JP7522424B2 - Biliary tract cancer or pancreatic cancer treatment - Google Patents

Description

The present disclosure relates to a biliary tract cancer therapeutic agent and a pancreatic cancer therapeutic agent. The contents of all documents described in this specification are incorporated herein by reference.

Bile duct cancer is common in Asia, including Japan, and is the second most difficult to treat after pancreatic cancer, with a poor prognosis (5-year survival rate of less than 20%, International Cancer Genome Consortium, August 3, 2017). Resection, chemotherapy, and radiation therapy can be selected alone or in combination, but since this cancer is one of the most severely clinically staged at the time of diagnosis, it is often difficult to resect at the time of initial presentation. The standard treatment for unresectable cases is gemcitabine/cisplatin. When this is unresponsive, there are no useful options, and the development of new drugs is awaited. Bile duct cancer positive for the important driver gene FGFR2 fusion gene (corresponding to 13% of all cases) progresses quickly, has a high recurrence rate, and has a poor prognosis. A treatment using the molecular targeted drug Infigratinib has been developed for patients with FGFR2 fusion gene-positive cholangiocarcinoma, but the effectiveness varies greatly from person to person, and there are cases where adverse events occur. The response rate of infigratinib was 39.3% in a second clinical trial (J Clin Oncol. 2018;36:276-82.), and is estimated to be about 5% for the entire disease. There is a need for more effective and broadly applicable therapeutic agents, either alone or in combination with existing drugs such as infigratinib.

J Clin Oncol. 2018;36:276-82.

The present inventors have conducted extensive research with the aim of providing a new treatment for biliary tract cancer (particularly bile duct cancer).

The inventors discovered that miRNA3648 may be related to bile duct cancer and have made further improvements.

The present disclosure includes, for example, the subject matter described in the following sections:
Item 1.
(i) a nucleic acid capable of binding complementarily to miRNA3648;
(ii) a nucleic acid encoding a protein having SKI function;
(iii) a vector incorporating at least one nucleic acid selected from the nucleic acids (i) to (iii) which is capable of binding to a complementary strand of the 3'UTR of the SKI gene and has an effect of enhancing the expression of the SKI protein, in an expressible manner;
(II) A therapeutic agent for biliary tract cancer or pancreatic cancer, comprising an antibody or a fragment thereof that recognizes a protein having SKI function, or (IV) miRNA3648.
Item 2.
(ia): a nucleic acid consisting of the base sequence of SEQ ID NO: 1;
(ib): a nucleic acid having a base sequence in which one or more bases are deleted, substituted, or added in the base sequence of (ia), and capable of binding complementarily to miRNA3648;
(ii-a): a nucleic acid consisting of the base sequence of SEQ ID NO: 2; or (ii-b): a nucleic acid consisting of the base sequence of (ii-a) in which one or more bases are deleted, substituted, or added, and encoding a polypeptide that inhibits the proliferation of cholangiocarcinoma cells;
(iii-a): a nucleic acid consisting of a sequence of 7 or more consecutive bases in the base sequence of SEQ ID NO: 3 and enhancing the expression of an SKI protein; or (iii-b): a nucleic acid consisting of a base sequence in which one or two or more bases are deleted, substituted, or added in the base sequence of (iii-a), and enhancing the expression of an SKI protein;
or,
(iv): A vector incorporating at least one nucleic acid selected from the group consisting of (ia), (ib), (ii-a), (ii-b), (iii-a), and (iii-b) in an expressible manner;
or (II-a): a polypeptide consisting of the amino acid sequence of SEQ ID NO: 3; or (II-b): a polypeptide consisting of the amino acid sequence of (II-a) in which one or more amino acids are deleted, substituted, or added, and which inhibits the proliferation of cholangiocarcinoma cells;
Or (IV): a therapeutic agent for biliary tract cancer or pancreatic cancer, comprising an antibody or a fragment thereof that recognizes miRNA3648.
Item 3.
A therapeutic agent for biliary tract cancer or pancreatic cancer, comprising a substance that suppresses miRNA3648 expression and/or a substance that promotes SKI protein expression.
Item 4.
Item 4. The therapeutic agent according to any one of Items 1 to 3, which is a therapeutic agent for bile duct cancer.
Item 5.
A nucleic acid comprising:
(ia): a nucleic acid consisting of the base sequence of SEQ ID NO: 1; (ib): a nucleic acid consisting of the base sequence of (ia) in which one or more bases have been deleted, substituted or added, and capable of complementarily binding to miRNA3648; or (ii-b): a nucleic acid consisting of the base sequence of SEQ ID NO: 2 in which one or more bases have been deleted, substituted or added, and encoding a polypeptide that suppresses the proliferation of cholangiocarcinoma cells.
Item 6.
A vector incorporating the nucleic acid according to item 5 in an expressible manner.
Item 7.
A polypeptide consisting of an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 4, and which inhibits the proliferation of bile duct cancer cells.
Item 8.
An antibody or a fragment thereof that recognizes miRNA3648.
Item 9.
A method for screening an anti-biliary tract cancer or pancreatic cancer substance, comprising a step of measuring expression of miRNA3648 and/or expression of SKI protein in biliary tract cancer or pancreatic cancer cells to which a test substance has been applied.

The present disclosure provides a novel means that is effective in treating biliary tract cancer and pancreatic cancer (particularly bile duct cancer).

1 shows the ratio of SKI mRNA expression levels in each cholangiocarcinoma cell line with different degrees of differentiation. The cell proliferation inhibitory effect of enhanced SKI gene expression in cholangiocarcinoma cell line (OZ). SKI: SKI-flag gene, siSKI: loss-of-function SKI gene, CNT: control, ** and ***: significant difference by t-test. The cell proliferation promoting effect of knockdown of SKI gene expression in cholangiocarcinoma cell line (OZ). SKI: SKI-flag gene, siSKI: loss-of-function SKI gene, CNT: control, ** and ***: significant difference by t-test. Suppression of SKI expression by miR 3648 mimic in cholangiocarcinoma cell line (OZ). GAPDH is used as a control. This shows the upregulation of SKI expression by miR 3648 inhibitor in a cholangiocarcinoma cell line (OZ). GAPDH is used as a control.

Each embodiment included in the present disclosure will be described in more detail below. The present disclosure preferably includes, but is not limited to, therapeutic agents for biliary tract cancer or pancreatic cancer, and the present disclosure includes everything disclosed in this specification that can be recognized by a person skilled in the art.

The therapeutic agent for biliary tract cancer or pancreatic cancer included in the present disclosure contains a specific nucleic acid, a vector incorporating the specific nucleic acid, or a specific polypeptide (protein), etc. Hereinafter, the therapeutic agent included in the present disclosure may be referred to as the "therapeutic agent of the present disclosure."

The therapeutic agent of the present disclosure preferably contains, in particular, (i): a nucleic acid capable of binding complementarily to miRNA (microRNA) 3648, (ii) a nucleic acid encoding a protein having SKI function, (iii): a nucleic acid capable of binding to a complementary strand of the 3'UTR of the SKI gene and having an effect of enhancing SKI protein expression, (iv): a vector incorporating at least one nucleic acid selected from the group consisting of (i) to (iii) in an expressible manner, (II): a protein having SKI function, or (IV): an antibody or a fragment thereof that recognizes miRNA 3648.

miRNA3648 is a microRNA consisting of the base sequence AGCCGCGGGGAUCGCCGAGGG (SEQ ID NO: 5). The present inventors have found that miRNA3648 is highly expressed in biliary tract cancer or pancreatic cancer cells (particularly cholangiocarcinoma cells), that the mRNA of the SKI gene (Homo sapiens SKI proto-oncogene: NM_003036) is a target molecule of miRNA3648, that the amount of SKI protein decreases when miR3648 mimic is introduced into the cancer cells, and that the amount of SKI protein increases when miR3648 inhibitor is introduced, and further, that SKI protein has a strong cell proliferation inhibitory effect in the cancer cells, and that SKI protein expression is enhanced by miR-3648 inhibitor. They also found that by enhancing SKI expression or decreasing miRNA3648 expression, the proliferation of the cancer cells can be suppressed and the cancer can be treated. From these findings, it is understood that the above-mentioned specific nucleic acid, the vector incorporating the specific nucleic acid, or the specific polypeptide (protein) is useful for treating the cancer.

More specifically, preferred examples of ingredients contained in the therapeutic agent disclosed herein include the following:

(i-a): A nucleic acid having the base sequence of SEQ ID NO: 1; (i-b): A nucleic acid having a base sequence in which one or more bases are deleted, substituted, or added in the base sequence of (i-a), and capable of binding complementarily to miRNA3648; (ii-a): A nucleic acid having the base sequence of SEQ ID NO: 2; (ii-b): A nucleic acid having a base sequence in which one or more bases are deleted, substituted, or added in the base sequence of (ii-a), and encoding a polypeptide that suppresses cholangiocarcinoma cell proliferation; (iii-a): A nucleic acid having a sequence of 7 or more consecutive bases in the base sequence of SEQ ID NO: 3, and enhancing SKI protein expression in cholangiocarcinoma cells; (iii-b): A nucleic acid having a base sequence of (iii-a). (iv) a nucleic acid that enhances SKI protein expression in cholangiocarcinoma cells, the nucleic acid comprising a base sequence in which one or more bases are deleted, substituted, or added; (ii) a vector incorporating at least one nucleic acid selected from the group consisting of (ii-a), (ii-b), (ii-a), (ii-b), (iii-a), and (iii-b) in an expressible manner; (ii-a) a polypeptide comprising the amino acid sequence of SEQ ID NO: 4; (ii-b) a polypeptide comprising an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence of (ii-a), the polypeptide suppressing cholangiocarcinoma cell proliferation; (iv) an antibody or a fragment thereof that recognizes miRNA3648

In this specification, the bases that can be deleted, substituted, or added are not particularly limited as long as they are bases that can form nucleic acids, but are preferably adenine (A), thymine (T), guanine (G), cytosine (C), or uracil (U).

The sequence of SEQ ID NO: 1 is a complementary DNA sequence (CCCTCGGCGATCCC CCGCGGC T) of the sequence of miR3648, which is a human microRNA (the underlined portion is a sequence portion common to the 3'UTR sequence of the human SKI gene, which is the target of miR3648). The nucleic acid of (i-a) is a nucleic acid consisting of this sequence. The nucleic acid of (i-b) is a nucleic acid consisting of a base sequence in which one or two or more bases are deleted, substituted, or added in the base sequence of (i-a). The number of bases deleted, substituted, or added is preferably 1 to 10 (1, 2, 3, 4, 5, 6, 7, 8, 9, or 10), more preferably 1 to 5, and even more preferably 1 to 3. The nucleic acid of (i-b) is a nucleic acid capable of binding complementarily to miRNA3648. The term "complementarily binding nucleic acid" includes not only nucleic acids with completely complementary base sequences, but also nucleic acids with non-completely complementary base sequences that can hybridize under stringent conditions. "Stringent conditions" refers to hybridization in 2xSSC containing 0.1% SDS at 50°C and not detached even after washing in 1xSSC containing 0.1% SDS at 60°C.

The sequence of SEQ ID NO:2 is a cDNA sequence encoding the human SKI protein.
The nucleic acid (ii-a) is a nucleic acid consisting of the base sequence of SEQ ID NO: 2. The nucleic acid (ii-b) is a nucleic acid consisting of the base sequence of (ii-a) in which one or more bases are deleted, substituted, or added. The number of deleted, substituted, or added bases is preferably 1 to 100 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100), more preferably 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, or 1 to 10. The nucleic acid of (ib) is a nucleic acid encoding a polypeptide that suppresses cholangiocarcinoma cell proliferation. Whether or not a nucleic acid encodes a polypeptide that inhibits cholangiocarcinoma cell proliferation can be determined by incorporating the nucleic acid into an expression vector, transfecting the expression vector into cholangiocarcinoma cells, and culturing them for 1 or 2 days, and examining whether cell proliferation is inhibited compared to a control (cholangiocarcinoma cells transfected with the vector itself that does not incorporate a gene to be expressed). Cell proliferation can be examined by the WST method using cholangiocarcinoma cell line OZ. If cell proliferation is inhibited, it can be determined that the nucleic acid encodes a polypeptide that inhibits cholangiocarcinoma cell proliferation.

The nucleic acid (iii-a) is a nucleic acid consisting of a sequence of 7 or more consecutive bases in the base sequence of SEQ ID NO: 3 and enhancing SKI protein expression in cholangiocarcinoma cells. The nucleic acid preferably consists of a sequence of about 7 to 3400 consecutive bases in the base sequence of SEQ ID NO: 3, and the upper limit may be, for example, about 3300, 3200, 3100, 3000, 2900, 2800, 2700, 2600, 2500, 2400, 2300, 2200, 2100, 2000, 1900, 1800, 1700, 1600, 1500, 1400, 1300, 1200, 1100, 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15, or 10.

SEQ ID NO: 3 is the 3'UTR (untranslated region) sequence of the human SKI gene. miRNAs bind to their target mRNAs with incomplete homology, and generally recognize the 3'UTR of the target gene in particular, destabilizing the target mRNA and suppressing translation, thereby suppressing protein production. For this reason, miR3648 is also thought to suppress SKI protein production by binding to the 3'UTR of the human SKI gene. In addition to miR3648, there are other miRNAs that are thought to potentially target the SKI gene, and these miRNAs are also thought to suppress SKI protein production by binding to the 3'UTR of the human SKI gene.

Therefore, a nucleic acid having a base sequence similar to that of the human SKI gene 3'UTR can competitively inhibit the binding of SKI protein production-inhibiting miRNAs such as miR3648 to the SKI gene 3'UTR, thereby inhibiting the inhibition of SKI protein production by miRNAs. This makes it possible to inhibit the proliferation of biliary tract cancer or pancreatic cancer (particularly cholangiocarcinoma) cells.

Whether a nucleic acid enhances SKI protein expression in cholangiocarcinoma cells can be determined by incorporating the nucleic acid into an expression vector, transfecting the expression vector into cholangiocarcinoma cells, and examining whether the cells are able to enhance SKI protein expression when cultured for 1 or 2 days, compared to a control (cholangiocarcinoma cells transfected with the vector itself that does not incorporate the gene to be expressed). This examination can be performed using the cholangiocarcinoma cell line OZ. Protein expression can also be detected by Western blotting.

The nucleic acid (iii-b) is a nucleic acid that consists of a base sequence in which one or more bases are deleted, substituted, or added in the base sequence of (iii-a), and enhances SKI protein expression in cholangiocarcinoma cells. The nucleic acid (iii-b) consists of a base sequence in which one or more bases are deleted, substituted, or added in the base sequence of (iii-a). The number of bases to be deleted, substituted, or added is preferably 1 to 100 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 108, 109, 110, 111, 112, 113, 114, 115, 8, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100), more preferably 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, or 1 to 10.

In addition, antisense nucleic acids of miRNAs other than miRNA3648 that target the 3'UTR of the SKI gene may also be included in the nucleic acids of (iii-a) or (iii-b). Examples of miRNAs other than miRNA3648 that target the 3'UTR of the SKI gene include miRNAs that are known or predicted to target the 3'UTR of the SKI gene, such as those listed at the following URL:

Preferably, the miRNA is predicted to target the 3'UTR of the SKI gene with a target score of 50 or more (preferably 60 or more, 70 or more, 80 or more, or 90 or more) using "TargetScan" published on the Internet. Examples of such miRNA include hsa-miR-8071 (CGGUGGACUGGAGUGGGUGG) and hsa-miR-3680-3p (UUUUGCAUGACCCUGGGAGUAGG), and examples of antisense nucleic acids thereof include antisense hsa-miR-8071 (CCACCCACTCCAGTCCACCG) and antisense hsa-miR-3680-3p (CCTACTCCCAGGGTCATGCAAAA).

The vector (iv) is a vector into which at least one nucleic acid selected from the group consisting of (i-a), (i-b), (ii-a), (ii-b), (iii-a), and (iii-b) (hereinafter also referred to as "(i-a) to (iii-b)") is incorporated in an expressible manner.

Expressibly incorporated includes not only the case where the nucleic acids (i-a) to (iii-b) are incorporated so as to be expressed (transcribed) from the expression vector in a cell (particularly a cholangiocarcinoma cell), but also the case where the nucleic acids (i-a) to (iii-b) are ultimately expressed in a cell after processing in the cell, even if they are not the nucleic acids (i-a) to (iii-b) at the stage of expression (transcription) from the expression vector. For example, a vector that contains the sequence of SEQ ID NO: 1 (a complementary DNA sequence of the sequence of miR3648) and is incorporated so as to express a nucleic acid that can form a hairpin structure can be said to be a vector in which the nucleic acid (i-a) or (i-b) is incorporated so as to be expressible, since the nucleic acid expressed from the vector can be processed in the cell to become the nucleic acid of SEQ ID NO: 1 or a nucleic acid consisting of a base sequence in which one or more bases have been added to the base sequence of the nucleic acid of SEQ ID NO: 1.

The vector into which the nucleic acids (i-a) to (iii-b) are incorporated is not particularly limited, and any known expression vector can be used. For example, a plasmid vector, a viral vector, etc. can be used.

(II-a) is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 4. SEQ ID NO: 4 is the amino acid sequence of human SKI protein. Moreover, the polypeptide (II-b) is a polypeptide consisting of the amino acid sequence of (II-a) in which one or more amino acids are deleted, substituted or added, and which inhibits the proliferation of cholangiocarcinoma cells. In the base sequence (II-b), the number of amino acids to be deleted, substituted, or added is preferably 1 to 100 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100), more preferably 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, or 1 to 10. In addition, whether the polypeptide inhibits cholangiocarcinoma cell proliferation can be determined by a method similar to that described for the nucleic acid of (ii-b) above.

(IV) is an antibody or a fragment thereof that recognizes miRNA3648. The fragment has the miRNA3648 recognition site of the antibody. Examples of the fragment include Fab and F(ab') ₂ of the antibody. The antibody is not particularly limited as long as it recognizes miRNA3648, and may be, for example, a polyclonal antibody or a monoclonal antibody. The antibody may be a chimeric antibody, a humanized antibody, or a fully humanized antibody. The method for producing the antibody is also not particularly limited, and may be produced by, for example, a known method or a method that can be easily conceived from a known method.

The therapeutic agent disclosed herein also includes a therapeutic agent for biliary tract cancer or pancreatic cancer that contains a substance that inhibits miRNA3648 expression and/or a substance that promotes SKI protein expression.

Examples of substances that suppress the expression of miRNA3648 include the above-mentioned nucleic acids (i-a) and (i-b), and the antibody (IV) or a fragment thereof. Examples of substances that promote the expression of SKI protein include the above-mentioned nucleic acids (ii-a), (ii-b), (iii-a), and (iii-b).

The present disclosure also includes a method for screening for anti-biliary tract cancer or pancreatic cancer substances using the expression of miRNA3648 and/or the expression of SKI protein as an indicator. The anti-biliary tract cancer or pancreatic cancer substance is useful as an active ingredient of a biliary tract cancer or pancreatic cancer therapeutic agent (particularly a bile duct cancer therapeutic agent).

The screening method includes a step of measuring the expression of miRNA3648 and/or the expression of SKI protein in biliary tract cancer or pancreatic cancer cells (particularly preferably bile duct cancer cells) to which a test substance (including both a compound and a composition) has been applied. In the measurement, if the expression of miRNA3648 is reduced by application of the test substance, the test substance can be selected as a candidate for an active ingredient of a biliary tract cancer or pancreatic cancer therapeutic agent (particularly a bile duct cancer therapeutic agent). In addition, in the measurement, if the expression of SKI protein is enhanced by application of the test substance, the test substance can be selected as a candidate for an active ingredient of a biliary tract cancer or pancreatic cancer therapeutic agent (particularly a bile duct cancer therapeutic agent). The method of these measurements is not particularly limited, and can be performed by a known method or a method that can be easily conceived from a known method. For example, quantitative PCR or the like can be used to measure the amount of nucleic acid, and Western blotting or the like can be used to measure the amount of protein.

The test substance here is not particularly limited. For example, it may be a compound or a composition. The compound may be, for example, a low molecular weight compound, a nucleic acid (e.g., DNA, RNA, etc.), a protein (e.g., an antibody or a part thereof, etc.), or a polymeric compound such as a polymer. The composition may also be an extract obtained from a living organism (e.g., an animal, a plant, a microorganism, etc.), or a combination of two or more compounds.

The biliary tract cancer or pancreatic cancer cells used may be human-derived cells or non-human mammal-derived cells. Examples of non-human mammals include monkeys, dogs, cats, mice, rats, Chinese hamsters, sheep, cows, and horses.

In this specification, the term "comprising" includes "consisting essentially of" and "consisting of." In addition, the present disclosure includes any combination of the constituent elements described in this specification.

Furthermore, the various characteristics (properties, structures, functions, etc.) described for each embodiment of the present disclosure above may be combined in any way to identify the subject matter encompassed by the present disclosure. In other words, the present disclosure encompasses all subject matter consisting of any combination of the combinable characteristics described in this specification.

Below, the embodiments of the present disclosure will be explained in more detail with examples, but the embodiments of the present disclosure are not limited to the following examples. Unless otherwise specified, reagents were purchased from Merck & Co. (USA) or Fujifilm Wako Pure Chemical Industries (Osaka). In addition, the following experiments were conducted with the approval of our hospital's IRB (Article 6 of the Human Genome Genetic Analysis Research) in accordance with the Declaration of Helsinki (revised in 2008).

Target specimen
Tissue samples from tumorous and non-tumorous areas of the biliary tract and liver from 24 surgically resected cases without chronic hepatitis due to HBV/HCV were used (surgeries were performed from July 2007 to June 2015 at the Department of Hepato-Biliary-Pancreatic Surgery, Osaka City University Hospital). The breakdown was 10 cases of bile duct cancer [57.6 years old, 8 males/2 females, and 14 control cases [10 hepatocellular carcinoma (66.8 years old, 8 males/2 females), 4 benign biliary tumors (74.3 years old, 2 males/2 females)]. MicroRNAs related to the development of bile duct cancer from these resected specimens were labeled using the 3D-Gene miRNA Labeling kit and extracted using the 3D-Gene Human miRNA Oligo Chip (Toray, Osaka).

Cell culture Three human cholangiocarcinoma cell lines (OZ, KKU156, and KKU100) were cultured in Dulbecco's modified Eagle's medium (low glucose) (#D5546-500ML, Sigma-Aldrich, USA) supplemented with 10% fetal bovine serum (#10270-098, Gibco, USA) and 1% penicillin-streptomycin (#15070-063, Gibco). Before gene transfection, the medium was free of 1% penicillin-streptomycin. The cell lines were maintained at 37°C in a humidified incubator with 5% _CO2 . The three lines were provided by different individuals (OZ: #1032, KKU-M156: #1561, and KKU-100: #1568) (JCRB Cell Bank, Osaka).

RNA analysis
RNA was extracted from cultured cells using TRIzol reagent (Invitrogen, USA) and Direct-zol RNA Miniprep (Zymo Research, USA). Quantitative PCR of SKI messenger RNA was performed using a PCR Thermal Cycler Dice Gradient TP600 (Takara Bio) with 500 ng of RNA/sample, 1 μl of dNTP mixture (#4030, Takara Bio, Shiga), 1 μl of random primers (#58875, Invitrogen), and 2 μl of diethylpyrocarbonate treated water. The cDNA solution was synthesized using a PCR Thermal Cycler Dice Gradient TP600 (Takara Bio) and diluted 5-fold for SKI and 500-fold for 18S with Elution Buffer (0.1×). The cDNA solution was diluted 5-fold for SKI and 500-fold for 18S with Elution Buffer (0.1×). The cDNA solution was mixed with 0.3 μl of forward primer, 0.3 μl of reverse primer (Sigma Aldrich), 5 μl of SYBR green PCR master mix (×2) (Applied Biosystem, USA), and 3.4 μl of ion-exchanged water, and then used in a Thermal Cycler Dice Real Time System II TP900 (Takara Bio). The primers used in this case are shown in the following table.

Introduction of DNA fragments or microRNA into cholangiocarcinoma cell lines
The SKI-flag gene was obtained by cloning the non-quantitative PCR product of a Myc-DDK-tagged human SKI plasmid (OriGene, USA) expressing human v-ski sarcoma viral oncogene homolog from Kanamycin resistance cDNA into E. coli DH5α competent cells (Takara Bio). Gene transfer was performed on cholangiocarcinoma cell line OZ (2x105 cells/well, 12-well plate) by adding 1) pCMV6 SKI-flag gene and its control pCMV6-Entry Empty vector (homemade), and 2) functionally defective SKI gene (siGENOME Human SKI (6497) siRNA #IM-003927-02-0010, Dharmacon) and its control pCMV6-Entry Empty vector (total amount of DNA for 1) to 2) was 500 ng/well) to each well. The DNA solution was prepared by adding 1 μl of P3000 Reagent (Invitrogen) and 50 μl of Opti-MEM (#31985-062, Gibco) to each well, and then adding 1 μl of Lipofectamine (Invitrogen) and 50 μl of Opti-MEM to each well. Similarly, 1) microRNA 3648 mimic (#c-301526-00-0005, Dharmacon, USA) and its control Negative Control siRNA (#AM4637, Ambion, USA), 2) microRNA 3648 hairpin inhibitor (#IH-301526-01-0005, Dharmacon, USA) and its control Hairpin Inhibitor Negative Control (#IN-001005-01-50, Dharmacon) (1) to 2) were prepared by adding 50 μl of Opti-MEM per well to a total amount of RNA (100 pmol/well), and then further adding 4 μl of RNAiMAX (Invitrogen) and 50 μl of Opti-MEM to the RNA solution.

Cell proliferation assay: Cell numbers were measured over time in the human cholangiocarcinoma cell line OZ under conditions of overexpression or inhibition of SKI gene expression. The proliferating cells were seeded onto a 96-well plate at 5000 cells/100μl/well and cultured overnight in a 5% _CO2 humidified incubator. Water soluble tetrazolium salt-8 solution was added at 10μl/well (Dojindo Laboratories, Kumamoto). After incubation for about 1 hour, the absorbance at 450 nm was measured (Varioskan Lux #VLBL00D0, Thermo Fisher Scientific, USA).

Protein expression analysis: In human cholangiocarcinoma cell line OZ, RNA dose-dependent protein expression analysis was performed by Western blotting under overexpression or suppression of microRNA 3648. Proteins were collected from OZ cell line using sample buffer (100 mM/pH 6.8 Tris-HCl buffer, 20% glycerin, 4% sodium dodecyl sulfate, 10% 2-mercaptoethanol). The collected samples were homogenized in non-denaturing cell lysis buffer [50 mM/pH 7.5 Tris-HCl buffer, 150 mM sodium chloride, 1% Triton X-100 (Nacalai Tesque, Kyoto), 1% sodium dodecyl sulfate] containing protease inhibitor (cOmplete Mini, # No. 4 693 124. Roche, Switzerland) and phosphatase inhibitor (1 mM sodium fluoride, 1 mM glycerol 2-phosphate, 1 mM sodium vanadate). Protein samples were separated by 8-15% sodium dodecyl sulfate polyacrylamide gel electrophoresis along with molecular weight markers [Precision Plus Protein Dual Color Standards (#1610374, Bio Rad, USA)] and transferred from the gel to a polyvinylidene difluoride membrane (PowerPac Basic Power Supply #1645050JA, Bio Rad, USA). The membrane was blocked with a mixture of 5% nonfat dry milk, Tris-buffered saline, and polysorbate 20 to prevent antibody binding to nonspecific proteins. Primary antibodies against SKI protein (#SC33693, Santa Cruz Biotechnology, USA) and glyceraldehyde phosphate dehydrogenase (GAPDH: endogenous control, #MAB374, Sigma-Aldrich) were reacted with enzyme-conjugated goat anti-mouse secondary antibody (#7074, Cell Signaling Technology, USA). The peroxidase labeled secondary antibody was reacted with a luminescent substrate (ImmunoStar Zeta or LD. Fujifilm Wako Pure Chemical Industries, Ltd.) to detect the bands of the target protein (ImageQuant LAS4000 mini. GE Healthcare, Inc., USA). If necessary, a stripping buffer (Nacalai Tesque) was used to remove the antibody bound to the membrane and detect other target proteins.

Statistical analysis Extraction of microRNAs related to cholangiocarcinoma onset from surgically resected specimens was performed using principal component analysis with the prcomp function in the statistical environment R (YH Taguchi and Y. Murakami. Principal component analysis based feature extraction approach to identify circulating microRNA biomarkers. PLoS ONE 2013,8:e66714.) and linear discriminant analysis with the lda function (R Core Team. R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria, 2014.). Comparison of groups (student t-test) and time-course changes (multiple student t-test) in human cholangiocarcinoma cell lines of the same disease were performed using Prism ver. 8 (GraphPad Software, USA).

<Exploration and confirmation of bile duct cancer-related factors>
Microarray gene profiling and principal component analysis were performed on the development of bile duct cancer, and miR3648 was extracted. Using the mRNA database, miR3648 and SKI mRNA with high targeting efficiency (high target score and low context++ score) were extracted (http://www.targetscan.org) (see table below).

Based on these results, it was expected that miR 3648 inhibitor would promote SKI expression and suppress cholangiocarcinoma cell proliferation. To confirm this, further experiments were performed. To observe the relationship between the action of SKI and the degree of pathological differentiation, the level of SKI mRNA expression was evaluated in three types of human cholangiocarcinoma cell lines (OZ, KKU156, and KKU100: high, medium, and low degrees of differentiation, respectively). Specifically, the amount of SKI mRNA was corrected for the amount of 18S rRNA and calculated. The results are shown in Figure 1a.

In addition, the SKI flag gene and the loss-of-function gene siSKI (SKI knockdown) were added to the OZ with the highest potential expression of SKI (highest degree of differentiation), and cell proliferation was evaluated using the WST method. Suppression and promotion of cell proliferation were observed over time (Figures 1b and 1c). Note that Empty in Figure 1b refers to cells transfected with the vector itself (self-ligated after restriction enzyme treatment).

Furthermore, miR-3648 mimic (specifically, double-stranded RNA designed to generate miR3648 sense RNA: AGCCGCGGGGAUCGCCGAGGG) and miR-3648 inhibitor (specifically, double-stranded RNA designed to generate miR3648 antisense RNA: CCCTCGGCGATCCCCGCGGCT) were added to cholangiocarcinoma cell lines, and SKI protein expression was evaluated by Western blotting. SKI expression was suppressed (Fig. 2a) and promoted (Fig. 2b) depending on the miR dosage. These results indicate that SKI has a strong cell proliferation inhibitory effect in cholangiocarcinoma cell lines, and SKI expression is enhanced by miR-3648 inhibitor.

Claims (3)

(i) a nucleic acid capable of binding complementarily to miRNA3648 and inhibiting the suppression of SKI protein production by miRNA3648 ;
(ii) a nucleic acid encoding an SK I protein ;
A vector incorporating at least one nucleic acid selected from ( i) to ( ii ) in an expressible manner;
(II) SK I protein ; or (IV) an antibody or a fragment thereof that recognizes miRNA3648;
To promote expression of SKI protein in a subject,
A drug for treating bile duct cancer. (ia): a nucleic acid consisting of the base sequence of SEQ ID NO: 1;
(ib): a nucleic acid having a base sequence in which one or two bases are deleted, substituted, or added in the base sequence of (ia), capable of binding complementarily to miRNA3648 , and capable of inhibiting the suppression of SKI protein production by miRNA3648 ;
(ii-a): a nucleic acid consisting of the base sequence of SEQ ID NO: 2, or (ii-b): a nucleic acid consisting of the base sequence of (ii-a) in which 1 to 100 bases are deleted, substituted, or added, and encoding a polypeptide that inhibits the proliferation of cholangiocarcinoma cells ;
or,
(iv): A vector incorporating at least one nucleic acid selected from the group consisting of (ia), (ib), (ii-a), and ( ii-b ) in an expressible manner;
or (II-a): a polypeptide consisting of the amino acid sequence of SEQ ID NO: 4; or (II-b): a polypeptide consisting of the amino acid sequence of (II-a) in which 1 to 72 amino acids are deleted, substituted or added, and which inhibits the proliferation of cholangiocarcinoma cells;
or (IV): containing an antibody or a fragment thereof that recognizes miRNA3648;
To promote expression of SKI protein in a subject,
A drug for treating bile duct cancer. A method for screening an anti-bile duct cancer substance, comprising a step of measuring expression of SKI protein in bile duct cancer cells to which a test substance has been administered ,
When the expression of SKI protein is enhanced by application of the test substance in the measurement, the test substance is selected as a candidate active ingredient of an anti-bile duct cancer substance.