Use of untranslated RNA RP11-252e2.2 for predicting liver cancer metastasis or treating liver cancer metastasis

Abstract

The present invention relates to a use of untranslated RNA RP11-252E2.2 for predicting liver cancer metastasis or treating liver cancer metastasis. lncRNA which specifically decrease in expression in liver cancer tissues and cells were identified by analyzing lncRNA which specifically decrease in expression in liver cancer as compared to normal cells with respect to lncRNA showing the potential to function as tumor suppressor genes, and RP11-252E2.2 was found to be involved in the metastasis of liver cancer. Therefore, the untranslated RNA RP11-252E2.2 discovered in the present invention is highly likely to be effectively used for predicting or treating liver cancer metastasis.

Description

Prediction of liver cancer metastasis or treatment of liver cancer metastasis using non-translated RNA RP11-252E2.2

[0001] The present invention relates to the use of non-translated RNA RP11-252E2.2 for predicting liver cancer metastasis or treating liver cancer metastasis.

[0002] Liver cancer has a relatively low incidence in Korea, but with a five-year relative survival rate of less than 40%, continuous efforts are needed to improve survival rates. Liver cancer is the leading cause of cancer deaths among socioeconomically active individuals in their 40s and 50s, posing a significant national challenge. The high mortality rate stems from the lack of biomarkers for early diagnosis and the fact that liver cancer is often diagnosed at an advanced stage, making it difficult to achieve curative treatment. Therefore, research into highly accurate and specific biomarkers to improve the early diagnosis rate of liver cancer is necessary.

[0003] Oncogenes promote cell proliferation, while tumor suppressor genes act as brakes to prevent excessive cell proliferation. Loss of tumor suppressor gene function, i.e., loss of cell growth suppression function, is a key element in the carcinogenesis process and appears to be an important phenomenon observed in most human tumors.

[0004] Therefore, restoring the function of tumor suppressor genes is a potential approach to cancer treatment, as these genes play a crucial role in suppressing cancer cell proliferation and regulating abnormal cell growth. Certain non-coding RNAs (lncRNAs) possess tumor suppressor functions, and they are being studied as new targets for cancer treatment. Because these non-coding RNAs play a crucial role in the development and progression of cancer, understanding and regulating them can play a crucial role in cancer treatment and prevention. In particular, examples of long non-coding RNAs (lncRNAs) functioning as tumor suppressor genes have been identified. For example, the lncRNA ACTA2-AS1 tends to be downregulated in thyroid cancer tissues, and its overexpression has been shown to inhibit the growth and proliferation of thyroid cancer cells. Furthermore, overexpression of this lncRNA significantly reduced the metastatic capacity of thyroid cancer.

[0005] Therefore, discovering new non-coding RNAs for predicting or treating liver cancer metastasis could be a new strategy for preventing liver cancer recurrence and developing treatments.

[0006] The purpose of the present invention is to provide a biomarker composition for predicting liver cancer metastasis, which comprises non-translated RNA RP11-252E2.2 or a methylated promoter region thereof as an active ingredient.

[0007] In addition, another object of the present invention is to provide a composition for predicting liver cancer metastasis, which comprises as an active ingredient an agent capable of measuring the expression level of non-translated RNA RP11-252E2.2 or the methylation level of the non-translated RNA RP11-252E2.2 promoter region, and a kit for predicting liver cancer metastasis comprising the same.

[0008] In addition, another object of the present invention is to provide a method for providing information necessary for predicting liver cancer metastasis, including a step of measuring the expression level of non-translated RNA RP11-252E2.2 or the methylation level of the non-translated RNA RP11-252E2.2 promoter region.

[0009] In addition, another object of the present invention is to provide a composition for preventing, improving or treating liver cancer metastasis, which comprises non-translated RNA RP11-252E2.2 as an active ingredient.

[0010] In addition, another object of the present invention is to provide a method for screening a liver cancer metastasis treatment agent by measuring the expression level of non-translated RNA RP11-252E2.2 or the methylation level of the non-translated RNA RP11-252E2.2 promoter region in isolated liver cancer cells.

[0011] To achieve the above purpose, the present invention provides a biomarker composition for predicting liver cancer metastasis, comprising non-translated RNA RP11-252E2.2 or a methylated promoter region thereof as an active ingredient.

[0012] In addition, the present invention provides a composition for predicting liver cancer metastasis, which comprises as an active ingredient an agent capable of measuring the expression level of non-translated RNA RP11-252E2.2 or the methylation level of the non-translated RNA RP11-252E2.2 promoter region.

[0013] In addition, the present invention provides a kit for predicting liver cancer metastasis comprising the composition.

[0014] In addition, the present invention provides a method for providing information necessary for predicting liver cancer metastasis, comprising the steps of: (1) measuring the expression level of non-translated RNA RP11-252E2. 2 from a sample isolated from a liver cancer patient; (2) comparing the measured expression level of non-translated RNA RP11-252E2. 2 with a control sample; and (3) predicting that the possibility of liver cancer metastasis is high if the measured expression level of non-translated RNA RP11-252E2. 2 is lower than that of the control sample.

[0015] In addition, the present invention provides a method for providing information necessary for predicting liver cancer metastasis, comprising the steps of: (1) measuring the methylation level of the non-translated RNA RP11-252E2. 2 promoter region from a sample isolated from a liver cancer patient; (2) comparing the measured methylation level of the non-translated RNA RP11-252E2. 2 promoter region with a control sample; and (3) predicting that the possibility of liver cancer metastasis is high if the measured methylation level of the non-translated RNA RP11-252E2. 2 promoter region is increased compared to the control sample.

[0016] In addition, the present invention provides a pharmaceutical composition for preventing or treating liver cancer metastasis, comprising non-translated RNA RP11-252E2.2 as an active ingredient.

[0017] In addition, the present invention provides a health functional food composition for preventing or improving liver cancer metastasis, which comprises non-translated RNA RP11-252E2.2 as an active ingredient.

[0018] In addition, the present invention provides a method for screening a liver cancer metastasis treatment agent, comprising the steps of (1) contacting a test substance with isolated liver cancer cells; (2) measuring the expression or activity level of non-translated RNA RP11-252E2.2 in liver cancer cells contacted with the test substance; and (3) selecting a test substance having an increased expression or activity level of non-translated RNA RP11-252E2.2 compared to a control sample.

[0019] In addition, the present invention provides a method for screening a liver cancer metastasis treatment agent, comprising the steps of (1) contacting a test substance with isolated liver cancer cells; (2) measuring the methylation level of the non-translated RNA RP11-252E2.2 promoter region in the liver cancer cells contacted with the test substance; and (3) selecting a test substance having a reduced methylation level of the non-translated RNA RP11-252E2.2 promoter region compared to a control sample.

[0020] The present invention relates to the use of non-translated RNA RP11-252E2.2 for predicting liver cancer metastasis or treating liver cancer metastasis. In this case, lncRNAs that show the possibility of playing a role as tumor suppressor genes whose non-translated RNAs specifically expressed in the liver decrease as liver cancer progresses are analyzed in liver disease genome data to identify lncRNAs whose expression is specifically decreased in liver cancer tissues and cells compared to normal ones, and it is confirmed through cell experiments and animal experiments that RP11-252E2.2 is involved in liver cancer metastasis. Therefore, the non-translated RNA RP11-252E2.2 discovered in the present invention has a very high possibility of being useful for predicting liver cancer metastasis or treating liver cancer metastasis.

[0021] Figure 1 shows a schematic diagram of an overexpression vector (VB221110-1563exy) containing the exon domain constituting RP11-252E2.2.

[0022] Figure 2 shows the results of data analysis for liver cancer-specific lncRNA screening.

[0023] Figure 3 shows the results of analysis of the expression and diagnostic value of RP11-252E2.2 in public omics data for liver cancer.

[0024] Figure 4 shows the results of RP11-252E2.2 specificity evaluation in cells and tissues.

[0025] Figure 5 shows the results of vector production and efficiency verification.

[0026] Figure 6 shows the results of confirming proliferation ability in liver cancer cell lines.

[0027] Figure 7 shows the results of confirming the effect on the motility of liver cancer cell lines.

[0028] Figure 8 shows the results of confirming the effect on motility in other long-term cancer cell lines.

[0029] Figure 9 shows the results of confirming the metastatic potential of RP11-252E2.2 through an animal model.

[0030] Figure 10 shows the methylation confirmation results of RP11-252E2.2.

[0031] Figure 11 shows the results of confirming the downstream signaling pathway of RP11-252E2.2.

[0032] The present invention provides a biomarker composition for predicting liver cancer metastasis, comprising non-translated RNA RP11-252E2.2 or a methylated promoter region thereof as an active ingredient.

[0033]

[0034] As used herein, the term "prediction" refers to the process of predicting the outcome of a treatment for a pathological condition by collating data on the progression of the condition and the treatment process. For the purposes of the present invention, such prediction may be interpreted as assessing the likelihood of recurrence or metastasis after liver cancer treatment, or the likelihood of death due to such treatment, but is not limited thereto.

[0035]

[0036] In the present invention, the term "methylation" refers to a phenomenon that occurs at a cytosine in a CpG island in the promoter region of a specific gene, thereby blocking the binding of a transcription factor and thus blocking the expression of the specific gene. For the purpose of the present invention, methylation refers to methylation in a CpG island in the promoter region of non-translated RNA RP11-252E2.2.

[0037]

[0038] In addition, the present invention provides a composition for predicting liver cancer metastasis, which comprises as an active ingredient an agent capable of measuring the expression level of non-translated RNA RP11-252E2.2 or the methylation level of the non-translated RNA RP11-252E2.2 promoter region.

[0039] Specifically, the agent capable of measuring the expression level of the non-translated RNA RP11-252E2.2 may be a primer or probe that specifically binds to the non-translated RNA RP11-252E2.2, but is not limited thereto.

[0040] Specifically, the agent capable of measuring the methylation level of the non-translated RNA RP11-252E2.2 promoter region may be at least one selected from the group consisting of, but is not limited to, a primer capable of amplifying a fragment within the methylated promoter region of the non-translated RNA RP11-252E2.2, a probe capable of hybridizing with the methylated promoter region of the non-translated RNA RP11-252E2.2, a methylation-specific binding protein capable of binding to the methylated promoter region of the non-translated RNA RP11-252E2.2, a methylation-specific binding antibody or aptamer of the non-translated RNA RP11-252E2.2, and a methylation-sensitive restriction enzyme.

[0041] Preferably, the liver cancer metastasis may be, but is not limited to, metastasis from liver cancer to lung cancer.

[0042]

[0043] In addition, the present invention provides a kit for predicting liver cancer metastasis comprising the composition.

[0044]

[0045] As used herein, the term "primer" refers to a short nucleic acid sequence having a short free 3' hydroxyl group, which can form base pairs with a complementary template and serves as a starting point for copying the template strand. The primer can initiate DNA synthesis in the presence of a polymerization reagent (i.e., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in an appropriate buffer and temperature. PCR conditions and the lengths of the sense and antisense primers can be appropriately selected according to techniques known in the art.

[0046] In addition, the primers of the present invention can be preferably designed according to the sequence of the CpG island to be analyzed for methylation, and can be a primer pair that can specifically amplify cytosine that is methylated and not modified by bisulfite, and a primer set that can specifically amplify cytosine that is not methylated and modified by bisulfite.

[0047] As used herein, the term "probe" refers to a nucleic acid fragment, such as RNA or DNA, ranging from a few bases to several hundred bases in length, capable of specifically binding to mRNA, and is labeled so that the presence or absence of a specific mRNA and its expression level can be confirmed. The probe can be produced in the form of an oligonucleotide probe, a single-stranded DNA probe, a double-stranded DNA probe, an RNA probe, etc. The selection of an appropriate probe and hybridization conditions can be appropriately selected according to techniques known in the art.

[0048] As used herein, the term "antibody" is a term known in the art and refers to a specific immunoglobulin directed against an antigenic site. The antibody in the present invention refers to an antibody that specifically binds to the PMVK of the present invention, and the antibody can be prepared according to a conventional method in the art. The form of the antibody includes a polyclonal antibody or a monoclonal antibody, and all immunoglobulin antibodies are included. The antibody refers to a complete form having two full-length light chains and two full-length heavy chains. The antibody also includes specialized antibodies such as humanized antibodies.

[0049] As used herein, the term "aptamer" refers to a type of polynucleotide composed of a special type of single-stranded nucleic acid (DNA, RNA, or modified nucleic acid) that has a stable tertiary structure in itself and the characteristic of being able to bind to a target molecule with high affinity and specificity. As described above, aptamers can specifically bind to an antigenic substance in the same way as antibodies, but are composed of polynucleotides that are more stable than proteins, have a simpler structure, and are easy to synthesize, and therefore can be used as a substitute for antibodies.

[0050] The term "methylation-sensitive restriction enzyme" as used herein refers to a restriction enzyme that can specifically detect methylation of a CpG island and may be a restriction enzyme containing CG as a recognition site of the restriction enzyme. Examples thereof include, but are not limited to, SmaI, SacII, EagI, HpaII, MspI, BssHII, BstUI, NotI, etc. Depending on methylation or unmethylation at C of the restriction enzyme recognition site, whether or not the restriction enzyme cuts is different and this can be detected through PCR or Southern Blot analysis. Other methylation-sensitive restriction enzymes other than the above restriction enzymes are well known in the art.

[0051]

[0052] In addition, the present invention provides a method for providing information necessary for predicting liver cancer metastasis, comprising the steps of: (1) measuring the expression level of non-translated RNA RP11-252E2. 2 from a sample isolated from a liver cancer patient; (2) comparing the measured expression level of non-translated RNA RP11-252E2. 2 with a control sample; and (3) predicting that the possibility of liver cancer metastasis is high if the measured expression level of non-translated RNA RP11-252E2. 2 is lower than that of the control sample.

[0053] Specifically, methods for measuring the expression level of the non-translated RNA RP11-252E2.2 include, but are not limited to, RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection assay (RPA), Northern blotting, DNA chip, Western blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, or protein chip.

[0054]

[0055] In addition, the present invention provides a method for providing information necessary for predicting liver cancer metastasis, comprising the steps of: (1) measuring the methylation level of the non-translated RNA RP11-252E2. 2 promoter region from a sample isolated from a liver cancer patient; (2) comparing the measured methylation level of the non-translated RNA RP11-252E2. 2 promoter region with a control sample; and (3) predicting that the possibility of liver cancer metastasis is high if the measured methylation level of the non-translated RNA RP11-252E2. 2 promoter region is increased compared to the control sample.

[0056] Specifically, the measurement of the methylation level of the non-translated RNA RP11-252E2.2 promoter region can be performed by a method selected from the group consisting of, but not limited to, PCR, methylation specific PCR, real time methylation specific PCR, PCR using a methylated DNA-specific binding protein, ChIP analysis using a methylated DNA-specific binding antibody or aptamer, DNA chip, pyrosequencing, and bisulfite sequencing.

[0057]

[0058] The term "sample isolated from a patient" as used herein includes, but is not limited to, samples such as tissue, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, or urine that differ from the control group in the expression level of the non-translated RNA RP11-252E2.2, which is the biomarker of the present invention.

[0059]

[0060] In addition, the present invention provides a pharmaceutical composition for preventing or treating liver cancer metastasis, comprising non-translated RNA RP11-252E2.2 as an active ingredient.

[0061] Preferably, the liver cancer metastasis may be, but is not limited to, metastasis from liver cancer to lung cancer.

[0062] Preferably, the non-translated RNA RP11-252E2.2 can inhibit Notch signaling, but is not limited thereto.

[0063]

[0064] The pharmaceutical composition of the present invention can be prepared using pharmaceutically suitable and physiologically acceptable adjuvants in addition to the active ingredient, and the adjuvants may include solubilizers such as excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, glidants, or flavoring agents. The pharmaceutical composition of the present invention can be preferably formulated as a pharmaceutical composition by additionally including one or more pharmaceutically acceptable carriers in addition to the active ingredient for administration. In the composition formulated as a liquid solution, acceptable pharmaceutical carriers are sterile and biocompatible, and may include saline solution, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and a mixture of one or more of these components. If necessary, other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added. In addition, diluents, dispersants, surfactants, binders and lubricants can be additionally added to formulate the composition into injectable formulations such as aqueous solutions, suspensions and emulsions, pills, capsules, granules or tablets.

[0065] The pharmaceutical formulation form of the pharmaceutical composition of the present invention may be granules, powders, coated tablets, tablets, capsules, suppositories, syrups, juices, suspensions, emulsions, drops or injectable solutions, and sustained-release formulations of the active compound, etc. The pharmaceutical composition of the present invention may be administered in a conventional manner via intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, transdermal, intranasal, inhalation, topical, rectal, oral, intraocular or intradermal routes. The effective amount of the active ingredient of the pharmaceutical composition of the present invention refers to the amount required for the prevention or treatment of a disease. Therefore, it can be adjusted according to various factors including the type of disease, the severity of the disease, the types and contents of the active ingredient and other ingredients contained in the composition, the type of formulation, and the patient's age, body weight, general health condition, sex and diet, administration time, administration route and secretion rate of the composition, treatment period, and concurrently used drugs. Although not limited thereto, for example, in the case of adults, when administered once to several times a day, the composition of the present invention may be administered at a dose of 0.1 ng / kg to 10 g / kg in the case of a compound, 0.1 ng / kg to 10 g / kg in the case of a polypeptide, protein or antibody, and 0.01 ng / kg to 10 g / kg in the case of an antisense nucleotide, siRNA, shRNA or miRNA, when administered once to several times a day.

[0066]

[0067] In addition, the present invention provides a health functional food composition for preventing or improving liver cancer metastasis, which comprises non-translated RNA RP11-252E2.2 as an active ingredient.

[0068] The health functional food composition of the present invention may be provided in the form of a powder, granules, tablets, capsules, syrup, or beverage. The health functional food composition may be used in combination with other foods or food additives in addition to the active ingredient, and may be appropriately used according to conventional methods. The amount of the active ingredient mixed may be appropriately determined depending on the intended use, for example, for preventive, health, or therapeutic treatment.

[0069] The effective dosage of the active ingredient contained in the above health functional food composition may be used in accordance with the effective dosage of the above pharmaceutical composition, but in the case of long-term intake for the purpose of health and hygiene or health control, it may be below the above range. It is certain that the active ingredient may be used in an amount exceeding the above range because there is no problem in terms of safety.

[0070] There are no special restrictions on the types of the above health foods, and examples include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes.

[0071]

[0072] In addition, the present invention provides a method for screening a liver cancer metastasis treatment agent, comprising the steps of (1) contacting a test substance with isolated liver cancer cells; (2) measuring the expression or activity level of non-translated RNA RP11-252E2.2 in liver cancer cells contacted with the test substance; and (3) selecting a test substance having an increased expression or activity level of non-translated RNA RP11-252E2.2 compared to a control sample.

[0073]

[0074] In addition, the present invention provides a method for screening a liver cancer metastasis treatment agent, comprising the steps of (1) contacting a test substance with isolated liver cancer cells; (2) measuring the methylation level of the non-translated RNA RP11-252E2.2 promoter region in the liver cancer cells contacted with the test substance; and (3) selecting a test substance having a reduced methylation level of the non-translated RNA RP11-252E2.2 promoter region compared to a control sample.

[0075]

[0076] The term "test substance" used in referring to the screening method of the present invention refers to an unknown candidate substance used in screening to determine whether it affects the expression level of a gene or the expression or activity of a protein. The sample includes, but is not limited to, chemicals, nucleotides, antisense RNA, siRNA (small interference RNA), and natural product extracts.

[0077] Hereinafter, to aid understanding of the present invention, examples will be given in detail. However, the following examples are intended only to illustrate the scope of the present invention and are not intended to limit its scope. These examples are provided to more fully explain the present invention to those of average skill in the art.

[0078]

[0079] <Experimental Example>

[0080] The following experimental examples are intended to provide experimental examples commonly applied to each embodiment according to the present invention.

[0081]

[0082] 1. Selection of liver-specific lncRNAs

[0083] We identified lncRNAs with uniquely high expression in the liver compared to other organs.

[0084] In TCGA data, it was confirmed that expression was higher in normal surrounding tissues compared to liver cancer, and the group with high expression was analyzed to have a good survival rate.

[0085]

[0086] 2. Collection of tissue and clinical information from a liver disease cohort for verification purposes.

[0087] After passing the IRB at Ajou University Hospital, we conducted research by receiving 100 pairs of normal peripheral tissue and liver cancer tissue and their clinical information through the Human Resources Bank.

[0088]

[0089] 3. RNA extraction from tissue

[0090] RNA was extracted from tissues using Qiazol Lysis Reagent (Cat#79306).

[0091]

[0092] 4. Measurement of RP11-252E2.2 expression in tissues of liver disease cohort patients using qRT-PCR analysis.

[0093] cDNA synthesis was performed using PrimeScript™ RT Master Mix (Perfect Real Time) (Cat #RR036A) (TaKaRa).

[0094] cDNA was synthesized in a total volume of 10 μl by adding tissue RNA, RNA-Free water, and PrimeScript™ RT Master Mix.

[0095]

[0096] The cDNA synthesis conditions were set as follows.

[0097] A. Stage 1: 37℃, 15 minutes

[0098] B. Stage 2: 85℃, 5 seconds

[0099] C. Stage 3: 4℃, -ing

[0100]

[0101] Primer information is as follows. Primers were purchased from M.biotech (Hanam, Korea) and used. qRT-PCR was performed using 5 μl of qPCR Master Mix (2X, High ROX) (Gendepot, Cat #Q5602) and 0.5 μl each of the forward and reverse primers, for a total volume of 10 μl.

[0102] Gene Accession No. Forward sequence Reverse sequence RP11-252E2.2ENST00000563098.15'- GATGCTGGCCAGTACAA-3' (SEQ ID NO: 1) 5'- TGCATGACCACTGGCTA-3' (SEQ ID NO: 2) HMBSNM_000190.45'- GCTCAGATAGCATACAAGAG-3' (SEQ ID NO: 3) 5'- ACGAGCAGTGATGCCTACC-3' (SEQ ID NO: 4)

[0103]

[0104] qRT-PCR conditions are as follows.

[0105] A. Stage 1: 95℃, 2 minutes

[0106] B. Stage 2: 95℃, 15 seconds

[0107] C. : 62℃, 34 seconds

[0108] D. : 72℃, 30 seconds

[0109] E. Repeat Stage 2 40 cycles

[0110]

[0111] 5. Cell culture

[0112] Human HCC cell lines (Hep3B, Huh-7, SNU449, PLC / PRF / 5) were obtained from the Korea Cell Line Bank (Seoul, Korea). Human lung cancer cell lines (A549), human breast cancer cell lines (MCF-7), and human colon cancer cell lines (HCT-116) were obtained from the Ajou University Medical Research Institute. Each cell line was cultured in RPMI-1640, DMEM, or MEM medium containing 10% FBS and 100 units / mL penicillin-streptomycin (Invitrogen, CA, USA). Culture conditions were maintained at 5% carbon dioxide and 37°C.

[0113]

[0114] 6. Vector production

[0115] An overexpression vector (VB221110-1563exy) containing the exon domain constituting RP11-252E2.2 was constructed using Vectorbuilder (Santa Clara, CA) (Fig. 1).

[0116] The sequence of RP11-252E2.2 contained in the vector is as follows.

[0117] RP11-252E2.2 sequence: TCTTCAGCCTGAAACCCATCCCTAGAGTCTTGCTGTGTCGCCAGGC TGGAGTGCAGTGGCACGATCTCGGTTCACTGCAACCCCCGCCTCCCAGGTTCAAGCGATTCTCCTGCCTCAGCCTCCCAAGTAGCTGGGACTACAGGCGTGCATCACCATGCCCAGAGTCAAGAAAACTTTTAAGCTATTTACAACTTTTAACAATTGAGTTAAGTATACTCAAGAACAAAATTCAGAGCATATTTGTTTCTCTCTACCTGATTTCTACAGATTTTGGAAAC TATTTGTGGAGATGCTGGCCAGTACAACTTTCTGCTCTGATGGAACCATGCTAGATCTGCGCTTCCCACTTTGTAGCCAGTGGTCATGCATTGCTATGGAGCATTCAAAATGTGGCTAAGGGAAAATTAATAGAGGACAATGTGGTTGTGAATTTTAGAAGGTCTTGAAAAGTACAGCCCACACACGTGGTGCTTACTCAGTGCTGTTACACATAAGGCGGTAAGGGGAATTT (SEQ ID NO: 5)

[0118]

[0119] 7. Transfection

[0120] Transfection was performed using Lipofectamine 2000 (Invitrogen) reagent.

[0121]

[0122] 8. Cell growth analysis

[0123] For cell growth analysis, cells were seeded in a 24-well plate at approximately 30% confluence and transfected. After treating with MTT solution (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and reacting for 1 hour, the formazan crystals were dissolved in DMSO and the absorbance was measured at 570 nm.

[0124]

[0125] 9. Wound healing analysis

[0126] Transfected cells were plated onto a 6-well plate, and when 100% confluent, a scratch was made using a micropipette tip. Images of the scratched area were observed over time.

[0127]

[0128] 10. Cell migration and invasion analysis

[0129] Cell migration was performed using a modified Boyden chamber (BD Bioscience) assay, and invasion was performed using Matrigel (BD Bioscience).

[0130] For invasion analysis, Matrigel was diluted to a concentration of 0.3 mg / mL and dispensed into inserts. Cells transfected with the overexpressing RP11-252E2.2 vector were spread onto the inserts and cultured in medium containing 2.5–5% FBS. Migrated and invaded cells were fixed, and after a certain period of time, the cells were stained with the Diff Quick staining kit (Sysmex Corporation) and observed under a microscope.

[0131]

[0132] 11. Liver cancer orthotopic transplantation mouse model

[0133] To determine the metastatic potential of liver cells, a 1.5 cm skin and peritoneal incision was made in the upper abdominal wall of 6-week-old BALB / c nude mice to expose the left lobe of the liver, which was then seeded in 1:1 RPMI1640 / Matrigel at a density of 5 × 10 5 Transfected hepatoma cell lines (Huh-7) were injected under the surface of the left lobe of the mouse liver, the left lobe of the liver was placed into the body, and the incision site was sutured.

[0134] After surgery, the surgical site was disinfected with povidone-iodine, and the mouse was kept warm with an infrared heater to allow recovery.

[0135] Nineteen days after cell injection, mice were sacrificed and the number and size of tumors that developed in the left lobe of the liver were determined.

[0136]

[0137] 12. Methylation analysis of RP11-252E2.2

[0138] qMSP experiments were performed, and tissues treated with bisulfite were used using the EZ DNA Methylation-Gold kit (ZYMO RESEATRCH). Primers for detecting methylated and unmethylated DNA were designed using MethylPrimer, and the primer information is as follows.

[0139] GeneForward sequenceReverse sequencemethylation5'-AGG ATT GGA GTT TGG TTA GTT GTC -3' (SEQ ID NO: 6)5'- CTA AAA ATC ACG AAA TAA ATA CGA A-3' (SEQ ID NO: 7)unmethylation5'-TTG GAG TTT GGT TAG TTG TTG G -3' (SEQ ID NO: 8)5'-ACT AAA AAT CAC AAA ATA AAT ACA AA-3' (SEQ ID NO: 9)

[0140]

[0141] To determine the effect of DNA methylation on the expression of RP11-252E2.2, demethylation was induced by treatment with 5-Aza-2'-deoxycytidine (5-Aza) (Sigma-Aldrich).

[0142] The expression changes of RP11-252E2.2 after 5-Aza treatment in Hep3B, SNU475, SNU449, and Huh7 hepatoma cell lines were analyzed using qRT-PCR.

[0143] To observe the DNA demethylation process by overexpressing RP11-252E2.2, changes in TET1 and TET2 were evaluated by qRT-PCR.

[0144]

[0145] The primer information is as follows:

[0146] Gene Accession No. Forward sequence Reverse sequence TET1NM_001406365.15'-CTC AAG ACC TTG CCT CTT CTC-3' (SEQ ID NO: 10) 5'- TGC TCA CTG TCT GAC CAA TAC-3' (SEQ ID NO: 11) TET2NM_001127208.35'-CAA ACT CTA CTC GGA GCT TAC C-3' (SEQ ID NO: 12) 5'-CTT CCT TGG GAT CTT GCT TCT-3' (SEQ ID NO: 13)

[0147]

[0148] <Example 1> Data analysis for liver cancer-specific lncRNA screening

[0149] We aimed to select lncRNAs that showed high expression compared to other organs, and identified lncRNAs that showed analysis results significantly related to survival rate (Fig. 2).

[0150]

[0151] <Example 2> Analysis of the expression and diagnostic value of RP11-252E2.2 in public omics data for liver cancer.

[0152] RP11-252E2.2 is a liver-specific lncRNA, and its expression was confirmed to be significantly reduced in hepatocellular carcinoma (HCC) (Fig. 3).

[0153] To evaluate the diagnostic value of RP11-252E2.2 for HCC, ROC analysis was performed on TCGA_LIHC and GEO datasets (GSE77314, GSE124535).

[0154] It showed high discriminatory power with AUC = 0.89 (95% CI: 0.86-0.92) in the TCGA_LIHC dataset.

[0155] In the GSE77314 dataset, it showed the best diagnostic accuracy with AUC = 0.92 (95% CI: 0.87-0.98).

[0156] The diagnostic value of RP11-252E2.2 was also replicated in the GSE124535 dataset with an AUC = 0.83 (95% CI: 0.73–0.93).

[0157] Consistent results were observed in three independent datasets, suggesting that RP11-252E2.2 has the potential to be used as a diagnostic biomarker for HCC.

[0158]

[0159] <Example 3> Evaluation of RP11-252E2.2 specificity in cells and tissues

[0160] Compared to the normal cell line MIHA, it was confirmed that the expression of RP11-252E2.2 was reduced in all HCC cell lines.

[0161] As a result of confirming the expression of RP11-252E2.2 in normal surrounding tissues and liver cancer tissues donated from the Ajou University Hospital Human Genome Resource Bank, it was confirmed that expression was lower in liver cancer tissues than in normal surrounding tissues in 93% of patients (Fig. 4).

[0162]

[0163] <Example 4> Vector production and efficiency verification

[0164] The efficiency of the RP11-252E2.2 overexpression vector was confirmed in four HCC cell lines by qRT-PCR (Fig. 5).

[0165]

[0166] <Example 5> Confirmation of proliferation ability in liver cancer cell lines

[0167] When cell growth (MTT) was performed in four HCC cell lines, it was confirmed that RP11-252E2.2 was not involved in cell proliferation (Fig. 6).

[0168]

[0169] <Example 6> Confirmation of the effect on the motility of liver cancer cell lines

[0170] To determine whether RP11-252E2.2 is involved in the metastasis of liver cancer cells, wound healing, cell migration, and cell invasion assays were performed. Treatment with a vector overexpressing RP11-252E2.2 revealed a decrease in wound healing, cell migration, and invasion compared to the NC group (Fig. 7).

[0171]

[0172] <Example 7> Confirmation of the effect on motility in other organ cancer cell lines

[0173] RP11-252E2.2 is a liver-specific lncRNA. In addition to studying its expression in hepatoma cell lines, experiments were conducted to determine the effects of RP11-252E2.2 expression in cell lines other than hepatoma cell lines (Fig. 8).

[0174] The cell lines used in the experiment are as follows.

[0175] A549: lung cancer cell line

[0176] HT-29: colon cancer cell line

[0177] Hep3B: liver cancer cell line

[0178]

[0179] Cell migration was observed by comparing EV and RP11-252E2.2 overexpressing (OE) cell lines in each cell line using a wound healing assay.

[0180] Results: There was no significant difference in the wound closure rate between each cell line (ns, p > 0.05).

[0181] Interpretation: Overexpression of RP11-252E2.2 did not significantly affect cell migration in non-hepatoma cell lines, such as A549 (lung cancer) and HT-29 (colon cancer). This confirms that RP11-252E2.2 is a liver-specific gene, suggesting that its effects may be limited in cancer cells other than hepatocytes.

[0182]

[0183] <Example 8> Confirmation of metastatic potential of RP11-252E2.2 through animal model

[0184] Overexpressed RP11-252E2.2 was transfected into Huh-7 mice and injected into the liver. No changes in tumor size or weight were observed, consistent with the results obtained in vitro.

[0185] As shown in Figure 9, it was confirmed that the number of tumors generated in the liver in the group transfected by overexpressing RP11-252E2.2 was less than that in the NC group.

[0186] IHC results showed that ZO-1 and E-cadherin expression increased, while Snail and Vimentin expression decreased. In addition, when overexpressed RP11-252E2.2 was transfected, the expression of CD31, VEGF, HIF-1a, and CD133 decreased, but there was no difference in Ki-67 and DNA-PKs. This showed a similar trend to the results from the cell experiment shown previously.

[0187] By confirming that the number of nodules in the lungs of mice overexpressing RP11-252E2.2 was significantly reduced, we confirmed that RP11-252E2.2 is involved in the metastasis of liver cancer.

[0188] Therefore, it was confirmed that the injection of the vector overexpressing RP11-252E2.2 produced by the present invention was effective in both in vitro and in vivo in suppressing motility and metastasis, although it was not related to the proliferation of liver cancer.

[0189]

[0190] <Example 9> Confirmation of methylation of RP11-252E2.2

[0191] qMSP analysis results showed that the promoter methylation level of RP11-2522E.2 was increased in 29 (50.87%) of 57 liver cancer patient samples compared to non-tumor tissues, suggesting that RP11-2522E.2 may be hypermethylated in liver cancer and its expression may be suppressed.

[0192] In addition, when RP11-252E2.2 was overexpressed in the liver cancer cell line Huh-7 and treated with siTET1 and siTET2, it was confirmed that the expression of RP11-252E2.2 was overexpressed compared to EV, and the effect of DNA demethylation process on RP11-252E.2 expression was evaluated.

[0193] To determine the effect of DNA methylation on the expression of RP11-252E2.2, hepatoma cell lines SNU475, Hep3B, SNU449, and Huh-7 were treated with 5-Aza-2'-deoxycytidine to induce a demethylation effect. When the expression of RP11-252E2.2 was analyzed by qRT-PCR, it was confirmed that the 5-Aza-treated group was overexpressed (Fig. 10).

[0194] This suggests that RP11-252E2.2 expression may be repressed by promoter methylation.

[0195]

[0196] <Example 10> Confirmation of the lower signaling pathway of RP11-252E2.2

[0197] The expression of proteins such as p-JAK1, p-JAK2, p-STAT3, p-ERK, and p-AKT was similar to that of the mock even after RP11-252E2.2 treatment, which provides evidence that RP11-252E2.2 does not affect the JAK / STAT and ERK / AKT pathways.

[0198] Notch (full length) and Cleaved Notch levels were altered by RP11-252E2.2, and Cleaved Notch, MAML1, RBPSUH, Cyclin D3, P21 Waf1 / Cip1, etc. were regulated (Fig. 11).

[0199] This suggests that RP11-252E2.2 acts as a suppressor that inhibits Notch signaling.

[0200]

[0201] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A biomarker composition for predicting liver cancer metastasis, comprising non-translated RNA RP11-252E2.2 or a methylated promoter region thereof as an active ingredient.

2. A composition for predicting liver cancer metastasis, comprising as an active ingredient an agent capable of measuring the expression level of non-translated RNA RP11-252E2.2 or the methylation level of the non-translated RNA RP11-252E2.2 promoter region.

3. A composition for predicting liver cancer metastasis, characterized in that the agent capable of measuring the expression level of the non-translated RNA RP11-252E2.2 in the second paragraph is a primer or probe that specifically binds to the non-translated RNA RP11-252E2.

2.

4. In the second paragraph, the agent capable of measuring the methylation level of the non-translated RNA RP11-252E2.2 promoter region is characterized in that at least one selected from the group consisting of a primer capable of amplifying a fragment within the methylated promoter region of the non-translated RNA RP11-252E2.2, a probe capable of hybridizing with the methylated promoter region of the non-translated RNA RP11-252E2.2, a methylation-specific binding protein capable of binding to the methylated promoter region of the non-translated RNA RP11-252E2.2, a methylation-specific binding antibody or aptamer of the non-translated RNA RP11-252E2.2; and a methylation-sensitive restriction enzyme.

5. A composition for predicting liver cancer metastasis, characterized in that the liver cancer metastasis in the second paragraph is metastasis from liver cancer to lung cancer.

6. A kit for predicting liver cancer metastasis comprising a composition according to any one of claims 2 to 5. 7.(1) A step of measuring the expression level of non-translated RNA RP11-252E2.2 from a sample isolated from a liver cancer patient; (2) a step of comparing the expression level of the measured non-translated RNA RP11-252E2.2 with that of the control sample; and (3) A method for providing information necessary for predicting liver cancer metastasis, including a step of predicting that there is a high possibility of liver cancer metastasis when the expression level of the measured non-translated RNA RP11-252E2.2 is lower than that of the control sample. 8.(1) A step of measuring the methylation level of the non-translated RNA RP11-252E2.2 promoter region from a sample isolated from a liver cancer patient; (2) a step of comparing the methylation level of the measured non-translated RNA RP11-252E2.2 promoter region with that of a control sample; and (3) A method for providing information necessary for predicting liver cancer metastasis, including a step of predicting that the possibility of liver cancer metastasis is high if the methylation level of the measured non-translated RNA RP11-252E2.2 promoter region is increased compared to a control sample.

9. A pharmaceutical composition for preventing or treating liver cancer metastasis, comprising non-translated RNA RP11-252E2.2 as an active ingredient.

10. A pharmaceutical composition for preventing or treating liver cancer metastasis, characterized in that the liver cancer metastasis in paragraph 8 is metastasis from liver cancer to lung cancer.

11. A pharmaceutical composition for preventing or treating liver cancer metastasis, characterized in that the non-translated RNA RP11-252E2.2 in claim 8 inhibits Notch signaling.

12. A health functional food composition for preventing or improving liver cancer metastasis, comprising non-translated RNA RP11-252E2.2 as an active ingredient. 13.(1) Step of contacting the test substance with the separated liver cancer cells; (2) a step of measuring the expression or activity level of non-translated RNA RP11-252E2.2 in liver cancer cells that have come into contact with the test substance; and (3) A method for screening a liver cancer metastasis treatment agent, comprising a step of selecting a test substance having an increased expression or activity level of the non-translated RNA RP11-252E2.2 compared to a control sample. 14.(1) Step of contacting the test substance with the separated liver cancer cells; (2) a step of measuring the methylation level of the non-translated RNA RP11-252E2.2 promoter region in liver cancer cells that have come into contact with the test substance; and (3) A method for screening a liver cancer metastasis treatment agent, comprising a step of selecting a test substance having a reduced methylation level in the non-translated RNA RP11-252E2.2 promoter region compared to a control sample.

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