KR20120089387A - Anti-Sense Nucleic Acid of Inhibiting Expression of Granulin-Epithelin-Precursor Gene and Pharmaceutical Composition Containing Thereof - Google Patents

Abstract

PURPOSE: An antisense nucleic acid to granulin-epithelin precursor(GEP) and a composition containing the same are provided to suppress and treat hepatocellular carcinoma. CONSTITUTION: An antisense nucleic acid which suppresses granulin-epithelin precursor(GEP) expression contains siRNA having a base selected from sequence numbers 1-3; or shRNA having oligonucleotide(complementary oligonucleotide) which is linked to the siRNA through a linker sequence from the 5'-end or 3'-end. The shRNA has a base selected sequence numbers 4-9. A pharmaceutical composition for treating granulin-epithelin precursor-derived diseases contains the antisense nucleic acid or recombinant vector containing the nucleic acid.

Description

Anti-Sense Nucleic Acid of Inhibiting Expression of Granulin-Epithelin-Precursor Gene and Pharmaceutical Composition Containing Thereof}

The present invention relates to nucleic acids that inhibit the expression of genes, and more particularly, to nucleic acids as antisense that inhibits the expression of granular-epiterin-precursors, vectors into which they are inserted and pharmaceutical compositions containing them.

Hepatocellular carcinoma (HCC) is a representative type of liver tumor and is one of the major causes of death. Hepatocellular carcinoma is known to be mainly caused by hepatitis B, hepatitis C, aflatoxin, alcoholism and the like. In particular, when hepatitis B virus or hepatitis C virus is continuously infected, the immune system of the human body continuously attacks the liver cells, causing cirrhosis, and thus hepatocellular carcinoma develops.

Meanwhile, granulin-epithelin-precursor (GEP) may be used for progranulin, acroranin, and PC-cell-derived growth factor. It is a kind of glycoprotein that is released outside the cell, also called proepithelin, and is known as a growth factor that promotes cell cycle progression and cell growth. Granulin-epiterin-precursor (hereinafter referred to as GEP) is a glycoprotein composed of 593 amino acids, and mRNA expressing it is expressed in epithelial cells and hematopoietic cells. In particular, GEP is one of the genes overexpressed in hepatocellular carcinoma (HCC) having the gene locus of human chromosome 17q21.32, Figure 1 schematically shows the GEP gene and expression forms.

As shown in FIG. 1, the GEP genes of mammals, including humans, contain the exon (N) encoding the N-terminus, the exon (C) encoding the C-terminus, and the N-terminus / C-terminus. It consists of 12 exons, including exons (CN), and when the 10th exon (X) is not expressed, it forms a hybrid grn / epi domain with grnC at the N-terminus and grnD at the C-terminus. . In particular, the cysteinyl motif or Granulin / Epithelin module (GEM), which contains a total of 12 cysteines that form disulfide bonds like other granulins, tandem 7.5 times Repeated (Bateman A, Bennett HP 1998. Granulins: the structure and function of an emerging family of growth factors " J. Endocrinology, 158, 145-51, Non-Patent Document 1). The expressed GEP is secreted approximately 68-88 kDa. It is glycosylated in many parts as a protein.

Regarding the intracellular signal transduction of GEP in mammalian cells, GEP is responsible for the shc and p44 / 42 mitogen activating protein phosphatase (p44 / 42 MAPK) in the extracellular regulated phosphatase signaling pathway. In addition to promoting phosphorylation, phosphatidyl itositol-3 phosphatase (PI-3K) and protein phosphatase B / AKT are known to activate. Through this mechanism, GEP increases the expression of cyclin B and cyclinD, thereby regulating wound healing, early embryonic development and brain differentiation through somatic cell division or post- wound cell proliferation, as well as inflammation. Inflammatory reactions as inhibitors of the cytokine tumor necrosis factor-α (TNF-α) and the secretion of cytokines such as interleukin 8 (IL-8) are involved in immune responses, as well as neurodegenerative diseases such as dementia And is overexpressed in a variety of cancer cell lines such as adenocarcinoma, sheep, ovarian cancer and prostate cancer, especially breast cancer, ovarian cancer and prostate cancer (Andrew Bateman and Hugh PJ Bennett. 2009. The Granulin gene family: from cancer to dementia ". BioEssays , 31, 1245-1254, Non-Patent Document 2).

In particular, recent studies involving hepatocellular carcinoma (HCC) have shown that GEP RNA concentrations and protein expression levels in hepatocellular carcinoma cells are significantly higher than normal GEP RNA concentrations and protein expression levels, and that GEP proteins are elevated. Up-regulation is closely associated with hepatocellular carcinoma (Siu Tim Chenung et al., 2004. Granulin-Epithelin Precursor Overexpression Promotes Growth and Invasion of Hepatocellular Carcinoma. Clinical Cancer Research . 10, 7629-36, Non-Patent Literature 3) In particular, anti-GEP monoclonal antibody (A23) reduces the expression of GEP in serum and inhibits tumor growth in a concentration dependent form, which is anti-GEP monoclonal antibody (p44 / 42 MAPK) and Akt. GEP may be a target for the treatment of hepatocellular carcinoma because it inhibits the growth of hepatocellular carcinoma such as Hep3B hepatocarcinoma cells by reducing the proliferation of tumor cells through the pathway (Jenny C. Ho et. al., 2008. Granulin-Epithelin Precursor as a Therapeutic Target for Hepatocellular Carcinoma. Hepatology 47 (5), 1524-32, Non-Patent Document 4).

Overexpression of GEP in HpeG2 cells increased the expression of p53, a tumor suppressor, whereas inhibition of the expression of GEP protein decreased the expression of p53. (Siu Tim Cheung et al., 2006. GEP associates with wild-type p53 in hepatocellular carcinoma. Oncology Reports , 15, 1507-1511, Non-Patent Document 5).

In addition, some patent documents related to GEP have been published. For example, Korean Patent No. 10-0896328 (Patent Document 1) uses a specific antibody to progranuline to measure the concentration of progranuline in the blood. As a method, a composition for detecting a marker for treating metabolic diseases such as obesity, diabetes, and cardiovascular diseases, and a kit for diagnosing metabolic diseases including the same are provided.

As such, research on GEP, which is known to cause various diseases by controlling various biological reactions or phenomena, needs to be further progressed. In particular, there is a need to develop a method for treating these diseases as a method of inhibiting the expression of GEP causing various types of cancer as well as hepatocellular carcinoma.

1. Korea Patent Registration No. 10-0896328 (Notice date 2009. 5. 7)

Bateman A, Bennett H.P. 1998. Granulins: the structure and function of an emerging family of growth factors "J. Endocrinology, 158, 145-51. 2. Andrew Bateman and Hugh P. J. Bennett. 2009. The Granulin gene family: from cancer to dementia ". BioEssays, 31, 1245-1254. 3. Siu Tim Chenung et al., 2004. Granulin-Epithelin Precursor Overexpression Promotes Growth and Invasion of Hepatocellular Carcinoma. Clinical Cancer Research. 10, 7629-36. 4. Jenny C. Ho et al., 2008. Granulin-Epithelin Precursor as a Therapeutic Target for Hepatocellular Carcinoma. Hepatology 47 (5), 1524-32. 5. Siu Tim Cheung et al., 2006. GEP associates with wild-type p53 in hepatocellular carcinoma. Oncology Reports, 15, 1507-1511.

The present invention has been proposed to solve the above problems, and an object of the present invention is to suppress the expression of granule-epiterin precursor (GEP) involved in various biological reactions, specific sequence of mRNA of GEP and It is an object to provide complementary antisense nucleic acids, for example short-interfering RNA (siRNA) or short-hairpin RNA (shRNA).

It is another object of the present invention to provide a vector or gene carrier to which the antisense nucleic acid described above is inserted.

It is still another object of the present invention to provide a cell which inhibits the expression of GEP by introducing an antisense nucleic acid described above or a vector or gene carrier into which the antisense nucleic acid is inserted, into a host cell, and a method of producing the same.

Still another object of the present invention is to provide a pharmaceutical composition for inhibiting GEP expression containing the antisense nucleic acid or vector or gene carrier as an active ingredient.

Other objects and advantages of the invention will become apparent from the following detailed description and claims.

The present invention relates to an antisense nucleic acid prepared to inhibit the expression of granule-epiterin-precursor (GEP) and a method of using the same.

That is, according to one aspect of the invention, i) short-interfering RNA (siRNA) having a base sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 3, or ii) SEQ ID NO: Oligonucleotides (complementary oligos) capable of complementarily binding to SEQ ID NO: 1 to SEQ ID NO: 3 through a linker sequence that is linked to the 5 'end or 3' end of any one nucleotide sequence selected from 1 to SEQ ID NO: 3 Antisense nucleic acid that inhibits the expression of Granulin-Epithelin precursor (GEP), consisting of short-hairpin RNA (shRNA) linked to the short-interfering RNA To provide.

In this case, the short-hairpin RNA is 1-20 oligonucleotides (first auxiliary oligonucleotides) connected to one end of the short-interfering RNA, and connected to one end of the complementary oligonucleotides, and the first auxiliary It may further have an oligonucleotide (secondary oligonucleotide) capable of complementarily binding to the oligonucleotide.

Specifically, the short-hairpin RNA may be composed of any one nucleotide sequence selected from the group consisting of SEQ ID NO: 4 to SEQ ID NO: 9.

Meanwhile, according to another aspect of the present invention, there is provided a recombinant vector into which the aforementioned anti-sex nucleic acid is inserted.

In this case, the recombinant vector may be a plasmid vector or a viral vector. For example, the viral vector may include baculovirus, SV40 virus, vaccinia virus, adenovirus, adeno-associated virus, retrovirus, chicken pox virus, and pseudorabies virus. May be a viral vector.

According to another aspect of the present invention, there is provided a pharmaceutical composition for treating a disease derived from Granulin-Epithelin precursor (GEP), which contains the aforementioned antisense nucleic acid or recombinant vector as an active ingredient. to provide. In this case, the composition may be for treating a disease of hepatocellular carcinoma.

According to another aspect of the present invention, a method for producing a cell in which the expression of Granulin-Epithelin precursor (GEP) is suppressed, wherein the antisense nucleic acid or recombinant vector described above is introduced into a mammalian-derived host cell. To provide. The host cell may be isolated or cultured and may be liver cancer cell.

In the present invention, siRNA and shRNA as antisense nucleic acids, which have been proposed to inhibit the expression of granulin-epiterin precursor (GEP), which is a causative factor of various diseases as well as liver diseases such as hepatocellular carcinoma, have been prepared.

These antisense nucleic acids can be directly injected into the appropriate host cell having the disease caused by GEP, or a recombinant vector containing the antisense nucleic acid can be injected into the host cell to suppress the disease caused by GEP. Can be.

That is, by applying the above-described antisense nucleic acid and the vector into which the antisense nucleic acid is inserted as an active ingredient, and in the form of a pharmaceutical composition containing an appropriate carrier or the like, diseases caused by overexpression of GEP, such as dementia Neurodegenerative diseases, as well as cancer diseases such as adenocarcinoma, sheep, ovarian cancer and prostate cancer, in particular liver cancer, hepatocellular cancer, breast cancer, ovarian cancer, prostate cancer can be treated.

1 is a schematic representation of genes comprising exons and introns of granule-epiterin-precursor (GEP), proteins expressed therefrom and characteristic amino acid sequences.
Figure 2a shows the nucleotide sequence of the cloned confirmed GEP according to an embodiment of the present invention, the site where the translation start and transcription termination site is shown in the form of a box, Figure 2b is used to express GEP Is a schematic cleavage-map of the plasmid vectors thus obtained.
3 is a view showing a state in which GEP is expressed according to an embodiment of the present invention, Figure 3a is a photograph after performing Western blotting, Figure 3b is a graph showing the measurement results according to the ELISA analysis.
Figure 4 schematically shows the structure of GEP mRNA to which the short-interference RNA identified in accordance with one embodiment of the present invention is bound.
5 is a measure of the degree of inhibition of GEP expression using three siRNA according to an embodiment of the present invention, Figure 5a is a graph measuring the degree of inhibition of GEP expression over time, Figure 5b is siRNA It is a graph measuring the degree of inhibition of GEP expression according to the change in concentration. In Figure 5a GEP-siRNA106 is a commercially available short-interference RNA (AC AAG CCU UGA AGA GAG A, SEQ ID NO: 11, Bioneer) used for comparison with antisense nucleic acids according to the present invention, GFP is green fluorescent The transfected plasmid containing the gene expressing the protein was used as transfection efficiency confirmation and negative control. pcDNA-GEP is a measure of the expression of GEP in the absence of antisense nucleic acid.
6 is a measure of the degree of suppression of GEP expression using shRNA, an antisense nucleic acid modified through a linker sequence and an additional complementary sequence, according to another embodiment of the present invention, Figure 6a is a Figure 6b is a graph measuring the degree of inhibition of expression, Figure 6b is a graph measuring the degree of inhibition of expression of GEP according to the change in the concentration of shRNA, GFP transfection of the plasmid containing the gene expressing the green fluorescent protein confirmed transfection efficiency and negative As a control, pcDNA-GEP is a measure of the expression of GEP in the absence of antisense nucleic acid.
Figure 7 is a graph measuring the results through real-time PCR for Hep3B cells to determine whether the modified shRNA reduces the mRNA level of GEP present in the cell according to another embodiment of the present invention.
8 is a graph measuring growth inhibition of liver cancer cells in order to determine whether RNAi effects inhibit GEP expression by targeting GEP acting as a growth factor of liver cancer.
Figure 9 is a measure of tumor reduction by the antisense nucleic acid according to the present invention in vivo, Figure 9a is a graph measuring the degree of inhibition of the tumor after injecting the shRNA synthesized according to the invention in the mouse, Figure 9b And FIG. 9C is a comparison of the results using luciferase-inhibited shRNA as a negative control.
Figure 10 shows the results of measuring the number of cells according to the cell cycle (cell cycle) of the Hep3B liver cancer cell line according to an embodiment of the present invention, the upper left (Luciferase-shRNA) is a liver cancer cell line treated with luciferase inhibitory shRNA As a result of measuring the cell number according to the cell cycle, the upper right (pcDNA-GEP) is a cell number measurement according to the cell cycle of the liver cancer cell line not treated with antisense nucleic acid, the lower left (GEP-shRNA) is the antisense nucleic acid according to the present invention. It is a graph showing the cell number measurement results according to the cell cycle of the treated hepatocellular carcinoma cell line, the bottom right is the result in a tabular form.

After recognizing the above-mentioned problems, the present inventors have studied methods for suppressing diseases caused by overexpression of granulin-epiterin-precursor (GEP), such as various tumors or liver cancer, The present invention was completed based on the fact that antisense nucleic acids capable of complementary hybridization with mRNA of GEP can be transfected into an appropriate host cell to prevent and treat liver disease caused by overexpression of GEP. . EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail, referring an accompanying drawing.

The present inventors first measured the expression level of GEP based on the gene sequence (SEQ ID NO: 1) of the known granule-epiterin-precursor (GEP). After cloning the GEP using the gene sequence (SEQ ID NO: 1) of the GEP shown in FIG. 2A and the expression vector shown in FIG. 2B, the degree of expression was confirmed (FIGS. 3A and 3B). Subsequently, three types of short-interfering RNAs (siRNA, SEQ ID NO: 2 to SEQ ID NO: 4) as antisense nucleic acids capable of complementarily binding to specific sequences of mRNA of GEP and inhibiting their activity were found.

As a result of analyzing the secondary structure of the GEP mRNA attacked by siRNAs of SEQ ID NO: 2 to SEQ ID NO: 4 confirmed in accordance with the present invention, the target sites of these antisense nucleic acids have a stem-loop structure and are free. The energy was expected to be quite high, and access to RNAi via the antisense nucleic acid according to the present invention was expected for the target site of GEP (FIG. 4).

Subsequently, the present inventors confirmed whether the expression level of the target gene, GEP, was suppressed against the antisense nucleic acid as the siRNA of SEQ ID NO: 2 to SEQ ID NO: 4. Expression of GEP by antisense nucleic acids (GEP-siRNA1, GEP-siRNA2, GEP-siRNA3) of SEQ ID NO: 2 to SEQ ID NO: 4 as these short-interfering RNAs was significantly inhibited (see FIG. 5A), in particular of the siRNA used It was confirmed that the expression of GEP was suppressed depending on the concentration (see FIG. 5B).

In addition, the present inventors examined whether the antisense nucleic acid in the form of shRNA inhibits the expression of GEP in the state in which the siRNA is modified to be in the form of shRNA in consideration of instability in vivo when transfected in the form of siRNA. As a result, like the siRNA described above, shRNA also inhibited the expression of GEP (see FIG. 6A), and similarly, it was confirmed that the expression of GEP was suppressed depending on the concentration (see FIG. 6B).

In addition, the antisense nucleic acid used in the present invention, as a result of injection into Hep3B cells, a liver cancer cell line, was found to not only reduce the mRNA level of GEP by 4 times (FIG. 7) but also significantly inhibit the growth of cancer cells (FIG. 8). ).

In addition, after forming a tumor by injecting a liver cancer cell line into the mouse, it was also confirmed that the tumor size is greatly reduced as a result of injecting antisense nucleic acids according to the present invention (see Figs. 9a to 9c). In particular, according to the present invention, in the case of liver cancer cells treated with the GEP antisense nucleic acid synthesized according to the present invention, the cell cycle is stopped by a specific cycle (S phase and G2 / M phase), the cell It was confirmed to suppress the growth of (see Fig. 10).

1. Antisense Nucleic Acids

Therefore, the basic starting point of the present invention is to design an antisense nucleic acid such as siRNA or shRNA capable of hybridizing with a specific site of mRNA of GEP for the purpose of treating or preventing a disease caused by overexpression of GEP. The antisense nucleic acid designed in this way is injected directly into a cell or in the form of a recombinant vector to inhibit GEP expression through diseases caused by GEP overexpression, neurodegenerative diseases such as dementia and liver cancer, hepatocellular cancer, breast cancer and ovary. Cancer, adenocarcinoma such as prostate cancer, sheep, ovarian cancer and various cancer diseases such as prostate cancer.

RNA interference (RNAi) is simply a translation of a specific protein by a nucleic acid that is complementary to an mRNA expressing a specific protein, that is, an antisense nucleic acid, thereby inhibiting the expression of the specific protein. Refers to a phenomenon that does not occur. RNAs involved in RNA interference include microRNAs (miRNAs) and short interfering RNAs (siRNAs), which miRNAs complementarily bind to the 3 'nontranscribed region (3' UTR) of the target mRNA. In addition, siRNA hybridizes with a specific site of the protein coding region of the target mRNA to inhibit expression of the protein due to the target mRNA. RNAi is particularly involved in the regulation of cellular immune response and gene expression against parasitic genes such as viruses and transposons.

Among RNA-related RNAs, particularly short interfering RNAs (siRNAs) have been identified in various eukaryotes and are also known as 'post-transcripiton gene silencing' (Andrew J. Hamilton, David C. Baulcombe (1999). "A Species of Small Antisense RNA in Posttranscriptional Gene Silencing in Plants", Science 286 (5441): 950-52; Sayda M. Elbashir, Jens Harborth, Winfried Lendeckel, Abdullah Yalcin, Klaus Weber & Thomas Tuschl (2001). "Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells". Nature 411 (6836): 494 ?? 98). The schematic mechanism of siRNA is as follows. First, externally-induced or internally produced double-stranded RNA (dsRNA) is cleaved by an enzyme called Dicer with ribonuclease activity and converted into short fragments consisting of approximately 20-30 nucleotides.

The short fragments of RNA thus formed are called 'short interfering RNAs (siRNAs), and the double-stranded siRNA fragments are separated into individual strands, and one strand of RNA (guide strand) is RNA-induced silencing binds to a complex (RISC). When siRNA bound to RISC pairs with the complementary sequence of mRNA, argonatue protein having endonuclease activity as a catalytic component of RISC is known to cleave the target mRNA strand complementary to siRNA.

Using such RNA interference, double stranded RNA having a sequence complementary to a specific gene is synthesized and introduced into a cell to be used for 'gene knock down' which suppresses the expression of a specific gene, or specifically inserts a synthesized siRNA into a vector. Then, the method may be used for gene therapy, which inhibits the expression of genes associated with a specific disease by injecting into a host cell or directly injecting siRNA.

However, siRNA provided in single-stranded form is degraded in a short time in vivo because of its low stability, there is a limit to the effect of inhibiting the expression of the target gene. In order to overcome this limitation, a method of modifying siRNA to short sequence RNA (shRNA) is used.

Recent studies show that short RNAs of approximately 25 nucleotides can be introduced into mammalian cells to induce RNAi in mammalian cells, and vectors expressing chemically synthesized siRNA and shRNA are expressed in vitro. ) And in vivo RNAi.

Thus, the term 'anti-sense' in the present specification is introduced into a cell directly or through expression of another nucleic acid introduced directly into the cell, thereby specifically hybridizing with a portion of the mRNA in the cell encoding the protein. By nucleic acid means blocking the translation of the protein to inhibit the expression of the target protein.

On the other hand, the term 'short-interference RNA (siRNA)' as used herein, refers to RNA having a base sequence complementary to a specific base sequence of the target mRNA. On the other hand, 'short / small hairpin RNA (shRNA)' refers to transfection into cells through a linker sequence of sufficient length selectively for such short-interfering RNA and, if necessary, through a vector. refers to RNA modified to be transfected. In this sense 'antisense nucleic acid' is understood to include short-interfering RNA, short-hairpin RNA.

In this sense, siRNA terminal structures according to the present invention can be blunt ends or cohesive ends as long as the expression of the target gene can be suppressed by the RNAi effect. The cohesive end structure may be a structure in which the 3 'end protrudes and / or a structure in which the 5' end protrudes. The number of protruding bases is not limited and may be, for example, 1 to 8 bases, preferably 2 to 6 bases.

In addition, the antisense nucleic acid of the present invention can maintain the expression inhibitory effect (RNAi) of GEP used as a target gene, for example, low molecular RNA (eg, tRNA, rRNA, Natural RNA molecules such as viral RNA or artificial RNA molecules). The siRNA terminal structures need not all have a cleavage structure, and one end portion of the siRNA may be modified to have a shRNA form having a stem-loop structure connected by a linker RNA. At this time, if the stem portion does not interfere with forming a base pair, the length of the linker is not particularly limited, but may have a length of about 1-20, preferably about 3-10. For example, in Table 1 below, the antisense nucleic acid according to the present invention, which can bind complementarily to mRNA of granule-epiterin-precursor (GEP) having the nucleotide sequence represented by SEQ ID NO: 1, inhibits its activity. An example of this is shown.

Antisense Nucleic Acids for Granulin-Epiterine-Precursors denomination Sequence (5 '-> 3') Remarks GEP-siRNA1 GGACACUUCUGCCAUGAUA (SEQ ID NO: 2) Target position 1578 GEP-siRNA2 GAGAGAUGUCCCCUGUGAU (SEQ ID NO: 3) Target position 1091 GEP-siRNA3 UCUAAGGCCUUCCCUGUCA (SEQ ID NO: 4) Target location 1934 GEP-shRNA_A GGACACUUCUGCCAUGAUA UCUCUC UAUCAUGGCAGAAGUGUCC
(SEQ ID NO: 5) Before and after the linker (UCUCUC) sequence can form a stem structure double bond GEP-shRNA_B GAGAGAUGUCCCCUGUGAU UCUCUC AUCACAGGGGACAUCUCUC
(SEQ ID NO: 6) GEP-shRNA-C UCUAAGGCCUUCCCUGUCA UCUCUC UGACAGGGAAGGCCUUAGA
(SEQ ID NO: 7) GEP-shRNA GGACACUUCUGCCAUGAUA ACCAGA UCUCUC UCUGGU
UAUCAUGGCAGAAGUGUCC (SEQ ID NO: 8) Auxiliary sequences (ACCAGA / UCUGGU) are inserted before and after the linker for stable double bond GEP-shRNA2 GAGAGAUGUCCCCUGUGAUACCAGA UCUCUC UCUGGU
AUCACAGGGGACAUCUCUC (SEQ ID NO: 9) GEP-shRNA3 UCUAAGGCCUUCCCUGUCAACCAGA UCUCUC UCUGGU
UGACAGGGAAGGCCUUAGA (SEQ ID NO: 10)

As representatively described in Table 1, the antisense nucleic acid of the present invention has a short interfering RNA (siRNA) of SEQ ID NO: 2 to SEQ ID NO: 4 having 19 bases, and a linker sequence so that the short interfering RNA can form a double bond. It may be a short hairpin RNA (shRNA) of, for example, SEQ ID NO: 5 to SEQ ID NO: 10 linked through. The linker sequence for forming the shRNA is not limited to that illustrated in Table 1 as long as it is for forming a so-called 'loop' structure. Thus, shRNAs according to the invention can be transformed into shRNAs with enhanced stability in vivo by other suitable linker sequences.

In particular, Table 1 describes an oligonucleotide (complementary oligonucleotide) that binds to the siRNA sequence complementarily through a linker sequence linked to the 3 'end of the siRNA. Also possible are shRNAs to which oligonucleotides are linked that can complementarily bind to siRNA sequences through linker sequences to which they are linked.

In addition, as can be seen in SEQ ID NO: 8 to SEQ ID NO: 10 of Table 1, for example, 1-10, preferably 1-6, between the siRNA and the linker sequence according to the present invention or to the other end of the siRNA to which the linker is not linked Add up to about bases (ACCAGA, first auxiliary oligonucleotide), correspondingly between the oligonucleotide and linker sequence capable of complementary binding to the siRNA or to the other end of the complementary oligonucleotide with no linker linked An shRNA added with a base (UCUGGU, a second auxiliary oligonucleotide) capable of complementarily binding to one first auxiliary nucleotide may also be incorporated into an antisense nucleic acid according to the present invention.

At this time, as the antisense nucleic acid of the present invention, at least one base as a siRNA of SEQ ID NO: 2 to 4 or shRNA of SEQ ID NO: 5 to SEQ ID NO: 10 as well as a functional equivalent having a mutation that does not degrade their biological activity are substituted, Variants with insertions, deletions, and combinations thereof. For example, such variants may have at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% homology with the siRNAs of SEQ ID NO: 2 to SEQ ID NO: 4. . Such homology can be readily determined by using computer algorithms well known in the art, such as Align or BLAST algorithms, to compare the sequence of nucleotides with the corresponding portions of the target nucleotides.

The method for producing the antisense nucleic acid according to the present invention is not particularly limited. For example, siRNA can be synthesized directly chemically (Sui G et al., Proc Natl Acad Sci USA 99: 5515-5520, 2002), or by using in vitro transcription (Brummelkamp TR et al., Science 296: 550-553, 2002), Methods of Enzymatically Cutting Long Double-Stranded RNA Synthesized by In Vitro Transcription (Paul CP et al., Nature Biotechnology 20: 505-508, 2002 ), expression by intracellular delivery of siRNA expression plasmids or viral vectors (Lee NS et al., Nature Biotechnology 20: 500-505, 2002) and PCR (polymerase chain reaction) induced cells of siRNA expression cassettes Expression via internal delivery (Castanotto D et al., RNA 8: 1454-1460, 2002), and the like. Determination of the method of preparing siRNA may vary depending on the purpose of the experiment and the cellular biological function of the target gene product.

2. Recombinant Vectors and Gene Carriers

The antisense nucleic acids described above in connection with the present invention can also be transfected directly into host cells, but plasmid vectors or viral vectors can be utilized. For example, antisense nucleic acids such as siRNAs according to the invention or shRNAs modified with appropriate linker sequences can be inserted so that they can be cloned into a suitable vector. In this sense, as a recombinant vector comprising an antisense nucleic acid according to the present invention, numerous expression systems such as chromosomes, episomes and induced viruses can be used. Recombinant vectors that can be used more specifically include bacterial plasmids, transposons, yeast episomes, insertion factors, yeast chromosomal factors, viruses such as baculovirus, papilloma viruses such as SV40, vaccinia virus, adenovirus, adeno -Derived from accessory viruses, retroviruses, chicken pox viruses, pseudorabies virus, but may be used. These recombinant vectors may also be cosmid or phagemid derivatives.

If a vector is used, a strong promoter capable of transcription can be used, and if a prokaryotic cell is used as a host, the vector can be used for the ribosome binding site (RBC) and transcription / Translation termination sequences. As a promoter that may be included in a vector produced for prokaryotic cells, any of generally known promoters such as a tac promoter, a lac promoter, an lpp promoter, a pLλ promoter, and a pRλ promoter may be used. As such plasmid vectors, plasmid vectors generally used for cloning shRNAs, for example, pSC101, pBR322, pUC19, pET-22, pcDNA 3.1, pGSH1-GFP, and the like can be used, but the present invention is limited thereto. no.

On the other hand, in the case of using a eukaryotic cell as a host, a promoter derived from mammalian cells (metallothianin promoter) or a promoter derived from a mammalian virus can be used. In other words, when a virus is to be used as a vector of the present invention, an antisense nucleic acid such as an antisense nucleic acid modified through an siRNA of SEQ ID NO: 2 to SEQ ID NO: 4 or an appropriate linker, for example, shRNA of SEQ ID NO: 5 to SEQ ID NO: 10 Attenuated viruses such as vaccinia or flowpox, as well as adenovirus vectors, Adeno-assocated virus vectors, retrovirus vectors, herpes simplex viruses, nontoxic anthrax toxin vectors It is inserted into a back. At this time, for example, the adenovirus late promoter, the vaccinia virus 7.5K promoter SV40 promoter, the four promoters of the cytomegalovirus promoter HSV may be used, and may include a polyadenylation sequence as a transcription termination sequence.

That is, in connection with the present invention, a vector used to deliver an antisense nucleic acid molecule of SEQ ID NO: 5 to SEQ ID NO: 10, which is an antisense nucleic acid modified by SEQ ID NO: 2 to SEQ ID NO: 4 or a suitable linker sequence, into a host cell is a kind of gene carrier. As such, the nucleic acid molecule of interest is operably linked to an expression control site. 'Operably linked' refers to the functional binding between expression control sites (promoter, signal sequence, enhancer, transcription regulator binding site) of a nucleic acid of interest and other nucleic acids.

Thus, according to the present invention, any gene carrier capable of introducing an antisense nucleic acid comprising the aforementioned siRNA into a host cell, which gene carrier is for example naked recombinant DNA, plasmid vector, viral vector or naked recombinant nucleic acid Or in the form of liposomes or niosomes containing plasmids, preferably in the form of a vector or naked nucleic acid.

On the other hand, in order to stably express the above-described vector according to the present invention can be used a host cell commonly used in the art. For example E. coli JM109, E. coli BL21 (DE3), E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus Thuringiensis , BT), Bacillus sp. Strains such as Bacillus subtilis , and enterobacteria and strains such as Salmonella typhimurium, Serratia marsonsons and various Pseudomonas species. As eukaryotic cells, yeast cells such as Saccharomyce cerevisiae , CHO cell lines (Chinese hamster ovary), W138, BHK, COS-7, 293, HepG2, 3T3, RIN and MDCK cell lines are used. Can be. This host cell may be an isolated cell or cultured cell capable of synthesizing the antisense nucleic acid modified by SEQ ID NO: 2 to SEQ ID NO: 4 or an appropriate linker. The host cell may be a germ cell or a somatic cell, may be a stem cell or a differentiated cell, for example a cell derived from the mammalian eye.

Meanwhile, in order to transport or deliver antisense nucleic acids (eg, SEQ ID NO: 5 to SEQ ID NO: 10) modified by SEQ ID NO: 2 to SEQ ID NO: 4 and appropriate linker sequences according to the present invention, various known methods may be used. Can be used. For example, in order to transport the antisense nucleic acid of SEQ ID NO: 2 to SEQ ID NO: 10 directly into the host cell in the form of naked recombinant DNA or through a plasmid vector or viral vector, microinjection, calcium phosphate precipitation, electroporation, liposomes Nucleic acid molecules can be introduced into cells through mediated transfection, DEAE-dextran treatment, and the like. As a technique for introducing nucleotides into cells, one may also consider using cationic liposomes (Felgner, P.L. et al., Proc Natl Acad Sci USA 84: 7413-7417 (1987)). . Commercially available cationic lipid preparations include Tfx 50 (Promega) or Lipofectamin 2000 (Life Technologies).

For example, the CaCl ₂ method (Cohen, SN et al., Proc. Natl. Acac. Sci. USA, 9: 2110-2114 (1973)) for the delivery of the vector according to the present invention into a host cell which is a prokaryotic cell. , Hanahan, D., J. Mol. Biol., 166: 557-580 (1983), electroporation (Dower, WJ et al. Nucleic Acids Res. 16: 6127-6145 (1988)) Or the like. On the other hand, if the host cell is a eukaryotic cell, calcium phosphate precipitation (Graham, FL & van der Eb, AJ Virology 52: 456 ?? 467 (1973)), electroporation (Neueumann, E., M. Schaefer-Ridder) , Y. Wang, and PH Hofschneider, EMBO (Eur. Mol. Biol. Organ.) J. 1: 841 ?? 845 (1982)), DEAE-dextran treatment (GOPAL, TV, Mol. Cell. Biol. 5 : 1188-1190 (1985)), gene bombardment (Yang et al., Proc. Natl. Acad. Sci., 87: 9568-9572 (1990)), and the like.

3. Pharmaceutical Application

According to another aspect of the invention for example the siRNA of SEQ ID NO: 2 to SEQ ID NO: 4 or shRNA modified with an appropriate linker sequence in view of stability in vivo, for example the antisense nucleic acid of SEQ ID NO: 5 to SEQ ID NO: 10 Also provided are pharmaceutical compositions containing this inserted gene carrier as an active ingredient. The pharmaceutical composition of the present invention may comprise a pharmaceutically acceptable carrier or excipient, in addition to the gene carrier having an antisense nucleic acid of SEQ ID NOS: 2-4 or an antisense nucleic acid modified by an appropriate linker sequence.

In other words, when administered to a living body as a pharmaceutical composition, the antisense nucleic acid modified by SEQ ID NO: 2 to SEQ ID NO: 4 or the appropriate linker sequence, for example, the antisense nucleic acid of SEQ ID NO: 5 to SEQ ID NO: 10 directly to the host cell or These antisense nucleic acids can be administered as gene carriers in the form of recombinant vectors inserted. In this case, the above-described plasmid vector or viral vector is used as the recombinant vector. In this case, the antisense nucleic acid according to the present invention may be included in the pharmaceutical composition at a concentration of 1 to 1000 pmol / ml, preferably 30 to 500 pmol / ml, more preferably 50 to 300 pmol / ml, but the present invention It is not limited to this.

As used herein, 'effective concentration' or 'effective amount' means the desired effect through the aforementioned antisense nucleic acid, such as antisense such as siRNA of SEQ ID NO: 2 to SEQ ID NO: 4 and / or shRNA of SEQ ID NO: 5 to SEQ ID NO: 10 It refers to an amount sufficient to reduce the expression of the target gene compared to the normal expression level of the target gene in the state that the nucleic acid has not been transfected.

The term 'pharmaceutically acceptable carrier' as used herein refers to various carriers known in the art. Delivery systems employing commonly used pharmaceutical carriers may be used. Incoming drug delivery systems include solutions, suspensions, gels, microspheres, polymeric materials, solubility-modifying materials (ethanol, propylene glycol, sucrose), polymers (polycapryllactone, PLGA), and the like.

Finally, according to another aspect of the present invention, the present invention comprises an antisense nucleic acid modified by the aforementioned SEQ ID NO: 2 to SEQ ID NO: 4 or a suitable linker, or a gene carrier in the form of a recombinant vector into which the antisense nucleic acid is inserted, Provided are pharmaceutical compositions capable of treating and preventing diseases caused by overexpression of PEP, including an acceptable carrier or excipient. In other words, this pharmaceutical composition can be considered as one of gene therapy.

Pharmaceutically acceptable carriers contained in the pharmaceutical composition according to the present invention may be used materials commonly used, for example, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, Alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil Including but not limited to. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. In particular, when used as a gene carrier of nucleic acid molecules, liposomes, preferably cationic liposomes, which can increase the efficiency of introducing the introduced nucleic acid molecules into target-cells can be used as carriers.

Pharmaceutically acceptable excipients are known in the art and are relatively inert substances which facilitate the administration of pharmaceutically active substances. For example, an excipient can impart a shape or viscosity or can also act as a diluent. Suitable excipients include, but are not limited to, stabilizers, humectants, and emulsifiers, salts capable of varying osmolality, encapsulating agents, buffers, skin penetration promoters.

In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, as well as in the form of extracts, powders, tablets or capsules, and may further include a dispersant or stabilizer.

If the antisense nucleic acid of SEQ ID NO: 2 to SEQ ID NO: 4 is directly transfected into a host cell or in the form of the recombinant vector described above, the pharmaceutical composition of the present invention may be formulated by a known method. It can be administered directly in the form of a drug. In this case, the pharmaceutical composition may appropriately include a carrier such as sterilized water, saline, phosphate buffer, and culture solution in addition to the antisense nucleic acid or recombinant vector described above. Other buffers, suspensions, stock solutions, surfactants, etc. may be included as needed. In the case of administration in the form of an antisense nucleic acid or recombinant vector, i.e., a pharmaceutical composition as a gene carrier, factors such as formulation method, mode of administration, age, weight, sex, morbidity, time of administration, route of administration, responsiveness, etc. It can be adjusted by a single method of administration or several minutes. The antisense nucleic acid of the present invention may be administered, for example, at a concentration of 1 to 1000 pmole, and may be administered once every few days to once every few months. Those skilled in the art can appropriately select an appropriate dosage or administration frequency.

The pharmaceutical compositions according to the present invention can treat the diseases caused by overexpression of GEP, such as cancer, such as liver cancer, by inhibiting the expression of granulin-epiterin-precursor (GEP). The pharmaceutical composition may be a therapeutic agent, such as an anticancer agent, for treating a disease resulting from overexpression of GEP. The pharmaceutical composition can be applied to humans as well as mammals, in particular commercially important animals or livestock animals, including mice, rats, rabbits, dogs, sheep, goats, pigs, cattle, horses and the like. Hepatocellular carcinoma may be cited as a disease caused by overexpression of GEP, but the present invention is not necessarily limited to such a disease. In addition, the pharmaceutical compositions of the present invention may contain other therapeutic agents such as anti-inflammatory substances, analgesics and the like.

Accordingly, in another aspect of the present invention, a cell capable of inhibiting overexpression of GEP may be prepared by directly introducing the aforementioned antisense nucleic acid into a host cell having a disease resulting from overexpression of GEP or by introducing a recombinant vector containing an antisense nucleic acid. It is about how to. This host cell may be an isolated cell or cultured cell capable of synthesizing the antisense nucleic acid modified by SEQ ID NO: 2 to SEQ ID NO: 4 or an appropriate linker. The host cell may be a germ cell or a somatic cell, may be a stem cell or a differentiated cell, for example a cell derived from the mammalian eye.

Hereinafter, the present invention will be described in more detail with reference to exemplary embodiments, but the present invention is not limited to the following examples.

Example 1: GEP cloning and expression confirmation

(1) FC tagging GEP gene cloning

GEP was expressed in mammalian cells, and FC-tagged GEP was cloned to confirm the efficacy of GEP-specific siRNA / shRNA. It was thought that the amount of GEP that is actually expressed can be easily measured by ELISA using FC tagged with GEP. First, restriction enzymes Hind III and XhoI were used as plasmids in the pcDNA 3.1 vector shown in FIG. 2B based on the nucleotide sequence of the GEP gene shown in FIG. 2A, and secretion signal sequence, Human IgG FC sequence, The full gene of GEP was inserted. Hereinafter, for convenience of explanation, the plasmid into which the FC / GEP is inserted will be described as pcDNA-GEP.

PCR was performed to amplify the GEP gene. In order to amplify the GEP of SEQ ID NO: 1 used as a template, a forward primer (gctcgaagcttgaccatgctctagtaaat; SEQ ID NO: 12) and a reverse primer (cctgcactcgagtcatttacccgggga; SEQ ID NO: 13) including a sequence recognized by a restriction enzyme were used.

For PCR reaction, 5 μl of 10 X pfu buffer, 1 μl of dNTP, 1 μl of template, 1 μl of forward primer, 1 μl of reverse primer, 1 μl of pfu polymerase, and 40 μl of DW were mixed well and PCR was performed. PCR was performed for 2 minutes at 95 ° C., followed by 30 cycles of 95 seconds at 95 ° C., 40 seconds at 55 ° C., and 30 seconds at 72 ° C., followed by enzyme inactivation at 72 ° C. for 5 minutes.

Enzyme cutting was done using Hind III and Xho I. The composition was mixed with 20 µl of pcDNA3.1 (+) vector and 20 µl of PCR product, 1 µl of HindIII and XhoI, 5 µl of 10 X NEB buffer and 23 µl of DW, respectively. The reaction was carried out for 3 hours, and the PCR product was reacted at 37 ° C. for 5 hours.

For ligation, add 7 μl of pcDNA3.1 (+), 7 μl of insert (GEP, SEQ ID NO: 1), RBC rapid ligation Kit buffer A and B so that the ratio of vector: insert is 1: 3 2 μl and 1 μl of T4 DNA ligase were mixed and synthesized overnight at 22 ° C. To transform the completed plasmid into E. coli , mix 20 µl of the reaction product ligated with 50 µl of Competent DH5α, react for 20 minutes on ice, then heat shock at 42 ℃ for 45 seconds and then again on ice. The reaction was carried out for 20 minutes. After that, the seeding was incubated overnight at 37 ℃ seeding on the LB plate containing ampicillin. After incubation, the colonies grown on the plate were inoculated with single colony in LB broth media to which ampicillin was added and incubated overnight at 37 ° C. Plasmid was extracted using RBC's HiYield plasmid mini kit. The extracted plasmids were sequencing to confirm that the desired gene was inserted into the vector.

(2) Confirmation of GEP Expression

In order to confirm the expression of the cloned GEP in mammalian cells, 2 μg of pcDNA-GEP cloned with lipofectamine 2000 was transfected into CHO cells, which are generally not known to express GEP. After 24 hours of transfection, Western blot was performed by extracting proteins in cells using M-per buffer. Western blot was electrophoresed on 8% SDS-gel and used an acrogranin antibody. As shown in FIG. 3a, GEP-Human IgG FC transfected into CHO cells was confirmed to have a size of 98.5 kDa.

(2) ELISA analysis

Another method of confirming the expression of GEP was using the Human IgG (FC) ELISA kit. This is because the tagging of the human IgG FC in the GEP in the present invention, it is possible to measure the amount of GEP expressed by detecting the IgG FC when GEP is expressed. For this purpose, ELISA kit for detecting human IgG was used. ELISA analysis was performed using the Human IgG ELISA Core Kit (KOMA BIOTECH). 0.53 g of Na ₂ CO ₃ , 0.42 g of NaHCO ₃ , 100 ml of DW, pH 9.6 were used, and the purified Goat anti-human IgG and the above-described coating buffer were identified as affinity at a ratio of 1: 1000. 100 μl of coating antibody was coated on each well. Hep3B cells were transfected with 2 μg of pcDNA-GEP using lipofectamine 2000. After transfection, ELISA was performed by sampling the media of the cells at 6, 12, 18, 24, 27, 30, 33, 48, and 72 hours. The measurement results according to the present example are shown in FIG. 3B, and it was confirmed that the expression of GEP was continuously increased after 24 hours.

Example 2 Identification of Antisense Nucleic Acids for Inhibition of GEP Expression

SiRNA sequences for GEP were retrieved using the CAPSID program according to the method described in Hui sun Lee et al., Biochemical and Biophysical Research Communications 384: 431-35 (2009). The CAPSID program found three candidate portions of siRNA for GEP and synthesized each siRNA for it. For convenience of description, these three siRNAs will be referred to as GEP-siRNA1 (GGACACUUCUGCCAUGAUA, SEQ ID NO: 2), GEP-siRNA2 (GAGAGAUGUCCCCUGUGAU, SEQ ID NO: 3), and GEP-siRNA3 (UCUAAGGCCUUCCCUGUCA, SEQ ID NO: 4). The secondary structure of GEP mRNA targeted by the three identified antisense nucleic acids was then analyzed using the CAPSID program.

The results are shown in FIG. In FIG. 4, 1 is the secondary structure for the target site of GEP-siRNA1, the target site of divalent GEP-siRNA2, and 3 is the target site of GEP-siRNA3. As shown, the loop structure is embedded in the target site of the siRNA identified according to the present invention, and thus, the free energy is expected to be high, and RNAi by the antisense nucleic acid according to the present invention may be easily generated. . In particular, the target position of GEP-siRNA1 is likely to be a biologically active site constituting a stem-loop structure, so RNAi effects are well represented, and GEP expression knock-down effects are expected to occur well.

Example 3 Effect of siRNA as Antisense Nucleic Acid

In order to confirm the effects of the three GEP-siRNA identified in Example 2 above, the degree of inhibition of expression of GEP was confirmed using Hep3B cells. After co-transfection of 2 μg of pcDNA-GEP and 100 pmol / ml of GEP-siRNA1 / GEP-siRNA2 / GEP-siRNA3 into Hep3B cells, the media of the cells were sampled at 24, 48 and 72 hours. FC) ELISA kit was used to detect the amount of pcDNA-GEP. GEP-siRNA1, GEP-siRNA2, GEP-siRNA3 all inhibited the expression of GEP, in particular, it was confirmed that GEP-siRNA1 has the greatest effect in inhibiting the expression of GEP. GEP-siRNA1 reduced the level of GEP by 38-fold compared to transfecting GEP after 72 hours (FIG. 5A).

Subsequently, in order to confirm the efficacy of GEP-specific siRNA according to the GEP-siRNA concentration, co-transfection of 2 μg of pcDNA-GEP and GEP-siRNA1 in Hep3B cells at different concentrations of 10, 50, 100, 200 pmol / ml was performed. After 24, 48 and 72 hours, the media of the cells were sampled and the amount of pcDNA-GEP was detected using the Human IgG (FC) ELISA kit. It was confirmed that the expression of GEP was inhibited in proportion to the concentration of siRNA used (FIG. 5B).

Example 4 Inhibition of GEP Expression in ShRNA Form

 In order to secure the instability of the siRNA shRNA was synthesized using the sequence of GEP-siRNA1. For this purpose, an auxiliary oligonucleotide (hereinafter referred to as GEP-shRNA) for improving the linker sequence and stability was inserted into the pENTR ™ H1 / TO (3869 bp) vector as shown in SEQ ID NO: 8. In order to confirm whether the synthesized GEP-shRNA shows the same effect as the GEP-siRNA, co-transfection of 2 μg of pcDNA-GEP and 2 μg of GEP-shRNA1 in Hep3B cells in the same manner as in Example 3, 24, 48, After 72 hours, the media of the cells were sampled and the expression level of GEP was measured using a human IgG (FC) ELISA kit. Like the siRNA confirmed in Example 3, it was confirmed that the shRNA synthesized according to this example also inhibits the expression of GEP (FIG. 6A).

Subsequently, in order to confirm the degree of suppression of GEP expression according to the concentration of GEP-shRNA, the amount of 2 μg of pcDNA-GEP and GEP-shRNA in Hep3B cells was 0.1, 0.5. 1.0. After co-transfection with 2.0 ㎍, the media of cells after 24, 48, and 72 hours were sampled, and the expression level of pcDNA-GEP was measured using a Human IgG (FC) ELISA kit. It was confirmed that shRNA also inhibits the expression of GEP in a concentration dependent form (FIG. 6B).

Example 5 Endogenous GEP Inhibitory Effect of GEP-shRNA

In order to confirm whether the GEP-shRNA synthesized in Example 4 decreased the level of GEP mRNA present in the endogenous cell, GEP-shRNA was transfected into Hep3B cells continuously expressing GEP, and the level of GEP mRNA was determined by real-time PCR. It was confirmed.

RNA was extracted from the transfected cells and GFP transfected with 2 ㎍ of GEP-shRNA synthesized in Example 4 in Hep3B cells using the TRI sol LS reagent method. First, 500 μl of TRI sol LS reagent per well in a 6-well plate, followed by tapping, followed by reaction at room temperature for 5 minutes, 135 μl of chloroform, vortexing for 15 seconds, and then reaction at room temperature for 10 minutes. Thereafter, centrifuge at 12000 g, 4 ° C. for 15 minutes, and transfer the transparent supernatant to a new tube. After adding 333 μl of isopropyl alcohol, the solution was reacted at room temperature for 10 minutes. Then, once again centrifuge for 12000 g, 4 ° C. for 10 minutes, discard the supernatant, wash the pellet with 400ul of 70% ethanol made from DW treated with DEPC, then centrifuge for 7500g, 4 ° C. for 5 minutes After removing the pellet and drying the pellet, RNA was suspended in DW 20ul treated with DEPC.

Synthesis of RNA into cDNA 1 μl of oligoDT primer, 2 μl of DTT, and 4 μl of DW were mixed with 5 μl of RNA extracted from the above cell, followed by reaction at 70 ° C. for 5 min in a PCR machine. React for a while. After that, the reaction was performed at 70 ° C. for 15 minutes for enzyme inactivation.

To perform real-time PCR using the synthesized cDNA, 1 μl of synthesized cDNA, GEP forward primer (tccacgtgctgtgttatggt, SEQ ID NO: 14) / reverse primer (ctgccctgtgttcctctgggg, SEQ ID NO: 15), respectively, 1 μl, DW 9.5 μl, SYBR 12.5 The wells were mixed well and real-time PCR was performed in the same procedure as in Example 1. Measurement results are shown in Figure 7, the transfection of GEP-shRNA according to the present invention confirmed the result of reducing the mRNA level of GEP by four times.

Example 6 Inhibitory Effect of shRNA on Liver Cancer Cell Growth

Since GEP acts as a growth factor for liver cancer, an experiment was performed to confirm cell growth by transfecting GEP-shRNA into liver cancer cell line to suppress GEP expression. To this end, the following three cell populations were used. 1) GEP expressed stable cell line for continuous expression of GEP using Hep3B cell, a liver cancer cell line, 2) transfection control of GFP to transfection control in general Hep3B cells, 3) transfection of GEP-shRNA into Hep3B cells as an experimental group Afterwards, cells remaining on days 1, 3 and 5 were stained with triphan blue and counted using a Haemacytometer to compare cell growth.

Cell growth inhibition effect measurement results according to the present embodiment is shown in FIG. From day 3, we observed the fastest cell growth in GEP stable cell line, followed by cell growth using GFP cell group as transfection control and Mock Hep3B cell group without any treatment and transfection of GEP-shRNA. One cell was confirmed that the growth is slow (Fig. 8). This result means that the antisense nucleic acid according to the present invention can inhibit the growth of liver cancer.

Example 7: Reduction of tumor volume of GEP-shRNA in vivo

Subcutaneous injection of 1 × 10 ⁷ Hep3B cells into the thighs of Balb / c-derived nude mice was performed after 10 days to determine tumor volume reduction by antisense nucleic acids according to the present invention.

Intratumoral injection of 30 μg of GEP-shRNA synthesized above was applied to mice with tumors, and electroporation was performed eight times with a voltage of 50 mV. After injection, the tumor size was measured daily (FIG. 7A). In addition, GEP-shRNA and Luciferase-shRNA (provided by Jerolution) as NC were compared. Measurement results according to the present embodiment are shown in Figs. 9A to 9C.

As shown in Figures 9a to 9b, it was confirmed that tumor growth of mice injected with GEP-shRNA, one of the antisense nucleic acids according to the present invention, was reduced compared to tumors of mice injected with Luciferase-shRNA. In particular, as shown in Fig. 9c, comparing the negative control and the relative tumor volume confirmed that more than twice the size difference occurs on the 8th day after GEP-shRNA injection and continuously observed until 31 days after the final tumor It was confirmed that the size is more different. Therefore, it was determined that the GEP-specific antisense nucleic acid synthesized according to the present invention can be used as a substance capable of inhibiting the growth of liver cancer caused by GEP.

Example 8 Confirmation of GEP-shRNA of Hepatocellular Carcinoma Cell Cycle Arrest

In this example, it was confirmed whether the antisense nucleic acid (GEP-shRNA) synthesized according to the present invention affects the cell cycle of the liver cancer cell line. The knockdown of GEP, known as the growth factor of liver cancer, was analyzed by FACS using propidium iodide (PI) staining to confirm the relationship with the cell cycle of liver cancer cells.

2 ㎍ each of pcDNA-GEP, GEP-shRNA, and Luciferase-shRNA in Hep3B cells were transfected using lipofectamin2000 and sampled 5 days later. The culture medium of the cell was removed, washed twice with PBS, treated with 200 μl of trypsin, and the cells were harvested and placed in an ep-tube. Thereafter, after centrifuge at 4000 rpm for 2 minutes, the supernatant was discarded, washed with PBS, centrifuge again at 4000 rpm for 2 minutes, and then the supernatant was discarded. Then, 200 μl of 70% ethanol was added to the cell, pipettng, and reacted at 4 ° C. for 1 hour. After 1 hour, centrifuge at 4000 rpm for 2 minutes, discard the supernatant, wash 70% ethanol with PBS, centrifuge for 2 minutes at 4000 rpm, and discard the supernatant. Then, 500 μl of PI stainign solution was treated in the cell, blocked with light, reacted for 15 minutes, and analyzed through FACS.

The results according to this example are shown in FIG. Compared to the cells transfected with Luciferase-shRNA used as a negative control, the cells transfected with pcDNA-GEP increased the G0 / G1 phase and decreased the M phase, whereas the G0 / G1 phase decreased and the S and G2 / levels decreased in the cells transfected with GEP-shRNA. It was confirmed that the M group is increased. Therefore, it was confirmed that knockdown of GEP through GEP-shRNA caused cell cycle arrest.

In the above, the present invention has been described based on the preferred embodiment of the present invention, but the present invention is not limited to the above-described embodiment. Rather, one of ordinary skill in the art to which the present invention pertains will readily conceive various modifications and changes to the present invention based on the above-described embodiments. However, the fact that such modifications and variations are all within the scope of the present invention, will be apparent from the claims below.

<110> CATHOLIC UNIVERSITY INDUSTRY ACADEMIC COOPERATION FOUNDATION <120> Anti-Sense Nucleic Acid of Inhibiting Expression of          Granulin-Epitherin-Precursor Gene and Pharmaceutical Composition          Containing thereof <160> 17 <170> Kopatentin 1.71 <210> 1 <211> 2111 <212> DNA <213> Grnauline-Epitherin-Precursor <400> 1 caggcagacc atgtggaccc tggtgagctg ggtggcctta acagcagggc tggtggctgg 60 aacgcggtgc ccagatggtc agttctgccc tgtggcctgc tgcctggacc ccggaggagc 120 cagctacagc tgctgccgtc cccttctgga caaatggccc acaacactga gcaggcatct 180 gggtggcccc tgccaggttg atgcccactg ctctgccggc cactcctgca tctttaccgt 240 ctcagggact tccagttgct gccccttccc agaggccgtg gcatgcgggg atggccatca 300 ctgctgccca cggggcttcc actgcagtgc agacgggcga tcctgcttcc aaagatcagg 360 taacaactcc gtgggtgcca tccagtgccc tgatagtcag ttcgaatgcc cggacttctc 420 cacgtgctgt gttatggtcg atggctcctg ggggtgctgc cccatgcccc aggcttcctg 480 ctgtgaagac agggtgcact gctgtccgca cggtgccttc tgcgacctgg ttcacacccg 540 ctgcatcaca cccacgggca cccaccccct 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catgctgtgt gctgcgagga 1440 tcgccagcac tgctgcccgg ctggctacac ctgcaacgtg aaggctcgat cctgcgagaa 1500 ggaagtggtc tctgcccagc ctgccacctt cctggcccgt agccctcacg tgggtgtgaa 1560 ggacgtggag tgtggggaag gacacttctg ccatgataac cagacctgct gccgagacaa 1620 ccgacagggc tgggcctgct gtccctaccg ccagggcgtc tgttgtgctg atcggcgcca 1680 ctgctgtcct gctggcttcc gctgcgcagc caggggtacc aagtgtttgc gcagggaggc 1740 cccgcgctgg gacgcccctt tgagggaccc agccttgaga cagctgctgt gagggacagt 1800 actgaagact ctgcagccct cgggacccca ctcggagggt gccctctgct caggcctccc 1860 tagcacctcc ccctaaccaa attctccctg gaccccattc tgagctcccc atcaccatgg 1920 gaggtggggc ctcaatctaa ggccttccct gtcagaaggg ggttgtggca aaagccacat 1980 tacaagctgc catcccctcc ccgtttcagt ggaccctgtg gccaggtgct tttccctatc 2040 cacaggggtg tttgtgtgtg tgcgcgtgtg cgtttcaata aagtttgtac actttcaaaa 2100 aaaaaaaaaa a 2111 <210> 2 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siRNA to granulin-epithelin-precursor <400> 2 ggacacuucu gccaugaua 19 <210> 3 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siRNA to granulin-epithelin-precursor <400> 3 gagagauguc cccugugau 19 <210> 4 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siRNA to granulin-epithelin-precursor <400> 4 ucuaaggccu ucccuguca 19 <210> 5 <211> 44 <212> RNA <213> Artificial Sequence <220> <223> shRNA to granulin-epithelin-precursor <400> 5 ggacacuucu gccaugauau cucucuauca uggcagaagu gucc 44 <210> 6 <211> 44 <212> RNA <213> Artificial Sequence <220> <223> shRNA to granulin-epithelin-precursor <400> 6 gagagauguc cccugugauu cucucaucac aggggacauc ucuc 44 <210> 7 <211> 44 <212> RNA <213> Artificial Sequence <220> <223> shRNA to granulin-epithelin-precursor <400> 7 ucuaaggccu ucccugucau cucucugaca gggaaggccu uaga 44 <210> 8 <211> 56 <212> RNA <213> Artificial Sequence <220> <223> shRNA to granulin-epithelin-precursor <400> 8 ggacacuucu gccaugauaa ccagaucucu cucugguuau cauggcagaa gugucc 56 <210> 9 <211> 56 <212> RNA <213> Artificial Sequence <220> <223> shRNA to granulin-epithelin-precursor <400> 9 gagagauguc cccugugaua ccagaucucu cucugguauc acaggggaca ucucuc 56 <210> 10 <211> 56 <212> RNA <213> Artificial Sequence <220> <223> shRNA to granulin-epithelin-precursor <400> 10 ucuaaggccu ucccugucaa ccagaucucu cucugguuga cagggaaggc cuuaga 56 <210> 11 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siRNA 106578 commercial siRNA <400> 11 cacaagccuu gaagagaga 19 <210> 12 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for amplifying granulin-epithelin-precursor <400> 12 gctcgaagct tgaccatgct actagtaaat 30 <210> 13 <211> 27 <212> DNA <213> Artificial Sequence <220> Reverse primer for amplifying granulin-epithelin-precursor <400> 13 cctgcactcg agtcatttac ccgggga 27 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Real-time PCR Forward primer for amplifying GEP cDNA <400> 14 tccacgtgct gtgttatggt 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Real-time PCR Reverse priemr for amplifying GEP cDNA <400> 15 ctgccctgtt agtcctctgg 20 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Real-time PCR for amplifying 18s rRNA <400> 16 aactttcgat ggtagtcgcc g 21 <210> 17 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Real-time PCR Reverse primer for amplifying 18s rRNA <400> 17 ggatgtggta gccgtttctc a 21

Claims (10)

Iii) short-interfering RNA (siRNA) having a base sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 3, or
Ii) capable of complementarily binding to nucleic acids of SEQ ID NO: 1 to SEQ ID NO: 3 through a linker sequence linked to either the 5 'end or the 3' end of any one nucleotide sequence selected from SEQ ID NO: 1 to SEQ ID NO: 3 Oligonucleotides (complementary oligonucleotides) consist of short-hairpin RNAs (shRNAs) linked to the short-interfering RNAs,
Antisense nucleic acids that inhibit the expression of Granulin-Epithelin precursor (GEP).
The method of claim 1,
The short-hairpin RNA is 1-20 oligonucleotides (first auxiliary oligonucleotides) linked to one end of the short-interfering RNA, and linked to one end of the complementary oligonucleotides and is linked to the first auxiliary oligonucleotides. An antisense nucleic acid further having an oligonucleotide (secondary oligonucleotide) capable of complementarily binding to a second auxiliary oligonucleotide.
The method of claim 1,
The short-hairpin RNA is an antisense nucleic acid consisting of any one base sequence selected from the group consisting of SEQ ID NO: 4 to SEQ ID NO: 9.
The recombinant vector in which the anti-sex nucleic acid of any one of Claims 1-3 is inserted.
The method of claim 4, wherein
The recombinant vector is a recombinant vector, characterized in that the plasmid vector or viral vector.
6. The method of claim 5,
The viral vector is a recombinant vector, characterized in that the viral vector derived from baculovirus, SV40 virus, vaccinia virus, adenovirus, adeno-associated virus, retrovirus, chicken pox virus, pseudorabies virus.
A granulin-epiterin containing as an active ingredient the antisense nucleic acid described in any one of claims 1 to 3, or the recombinant vector described in any one of claims 4 to 6. -Pharmaceutical compositions for treating diseases derived from Granulin-Epithelin precursor (GEP).
8. The method of claim 7,
The composition is a pharmaceutical composition for treating a disease of hepatocellular carcinoma.
Granulin-epiterin, wherein the antisense nucleic acid according to any one of claims 1 to 3 or the recombinant vector according to any one of claims 4 to 6 is introduced into a mammalian-derived host cell. -Method for producing cells in which the expression of Granulin-Epithelin precursor (GEP) is suppressed.
The method of claim 9,
The host cell is characterized in that the liver cancer cell.

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