KR20200119538A - Composition for preventing or treating of liver cancer - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating liver cancer containing an expression inhibitor of let-7i-5p as an active component. The expression inhibitor of let-7i-5p according to the present invention inhibits tumor cell growth, proliferation, migration, and invasion, microtubule formation, and angiogenic activity in hepatocellular carcinoma, thereby being able to be used as the pharmaceutical composition for preventing or treating liver cancer. In addition, the expression inhibitor of let-7i-5p can be used for a method for providing information for diagnosis or prognosis of liver cancer according to expression control thereof, and screening liver cancer therapeutic agent candidate materials.

Description

Composition for preventing or treating liver cancer {COMPOSITION FOR PREVENTING OR TREATING OF LIVER CANCER}

The present invention relates to a pharmaceutical composition for preventing or treating liver cancer, and specifically, to a pharmaceutical composition for preventing or treating liver cancer comprising an inhibitor of let-7i-5p expression as an active ingredient.

Liver cancer is the fifth most frequent cancer in the world, but its mortality is the third most aggressive cancer (Ahn J, Flamm SL Hepatocellular carcinoma Dis Mon 2004; 50:556-573) Therapeutic surgery is only 15%. Only about 25% of patients die, and most liver cancer patients die within a relatively short period of time from diseases that progress or spread locally (Roberts LR, Gores GJ Hepatocellular carcinoma: molecular pathways and new therapeutic targets Semin Liver Dis 2005;25: 212-225) Hepatitis B virus (hepatitis B virus), hepatitis C virus (hepatitis C virus), and aflatoxin B1 (aflatoxin B1) are well known as major causes of liver cancer. However, the overall survival rate of liver cancer patients has not increased significantly over the past 20 years, and the mechanisms of development and progression of liver cancer are still unknown (Bruix J, et al Focus on hepatocellular carcinoma Cancer Cell 2004; 5:215-219) So far, molecular targeted therapy has been shown to be effective in the treatment of mature liver cancer (Shen YC, Hsu C, Cheng AL Molecular targeted therapy for advanced hepatocellular carcinoma: current status and future perspectives J Gastroenterol; 45:794-807), it is unclear how these genetic changes cause the clinical characteristics observed in individuals with liver cancer.

HDACs (Histone deacetylases) can often be attached to gene promoters by corepressors or multi-protein transcriptional complexes, where they do not bind directly to DNA and are modified with chromatin. (Thiagalingam S, Cheng KH, Lee HJ, Mineva N, Thiagalingam A, Ponte JF Histone deacetylases: unique players in shaping the epigenetic histone code Ann NY Acad Sci 2003; 983:84-100) There are 18 HDACs, which are Class I (HDAC 1, 2, 3 and 8), Class II (HDAC 4, 5, 6, 7, 9 and 10), Class III (SIRT 1-7), and Class IV ( HDAC11) is classified as enzymes (Yang XJ, Seto E The Rpd3/Hda1 family of lysine deacetylases: from bacteria and yeast to mice and men Nat Rev Mol Cell Biol 2008;9:206-218) Histone acetyltransferases And HDACs are known to be involved in cell proliferation, differentiation, and cell cycle regulation (Witt O, Deubzer HE, Milde T, Oehme I HDAC family: What are the cancer relevant targets? Cancer Lett 2009;277:8-21). In addition, it has been reported that pathological activity and deregulation of HDACs can lead to various diseases such as cancer, immune disease, and muscular dystrophy (Yang XJ, Seto E HATs and HDACs: from structure. , funct ion and regulation to novel strategies for therapy and prevention Oncogene 2007;26:5310-5318). HDAC6 is a member of the class IIb family of HDACs, acts as a cytoplasmic deacetylase, which is associated with microtubules (MTs)[0004], and deacetylates alpha-tubulin ( Hubbert C, Guardiola A, Shao R, Kawaguchi Y, Ito A, Nixon A, et al HDAC6 is a microtubule-associated Mis18α.Nature 2002;417:455-458).

On the other hand, miRNA is a type of endogenous small RNA (endogenous small RNA) with a length of 20-25 nucleotides that exists in cells, and is derived from DNA that does not synthesize proteins and is produced from hairpin-shaped transcripts. do. miRNA binds to the complementary sequence of the 3'-UTR of the target mRNA and induces translational inhibition or destabilization of the mRNA, and ultimately acts as a repressor to inhibit protein synthesis of the target mRNA. One miRNA targets multiple mRNAs, and it is known that mRNA can also be regulated by multiple miRNAs.

An object of the present invention is to provide a pharmaceutical composition for preventing or treating liver cancer.

In addition, an object of the present invention is to provide a method of providing information for diagnosis or prognosis of liver cancer.

In addition, an object of the present invention is to provide a method for screening a candidate substance for treating liver cancer.

In order to solve the above problems, the present invention provides a pharmaceutical composition for preventing or treating liver cancer.

In addition, the present invention provides a method of providing information for diagnosis or prognosis of liver cancer.

In addition, the present invention provides a method for screening a candidate substance for treating liver cancer.

Since TSP1 according to the present invention inhibited tumor cell growth, proliferation, migration and invasion, microtubule formation, and angiogenesis activity in hepatocellular carcinoma, it can be used as a pharmaceutical composition for the prevention and treatment of liver cancer, and its expression control Accordingly, it can be used to provide information for diagnosis or prognosis of liver cancer and to screen for candidates for liver cancer treatment.

1 is a diagram identifying miRNAs regulated by HDAC6 in hepatocellular carcinoma.
Figure 2 is a diagram confirming the Let-7i-5p significantly regulated in the HCC cell line.
3 is a diagram showing that HDAC6 inhibits let-7i-5p in a JNK-c Jun-miR-221-dependent manner in HCC.
4 is a diagram illustrating the functional characteristics of oncomiR let-7i-5p in HCC.
5 is a diagram illustrating THBS1 targeted by let-7i-5p and directly down-regulated in HCC cell lines.
6 is a diagram confirming the TSP1 inhibitory activity of let-7i-5p.
7 is a diagram illustrating the function of TSP1 as a tumor suppressor in hepatocellular carcinoma.
8 is a diagram confirming the anti-angiogenic effect of TSP1 through the CD47 receptor.
9 is a diagram confirming the anti-angiogenic and anti-metastatic effects of HDAC6-let-7i-5p-TSP1 signaling axis through CD47 in HCC.
10 is a diagram confirming the weakening of the anti-metastatic potential of the HDAC6-let-7i-5p-TSP1 axis through CD47.
11 is a diagram confirming the effect of CD47-TSP1 interaction on macrophage phagocytosis and exosome interaction of HCC cells.
12 is a diagram confirming the anti-phagocytic effect of the CD47-TSP1 interaction in macrophage phagocytosis of HCC cells.
13 is a diagram confirming in vivo that the HDAC6-let-7i-5p-TSP1 axis regulates HCC tumor formation.
14 is a diagram confirming in vivo that the HDAC6-let-7i-5p-TSP1 axis regulates HCC tumor formation.

Hereinafter, the present invention will be described in detail as an embodiment of the present invention with reference to the accompanying drawings. However, the following embodiments are presented as examples of the present invention, and if it is determined that a detailed description of a technique or configuration well known to those skilled in the art may unnecessarily obscure the subject matter of the present invention, the detailed description may be omitted. However, the present invention is not limited thereby. The present invention is capable of various modifications and applications within the scope of equivalents interpreted from the description of the claims to be described later and therefrom.

In addition, terms used in the present specification are terms used to properly express preferred embodiments of the present invention, which may vary depending on the intention of users or operators, or customs in the field to which the present invention belongs. Accordingly, definitions of these terms should be made based on the contents throughout the present specification. Throughout the specification, when a part "includes" a certain component, it means that other components may be further included rather than excluding other components unless specifically stated to the contrary.

In one aspect, the present invention relates to a pharmaceutical composition for the prophylaxis or treatment of liver cancer comprising an inhibitor of let-7i-5p expression as an active ingredient.

In one embodiment, let-7i-5p may be microRNA (miRNA) including the nucleotide sequence of SEQ ID NO: 1.

In one embodiment, the inhibitor of let-7i-5p expression may be HDAC6 (Histone deacetylase 6).

In one embodiment, the let-7i-5p expression inhibitor may be a siRNA (small interfering RNA), shRNA (small hairpin RNA), miRNA, antisense oligonucleotide, or nucleic acid aptamer that inhibits the expression of let-7i-5p, It may be an antisense let-7i-5p of SEQ ID NO: 2.

In one embodiment, the inhibitor of let-7i-5p expression may increase the secretion of TSP1.

In one embodiment, the liver cancer may be let-7i-5p high-expression liver cancer, may be hepatocellular carcinoma, and may be stage I, II, III, IVA or IVB stage hepatocellular carcinoma, It is more preferable that it is stage III to stage IV hepatocellular carcinoma, which is more difficult to treat than the initial stage.

In one embodiment, the inhibitor of expression of let-7i-5p can inhibit tumor cell growth, proliferation, migration and invasion.

In one aspect, the present invention relates to a pharmaceutical composition for the prevention or treatment of liver cancer comprising TSP1 (thrombospondin-1) as an active ingredient.

As used herein, the term “expression inhibition” refers to causing a decrease in the expression (to mRNA) or translation (to a protein) of a target gene, preferably thereby making the target gene expression undetectable or at an insignificant level. It means to exist as.

In one embodiment, the expression level can be determined by checking the level of expression of the mRNA or the level of the protein encoded by the gene. The amount of mRNA can be confirmed using a primer pair or probe, and analysis methods for this include RT-PCR, competitive RT-PCR, real-time RT-PCR, and RNase protection. Analytical methods (RNase protection assay, RPA), Northern blotting, DNA chips, and the like, but are not limited thereto. The amount of protein encoded by the gene can be determined by using an antibody that specifically binds to the protein, and analysis methods for this include western blot, ELISA (enzyme linked immunosorbent assay), and radioimmunoassay. (RIA: Radioimmunoassay), radioimmunodiffusion, Ouchterlony immune diffusion method, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay (Immunoprecipitation assay), complement fixation assay (Complement Fixation) Assay), FACS, protein chips, and the like, but are not limited thereto.

In one embodiment, TSP1 may be a protein comprising the amino acid sequence of SEQ ID NO: 3.

In one embodiment, the liver cancer may be TSP1 low-expression liver cancer, may be hepatocellular carcinoma, and may be stage I, II, III, IVA or IVB stage hepatocellular carcinoma, and it is difficult to treat than the initial stage. More preferably, it is stage IV hepatocellular carcinoma.

In one embodiment, TSP1 can inhibit tumor cell growth, proliferation, migration and invasion, microtubule formation, and angiogenic activity.

In one embodiment, TSP1 is capable of interacting with CD47.

In one aspect, the present invention relates to a pharmaceutical composition for preventing or treating liver cancer comprising a polynucleotide encoding TSP1, or a cell containing the vector as an active ingredient.

In one embodiment, the vector may be a linear DNA, a plasmid DNA or a recombinant viral vector.

In one embodiment, the recombinant virus may be any one selected from the group consisting of retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, and lentivirus.

In one embodiment, the cells may be any one selected from the group consisting of hematopoietic stem cells, dendritic cells, autologous tumor cells, and established tumor cells.

In one embodiment, in the case of a vector comprising a polynucleotide encoding TSP1, it is preferable to contain 0.05 to 500 mg, more preferably 0.1 to 300 mg, and a recombinant comprising a polynucleotide encoding TSP1 In the case of a virus, it is preferable to contain 10 ³ to 10 ¹² IU (10 to 10 ¹⁰ PFU), more preferably 10 ⁵ to 10 ¹⁰ IU, but is not limited thereto.

In one embodiment, in the case of a cell containing a polynucleotide encoding TSP1 of the present invention, it is preferable to contain 10 ³ to 10 ^8, more preferably 10 ⁴ to 10 ⁷ , but limited thereto. It doesn't work.

In one embodiment, the effective dose of the vector containing the polynucleotide encoding TSP1 of the present invention or the composition containing the cell as an active ingredient is 0.05 to 12.5 mg/kg per 1 kg body weight, in the case of recombinant virus In the case of 10 ⁷ to 10 ¹¹ virus particles (10 ⁵ to 10 ⁹ IU)/kg, in the case of a cell, it is 10 ³ to 10 ⁶ cells/kg, preferably in the case of a vector, 0.1 to 10 mg/kg, 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, and may be administered 2 to 3 times a day. The composition as described above is not necessarily limited thereto, and may vary depending on the condition of the patient and the degree of onset of the disease.

In the present invention, the term "prevention" as used refers to all actions of inhibiting or delaying the occurrence, development, and recurrence of liver cancer by administration of the composition according to the present invention.

The term "treatment" as used in the present invention refers to any act of improving or beneficially changing the symptoms of liver cancer and complications resulting from the administration of the composition according to the present invention. Those of ordinary skill in the art to which the present invention pertains can know the exact criteria of the disease in which the composition of the present invention is effective, and determine the degree of improvement, improvement and treatment by referring to data presented by the Korean Medical Association. will be.

The therapeutically effective amount of the composition of the present invention may vary depending on various factors, for example, the method of administration, the site of interest, and the condition of the individual.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. As used herein, the term "pharmaceutically effective amount" refers to an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment and not cause side effects, and the effective dose level is Health status, type of infectious disease, severity, drug activity, sensitivity to drugs, method of administration, time of administration, route of administration and rate of excretion, duration of treatment, factors including drugs used in combination or concurrently, and other factors well known in the medical field Can be determined according to. The composition of the present invention may be administered as an individual therapeutic agent or administered in combination with other therapeutic agents, may be administered sequentially or simultaneously with a conventional therapeutic agent, and may be administered single or multiple. In consideration of all the above factors, it is important to administer an amount capable of obtaining the maximum effect in a minimum amount without side effects, which can be easily determined by a person skilled in the art.

In one embodiment, the pharmaceutical composition may be one or more formulations selected from the group including oral dosage forms, external preparations, suppositories, sterile injectable solutions and sprays.

The compositions of the present invention may also contain carriers, diluents, excipients or combinations of two or more commonly used in biological preparations. The pharmaceutically acceptable carrier is not particularly limited as long as it is suitable for delivery of the composition in vivo. For example, Merck Index, 13th ed., Merck & Co. Inc. Compounds described in, saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components can be mixed and used, and antioxidants, buffers, bacteriostatic agents, etc. Conventional additives can be added. In addition, a diluent, a dispersant, a surfactant, a binder, and a lubricant may be additionally added to form an injectable formulation such as an aqueous solution, a suspension, an emulsion, and a pill, capsule, granule, or tablet. Further, it may be preferably formulated according to each disease or component by an appropriate method in the art or by using a method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).

In addition to the composition of the present invention, it may contain one or more active ingredients exhibiting the same or similar functions. The composition of the present invention contains 0.0001 to 10% by weight of the protein, preferably 0.001 to 1% by weight, based on the total weight of the composition.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable additive, wherein the pharmaceutically acceptable additives include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, Lactose, mannitol, syrup, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, opadry, sodium starch glycolate, lead carnauba, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, stearic acid Calcium, sucrose, dextrose, sorbitol, talc, and the like can be used. The pharmaceutically acceptable additive according to the present invention is preferably contained in an amount of 0.1 parts by weight to 90 parts by weight based on the composition, but is not limited thereto.

The composition of the present invention may be parenterally administered (for example, intravenously, subcutaneously, intraperitoneally or topically applied) or orally according to a desired method, most preferably oral administration. The dosage range varies depending on the individual's weight, age, sex, health status, diet, administration time, administration method, excretion rate, and disease severity.

Liquid formulations for oral administration of the composition of the present invention include suspensions, liquid solutions, emulsions, syrups, etc., and various excipients, such as wetting agents, sweetening agents, fragrances, preservatives, in addition to water and liquid paraffin, which are commonly used simple diluents. Etc. may be included together. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, suppositories, and the like.

In one aspect, the present invention relates to a method of providing information for diagnosis or prognosis of liver cancer comprising confirming the expression of let-7-5p or TSP1 in a sample isolated from a subject.

In one embodiment, it is determined that the risk of developing liver cancer is high when let-7-5p is overexpressed by 1.18 times or more compared to the normal control group; Alternatively, it may further include determining that the risk of developing liver cancer is high if the expression of SP1 is reduced to -1.40 times or less compared to the normal control group.

In one embodiment, the sample may include a sample such as tissue, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid or urine.

In the present invention, the term "diagnosis" is used to determine the susceptibility of an object to a specific disease or disease, to determine whether an object currently has a specific disease or disease, a specific disease or Determining the prognosis (e.g., identification of a pre-metastatic or metastatic cancer state, determining the stage of the cancer, or determining the responsiveness of the cancer to treatment) of a subject with the disease, or therametrics (e.g., Monitoring the condition of the subject to provide information on treatment efficacy).

In one aspect, the present invention relates to treatment of a test compound or composition to a cell line expressing let-7-5p or TSP1; Measuring the expression level of let-7-5p or TSP1 in the cell line treated with the test compound or composition; And selecting a test compound or composition in which the expression level of let-7-5p is decreased or the level of expression or secretion of TSP1 is increased compared to the control cell line without treatment of the test compound or composition. It's about the room.

The present invention will be described in more detail through the following examples. However, the following examples are only for embodiing the contents of the present invention and the present invention is not limited thereto.

Example 1. Identification of target miRNA of HDAC6 that inhibits HCC

1-1. Search for miRNA regulated by HDAC6

To search for miRNA regulated by HDAC6 (Histone deacetylase 6) in HCC (hepatocellular carcinoma), Hep3B cells transfected with HDAC6 were subjected to miRNA microarray analysis and compared with Hep3B cells transfected with empty vector. I did. Specifically, Hep3B cells were transfected with pcDNA3.1_Mock (mock) or pcDNA3.1_HDAC6 (HDAC6), and the data of each sample were extracted with Genome Studio software (Illumina, San Diego, CA) using basic parameters. Primary microarray data can be obtained from the NCBI GEO public database (accession number: GSE100017). As a result, 129 miRNAs were shown to be down-regulated in HDAC6-transfected Hep3B cells (GSE100017; <-1.5 fold change, p <0.05). Thereafter, the miRNAs were compared with those known to be abnormally overexpressed in HCC (GSE39678;> 1.5-fold change, p <0.05) by performing a band diagram analysis (FIG. 1A ). Through this, 18 miRNAs regulated by HDAC6 in HCC were selected as candidates (Fig. 1B). Among the top 5 miRNAs that were most inhibited, miR-335-3p, miR-17-5p, and miR-338-3p were the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) databases (GSE36915, GSE31384 and GSE76903), a cohort study of HCC patients was excluded from the candidate because it was not consistently overexpressed. In addition, in the Capulan-Meyer survival analysis of these five miRNAs obtained from Cancer Genome Atlas liver hepatocellular carcinoma project (TCGA_LIHC), HCC with high expression of the remaining let-7i-5p and miR-182-5p. The 5-year survival rate of the patients was significantly lower than that of HCC patients with low expression of let-7i-5p and miR-182-5p (FIG. 2A ).

In addition, total RNA was isolated from HCC cell lines (SNU-182, SNU-449 and SNU-475) using a TRIzol reagent (Invitrogen, Carlsbad, CA), and then the two miRNAdp-specific cDNAs were separated from the micscipt II RT kit (Qiagen , Manchester, UK) and Tetro cDNA synthesis kit (Bioline, London, UK). qRT-PCR was performed with SensiFASTTM SYBR® NoROX Kit (Bioline). As a result of qRT-PCR analysis of the two miRNAs in HCC cell lines (SNU-182, SNU-449 and SNU-475), only let-7i-5p was continuously down-activated by re-activation of HDAC6 in all HCC cell lines- It was confirmed that it was adjusted (FIG. 2B).

In addition, of the 8 let-7-5p family miRNAs, only let-7i-5p was overexpressed in 7 cohorts of HCC patients in the NCBI GEO database (GSE10694, GSE31384, GSE36915, GSE39678, GSE40744, GSE76903 and GSE67882). Appeared (Table 1).

1-2. Confirmation of let-7i-5p expression in HCC

Thus, to confirm the abnormal expression of let-7i-5p in HCC, 40 matched-pairs (HCC tissue vs non-cancerous peripheral liver tissue) of HCC tissue and 11 HCC cell lines were used as described above. QRT-PCR analysis was performed in the same way. The 40 comparison-paired HCC tissues and corresponding non-cancerous peripheral liver tissues were obtained from the National Biobank of Korea, and written consent was obtained from each subject according to the declaration of Helsinki, and It was approved by the Institutional Review of Board (IRB) (approval number: MC12EISI0106). As a result, 20 out of 40 HCC patients (50%) showed marked overexpression of let-7i-5p compared to non-cancerous liver tissue (FIG. 3A ). In addition, 9 of the 11 HCC cell lines showed marked overexpression of let-7i-5p compared to the immortalized non-transformed hepatocyte cell line (MIHA) that was not transformed in 9 HCC cell lines (Fig. 3B).

1-3. Confirmation of let-7i-5p expression regulation by HDAC6 signaling pathway

Thereafter, ectopic HDAC6 was introduced into HCC cells (SNU-387 and SNU-423) to confirm that let-7i-5p expression is regulated by HDAC6 and its upstream regulatory signaling pathway. As a result, ectopic overexpression of HDAC6 markedly inhibited let-7i-5p, and when the HDAC6 specific inhibitor Tubastatin A was treated in the same cells, the expression of let-7i-5p was restored (FIG. 3C ). As a result of using JNK-c-Jun pathway-dependent oncogenic miR-221-3p, which directly inhibits HDAC6 in HCC, as a positive control, miR-221- in non-transgenic hepatocytes MIHA and L-02 cell lines. Ectopic expression of the 3p analog inhibited HDAC6 and simultaneously caused the induction of let-7i-5p (Fig. 3D). In addition, JNK inhibition by treatment with c-Jun knockdown and the JNK-specific inhibitor SP600125 restored HDAC6 expression, thereby inhibiting let-7i-5p in HCC cells. In addition, HDAC6 knockdown restored let-7i-5p expression in the same cells (Figs. 3E and F).

Through this, it was found that let-7i-5p expression is located below the JNK/c-Jun-miR-221-3p-HDAC6 regulatory axis.

Example 2. Characterization of let-7i-5p for HCC

2-1. Confirmation of the characteristics of let-7i-5p on tumor formation through MTT and cell survival analysis

To confirm the tumorigenic properties of let-7i-5p in liver cancer, to confirm in vitro tumorigenesis, MTT using antisense let-7i-5p (AS-let-7i-5p) Assays and cell viability assays were performed. Specifically, for MTT analysis, SNU-387 and SNU-423 cells were dispensed into a 12-well plate and transfected with AS-let-7i-5p, followed by MTT [3-(4,5-dimethylthiazol-2). After incubation with 0.5 mg/ml of -yl)-2,5-diphenyltetrazolium bromide] solution (Sigma) for 1 hour, absorbance was measured using a VICTOR3 Multilabel plate reader (PerkinElmer, Waltham, MA). In addition, for cell viability analysis, cells were dispensed into a 6-well plate and cultured for 72 hours, and then cells were obtained with trypsin and stained with trypan blue solution (Sigma, St. Louis, MO). After staining, cells were counted with a hemocytometer (Marienfeld Superior, Lauda-Koonigshofen, Germany) to confirm cell survival. As a result, it was confirmed that ectopic expression of let-7i-5p remarkably suppressed tumor cell growth (Fig. 4A).

2-2. Confirmation of the effect of let-7i-5p on tumor cell proliferation

In addition, to confirm cell proliferation, BrdU (Bromodeoxyuridine) analysis and Clonogenic analysis were performed. Specifically, for BrdU analysis, SNU-387 and SNU-423 cells were distributed in a 24-well plate at a density of 40%-50%, and then confirmed using a BrdU cell proliferation assay kit (Millipore). For clonogenic analysis, the above cells were dispensed into a 60 mm ² cell culture plate and then transfected with AS-let-7i-5p. After 24 hours of transfection, 2 × 10 ³ and 4 × 10 ³ cells were dispensed into a 6-well plate and incubated for 2 weeks. Thereafter, the cells were washed with PBS and fixed with 1% paraformaldehyde at room temperature for 30 minutes. After staining the fixed cells with 0.5% crystal violet for 1 hour at room temperature, colonies were counted by a clono-counting program. As a result, it was confirmed that both BrdU analysis and Clonogenic analysis significantly inhibited tumor cell proliferation in AS-let-7i-5p-transfected cells (FIG. 4B).

2-3. Confirmation of the effect of let-7i-5p on tumor cell death

In addition, the degree of death of SNU-387 and SNU-423 cells by AS-let-7i-5p transfection was confirmed using Annexin V-FITC Apoptosis Detection Kit I (BD Biosciences, San Jose, CA). Specifically, cells were transfected with AS-let-7i-5p, and 72 hours later, detached with trypsin and resuspended with 1 × binding buffer (1 × 10 ⁶ cells/mL), containing 1 × 10 ⁵ cells. So, 10 ul was transferred to a 5 ml culture tube. Thereafter, 5 ul of Annexin V-FITC and 10 ul of PI (propidium iodide) solution were added, and the cells were incubated for 15 minutes under dark conditions at room temperature. After incubation, 400 ul of 1 × binding buffer was added to each tube, and the dead fraction was detected using a FACSCalibur flow cytometer (BD Biosciences). As a result, it was found that AS-let-7i-5p transfection remarkably induces the death of HCC cells (Fig. 4C). This result was the same in the result of confirming caspase-3 and PARP cleavage by Western blot analysis (Fig. 4D).

2-3. Confirmation of the effect of let-7i-5p on tumor cell migration and invasion

In addition, since epithelial-tomesenchymal transition (EMT) has been suggested as a key process in cancer progression, a scratch wound healing analysis was conducted to confirm the role of let-7i-5 in the malignant action of HCC cells. Performed. Specifically, SNU-387 and SNU-423 cells were transfected with AS-let-7i-5p and incubated for 24 hours, and the cells were removed and dispensed into 6-well plates at 1×10 ⁶ cells per well. After incubation overnight, the cell monolayer was scraped off with a sterile micropipette tip. The initial scratch interval after 0 hours of scratch and the interval after 24 hours were photographed using IX71 photomicrograph (Olympus, Tokyo, Japan). As a result, it was found that transfection of AS-let-7i-5p significantly reduced the wound healing effect of HCC cells (FIG. 4E ).

In addition, in order to confirm the migration and invasive characteristics of cancer cells, cell mobility was confirmed using a modified Boyden chamber assay (BD Biosciences, San Jose, CA). Specifically, for invasive analysis, Matrigel (BD Biosciences) was diluted with a coating buffer to a concentration of 0.3 mg/ml. Thereafter, the upper surface of a transwell cell culture insert was coated with 100 ul of Matrigel. After incubation at 37° C. for 2 hours, cells were dispensed on the surface of the insert and mounted on a 24-well plate containing a medium containing 2% FBS as a chemical stimulating substance. After incubating the plate overnight, it was stained using a Diff-Quik staining kit (Sysmex, Kobe, Japan). Cell images were confirmed with an Axiovert 200 inverted microscope (Zeiss, Oberkochen, Germany) at ×200 magnification, and the number of cells was randomly counted at three locations. As a result, it was found that inhibition of let-7i-5p significantly inhibited chemo-stimulatory migration and invasive reactions in both SNU-387 and SNU-423 cell lines (FIG. 4F).

Through the above results, it was found that the abnormal expression of let-7i-5p contributes to the anti-killing and metastatic properties of HCC.

Example 3. Screening of target molecules of let-7i-5p

3-1. Exploring the target molecule of let-7i-5p

To identify the target molecule of let-7i-5p, TargetScan ( http://www.targetscan.org/vert_72/ ), a target prediction program, was used. Through this, 1,207 genes were selected as target molecules for let-7i-5p (data not shown). Among the selected molecules, molecules related to apoptosis process were selected using DAVID Functional Annotation Tool 6.7 ( https://david.ncifcrf.gov/ ). Of the 54 genes selected for this (GO: 0006915, GO: 0012501), 10 genes down-regulated in the HCC dataset (Catholic_LIHC) were selected and additionally three large HCC cohort studies (TCGA_LIHC, ICGC_LIRI-JP and GSE77314). ) And compared. To select the target gene for let-7i-5p in HCC, the fold change (< -1.5 fold, p <4 cohort studies (Catholic_LIHC, TCGA_LIHC, ICGC_LIRI-JP and GSE77314) of the 10 genes selected above. The importance score at 0.001) was calculated (Table 2). As a result. THBS1 (thrombospondin-1 gene) was shown to be the most markedly down-regulated gene in four cohort studies of HCC patients. In addition, THBS1 was also found to be significantly down-regulated in HCC of 50 comparison-pairs (HCC tissue vs non-cancerous peripheral liver tissue) of the TCGA_LIHC dataset (FIG. 5A ). In addition, qRT-PCR analysis of 40 comparison-pairs of the HCC subset showed that THBS1 was significantly down-regulated in 26 comparison-pairs (65%) of 40 comparison-pairs of the HCC subset (< -2.0 Fold, p <0.001) (left panel in Figure 5B). In addition, in the same HCC subset, let-7i-5p was found to have a significantly negative correlation (r = -0.55, P <0.001) with THBS1 (right panel of FIG. 5B).

3-2. Confirmation of TSP1 inhibitory activity of let-7i-5p

Since TSP1 (thrombospondin-1) is a glycoprotein secreted from several cell types, the degree of secretion of TSP1 in the culture medium of SNU-387 and SNU-423 cell lines and MIHA was confirmed by Western blot analysis. As a result, unlike MIHA, secretion of TSP1 was not detected in the SNU-387 and SNU-423 cell lines (Fig. 6A).

In addition, for luciferase analysis, 3'-UTR of TSP-1 mRNA was amplified from cDNA of SNU-387 and SNU-423 cell lines by PCR, and then XhoI/NotI of psiCHECK-2 vector (Promega, Madison, WI) The reporter vector psiCHECK2-THBS1_3' UTR was constructed by cloning in position. After that, the dual luciferase reporter analysis of psiCHECK2-THBS1-3' UTR and antisense let-7i-5p (ASlet-7i-5p) was performed using the Dual-Luciferase Reporter Assay System (Promega), and the results were evaluated as a negative control. It was compared with the results of the control antisense inhibitor treatment group (NC). As a result, in both SNU-387 and SNU-423 cell lines, luciferase activity was significantly increased compared to the control group due to AS-let-7i-5p treatment (FIG. 6B).

In addition, after transfecting the two cell lines with Dicer siRNA (siDicer) and/or AS-let-7i-5p, the induced TSP1 was confirmed by Western blot analysis, and Dicer knockdown induced TSP1 induction, Let-7i-5p treatment attenuated the Dicer knockdown effect (top panel in Figure 6C). In addition, after transfection with AS-let-7i-5p, the degree of TSP1 secretion in the two cell lines was confirmed by Western blot, and as a result, AS-let-7i-5p was found to increase the secretion of TSP1 (Fig. 6C). Bottom panel).

3-3. Confirmation of the effect of TSP1 on tumor cell migration and invasion

As a result of confirming the wound healing effect of the treatment of partially purified TSP1 (MIHA_ppTSP1) from MIHA cells, it was found that AS-let-7i-5p was similar to that of inhibiting the wound healing effect of HCC (FIG. 6D ).

In addition, in order to confirm the migration and invasion characteristics of cancer cells, as a result of confirming cell mobility using a modified Boyden chamber assay (BD Biosciences, San Jose, CA), both AS-let-7i-5p treatment and MIHA_ppTSP1 treatment were performed on HCC cells. It was found to significantly inhibit the migration and invasion of (Fig. 6E).

In order to confirm the regulatory effects of let-7i-5p and MIHA_ppTSP1 on epithelial-mesenchymal transition, the expression levels of N-cadherin, Fibronectin, Slug and Snail, which are characteristic of EMT, and E-cadherin, which are epithelial markers, as EMT regulatory molecules Confirmed by Western blot analysis. As a result, the expression of N-cadherin, Fibronectin, Slug and Snail was significantly decreased in both the AS-let-7i-5p treatment group and the MIHA_ppTSP1 treatment group, and the expression of E-cadherin was significantly increased (FIG. 6F).

Through the above results, it can be seen that let-7i-5p directly inhibits TSP1 and contributes to tumor formation of HCC.

Example 4 Confirmation of the effect of TSP1 on HCC

4-1. Effect of TSP1 on tumor growth and proliferation

In the above examples, let-7i-5p directly regulated TSP1, so in order to confirm the tumor suppressor role of TSP1 in liver cancer, recombinant TSP1 (rTSP1), 293T cell culture medium was used in SNU-387 and SNU-423 cell lines. After the partially purified TSP1 (293T_ppTSP1) and MIHA_ppTSP1 were treated, respectively, as in Example 2, cell growth and proliferation assays were confirmed for tumor formation in vitro. As a result of cell growth and proliferation analysis, cell growth and proliferation were significantly increased in all of the rTSP1, 293T_ppTSP1 and MIHA_ppTSP1 treatment groups (FIGS. 7A and B).

4-2. Effect of TSP1 on tumor cell death

To confirm the killing effect of rTSP1, 293T_ppTSP1 and MIHA_ppTSP1 on HCC cell lines, the degree of killing of SNU-387 and SNU-423 cells by treatment with rTSP1, 293T_ppTSP1 and MIHA_ppTSP1 was measured by Annexin V-FITC Apoptosis Detection Kit I (BD Biosciences, San Jose, CA), and cells related to cell death (cleaved caspase-3 and PARP) were confirmed by Western blot analysis. As a result, in both SNU-387 and SNU-423 cell lines, apoptosis was significantly increased due to the treatment of rTSP1, 293T_ppTSP1 and MIHA_ppTSP1, and it was found that cleaved caspase-3 and PARP were significantly induced (Figs. 7C and D ).

4-3. Confirmation of the effect of TSP1 on tumor cell migration and invasion

The effect on the migration and invasion of tumor cells due to treatment with rTSP1, 293T_ppTSP1 and MIHA_ppTSP1 in HCC cell lines was confirmed by the method of Example 2-3. As a result, rTSP1, 293T_ppTSP1 and MIHA_ppTSP1 were found to significantly inhibit the invasive response in both SNU-387 and SNU-423 cell lines, which was attenuated by co-treatment of A6.1, a TSP1-specific neutralizing antibody (Fig. 7E). In addition, HDAC6 expression using pcDNA3.1_HDAC6, let-7i-5p inhibition using AS-let-7i-5p, cytoplasmic TSP1 (293T_ppTSP1 and MIHA_ppTSP1) and recombinant TSP1 (rTSP1) treatment also inhibited the invasive response of HCC cells. It was found to be recovered by co-treatment of A6.1, a TSP1-specific neutralizing antibody (Fig. 7F).

Through the above results, it was found that the effect of let-7i-5p on the anti-kill and metastasis properties of HCC is due to TSP1.

Example 5. Confirmation of sub-mechanism of HDAC6-LET-7I-5P-TSP1 signaling axis

5-1. Confirmation of the effect of CD47 on the anti-microtubule formation activity of TSP1

In order to confirm whether secreted TSP1 mediates angiogenesis through cell surface receptor CD47, in vitro microtubules for rTSP1 and MIHA_ppTSP1 in Ras-transformed mouse fibroblasts (Ras-NIH3T3) and human umbilical vascular endothelial cells (HUVECs). A microtubule formation assay was performed. Specifically, Matrigel Basement Membrain Matrix (BD Biosciences) was coated on a 24-well plate at 37° C. for 1 hour, and Ras-NIH3T3, HUVEC, or SNU-423 cells were dispensed and cultured. Anti-TSP1 antibodies (3F352, US Biologicals, Salem, MA) containing MIHA_ppTSP1, rTSP1 and CD47 binding motifs were treated and incubated with O/N. After that, the number of nodes in the tube structure was quantified. As a result, treatment of MIHA_ppTSP1 and rTSP1 in Ras-NIH3T3 and HUVEC remarkably inhibited microtubule formation, and blocking of the C-terminal domain against the anti-TSP1 antibody (3F352) interacting with the CD47 receptor binding motif was prevented by TSP1. -Significantly reduced microtubule formation activity (Figs. 8A and B).

5-2. Confirmation of tumor angiogenesis regulation of HDAC6-let-7i-5p-TSP1 signaling axis through CD47 receptor

To confirm whether the HDAC6-let-7i-5p-TSP1 signaling axis regulates tumor angiogenesis through the CD47 receptor, SNU-423 cells transfected with pcDNA3.1_HDAC6 or AS-let-7i-5p were In vitro microtubule formation analysis was performed by co-culture with a number of Ras-NIH3T3 or HUVECs. As a result, microtubule formation activity was remarkably suppressed in Ras-NIH3T3 and HUVEC co-cultured with SNU-423 cells transfected with pcDNA3.1_HDAC6 or AS-let-7i-5p. Treatment with 3F352 showed microtubule formation activity. It can be seen that this recovers again, and the same results were also found in the co-culture medium treated with MIHA_ppTSP1 and rTSP1 (FIGS. 9A and B).

The above results indicate that the HDAC6-let-7i-5p-TSP1 signaling axis exhibits anti-angiogenic activity through interaction with the CD47 cell surface receptor.

5-3. Confirmation of the metastatic effect of HCC cells by the interaction of TSP1 and CD47

As a result of confirming the effect on the migration and invasiveness of HCC cells by treatment with cDNA3.1_HDAC6, AS-let-7i-5p, MIHA_ppTSP1 or rTSP1, the migration and invasion of HCC cells were significantly reduced by the treatment of the above substances. It was found that the same effect was recovered by the 3F352 treatment. Through this, it can be seen that the TSP1-CD47-mediated malignant trait of HCC cells represents an autocrine and/or paracrine mechanism (Figs. 9C and D, and Figs. 10A to D).

5-4. Confirmation of the effect of TSP1 on CD47 in the macrophage phagocytic regulatory network

In the interaction of CD47 and signal regulatory protein α (SIRPα) in the macrophage phagocytosis regulatory network, it was confirmed whether TSP1 occupies the CD47 receptor and interferes with the interaction of CD47-SIRPα, allowing macrophages to phagocytosis HCC. In order to do so, phagocytosis analysis was performed in vitro. Specifically, HCC cell lines SNU-387 and SNU-423 were each made into a single cell suspension, and then labeled with CFSE (abcam, Cambridge, UK). Thereafter, peritoneal macrophages were obtained from C57BL/6 mice and co-cultured with SNU-387 and SNU-423 for 2 hours, and then HCC treated with pcDNA3.1_HDAC6, AS-let-7i-5p, MIHA_ppTSP1 and rTSP1, respectively. Compared with cells. The phagocytic index was calculated by dividing the number of macrophages capturing tumor cells by the total number of macrophages. As a result, pcDNA3.1_HDAC6, AS-let-7i-5p, MIHA_ppTSP1 or rTSP1 treated HCC cells significantly increased macrophage phagocytosis activity, which was significantly attenuated due to the treatment of 3F352 (FIGS. 11A and 12 ).

Through this, the HDAC6-let-7i-p-TSP1 signaling axis converts the CD47-SIRPα interaction between macrophages and HCC into the CD47-TSP1 interaction, thereby re-activating the phagocytosis of macrophages on HCC cells. Able to know.

5-5. Effect of CD47-TSP1 on exosome interactions in HCC cells

Since tumor-derived exosome elements, especially various microRNAs, promote the malignant properties of HCC, in order to determine what role tumor cell-derived nanovesicles play in the regulation of cytoplasmic TSP1 in hepatocytes, exosomes from the culture medium of HCC cells Was separated and purified. Specifically, the culture medium of HCC was collected and centrifuged, and then Exoquick Exosome Precipitation Solution (System Biosciences, Palo Alto, CA) was added and mixed well. After incubation with O/N at 4° C., the mixture was centrifuged at 1500 g for 30 minutes and the supernatant was removed, and the obtained exosome pellet was resuspended in 50 ul of PBS. The cup-shaped structure and size of the separated exosomes were confirmed with an electron microscope (FIG. 11B). Thereafter, the presence of the exosome-specific markers CD81, CD9 and TSG101 and the absence of HSP70 and HSP90 were confirmed in the HCC-derived exosomes (FIG. 11C). The comparison of let-7i-5p qRT-PCR analysis Ct (threshold cycle) values between exosomes and donor cell fractions revealed that let-7i-5p is predominantly present in exosomes than in whole cell lysates (Fig. 11D). Thereafter, in order to confirm whether non-malignant transformed hepatocytes can take HCC-derived exosomes, PKH67 dye (green fluorescence)-labeled exosomes were incubated with MIHA. As a result, green fluorescence was observed with a confocal microscope in the recipient cells (MIHA) (Fig. 11E). Thus, the relative expression of let-7i-5p in MIHA cells incubated with HCC-derived exosomes was measured, and as a result of comparison with non-treated MIHA, let-7i-5p in HCC-derived exosomes-treated cells Was induced, thereby inhibiting TSP1 in MIHA cells (Fig. 11F).

Through this, it can be seen that the intercellular crossover between HCC cells and non-transgenic hepatocytes is mediated by HCC-derived exosomes that enable the growth of cancer cells in malignant transformation and hepatocellular carcinoma.

5-6. In vivo confirmation of cancer-preventing effects of HDAC6-let-7i-5p-TSP1 signaling activation

In order to confirm the above contents in vivo, pcDNA3 every week from 13 weeks of age to H- ras transgenic mice (Ras-Tg mice) (Laboratory of Human Genomics, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea). 1_HDAC6 50 ug (intravenous injection with Turbofect in vivo Transfection Reagent (Invitrogen, Carlsbad, CA)), AS-let-7i-5p 0.25 mg/kg (intravenous injection with Invivofectamine 3.0 (Invitrogen)), or recombinant TSP1 (R&D Systems) 0.5 ug (tail vascular injection with Invivofectamine) was delivered specifically to the liver (top panel of FIG. 13A). Thereafter, ultrasound images were taken with an ultrasound machine (Philips, Amsterdam, Nederland) at 17 weeks, 19 weeks, 21 weeks and 23 weeks of age, and the liver was obtained at 25 weeks of age to confirm HCC. HCC between negative control (NC) mice was detected by ultrasound from 17 weeks of age, and all 4 out of 4 developed large and multiple HCC. However, in the mice overexpressing HDAC6, HCC was detected at 21 weeks of age (except 1 mouse), and 1 to 4 of 4 mice in the let-7i-5p inhibitory group (AS-let-7i-5p) and the rTSP1-treated group A relatively small HCC was developed in (Fig. 13A bottom panel, and Figs. 14A-E). In addition, as a result of Western blot analysis, overexpression of HDAC6 and TSP1 was confirmed in adjacent normal livers of the pcDNA3.1_HDAC6 group, and TSP1 expression was increased in adjacent normal livers of the AS-let-7i-5p group (FIG. 13B). . In addition, let-7i-5p down-regulation was consistently observed in the adjacent normal liver of mice in the pcDNA3.1_HDAC6 group (Fig. 13C).

<110> THE CATHOLIC UNIVERSITY OF KOREA INDUSTRY-ACADEMIC COOPERATION FOUNDATION <120> COMPOSITION FOR PREVENTING OR TREATING OF LIVER CANCER <130> PN1903-183 <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> let-7i-5p <400> 1 ugagguagua guuugugcug uu 22 <210> 2 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> antisense let-7i-5p <400> 2 aacagcacaa acuacuaccu ca 22 <210> 3 <211> 1112 <212> PRT <213> Artificial Sequence <220> <223> TSP1 <400> 3 Met Gly Leu Ala Trp Gly Leu Gly Val Leu Phe Leu Met His Val Cys 1 5 10 15 Gly Thr Asn Arg Ile Pro Glu Ser Gly Gly Asp Asn Ser Val Phe Asp 20 25 30 Ile Phe Glu Leu Thr Gly Ala Ala Arg Lys Gly Ser Gly Arg Arg Leu 35 40 45 Val Lys Gly Pro Asp Pro Ser Ser Pro Ala Phe Arg Ile Glu Asp Ala 50 55 60 Asn Leu Ile Pro Pro Val Pro Asp Asp Lys Phe Gln Asp Leu Val Asp 65 70 75 80 Ala Val Arg Ala Glu Lys Gly Phe Leu Leu Leu Ala Ser Leu Arg Gln 85 90 95 Met Lys Lys Thr Arg Gly Thr Leu Leu Ala Leu Glu Arg Lys Asp His 100 105 110 Ser Gly Gln Val Phe Ser Val Val Ser Asn Gly Lys Ala Gly Thr Leu 115 120 125 Asp Leu Ser Leu Thr Val Gln Gly Lys Gln His Val Val Ser Val Glu 130 135 140 Glu Ala Leu Leu Ala Thr Gly Gln Trp Lys Ser Ile Thr Leu Phe Val 145 150 155 160 Gln Glu Asp Arg Ala Gln Leu Tyr Ile Asp Cys Glu Lys Met Glu Asn 165 170 175 Ala Glu Leu Asp Val Pro Ile Gln Ser Val Phe Thr Arg Asp Leu Ala 180 185 190 Ser Ile Ala Arg Leu Arg Ile Ala Lys Gly Gly Val Asn Asp Asn Phe 195 200 205 Gln Gly Val Leu Gln Asn Val Arg Phe Val Phe Gly Thr Thr Pro Glu 210 215 220 Asp Ile Leu Arg Asn Lys Gly Cys Ser Ser Ser Thr Ser Val Leu Leu 225 230 235 240 Thr Leu Asp Asn Asn Val Val Asn Gly Ser Ser Pro Ala Ile Arg Thr 245 250 255 Asn Tyr Ile Gly His Lys Thr Lys Asp Leu Gln Ala Ile Cys Gly Ile 260 265 270 Ser Cys Asp Glu Leu Ser Ser Met Val Leu Glu Leu Arg Gly Leu Arg 275 280 285 Thr Ile Val Thr Thr Leu Gln Asp Ser Ile Arg Lys Val Thr Glu Glu 290 295 300 Asn Lys Glu Leu Ala Asn Glu Leu Arg Arg Pro Pro Leu Cys Tyr His 305 310 315 320 Asn Gly Val Gln Tyr Arg Asn Asn Glu Glu Trp Thr Val Asp Ser Cys 325 330 335 Thr Glu Cys His Cys Gln Asn Ser Val Thr Ile Cys Lys Lys Val Ser 340 345 350 Cys Pro Ile Met Pro Cys Ser Asn Ala Thr Val Pro Asp Gly Glu Cys 355 360 365 Cys Pro Arg Cys Trp Pro Ser Asp Ser Ala Asp Asp Gly Trp Ser Pro 370 375 380 Trp Ser Glu Trp Thr Ser Cys Ser Thr Ser Cys Gly Asn Gly Ile Gln 385 390 395 400 Gln Arg Gly Arg Ser Cys Asp Ser Leu Asn Asn Arg Cys Glu Gly Ser 405 410 415 Ser Val Gln Thr Arg Thr Cys His Ile Gln Glu Cys Asp Lys Arg Phe 420 425 430 Lys Gln Asp Gly Gly Trp Ser His Trp Ser Pro Trp Ser Ser Cys Ser 435 440 445 Val Thr Cys Gly Asp Gly Val Ile Thr Arg Ile Arg Leu Cys Asn Ser 450 455 460 Pro Ser Pro Gln Met Asn Gly Lys Pro Cys Glu Gly Glu Ala Arg Glu 465 470 475 480 Thr Lys Ala Cys Lys Lys Asp Ala Cys Pro Asn Gly Cys Leu Ser Asn 485 490 495 Pro Cys Phe Ala Gly Val Lys Cys Thr Ser Tyr Pro Asp Gly Ser Trp 500 505 510 Lys Cys Gly Ala Cys Pro Pro Gly Tyr Ser Gly Asn Gly Ile Gln Cys 515 520 525 Thr Asp Val Asp Glu Cys Lys Glu Val Pro Asp Ala Cys Phe Asn His 530 535 540 Asn Gly Glu His Arg Cys Glu Asn Thr Asp Pro Gly Tyr Asn Cys Leu 545 550 555 560 Pro Cys Pro Pro Arg Phe Thr Gly Ser Gln Pro Phe Gly Gln Gly Val 565 570 575 Glu His Ala Thr Ala Asn Lys Gln Val Cys Lys Pro Arg Asn Pro Cys 580 585 590 Thr Asp Gly Thr His Asp Cys Asn Lys Asn Ala Lys Cys Asn Tyr Leu 595 600 605 Gly His Tyr Ser Asp Pro Met Tyr Arg Cys Glu Cys Lys Pro Gly Tyr 610 615 620 Ala Gly Asn Gly Ile Ile Cys Gly Glu Asp Thr Asp Leu Asp Gly Trp 625 630 635 640 Pro Asn Glu Asn Leu Val Cys Val Ala Asn Ala Thr Tyr His Cys Lys 645 650 655 Lys Asp Asn Cys Pro Asn Leu Pro Asn Ser Gly Gln Glu Asp Tyr Asp 660 665 670 Lys Asp Gly Ile Gly Asp Ala Cys Asp Asp Asp Asp Asp Asn Asp Lys 675 680 685 Ile Pro Asp Asp Arg Asp Asn Cys Pro Phe His Tyr Asn Pro Ala Gln 690 695 700 Tyr Asp Tyr Asp Arg Asp Asp Val Gly Asp Arg Cys Asp Asn Cys Pro 705 710 715 720 Tyr Asn His Asn Pro Asp Gln Ala Asp Thr Asp Asn Asn Gly Glu Gly 725 730 735 Asp Ala Cys Ala Ala Asp Ile Asp Gly Asp Gly Ile Leu Asn Glu Arg 740 745 750 Asp Asn Cys Gln Tyr Val Tyr Asn Val Asp Gln Arg Asp Thr Asp Met 755 760 765 Asp Gly Val Gly Asp Gln Cys Asp Asn Cys Pro Leu Glu His Asn Pro 770 775 780 Asp Gln Leu Asp Ser Asp Ser Asp Arg Ile Gly Asp Thr Cys Asp Asn 785 790 795 800 Asn Gln Asp Ile Asp Glu Asp Gly His Gln Asn Asn Leu Asp Asn Cys 805 810 815 Pro Tyr Val Pro Asn Ala Asn Gln Ala Asp His Asp Lys Asp Gly Lys 820 825 830 Gly Asp Ala Cys Asp His Asp Asp Asp Asn Asp Gly Ile Pro Asp Asp 835 840 845 Lys Asp Asn Cys Arg Leu Val Pro Asn Pro Asp Gln Lys Asp Ser Asp 850 855 860 Gly Asp Gly Arg Gly Asp Ala Cys Lys Asp Asp Phe Asp His Asp Ser 865 870 875 880 Val Pro Asp Ile Asp Asp Ile Cys Pro Glu Asn Val Asp Ile Ser Glu 885 890 895 Thr Asp Phe Arg Arg Phe Gln Met Ile Pro Leu Asp Pro Lys Gly Thr 900 905 910 Ser Gln Asn Asp Pro Asn Trp Val Val Arg His Gln Gly Lys Glu Leu 915 920 925 Val Gln Thr Val Asn Cys Asp Pro Gly Leu Ala Val Gly Tyr Asp Glu 930 935 940 Phe Asn Ala Val Asp Phe Ser Gly Thr Phe Phe Ile Asn Thr Glu Arg 945 950 955 960 Asp Asp Asp Tyr Ala Gly Phe Val Phe Gly Tyr Gln Ser Ser Ser Arg 965 970 975 Phe Tyr Val Val Met Trp Lys Gln Val Thr Gln Ser Tyr Trp Asp Thr 980 985 990 Asn Pro Thr Arg Ala Gln Gly Tyr Ser Gly Leu Ser Val Lys Val Val 995 1000 1005 Asn Ser Thr Thr Gly Pro Gly Glu His Leu Arg Asn Ala Leu Trp His 1010 1015 1020 Thr Gly Asn Thr Pro Gly Gln Val Arg Thr Leu Trp His Asp Pro Arg 1025 1030 1035 1040 His Ile Gly Trp Lys Asp Phe Thr Ala Tyr Arg Trp Arg Leu Ser His 1045 1050 1055 Arg Pro Lys Thr Gly Phe Ile Arg Val Val Met Tyr Glu Gly Lys Lys 1060 1065 1070 Ile Met Ala Asp Ser Gly Pro Ile Tyr Asp Lys Thr Tyr Ala Gly Gly 1075 1080 1085 Arg Leu Gly Leu Phe Val Phe Ser Gln Glu Met Val Phe Phe Ser Asp 1090 1095 1100 Leu Lys Tyr Glu Cys Arg Asp Pro 1105 1110

Claims (8)

A pharmaceutical composition for the prevention or treatment of liver cancer comprising an inhibitor of let-7i-5p expression as an active ingredient. The method of claim 1, wherein the let-7i-5p expression inhibitor is HDAC6 (Histone deacetylase 6), or siRNA (small interfering RNA), shRNA (small hairpin RNA), miRNA, antisense that inhibits the expression of let-7i-5p. Oligonucleotide or nucleic acid aptamer, a pharmaceutical composition for the prevention or treatment of liver cancer. The pharmaceutical composition for preventing or treating liver cancer according to claim 1, wherein the let-7i-5p expression inhibitor increases the secretion of TSP1. According to claim 1, wherein the liver cancer is let-7i-5p high expression liver cancer, a pharmaceutical composition for preventing or treating liver cancer. The pharmaceutical composition for preventing or treating liver cancer according to claim 1, wherein the let-7i-5p expression inhibitor inhibits tumor cell growth, proliferation, migration and invasion. A method of providing information for diagnosis or prognosis of liver cancer comprising confirming the expression of let-7-5p in a sample isolated from the subject. The method of claim 6, further comprising determining that the risk of developing liver cancer is high when let-7-5p is overexpressed by 1.18 times or more compared to the normal control group. 1) treating the test compound or composition to a cell line expressing let-7-5p;
2) measuring the expression level of let-7-5p in the cell line treated with the test compound or composition; And
3) A method for screening a candidate material for treating liver cancer comprising selecting a test compound or composition having a reduced expression level of let-7-5p compared to a control cell line without treatment of the test compound or composition.

Images