCF7-ADR cells resulted in decreased LY6K expression, but AGO2 knockdown no longer caused decreased LY6K expression (FIG. 4A (white bar: siCon / Mock, black bar: siCon / Mock, gray bar: siAgo2 / ERα, B) Therefore, it has been suggested that upregulated miRNAs regulated by ERα in ERα negative and LY6K-positive breast cancer cells MCF7-ADR affect LY6K mRNA and protein expression.

3. LY6K affects miRNAs expression in ERα positive cells.

To identify miRNAs involved in ERα regulation, miRNA microarray analysis was performed on T47D, an ERα positive cell as compared to T47D / LY6Ks. Unsupervised hierarchical clustering showed a clear expression pattern between T47D / Mock and T47D / LY6K (FIG. 5A). In miRNAs upregulated in T47D / LY6K using miRanda, hsa-miR-29-3p, hsa-miR-29c-5p and hsa-miR-192-5p bind to the 3'UTR of the human ESR1 gene ( 5B).

Expression of selected miRNAs was confirmed to confirm miRNA microarray analysis. In animal miRNAs, miRNA genes are transcribed by RNA polymerase II and primary miRNAs (pri-miRNAs) are activated by binding of polymerase to the DNA sequence of interest (Lee, Kim et al. 2004). To investigate the regulatory mechanism of LY6K on miRNAs, the expression of pri-miR-29a / b1, pri-miR-29b2 / c and pri-miR-194-2 / 192 was first confirmed by qRT-PCR (FIG. 6A). . All three pri-miRNAs were upregulated by the overexpressed LY6K gene. In addition, it was confirmed that mature miRNA expression transiently transfects LY6K for 48 hours in ERα positive cell lines MCF7 and T47D (FIG. 6B). Expression of miRNAs increased in MCF7 / LY6K and T47D / LY6K. Taken together, miR-29-3p, miR-29-5p and miR-192-5p were induced by LY6K in ERα positive cells.

4. ERα  In negative breast cancer cells miR -34a, miR -194-5p and miR -500a-3p Of ERα  Upregulated by ectopic expression.

To identify miRNAs affected by ERα overexpression, miRNA microarray analysis was performed by comparing MCF7-ADR, an ERα negative cell, with MCF7-ADR / ERα, which transiently overexpresses ERα. As a result, several miRNAs were found to be affected and upregulated by ERα (FIG. 7). Of these miRNAs, three miRNAs, has-miR-34a, has-miR194-5p and has-miR-500a-3p, not only increase significantly in MCF7-ADR overexpressing ERα but also in silico analysis (http: // www.microrna.org) is expected to bind to human LY6K 3'UTR.

To verify miRNA microarray analysis, the possibility that three selected miRNAs were activated by transient ERα overexpression of MCF7-ADR cells was studied. As a result, we found that all three primary miRNAs (primary-miRNAs) are activated by RNA polymerase II, which binds to the DNA sequence of interest in miRNA biosynthesis, significantly increasing (FIG. 8A, white bars). : MCF7-ADR / Mock, black bar: MCF7-ADR / ERα), mature miRNA expression was also confirmed to increase (Fig. 8B, white bar: MDA-MB-468 / Mock, black bar: MDA-MB- 468 / ERα). This result indicates that the selected miRNAs were activated at the transcriptional stage by ERα.

5. miR-192-5p directly targets the ESR1 gene.

To determine whether the reduction in ERα expression is due to direct targeting in miR-29b-3p, miR-29c-5p and miR-192-5p, the luciferase reporter construct ESR1 3'UTR containing each miRNA predicted binding site was pGL3-controlled. The vector was cloned (FIG. 9A). Luciferase activity in MCF7 cells was reduced by transpotent expression of LY6K, transfected luciferase reporter construct wild type or miR-192-5p mutant type (FIG. 9B). These results indicate that LY6K indirectly targets ESR1 3'UTR.

Next, in order to confirm that ESR1 is a direct target of miR-192-5p, the miR-192-5p mimic and the wild type luciferase reporter construct or mutant were transfected together in MCF7 cells. Transfection performed with miR-192-5p mimics significantly reduced relative luciferase activity in wild-type constructs, suggesting that ESR1 is the target of miR-192-5p. Furthermore, ESR1 mutations at the expected miR-192-5p target sites did not affect relative luciferase activity (FIG. 9C). In contrast, luciferase activity was restored in the mutant ESR1 luciferase reporter construct. In the case of miR-29b-3p and miR-29c-5p, there was no difference in luciferase activity between the wild-type reporter construct and the mutation of the expected binding site by transfection with each miRNA (FIGS. 9D and E). In conclusion, luciferase analysis shows that miR-192-5p is a direct target of ESR1.

6. phosphorus In beats ERα  Induced by miR -500a-3p is human LY6K  Regulate by targeting genes.

The 3'UTR portion of the LY6K gene was examined to find miRNA binding motifs using the miRnada program to determine if the decrease in LY6K expression was due to direct targeting among three selected miRNAs. Luciferase reporter constructs were prepared in which human LY6K 3′UTR containing each miRNA predicted binding site was inserted into a psiCHECK-2 vector (FIGS. 10A and 11A). Only one miR-500a-3p overexpression of these miRNAs reduced luciferase activity (FIG. 10B, right). Luciferase activity was also decreased by ectopic expression of ERα (FIG. 10B, left).

This reaction was stopped by mutation of two seed sequences targeting miR-500a-3p (FIG. 10B). In the case of miR-34a and miR-194-5p, there was no difference in luciferase activity between wild-type reporter construct and mutant construct of expected binding site by each miRNA transfection (FIG. 11B).

We further identified possible functions of LY6K degradation regulation by transient overexpression of miR-500a-3p mimics (mimics). The expression of LY6K mRNA and protein was markedly reduced by ectopic miR-500a-3p in both ERα negative and LY6K positive breast cancer cells, MCF7-ADR and MDA-MB-468 (FIG. 10C). Taken together, miR-500a-3p induced by ERα directly inhibited LY6K expression levels.

7. miR-192-5p induced by LY6K modulates ERα.

To assess the possible role of miR-192-5p in the regulation of ERα degradation, ERα positive cell lines MCF7 and T47D were transiently transfected for 48 hours with miR-192-5p mimic and negative control mock (NC). Sean. qRT-PCR confirmed the transfection efficiency of miR-192-5p mimics in MCF7 and T47D cells (FIG. 12A, top). In both MCF7 and T47D cells, ERα mRNA was significantly reduced by overexpression of miR-192-5p (FIG. 12A, bottom). Furthermore, results similar to ERα protein expression for overexpression of miR-192-5p mimics in MCF7 and T47D cells were confirmed (FIG. 12B).

In addition, to observe that miR-192-5p expression inhibition restored ERα mRNA expression, a T47D stable cell line overexpressing the human LY6K gene was prepared by G418 selection. It was confirmed by qRT-PCR and Western blot that these stable cell lines express large amounts of LY6K mRNA and protein (FIG. 12C). We inhibited the expression of miR-192-5p using T47D / hLY6K cells. In conclusion, miR-192-5p inhibition restored ERα mRNA expression (FIG. 12D). Similarly, ERα protein expression in T47D / hLY6K stabilized cells was upregulated by inhibiting miR-192-5p expression (FIG. 12E). Taken together, miR-192-5p induced by LY6K inhibited ERα expression levels.

8. miR-192-5p inhibition in breast cancer cells restores tamoxifen sensitivity.

We found that miR-192-5p downregulated ERα expression by directly targeting the 3′UTR. From this, it can be hypothesized that miR-192-5p inhibition can restore tamoxifen resistance in cells overexpressing LY6K. This is because tamoxifen is well known as a drug targeting ERα. In order to confirm that T47D / LY6K cells exhibited tamoxifen resistance, T47D and T47D / LY6K cells were treated with tamoxifen by concentration and cell viability was observed. As a result, tamoxifen treatment significantly reduced cell viability in T47D cells, which are ERα positive breast cancer cells. On the other hand, in T47D / LY6K cells, when treated with tamoxifen, cell survival was hardly reduced due to downregulation of ERα expression (FIG. 13A). In addition, inhibition of miR-192-5p expression compared to negative miRNA inhibitors (NC) resulted in T47D / LY6K cells becoming more sensitive to tamoxifen (FIG. 13B). From this result, it can be seen that miR-192-5p regulates tamoxifen resistance through cell proliferation.

9. In breast cancer cells miR -500a-3p Apoptosis  By promoting tamoxifen sensitivity.

We found that miR-500a-3p lowered LY6K expression by directly targeting the 3′UTR of LY6K. In the present invention, whether miR-500a-3p enhances tamoxifen sensitivity through apoptosis activation in ERα negative breast cancer cells. Tamoxifen is a selective estrogen antagonist and is well known as a drug targeting ERα in ERα positive breast cancer patients, but only about 10-15% of patients with ERα negative breast cancer respond to tamoxifen.

The present inventors treated 4-OHT (4-hydroxytamoxifen) by concentration in MCF7-ADR cells transiently overexpressing ERα and observed cell viability. Cell viability in MCF7-ADR was significantly reduced by ectopic expression of miR-500a-3p (FIG. 14). In addition, tamoxifen in breast cancer cells not only inhibited cell proliferation but also induced apoptosis. We tested whether miR-500a-3p reexpression enhances tamoxifen sensitivity through apoptosis. As a result, miR-500a-3p significantly increased caspase-3 activation when tamoxifen treated as compared to the negative control. Taken together, miR-500a-3p induced by ERα regulates tamoxifen sensitivity by targeting LY6K in ERα negative breast cancer cells.

Claims (27)

1. A biomarker composition for diagnosing anticancer sensitivity of anticancer agent resistant breast cancer, comprising LY6K.
2. An anticancer drug sensitivity diagnostic kit for anticancer drug resistant breast cancer, comprising an agent for detecting LY6K.
3. Analyzing LY6K expression in a sample isolated from the individual; And

   Comparing the expression level with LY6K expression level in the normal group

   Comprising, anticancer drug sensitivity diagnosis of anticancer drug resistant breast cancer.
4. A pharmaceutical composition for enhancing the anticancer effect, comprising a LY6K inhibitor and enhancing the anticancer effect of the anticancer agent against anticancer drug resistant breast cancer.
5. The method according to claim 4,

   The LY6K inhibitor is at least one selected from the group consisting of a LY6K gene-specific siRNA, a LY6K gene-specific shRNA, a recombinant expression vector comprising a LY6K gene-specific siRNA, and a recombinant expression vector comprising a LY6K gene-specific shRNA. To, anti-cancer effect for improving pharmaceutical composition.
6. The method according to claim 5,

   The LY6K gene-specific siRNA is characterized in that any one of SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17, anti-cancer effect enhancing pharmaceutical composition.
7. The method according to claim 5,

   The LY6K gene-specific shRNA is characterized in that any one of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21, a pharmaceutical composition for enhancing anticancer effect.
8. The method according to claim 5,

   The expression vector is any one selected from the group consisting of lentiviral vectors, retroviral vectors and adenovirus vectors.
9. A pharmaceutical composition for preventing or treating cancer disease, comprising a LY6K inhibitor and an anticancer agent, and enhancing an anticancer effect of the anticancer agent against the anticancer drug resistant breast cancer.
10. The method according to claim 9,

    The anticancer agent is tamoxifen, 5-Fluorouracil, doxorubicin, doxorubicin, mitomycin, cisplatin, paclitaxel, docetaxel, docetaxel, irinotecan, Irinotecan. Xeloda (Xeloda), oxalopatin (Oxalopatin) and etoposide (etoposide) is any one or more selected from the group consisting of a pharmaceutical composition for preventing or treating cancer diseases.
11. A biomarker composition for diagnosing anticancer sensitivity of anticancer agent resistant breast cancer, comprising miR-192-5p.
12. An anticancer drug sensitivity diagnostic kit for anticancer drug resistant breast cancer, comprising an agent for detecting miR-192-5p.
13. Analyzing miR-192-5p expression in a sample isolated from the subject; And

    Comparing the expression level with miR-192-5p expression level in the normal group

    Comprising, anticancer drug sensitivity diagnosis of anticancer drug resistant breast cancer.
14. A pharmaceutical composition for preventing or treating cancer diseases, comprising a miR-192-5p inhibitor and enhancing an anticancer effect of an anticancer agent against anticancer drug resistant breast cancer.
15. The method according to claim 14,

    The miR-192-5p inhibitor is an anti-miR-192-5p oligonucleotide or a pharmaceutical composition for preventing or treating cancer disease, characterized in that any one of the expression vectors comprising the same.
16. The method according to claim 15,

    The anti-miR-192-5p oligonucleotide is a pharmaceutical composition for enhancing anticancer effect, characterized in that represented by SEQ ID NO: 22.
17. The method according to claim 15,

    The expression vector is any one selected from the group consisting of lentiviral vectors, retroviral vectors and adenovirus vectors.
18. A pharmaceutical composition for preventing or treating cancer disease, comprising a miR-192-5p inhibitor and an anticancer agent, and enhancing an anticancer effect of the anticancer agent against the anticancer drug resistant breast cancer.
19. The method according to claim 18,

    The anticancer agent is tamoxifen, 5-Fluorouracil, doxorubicin, doxorubicin, mitomycin, cisplatin, paclitaxel, docetaxel, docetaxel, irinotecan, Irinotecan. Xeloda (Xeloda), oxalopatin (Oxalopatin) and etoposide (etoposide) is any one or more selected from the group consisting of a pharmaceutical composition for preventing or treating cancer diseases.
20. Biomarker composition for diagnosing anticancer agent sensitivity of anticancer agent resistant breast cancer, comprising miR-500a-3p.
21. An anticancer drug sensitivity diagnostic kit for anticancer drug resistant breast cancer, comprising an agent for detecting miR-500a-3p.
22. Analyzing miR-500a-3p expression in a sample isolated from the subject; And

    Comparing the expression level with miR-500a-3p expression level in the normal group

    Comprising, anticancer drug sensitivity diagnosis of anticancer drug resistant breast cancer.
23. A pharmaceutical composition for enhancing anticancer effect, comprising miR-500a-3p or an expression promoter thereof and enhancing the anticancer effect of an anticancer agent against anticancer drug resistant breast cancer.
24. The method according to claim 23,

    The miR-500a-3p is a pharmaceutical composition for enhancing anticancer effect, characterized in that represented by SEQ ID NO: 23.
25. The method according to claim 23,

    The expression promoter is any one of the expression vector selected from the group consisting of lentiviral vector, retrovirus vector and adenovirus vector expressing miR-500a-3p anti-cancer effect enhancement pharmaceutical composition.
26. A pharmaceutical composition for preventing or treating cancer diseases comprising any one of miR-500a-3p or an expression promoter thereof and an anticancer agent, and promoting an anticancer effect of the anticancer agent against the anticancer drug resistant breast cancer.
27. The method of claim 26,

    The anticancer agent is tamoxifen, 5-Fluorouracil, doxorubicin, doxorubicin, mitomycin, cisplatin, paclitaxel, docetaxel, docetaxel, irinotecan, Irinotecan. Xeloda (Xeloda), oxalopatin (Oxalopatin) and etoposide (etoposide) is any one or more selected from the group consisting of a pharmaceutical composition for preventing or treating cancer diseases.

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