JP7319688B2 - CANCER THERAPY AGENT AND CANCER DIAGNOSTIC METHOD TARGETING LONG NON-CODED RNA - Google Patents

Description

TECHNICAL FIELD The present invention relates to cancer therapeutic agents and cancer diagnostic methods that target long non-coding RNAs.

Long non-coding RNAs (lncRNAs) are RNAs with a length of 200 bases or more that do not code for proteins. lncRNAs are involved in various life phenomena such as cell proliferation, apoptosis, cell differentiation, and diseases (cancer, heart disease, neurodegenerative disease, etc.). It is known that the expression pattern of lncRNA shows tissue specificity and disease specificity.
lncRNAs show organ- or disease-specific expression patterns, and their association with cancer has also attracted attention in recent years. Although the functions of most of the more than 10,000 types of lncRNAs are still unknown, it is expected that clarifying the functions of lncRNAs in carcinogenesis will lead to the development of new diagnostic and therapeutic methods. However, there are currently no clinical applications targeting lncRNAs.

Patent Document 1 describes a method for predicting the onset or recurrence of acute myeloid leukemia using TM4SF1 as a leukemic stem cell marker gene. are not described and their sequences are also different.
Non-Patent Document 1 describes the identification of a long noncoding RNA associated with chronic gastritis and gastric cancer, but does not describe its specific gene or its function.

JP 2015-231375 A

Hiroshi Kitajima, P-1225 Identification of long noncoding RNA associated with chronic gastritis and gastric cancer, The 75th Annual Meeting of the Japanese Cancer Society, October 6, 2016

An object of the present invention is to provide new diagnostic and therapeutic methods for cancer by clarifying the function of lncRNA in carcinogenesis.

In order to solve the above problems, the present inventors have conducted intensive studies and found that the transcript RNA of TM4SF1AS1 is expressed from the stage of precancerous lesions of gastric cancer, and that this expression is suppressed. As a result, the inventors have found that the cell growth inhibitory effect is exhibited, and have completed the present invention.

That is, the present invention relates to the following.
(1) A therapeutic agent for cancer comprising at least one of a TM4SF1AS1 expression inhibitor, a PURA expression inhibitor, and a YB1 expression inhibitor.
(2) The cancer therapeutic agent according to (1), which contains an inhibitor of TM4SF1AS1 expression.
(3) The cancer therapeutic agent according to (2), wherein the inhibitor of TM4SF1AS1 expression is siRNA, antisense nucleic acid, shRNA, or miRNA against transcript RNA of TM4SF1AS1.
(4) The cancer therapeutic agent according to (3), wherein the suppressor of TM4SF1AS1 expression is siRNA.
(5) The cancer therapeutic agent according to any one of (2) to (4), further comprising a PURA expression inhibitor and a YB1 expression inhibitor.
(6) The cancer therapeutic agent according to any one of (1) to (5), wherein the cancer is gastric cancer, breast cancer, liver cancer, colon cancer, or pancreatic cancer.
(7) A vector expressing siRNA, antisense nucleic acid, shRNA or miRNA against transcript RNA of TM4SF1AS1.
(8) A cell into which the vector of (7) has been introduced.
(9) A pharmaceutical composition for treating cancer, comprising the therapeutic agent for cancer according to any one of (1) to (6), the vector according to (7), or the cell according to (8). thing.
(10) A method for measuring the expression level of transcript RNA of TM4SF1AS1 as an index for predicting whether a human subject will develop cancer, comprising:
(a) determining the expression level of TM4SF1AS1 transcript RNA in a test sample from a human subject;
(b) determining the expression level of TM4SF1AS1 transcript RNA in a control sample from a healthy human subject; and (c) determining the expression level of TM4SF1AS1 transcript RNA in a test sample from a human subject. predicting that a human subject will develop cancer if the amount of transcript RNA of TM4SF1AS1 expressed in a control sample from which it is derived is greater than two-fold;
A method, including
(11) In (c), when the expression level of TM4SF1AS1 transcript RNA in a test sample derived from a human subject exceeds 3 times the expression level of TM4SF1AS1 transcript RNA in a control sample derived from a healthy human subject. (10), wherein the human subject is predicted to develop cancer.
(12) The method according to (10) or (11), wherein the cancer is gastric cancer, breast cancer, liver cancer, colon cancer, or pancreatic cancer.
(13) The method of any of (10)-(12), wherein the human subject is precancerous.

By suppressing the expression of TM4SF1AS1, the cancer therapeutic agent and pharmaceutical composition of the present invention exhibit cell growth inhibitory effects in cancers such as gastric cancer, breast cancer, liver cancer, colon cancer, and pancreatic cancer. can do.
According to the method of the present invention, the possibility of developing cancer can be predicted in the precancerous stage.

The left panel of FIG. 1 shows histone modification (H3K4me3) profiles (ChIP-Seq method) of background mucosa derived from healthy subjects and gastric cancer patients. The middle panel in FIG. 1 shows the gene annotation. Of the 35,387 peaks detected in the H3K4me3 profile, 1512 were upregulated, 3140 were downregulated and 11398 were unchanged. The right figure of FIG. 1 shows an example of the active region of the transcription initiation site by H3K4me3. TM4SF1AS1 was activated in cancer patients and not in healthy controls. The left panel of FIG. 2 shows the results of quantitative RT-PCR performed on total RNA obtained from each gastric cancer cell line. TCONS — 00006264 corresponding to TM4SF1AS1 was highly expressed in gastric cancer cells (MKN45, HSC45, JRST) compared to normal gastric mucosal cells. The upper right diagram of FIG. 2 shows the positional relationship on the genome between TM4SF1AS1 and its mRNA, TM4SF1. The lower right diagram of FIG. 2 shows the results of expression analysis of TM4SF1 and TM4SF1AS1 in each gastric cancer cell line. TM4SF1AS1 (right side) was expressed more than TM4SF1 (left side) except for a few gastric cancer cell lines. The vertical axis indicates the relative expression of gastric cancer cells compared to normal stomach, and the horizontal axis indicates the names of gastric cancer cells. FIG. 2B shows the results of quantitative RT-PCR performed on total RNA obtained from cancer tissues (clinical specimens) of each gastric cancer stage. The left figure in FIG. 2B shows the expression of TM4SF1AS1 in cancer tissues (clinical specimens) of each gastric cancer stage and normal gastric mucosa of healthy subjects. The expression of TM4SF1AS1 is shown in the section. FIG. 3 shows the effect of TM4SF1AS1 (lncRNA) knockdown (KD) on cell proliferation using MTT assay. The left figure shows gastric cancer cell line HSC45, the middle figure shows MKN45, and the right figure shows SNU1. In each figure, the vertical axis indicates the cell viability (OD450), and the horizontal axis indicates the number of days after knockdown.

FIG. 4 shows the results of cell migration assay (left panel) and matrigel invasion assay (right panel) for cells knocked down TM4SF1AS1 by transfection. In the left figure, the vertical axis indicates migratory cells (%), and the horizontal axis indicates the type of RNA used for knockdown. The vertical axis in the right figure indicates invading cells (%), and the horizontal axis indicates the type of RNA used for knockdown. Figure 5 shows evaluation of tumorigenicity by TM4SF1AS1 overexpression in mouse xenografts. Upper left panel shows a tumor removed 31 days after implantation of SNU638 stable cell line (4×10 ⁶ cells) (photograph). The vertical axis in the upper right figure indicates the tumor volume (mm ³ ) of the TM4SF1AS1 overexpressing strain and the control strain (GFP), and the horizontal axis indicates the number of days after cell line transplantation. The lower left panel shows a tumor removed 30 days after implantation of the SNU638 stable cell line (1×10 ⁷ cells) (photograph). The vertical axis of the lower right graph indicates the tumor volume (mm ³ ) of the TM4SF1AS1 overexpressing strain and the control strain (GFP), and the horizontal axis indicates the number of days after cell line transplantation. Figure 6 shows the tumor suppressive effect of TM4SF1AS1 knockdown (KD) in mouse xenografts. The left figure shows a tumor removed 30 days after transplantation of the HSC45 cell line (2.7×10 ⁶ cells) (photograph). The vertical axis in the right figure indicates the tumor volume (mm ³ ) of the TM4SF1AS1 knockdown cell line and the non-knockdown cell line, and the horizontal axis indicates the number of days after transplantation of the cell line. Figure 7 shows Western blotting against PURA or YB1. TM4SF1AS1, PURA and YB1 formed a complex to increase band intensity (left panel), and the band disappeared in the absence of TM4SF1AS1 (right panel). FIG. 8 shows the effect of PURA or YB1 knockdown on cell proliferation. The vertical axis in the left figure indicates cell viability compared to normal cells, and the horizontal axis indicates the number of days after knockdown.

FIG. 9 shows the results of microarray experiments after synthesizing cRNA of RNA extracted from TM4SF1AS1-knockdown gastric cancer cell line HSC45. FIG. 10 shows that knockdown of TM4SF1AS1 suppresses the expression of immune response genes. The vertical axis indicates the relative expression of gastric cancer cells (TM4SF1AS1 siRNA administration) compared to gastric cancer cells (control siRNA administration), and the horizontal axis indicates the names of immune response genes. FIG. 11 shows that overexpression of TM4SF1AS1 induces the expression of immune response genes. The vertical axis indicates the relative expression of gastric cancer cells (TM4SF1AS1 overexpression) compared to gastric cancer cells (GFP expression), and the horizontal axis indicates the names of immune response genes. FIG. 12 shows that knockdown of PURA suppresses the expression of immune response genes in TM4SF1AS1 overexpressing cell lines. The vertical axis indicates the relative expression of gastric cancer cells (PURA siRNA administration) compared to gastric cancer cells (control siRNA administration), and the horizontal axis indicates the names of immune response genes.

FIG. 13 shows the results of expression analysis of TM4SF1AS1 in each cancer cell line. TM4SF1AS1 was highly expressed in various breast cancer cells (left panel), various liver cancer cells (middle panel), and various colon cancer cells (right panel). The vertical axis indicates the relative expression of TM4SF1AS1 compared to actin, and the horizontal axis indicates the names of various cancer cells. The left panel of FIG. 14 shows that knockdown of TM4SF1AS1 suppresses the expression of TM4SF1AS1 in breast cancer cell lines two days later. The vertical axis indicates the relative expression of breast cancer cells (TM4SF1AS1 siRNA administered) compared to breast cancer cells (control siRNA administered), and the horizontal axis indicates the name of the breast cancer cell line. The right panel of FIG. 14 shows the effect of knockdown (KD) of TM4SF1AS1 (lncRNA) on cell proliferation using MTT assay. The upper right figure shows the breast cancer SK-BR-3 cell line, and the lower right figure shows the MDA-MB-435S cell line. In each figure, the vertical axis indicates the cell viability (OD450), and the horizontal axis indicates the number of days after knockdown.

The left panel of FIG. 15 shows that knockdown of TM4SF1AS1 suppresses the expression of TM4SF1AS1 in hepatoma cells two days later. The vertical axis indicates relative expression in liver cancer cells (TM4SF1AS1 siRNA administration) compared to liver cancer cells (control siRNA administration), and the horizontal axis indicates the name of the liver cancer cell line. The right panel of FIG. 15 shows the effect of TM4SF1AS1 (lncRNA) knockdown (KD) on cell proliferation using MTT assay. The upper right figure shows the liver cancer Huh7 cell line, and the lower right figure shows the HT17 cell line. In each figure, the vertical axis indicates the cell viability (OD450), and the horizontal axis indicates the number of days after knockdown. The left panel of FIG. 16 shows that knockdown of TM4SF1AS1 suppresses the expression of TM4SF1AS1 in hepatoma cells two days later. The vertical axis indicates relative expression in liver cancer cells (TM4SF1AS1 siRNA administration) compared to liver cancer cells (control siRNA administration), and the horizontal axis indicates the name of the liver cancer cell line. The upper right panel of FIG. 16 shows the effect of TM4SF1AS1 (lncRNA) knockdown (KD) on cell proliferation using the MTT assay. The upper right figure used the liver cancer HLF cell line. The vertical axis indicates cell viability (OD450), and the horizontal axis indicates days after knockdown. The lower right panel of FIG. 16 shows the results of a cell migration assay (left panel) and Matrigel invasion assay (right panel) for cells in which TM4SF1AS1 was knocked down by transfection. In the left figure, the vertical axis indicates migratory cells (%), and the horizontal axis indicates the type of RNA used for knockdown. The vertical axis in the right figure indicates invading cells (%), and the horizontal axis indicates the type of RNA used for knockdown. The left panel of FIG. 17 shows that knockdown of TM4SF1AS1 suppresses the expression of TM4SF1AS1 in pancreatic cancer cells two days later. The vertical axis indicates relative expression in pancreatic cancer cells (TM4SF1AS1 siRNA administration) compared to pancreatic cancer cells (control siRNA administration), and the horizontal axis indicates the names of pancreatic cancer cell lines. The right panel of FIG. 17 shows the effect of knockdown (KD) of TM4SF1AS1 (lncRNA) on cell proliferation using MTT assay. The vertical axis indicates cell viability (OD450), and the horizontal axis indicates days after knockdown.

As used herein, therapeutic agents for suppressing at least one of expression of TM4SF1AS1, expression of PURA, and expression of YB1, e.g., therapeutic agents, compositions, methods and kits for suppressing expression of TM4SF1AS1, and A cancer detection method comprising measuring the expression level of TM4SF1AS1 transcript RNA is provided.
The therapeutic agents, compositions, methods and kits may include nucleic acid molecules (e.g., short interfering nucleic acids (siNA), short interfering RNA) that bind TM4SF1AS1 transcript RNA, e.g., TM4SF1AS1 transcript RNA exemplified by SEQ ID NO:1. (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA) or short hairpin RNA (shRNA)).
Also provided herein are therapeutic agents, compositions, methods and kits for treating and/or preventing diseases, conditions or disorders associated with high expression of TM4SF1AS1, such as cell proliferative diseases, tumors, cancers, etc. be done.
Further provided herein are methods for diagnosing diseases, conditions or disorders associated with high expression of TM4SF1AS1, such as cell proliferative diseases, tumors, cancers, and the like.

1. Cancer therapeutic agent of the present invention One aspect of the present invention is a cancer therapeutic agent containing at least one of a TM4SF1AS1 expression inhibitor, a PURA expression inhibitor, and a YB1 expression inhibitor, such as TM4SF1AS1. The present invention relates to a cancer therapeutic agent containing an expression inhibitor.
The cancer therapeutic agent of the present invention includes, for example, an inhibitor of TM4SF1AS1 expression. Annotation data for the long non-coding RNA TM4SF1AS1 can be obtained from the Human Body Map (Broad Institute). The transcript RNA of TM4SF1AS1 is represented herein by SEQ ID NO:1.

In the present invention, the transcript RNA of TM4SF1AS1 is, for example, human-derived, and includes the nucleotide sequence shown in SEQ ID NO: 1, or deletion, substitution, addition, or insertion of one or several nucleotides in each nucleotide sequence. A sequence, or a base sequence having a sequence identity of 70% or more, 80% or more, or 90% or more, preferably 95% or more, more preferably 98% or more or 99% or more with each of the above base sequences good.
As used herein, "several" means 2 to 10 bases, preferably 2 to 5 bases, more preferably 2 to 3 bases. Sequence identity can also be determined using known algorithms such as BLAST.

In the present invention, the inhibitor of TM4SF1AS1 expression is, for example, an siRNA (for example, 5'-CCAUCAGUUGGGAGUUGAAGA-3' (SEQ ID NO: 2) or 5'-GCCAAGUGUCCGAGAUGCAAC-3' (SEQ ID NO: 2) having an RNA interference (RNAi) effect. 3)) or a precursor RNA thereof, modified RNA thereof, or a vector containing a DNA encoding an siRNA directed against the transcript RNA of the TM4SF1AS1 gene. In the present invention, the inhibitor of TM4SF1AS1 expression may be, for example, shRNA, miRNA, or the like. The vector may contain antisense RNA or antisense DNA, DNA encoding said antisense RNA or said antisense DNA.

The siRNA is a sense RNA consisting of 18-25 nucleotides, preferably 20-24 nucleotides, more preferably 21-23 nucleotides complementary to a part of the transcript RNA of the TM4SF1AS1 gene, and having an RNAi (RNA interference) effect. and antisense RNA. Each 3' end of sense RNA and antisense RNA may have a protruding end of 2 to 5 nucleotides, preferably 2 nucleotides.
In the present invention, the inhibitor of TM4SF1AS1 gene expression may be, for example, a combination of siRNA containing a sense strand and an antisense strand. ) and antisense strand 5′-UUCAACUCCCAACUGAUGGAA-3′ (SEQ ID NO: 4), or sense strand 5′-GCCAAGUGUCCGAGAUGCAAC-3′ (SEQ ID NO: 3) and antisense strand 5′-UGCAUCUCCGGACACUUGGCAG-3′ (SEQ ID NO: 5) combinations are preferred.

In one aspect of the present invention, the therapeutic agent for cancer of the present invention comprises an inhibitor of PURA (purine-rich element binding protein A) expression and YB1 (Y box binding protein) without containing an inhibitor of TM4SF1AS1 expression. may contain both inhibitors of the expression of, or may contain either one of them alone.
In another aspect of the present invention, the therapeutic agent for cancer of the present invention may contain a PURA expression inhibitor and a YB1 expression inhibitor in addition to the TM4SF1AS1 expression inhibitor.
A complex of PURA, PURB and YB1 has been reported to be involved in transcriptional regulation (J Biol Chem, 2008; Mol Cell Biol, 2007). The inhibitor of PURA expression and the inhibitor of YB1 expression are each, for example, an siRNA (for example, 5′-CCACCUAUCGCAACUCCAUTT-3′ (SEQ ID NO: 6) or 5′ of siRNA against PURA) having RNA interference (RNAi) action '-GCUACUGCAGGGUGAGGAATT-3' (SEQ ID NO: 7), 5'-CCUAUGGGCGUCGACCACATT-3' (SEQ ID NO: 10) or 5'-GUUCCAGUUCAAGGCAGUATT-3' (SEQ ID NO: 11) of siRNA against YB1, or precursor RNA thereof, or or vectors containing DNA encoding siRNA directed against transcript RNAs of the PURA and YB1 genes. In the present invention, the inhibitor of PURA or YB1 expression may be, for example, shRNA. The vector may contain antisense RNA or antisense DNA, DNA encoding said antisense RNA or said antisense DNA.

The siRNA against PURA and YB1 consists of 18-25 nucleotides, preferably 20-24 nucleotides, more preferably 21-23 nucleotides complementary to a portion of the transcript RNA of the PURA and YB1 genes, and RNAi (RNA interference) A double-stranded RNA consisting of a sense RNA and an antisense RNA having an action may also be used. Each 3' end of sense RNA and antisense RNA may have a protruding end of 2 to 5 nucleotides, preferably 2 nucleotides.
In the present invention, the inhibitor of PURA and YB1 gene expression may be, for example, a combination of siRNA containing a sense strand and an antisense strand. A combination of 3′ (SEQ ID NO: 6) and 5′-AUGGAGUUGCGAUAGGUGGTT-3′ (SEQ ID NO: 8) on the antisense strand, or 5′-GCUACUGCAGGGUGAGGAATT-3′ (SEQ ID NO: 7) on the sense strand and 5 on the antisense strand A combination of '-UUCCUCACCCUGCAGUAGCTT-3' (SEQ ID NO: 9), or 5'-CCUAUGGGCGUCGACCACATT-3' (SEQ ID NO: 10) of the sense strand of the siRNA against YB1 and 5'-UGUGGUCGACGCCCAUAGGTT-3' of the antisense strand (SEQ ID NO: 9) 12), or a combination of 5'-GUUCCAGUUCAAGGCAGUATT-3' (SEQ ID NO: 11) on the sense strand and 5'-UACUGCCUUGAACUGGAACTT-3' (SEQ ID NO: 13) on the antisense strand.

The therapeutic agent of the present invention can be introduced into cells by any known introduction technique, for example, without limitation, lipofectamine method, lipofection method, calcium phosphate method, ultrasonic transduction method, electroporation method, particle gun method, viral Methods using vectors (eg, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, etc.) or microinjection methods can be used.
When using a viral vector, the virus titer is 1×10 ³ to 1×10 ¹⁵ p.o. f. u. (plaque-forming units), preferably 1×10 ⁵ to 1×10 ¹³ , more preferably 1×10 ⁷ to 1×10 ¹¹ , still more preferably 1×10 ⁸ to 1×10 ¹⁰ be able to.

The nucleic acid molecules can be used as naked nucleic acids or incorporated into various nucleic acid constructs or vectors. As a vector, any known vectors such as plasmid vectors, phage vectors, phagemid vectors, cosmid vectors and virus vectors can be used. Nucleic acid constructs or vectors preferably include at least suitable transcriptional or translational control sequences, eg, derived from mammalian, microbial, viral, or insect genes. Such regulatory sequences include sequences that have a regulatory role in gene expression, such as transcriptional promoters or enhancers, operator sequences for regulating transcription, sequences encoding ribosome binding sites within messenger RNAs, and transcription, translation initiation. or include appropriate sequences that regulate transcription termination.

2. Pharmaceutical Compositions of the Invention The present invention also relates to pharmaceutical compositions comprising the therapeutic agents described above. The therapeutic agents described above are useful as active ingredients of pharmaceutical compositions because they can suppress the growth of cancer cells. The pharmaceutical composition of the present invention may contain a PURA expression inhibitor and a YB1 expression inhibitor in addition to the TM4SF1AS1 expression inhibitor, or may contain no TM4SF1AS1 expression inhibitor, Both the inhibitor of PURA expression and the inhibitor of YB1 expression may be contained, or either one of them may be contained alone. Pharmaceutical compositions of the present invention may comprise a therapeutic agent as described above and one or more pharmaceutically acceptable surfactants, carriers, diluents and/or excipients. Pharmaceutically acceptable carriers, diluents and the like are well known in the pharmaceutical art, see, for example, Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, PA (1990), which is incorporated herein in its entirety. ), etc.

The pharmaceutical composition can be used, without limitation, to treat diseases associated with high expression of TM4SF1AS1, PURA, and YB1, cell proliferative diseases, and the like. Accordingly, the present invention also relates to a pharmaceutical composition comprising an effective amount of an inhibitor of the transcript RNA of TM4SF1AS1, PURA and YB1 described above for the treatment of diseases associated with high expression of TM4SF1AS1, PURA and YB1.

Cell proliferative disorders include, but are not limited to, benign or malignant tumors, and the like. In one aspect of the invention, the cell proliferative disorder is cancer. In one aspect of the present invention, the pharmaceutical composition of the present invention can suppress the cell proliferation ability of cancer cells to, for example, 1/2 or less, or 1/3 or less of that of normal cells in the MTT assay. .

Cancers targeted by the present invention are not limited as long as transcript RNAs of TM4SF1AS1, PURA, and YB1 are expressed. Examples include fibrosarcoma, malignant fibrous histiocytoma, liposarcoma, and rhabdomyosarcoma. , leiomyosarcoma, angiosarcoma, Kaposi's sarcoma, lymphangiosarcoma, synovial sarcoma, chondrosarcoma, osteosarcoma, brain tumor, head and neck cancer, breast cancer, lung cancer, esophageal cancer, gastric cancer, duodenal cancer, appendix cancer, colon Cancer, Rectal cancer, Liver cancer, Pancreatic cancer, Gallbladder cancer, Bile duct cancer, Anal cancer, Kidney cancer, Ureter cancer, Bladder cancer, Prostate cancer, Penis cancer, Testicular cancer, Uterine cancer, Ovarian cancer, Vulvar cancer, Vaginal cancer, Skin Examples include carcinoma such as cancer, leukemia, and malignant lymphoma.

Whether cancer cells express TM4SF1AS1, PURA, and YB1 can be determined by detecting the expression of TM4SF1AS1, PURA, and YB1 transcript RNAs at the gene level. Specifically, at the gene level, for example, any known gene expression analysis method such as Northern blotting, RNase protection assay, RT-PCR, PCR methods such as real-time PCR, in situ hybridization method, in vitro transcription method, etc. can be detected by In one aspect of the present invention, the expression levels of TM4SF1AS1, PURA, and YB1 transcript RNAs are 2-fold or more, preferably 3-fold or more, more preferably 5-fold or more, and still more preferably 10-fold or more, compared to normal cells. Particularly preferably, the increase is 20 times or more.

The pharmaceutical compositions of the invention may further comprise other agents useful in treating the disease of interest, such as anti-tumor agents, anti-inflammatory agents, and the like.
Antitumor agents include, but are not limited to, ifosfamide, nimustine (e.g., nimustine hydrochloride), cyclophosphamide, dacarbazine, melphalan, alkylating agents such as ranimustine, gemcitabine (e.g., gemcitabine hydrochloride), enocitabine, Cytarabine/Oxphosphate, Cytarabine preparations, Tegafur/Uracil, Tegafur/Gimeracil/Oteracil potassium combination (e.g., TS-1), antimetabolites such as doxifluridine, hydroxycarbamide, fluorouracil, methotrexate, mercaptopurine, idarubicin (e.g., hydrochloric acid) idarubicin), epirubicin (e.g. epirubicin hydrochloride), daunorubicin (e.g. daunorubicin hydrochloride, daunorubicin citrate), doxorubicin (e.g. doxorubicin hydrochloride), pirarubicin (e.g. pirarubicin hydrochloride), bleomycin (e.g. bleomycin hydrochloride), peplomycin (e.g. , peplomycin sulfate), mitoxantrone (e.g. mitoxantrone hydrochloride), antineoplastic antibiotics such as mitomycin C, etoposide, irinotecan (e.g. irinotecan hydrochloride), vinorelbine (e.g. vinorelbine tartrate), docetaxel (e.g. docetaxel hydrate), alkaloids such as paclitaxel, vincristine (e.g. vincristine sulfate), vindesine (e.g. vindesine sulfate), vinblastine (e.g. vinblastine sulfate), anastrozole, tamoxifen (e.g. tamoxifen citrate), toremifene (e.g. , toremifene citrate), hormone therapy agents such as bicalutamide, flutamide, estramustine (e.g., estramustine phosphate), platinum complexes such as carboplatin, cisplatin (CDDP), nedaplatin, blood vessels such as thalidomide, neovastat, bevacizumab, etc. Neogenesis inhibitors, L-asparaginase and the like can be mentioned.

Anti-inflammatory agents include, but are not limited to, steroidal anti-inflammatory agents (prednisolone, beclomethasone, betamethasone, fluticasone, dexamethasone, hydrocortisone, etc.) and non-steroidal anti-inflammatory agents (acetylsalicylic acid, loxoprofen, acetaminophen, ketoprofen, tiaprofen, etc.). acid, suprofen, tolmetin, carprofen, benoxaprofen, piroxicam, benzydamine, naproxen, diclofenac, ibuprofen, diflunisal, azapropazone, etc.), RNAi molecules that suppress the expression of inflammatory cytokines (e.g., siRNA, shRNA, ddRNA, miRNA, piRNA, rasiRNA, etc.), substances such as antisense nucleic acids, and/or drugs that suppress the action of inflammatory cytokines, such as antibodies to inflammatory cytokines and inflammatory cytokine receptor antagonists.

When two or more TM4SF1AS1, PURA, and YB1 expression inhibitors are used in combination in the cancer therapeutic agent, pharmaceutical composition, etc. of the present invention, two or more molecules may be contained in a single composition. , may be included separately in multiple compositions and used in combination. For example, in the case of nucleic acid molecules, two or more types of nucleic acid molecules may be incorporated into a single nucleic acid construct or vector, or a combination of multiple types of nucleic acid constructs or vectors each containing one type of nucleic acid molecule may be prepared. .

In various embodiments of the present invention, the above cancer therapeutic agents can be used by being supported on various drug delivery carriers. Examples of such carriers include, but are not limited to, polymeric nanoparticles, polymeric micelles, dendrimers, liposomes, viral nanoparticles, carbon nanotubes and the like (Cho K. et al., Clin Cancer Res. 2008 Mar 1;14 (5):1310-6, etc.).

Therapeutic agents and pharmaceutical compositions of the present invention can be administered by a variety of routes, including both oral and parenteral, including, but not limited to, oral, intravenous, intramuscular, subcutaneous, topical, rectal, intratumoral, intraarterial. , intraportal vein, intramedullary, intramedullary, sublingual, intraoral, intraventricular, transmucosal, transdermal, intranasal, intraperitoneal, intrapulmonary, intrauterine, and the like. may be formulated into dosage forms suitable for Any known dosage form and formulation method can be appropriately employed (see, for example, Standard Pharmacology, Edited by Yoshiteru Watanabe, Nankodo, 2003, Remington's Pharmaceutical Sciences, etc.).

For example, dosage forms suitable for oral administration include, without limitation, powders, granules, tablets, capsules, liquids, suspensions, emulsions, gels, syrups and the like; Suitable dosage forms include injections such as solution injections, suspension injections, emulsion injections, and injections prepared just before use. Formulations for parenteral administration can be in the form of aqueous or non-aqueous isotonic sterile solutions or suspensions.

3. Kits of the Invention The invention provides kits for the preparation of compositions comprising one or more containers containing, singly or in combination, the active ingredients that can be included in various agents or compositions of the invention, as well as kits for preparing such compositions. It also relates to necessary components of various agents or compositions that are provided in a convenient kit form. In addition to the above, the kit of the present invention includes instructions describing the preparation method and administration method of the therapeutic agent or pharmaceutical composition of the present invention, such as instructions, electronic recording media such as CDs and DVDs, and the like. good too.

4. Cancer Diagnosis Method of the Present Invention One aspect of the present invention relates to a method of diagnosing cancer that targets TM4SF1AS1 transcript RNA.

One aspect of the present invention is a method for measuring the expression level of transcript RNA of TM4SF1AS1 as an index for predicting whether a human subject will develop cancer, comprising:
(a) determining the expression level of TM4SF1AS1 transcript RNA in a test sample from a human subject;
(b) determining the expression level of TM4SF1AS1 transcript RNA in a control sample from a healthy human subject; and (c) determining the expression level of TM4SF1AS1 transcript RNA in a test sample from a human subject. predicting that a human subject will develop cancer if the amount of transcript RNA of TM4SF1AS1 expressed in a control sample from which it is derived is greater than two-fold;
including.

In another aspect of the present invention, in (c), the expression level of TM4SF1AS1 transcript RNA in a test sample derived from a human subject is higher than the expression level of TM4SF1AS1 transcript RNA in a control sample derived from a healthy human subject. is predicted to develop cancer in a human subject if, for example, more than 3-fold, 5-fold, 10-fold, or 20-fold.

In the diagnostic method of the present invention, the cancer is not limited as long as it expresses transcript RNA of TM4SF1AS1, but is preferably gastric cancer, breast cancer, liver cancer, colon cancer, or pancreatic cancer. , the human subject is a precancerous human subject.

5. The cancer treatment method of the present invention A further aspect of the present invention is a therapeutic agent for cancer comprising at least one of the TM4SF1AS1 expression inhibitor, the PURA expression inhibitor, and the YB1 expression inhibitor of the present invention. , relates to a method of treating diseases using a cancer therapeutic agent or pharmaceutical composition comprising an inhibitor of TM4SF1AS1 expression. In the treatment method of the present invention, a therapeutic agent for cancer or a pharmaceutical composition containing at least one of the TM4SF1AS1 expression inhibitor, the PURA expression inhibitor, and the YB1 expression inhibitor of the present invention is administered to a subject. Including process. The subject to be administered is typically in need of treatment with the TM4SF1AS1 expression inhibitor, the PURA expression inhibitor, the YB1 expression inhibitor, and the like of the present invention. Such subjects include, but are not limited to, diseases associated with high expression of TM4SF1AS1, PURA, and YB1, cell proliferative diseases, and the like, or have been diagnosed with these diseases. is a subject at high risk of developing the disease of Therefore, the treatment method of the present invention requires treatment with a therapeutic agent or pharmaceutical composition comprising at least one of the TM4SF1AS1 expression inhibitor, the PURA expression inhibitor, and the YB1 expression inhibitor of the present invention. The step of identifying the subject in question may be further included. Specific examples of cell proliferative disorders are as described above for pharmaceutical compositions.

In the treatment methods of the invention, the therapeutic agents or pharmaceutical compositions of the invention can be used in combination with other agents or treatment methods useful for treating the disease of interest. The therapeutic agent or pharmaceutical composition of the present invention is expected to improve cancer sensitivity to chemotherapy and radiotherapy. Agents, such as anti-tumor agents, anti-inflammatory agents, etc., that may be used in combination with the therapeutic agents or pharmaceutical compositions of the present invention in the treatment of disease are as described above for pharmaceutical compositions. Moreover, the treatment method of the present invention can be used in combination with physical therapy such as radiation therapy, surgical treatment such as surgery, and the like.

An effective amount in the treatment method of the present invention is, for example, an amount that reduces symptoms of a disease or delays or halts progression of the disease, preferably an amount that suppresses or cures the disease. Also, an amount that does not cause adverse effects that exceed the benefits of administration is preferred. Such amount can be appropriately determined by in vitro tests using cultured cells or the like, or tests in model animals such as mice, rats, dogs or pigs, and such test methods are well known to those skilled in the art. . In addition, the dose of the drug used in the treatment method of the present invention is known to those skilled in the art, or can be determined as appropriate by the above tests and the like.

Specific doses of the active ingredient to be administered in the treatment methods of the invention described herein will vary depending on the various conditions associated with the subject requiring treatment, such as severity of symptoms, general health of the subject, age, weight, etc. , the sex of the subject, diet, timing and frequency of administration, concomitant drugs, responsiveness to treatment, dosage form, compliance with treatment, and the like. The dose of the drug used in the treatment method of the present invention is preferably about 0.01 mg to about 1,000 mg of siRNA molecules per dose per 1 kg body weight of an adult human. The concentration of the drug used in the treatment method of the present invention is preferably 1 nM to 200 μM, more preferably 5 nM to 180 μM, particularly preferably 10 nM to 160 μM, in the case of siRNA.
The routes of administration include various routes including both oral and parenteral, for example, oral, intravenous, intramuscular, subcutaneous, topical, intratumoral, rectal, intraarterial, intraportal, intramedullary, intradental, Included are routes such as sublingual, buccal, intraventricular, transmucosal, transdermal, intranasal, intraperitoneal, intrapulmonary and intrauterine.
The administration frequency varies depending on the properties of the agent or composition to be used and the conditions of the subject including those mentioned above. It may be once, every few days (ie, every 2, 3, 4, 5, 6, 7 days, etc.), every week, every few weeks (ie, every 2, 3, 4 weeks, etc.).

As used herein, the term "subject" means any living individual, preferably an animal, more preferably a mammal, and more preferably a human individual. In the present invention, the subject may be healthy or suffering from any disease, although when treatment of a particular disease is contemplated, typically the subject is suffering from such disease. , means a subject at risk of being affected.
Also, the term "treatment" as used herein encompasses all types of medically acceptable prophylactic and/or therapeutic interventions aimed at curing, temporary remission or prevention of disease, etc. shall be For example, the term "treatment" encompasses medically acceptable interventions for various purposes, including slowing or arresting disease progression, regression or disappearance of lesions, preventing onset or recurrence, and the like.

This time, the present inventors have found that the inhibitor of the present invention can suppress cell proliferation in gastric cancer, breast cancer, liver cancer, colon cancer, and pancreatic cancer cell lines by suppressing the expression of TM4SF1AS1 (SEQ ID NO: 1). Then, the binding of PURA, which is reported to be a DNA- or RNA-binding protein, to TM4SF1AS1 (SEQ ID NO: 1) is inhibited, resulting in decreased expression of gene clusters involved in immune response. It was revealed.
Therefore, the inhibitor of the present invention can induce or maintain an immune response in addition to inhibiting cell proliferation. Accordingly, the present invention also provides a composition for providing said action comprising said inhibitor, the use of said inhibitor in the manufacture of a composition for providing said action, said inhibitor for providing said action. composition comprising an effective amount of said inhibitor to provide said effect, as well as the use of said inhibitor to provide said effect.

The present invention will be described more specifically by the following examples, but the technical scope of the present invention shall not be limited to these examples.

Example 1: Acquisition of lncRNA from gastric cancer cell lines and clinical samples SNU1, SNU638), liver cancer cell lines (Hep3B, HepG2, Huh1, Huh7, HT17, Li-7, HLE, HLF, PLC/PRF/5, JHH4), colon cancer cell lines (CaCO2, Colo320, DLD1, HCT116 , HT29, Lovo, RKO, SW48, SW480, SW620, T84, WiDr), breast cancer cell lines (MCF7, MDA-MB-157, MDA-MB-231, MDA-MB361, MDA-MB-435S, MDA-MB- 468, SK-BR-3, T47D) and a pancreatic cancer cell line (Panc-1) were cultured and used for analysis. Regarding clinical specimens, ducts were isolated by the duct isolation method from a part of the tissue obtained by biopsy by upper gastrointestinal endoscopy (Nakamura S, Goto J, Kitayama M, Kino I (1994) Application of the crypt-isolation technique to flow-cytometric analysis of DNA content in colorectal neoplasms. Gastroenterology 106 (1): 100-107). Analysis of clinical specimens was approved by Sapporo Medical University.

Example 2: ChIP-seq experiment and data analysis A ChIP (chromatin precipitation) experiment was performed using ductal components obtained by the duct isolation method from the normal gastric mucosa of healthy subjects and the non-cancerous background mucosa of gastric cancer patients ( Maruyama R, Choudhury S, Kowalczyk A, Bessarabova M, Beresford-Smith B, Conway T, Kaspi A, Wu Z, Nikolskaya T, Merino VF, Lo PK, Liu XS, Nikolsky Y, Sukumar S, Haviv I, Polyak K ( 2011) Epigenetic regulation of cell type-specific expression patterns in the human mammary epithelium. PLoS genetics 7 (4):e1001369. doi:10.1371/journal.pgen.1001369). An antibody directed against the histone modification H3K4me3, an activation marker for transcription initiation sites, was used. An adapter sequence was added to the DNA obtained by the ChIP experiment, amplified by PCR, and then analyzed using a next-generation sequencer SOLiD4 (Life Technologies). Analysis software MACS 1.4 was used, and lncRNA annotation data were obtained from the Human Body Map (Broad Institute). The results are shown in FIG.
As a result of comparing healthy individuals and gastric cancer (GC) patients, 1512 annotations were upregulated, and TM4SF1AS1 (SEQ ID NO: 1) was found as an example of the active region of the transcription start site by H3K4me3.

Example 3: RNA extraction and expression analysis Total RNA was extracted from each gastric cancer cell line using Trizol and subjected to reverse transcription reaction. Quantitative RT-PCR was performed by the SYBR method using primers designed for each gene. A commercially available RNA of normal gastric mucosa was used. The results are shown in Figure 2A.
It was found that TCONS — 00006264 corresponding to TM4SF1AS1 was more expressed in gastric cancer cells than in normal gastric mucosa cells (left panel in FIG. 2A). In addition, TM4SF1AS1, a long non-coding RNA, and its mRNA, TM4SF1, were predicted to be located proximally on the genome and co-expressed (upper right panel of FIG. 2A).
In addition, expression analysis of TM4SF1 and TM4SF1AS1 in each gastric cancer cell line revealed that TM4SF1AS1 and TM4SF1 were both highly expressed in many cell lines except for a very small number of gastric cancer cell lines (lower right figure in FIG. 2A).
Furthermore, total RNA was extracted from cancer tissues (clinical specimens) of each gastric cancer stage using Trizol and subjected to reverse transcription reaction. Quantitative RT-PCR was performed by the SYBR method using primers designed for each gene. A commercially available RNA of normal gastric mucosa was used. The results are shown in Figure 2B. Comparison with normal gastric mucosa of healthy subjects or non-cancerous sites of gastric cancer patients suggested that the expression of TM4SF1AS1 is enhanced in cancerous sites. In gastric cancer, the expression of TM4SF1AS1 is enhanced in each stage (I-IV) compared to normal tissues, so it is suggested that it is effective for early detection of gastric cancer, prevention of gastric cancer, and early treatment. rice field.

Example 4: Design of siRNAs targeting TM4SF1AS1 Design algorithms of Rossetta Inpharmatics and siDirect were used to design siRNAs targeting TM4SF1AS1. This results in a sense strand (e.g., 5'-CCAUCAGUUGGGAGUUGAAGA-3' (SEQ ID NO: 2), 5'-GCCAAGUGUCCGAGAUGCAAC-3' (SEQ ID NO: 3)) and an antisense strand (e.g., 5'-UUCAACUCCCAACUGAUGGAA-3' (sequence No. 4) and 5'-UGCAUCUCCGGACACUUGGCAG-3' (SEQ ID NO: 5)) were used.
Mission Negative control SIC-001 manufactured by Sigma was used as control siRNA (sequence not disclosed).

Example 5: Knockdown experiment and functional analysis Gastric cancer cell lines were transfected with siRNA designed to target TM4SF1AS1 by the Lipofectamine method. Quantitative RT-PCR, MTT assay, cell migration assay, and matrigel invasion assay were performed on cells in which TM4SF1AS1 was knocked down by transfection. The results are shown in FIGS.
Knockdown of TM4SF1AS1 in gastric cancer cells (HSC45, MKN45, SNU1) using siRNAs (si-TM4SF1AS1#2 (SEQ ID NOS: 2, 4) and si-TM4SF1AS1#3 (SEQ ID NOS: 3, 5)) (20 nM) By doing so, the cell proliferation ability was significantly reduced (Fig. 3). In addition, in the HSC45 gastric cancer cell line, cell migration ability and invasion ability were significantly reduced (Fig. 4), suggesting that TM4SF1AS1 functions as an oncogene.

Example 6: Establishment of TM4SF1AS1 stable expression strain A TM4SF1AS1-GFP expression vector or control GFP vector was constructed. For TM4SF1AS1, synthetic DNA was purchased from DNA synthesis service of SGI-DNA, and for plasmid vector, pBI-CMV2 (Clontech) was purchased. pBI-CMV2 NeoR, in which a PCR-amplified neomycin resistance gene (NeoR) cassette was incorporated into pBI-CMV2 by In-fusion reaction (Takara), was used as a control GFP vector. Further, TM4SF1AS1 amplified by PCR based on the synthetic DNA was incorporated into the MCS site of pBI-CMV2 NeoR by in-fusion reaction to construct a TM4SF1AS1-GFP expression vector.
Gastric cancer cell line SNU638 was transfected with TM4SF1AS1-GFP expression vector or control GFP vector. After selection for 3 weeks with the addition of antibiotic neomycin, GFP-positive cells were sorted using BD FACS Aria (BD Bioscience).

Example 7: Mouse Xenograft Experiment Gastric cancer cell line SNU638 transfected with TM4SF1AS1-GFP expression vector or control GFP vector was subcutaneously implanted into the left and right dorsal regions of nude mice (BALB/cAJcl-nu). Measurements of tumor size were performed periodically after implantation. The results are shown in FIG.
In addition, gastric cancer cell line HSC45 was subcutaneously implanted into the left and right dorsal regions of nude mice (BALB/cAJcl-nu). After the transplantation, the tumor size was periodically measured, and at the same time, TM4SF1AS1 (SEQ ID NOS: 2, 4) or control siRNA (50 µM) was injected into the tumor using AteloGene in vivo Transfection kit (KOKEN), and mouse subcutaneous tumor was grown. was knocked down. The TM4SF1AS1 stably expressing strain and the control GFP strain were similarly subcutaneously implanted into nude mice, and the tumor size was measured. The results are shown in FIG.
In the SNU638 strain, which is a TM4SF1AS1 stable expression strain, the tumor size increased significantly (Fig. 5), indicating that overexpression of TM4SF1AS1 promotes tumorigenesis. It was also found that knockdown of TM4SF1AS1 in the HSC45 gastric cancer cell line suppresses tumorigenesis (Fig. 6).

Example 8: Microarray analysis RNA was extracted from gastric cancer cell line HSC45 in which TM4SF1AS1 was knocked down, cRNA was synthesized, and microarray experiments were performed on SurePrint G3 Human GE 8x60K V2 microarray (Agilent Technologies). For data analysis, GeneSpring GX version 13 (Agilent Technologies) was used.

Example 9: Identification of TM4SF1AS1 binding proteins
RNA pull-down experiments were performed using 5-bromo UTP-labeled TM4SF1AS1 RNA synthesized by in vitro transcription reaction and RiboCluster Profiler RiboTrap Kit (MBL). A protein band specific to TM4SF1AS1 RNA was cut out by SDS-PAGE and subjected to mass spectrometry.

Example 10: Western Blot After subjecting the proteins to SDS-PAGE and transferring the proteins from the gel to a PVDF membrane, the membrane was treated with a blocking agent. A primary antibody reaction was performed with an antibody against each protein, and a secondary antibody reaction was performed after washing the membrane. After washing the membrane again, proteins were developed using Clarity Western ECL substrate (BIORAD), and fluorescence was detected with ImageQuant LAS4000 mini (GE Heathcare). Primary antibodies were anti YB1 antibody (RN015P, MBL), anti Myc tag mAb (M192-3S, MBL), anti PURA antibody (ab79936, abcam), secondary antibody was mouse IgG antibody (CST, 7076) or Anti- Rabbit IgG antibody conjugated with horseradish peroxidase (CST, 7074) was used.
A complex of PURA, PURB and YB1 has been reported to be involved in transcriptional regulation (J Biol Chem, 2008; Mol Cell Biol, 2007).
As a result of Western blotting, TM4SF1AS1, PURA, and YB1 formed a complex to increase band intensity (left panel in FIG. 7), and the band disappeared in the absence of TM4SF1AS1 (right panel in FIG. 7). Therefore, it was presumed that PURA and YB1 interact via TM4SF1AS1 RNA.

Example 11: Effect of Knockdown of PURA and YB1 on Cell Proliferation Ability Using siRNA Design algorithms of Rossetta Inpharmatics and siDirect were used to design siRNAs targeting PURA and YB1. This results in a sense strand (e.g., 5'-CCACCUAUCGCAACUCCAUTT-3' (SEQ ID NO: 6) or 5'-GCUACUGCAGGGUGAGGAATT-3' (SEQ ID NO: 7) of siRNA against PURA, 5'-CCUAUGGGCGUCGACCACATT-3' of siRNA against YB1 (sequence No. 10) or 5'-GUUCCAGUUCAAGGCAGUATT-3' (SEQ ID NO: 11)) and an antisense strand (e.g., 5'-AUGGAGUUGCGAUAGGUGGTT-3' (SEQ ID NO: 8) or 5'-UUCCUCACCCUGCAGUAGCTT-3' (SEQ ID NO: 8) of the siRNA against PURA SEQ ID NO: 9), 5'-UGUGGUCGAGCCCAUAGGTT-3' (SEQ ID NO: 12) or 5'-UACUGCCUUGAACUGGAACTT-3' (SEQ ID NO: 13)) of siRNA against YB1 was used.
Mission Negative control SIC-001 manufactured by Sigma was used as control siRNA (sequence not disclosed).
Gastric cancer cell line HSC45 was transfected with siRNA designed to target PURA or YB1 by the lipofectamine method. MTT assay was performed on PURA- or YB1-knockdown cells by transfection. The results are shown in FIG.
In gastric cancer cells (HSC45), siRNA (siPURA_0411 (SEQ ID NOS: 6, 8), siPURA_0412 (SEQ ID NOS: 7, 9), YBX_7064 (SEQ ID NOS: 10, 12), and YBX_7065 (SEQ ID NOS: 11, 13)) (20 nM) Knockdown of PURA (left panel) or YB1 (right panel) by using PURA (left panel) significantly decreased cell proliferation ability.

Example 12: Search for downstream genes of TM4SF1AS1 RNA was extracted from gastric cancer cell line HSC45 in which TM4SF1AS1 was knocked down, cRNA was synthesized, and microarray experiments were performed on SurePrint G3 Human GE 8x60K V2 microarray (Agilent Technologies) (Fig. 9). . For data analysis, GeneSpring GX version 13 (Agilent Technologies) was used. Among the 649 genes whose expression was suppressed, for example, immune response genes listed in Table 1 below were accumulated (Gene Ontology analysis).

In addition, using siRNA (si-TM4SF1AS1#2 (SEQ ID NOS: 2, 4) and si-TM4SF1AS1#3 (SEQ ID NOS: 3, 5)) (20 nM), TM4SF1AS1 was knocked down to suppress immune response gene expression. expression analysis (RT-PCR) of the gene showed that the expression of the immune response gene was actually suppressed (Fig. 10). In contrast, in the TM4SF1AS1 stable expression cell line, the expression of immune response genes was actually induced (Fig. 11). In addition, when both TM4SF1AS1 and PURA were knocked down using siRNA (si-PURA_#1 (SEQ ID NOS: 6, 8) and si-PURA_#2 (SEQ ID NOS: 7, 9)) (20 nM), Expression of immune response genes was further reduced compared to knockdown of TM4SF1AS1 alone (Fig. 12). Therefore, it was suggested that TM4SF1AS1 and PURA are involved in the expression of immune response genes as a lncRNA-protein complex.

Example 13 Expression of TM4SF1AS1 in Other Cancer Cells When TM4SF1AS1 RNA expression was analyzed in the same manner as in Example 3, TM4SF1AS1 was highly expressed also in breast cancer cells, liver cancer cells, and colon cancer cells. (Fig. 13). Therefore, the expression of TM4SF1AS1 was enhanced also in cancer cell lines of other tissues, suggesting that it functions as an oncogene.
In the same manner as in Example 5, among the above cell lines, breast cancer cell lines (SK-BR-3, MDA-MB-231, MDA-MB-435S), liver cancer cell lines (Huh7, HLF, HT17, Li- 7, PLC/PRF/5), colon cancer cell lines (SW620, HT29, DLD1, T84, WiDr) and pancreatic cancer cell lines (Panc-2) were used for knockout experiments. Using siRNA (siTM4SF1AS102 (SEQ ID NOS: 2, 4)) (20 nM), breast cancer cell lines showed SK-BR-3 and MDA-MB-435S, and liver cancer cell lines Huh7, HLF, and Suppression of cell proliferation was confirmed for HT17 (Figs. 14 to 16), suppression of migration and invasion ability was confirmed for HLF, a liver cancer cell line (Fig. 16), and Panc-1, a pancreatic cancer cell line, was confirmed. , Suppression of cell proliferation was confirmed (FIG. 17).
Therefore, even in cancer cell lines other than gastric cancer, knockdown of TM4SF1AS1 was found to have an effect of suppressing cell growth.

Claims (11)

A cancer therapeutic agent comprising at least one of a TM4SF1AS1 expression inhibitor and a PURA expression inhibitor ,
the inhibitor of TM4SF1AS1 expression is siRNA, antisense nucleic acid, shRNA or miRNA against transcript RNA of TM4SF1AS1;
The above cancer therapeutic agent, wherein the inhibitor of PURA expression is siRNA, antisense nucleic acid, shRNA, or miRNA against transcript RNA of PURA. 2. The therapeutic agent for cancer according to claim 1, comprising an inhibitor of TM4SF1AS1 expression. 3. The therapeutic agent for cancer according to claim 2 , wherein the inhibitor of TM4SF1AS1 expression is siRNA. 2. The therapeutic agent for cancer according to claim 1, comprising a PURA expression inhibitor. 5. The method according to any one of claims 1 to 4, further comprising an inhibitor of YB1 expression, wherein the inhibitor of YB1 expression is siRNA, antisense nucleic acid, shRNA or miRNA against YB1 transcript RNA. cancer drug. The cancer therapeutic agent according to any one of claims 1 to 5, wherein the cancer is gastric cancer, breast cancer, liver cancer, colon cancer, or pancreatic cancer. (1) The cancer therapeutic agent according to any one of claims 1 to 6, (2) a vector expressing siRNA, antisense nucleic acid, shRNA or miRNA against transcript RNA of TM4SF1AS1 , or (3) TM4SF1AS1 A pharmaceutical composition for treating cancer, comprising a cell introduced with a vector that expresses an siRNA, an antisense nucleic acid, an shRNA, or a miRNA against the transcript RNA of . A method for measuring the expression level of transcript RNA of TM4SF1AS1 as an index for predicting whether a human subject will develop cancer, comprising:
(a) determining the expression level of TM4SF1AS1 transcript RNA in a test sample from a human subject;
(b) determining the expression level of TM4SF1AS1 transcript RNA in a control sample from a healthy human subject; and (c) determining the expression level of TM4SF1AS1 transcript RNA in a test sample from a human subject. predicting that a human subject will develop cancer if the amount of transcript RNA of TM4SF1AS1 expressed in a control sample from which it is derived is greater than two-fold;
A method, including In (c), when the expression level of TM4SF1AS1 transcript RNA in a test sample derived from a human subject exceeds 3 times the expression level of TM4SF1AS1 transcript RNA in a control sample derived from a healthy human subject, the human 9. The method of claim 8 , wherein the subject is predicted to develop cancer. 10. The method of claim 8 or 9 , wherein the cancer is gastric cancer, breast cancer, liver cancer, colon cancer, or pancreatic cancer. 11. The method of any one of claims 8-10 , wherein the human subject is precancerous.