JP2015163590A - Colon cancer therapeutic agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an effective colon cancer therapeutic agent.SOLUTION: The invention provides a colon cancer therapeutic agent or a method of collecting data for evaluating the risk of onset or recurrence of colon cancer by finding that (1) the proliferation of colon cancer cell line is suppressed, (2) the apoptosis of colon cancer cell line is induced, and (3) the motor function of colon cancer cell is suppressed by administering a silencing substance of FAM83H protein, such as siRNA to FAM83H known as a protein involved in tooth enamel formation, or a functional inhibitor of FAM83H protein, such as an anti-FAM83H protein antibody.

Description

  The present invention relates to a colorectal cancer therapeutic agent containing an FAM83H protein expression inhibitor or function inhibitor as an active ingredient, and a method for collecting data for evaluating the risk of developing or recurrence of colorectal cancer.

  FAM83H protein is known as a protein involved in tooth enamel formation, and its gene mutation has been reported to cause failure of enamel formation (see, for example, Non-Patent Document 1), so FAM83H protein is a stage of tooth development. It was thought that it only had important functions.

  In recent years, a paper in which the FAM83H gene has been reported as a candidate for a new blood vessel marker for uterine cancer has been published (for example, see Non-Patent Document 2). Therefore, it can be said that the FAM83H protein may play a predetermined role not only in tooth enamel formation but also in other tissues, but there is no report about having an inhibitory effect on colorectal cancer.

Int J Paediatr Dent 21 (6), 407-412 (2011) Cancer Biology & Therapy 12: 3, 1-12; August 1, 2011

  An object of the present invention is to provide a therapeutic agent for colorectal cancer that is more effective and has fewer side effects.

  The present inventors perform gene expression analysis of colorectal cancer tissue and identify a gene whose expression is elevated at the transcriptional level in colorectal cancer and a protein that is a product thereof, thereby suppressing the growth / metastasis of colorectal cancer. Although the mechanism was continuously examined, it was confirmed by chance that the expression of FAM83H protein known as a protein involved in dental enamel formation was increased at the transcriptional level in cancer tissues of colon cancer patients. The inventors continue to further examine the function of FAM83H protein in colon cancer cells and its application to cancer treatment, and administration of FAM83H siRNA suppresses the growth of (1) colon cancer cell lines. It was found that apoptosis of cancer cell lines is induced, and (3) suppresses the motor function of colon cancer cell lines. The present invention has been completed based on the above findings.

  That is, the present invention is characterized in that [1] a therapeutic agent for colorectal cancer containing an FAM83H protein expression inhibitor or function inhibitor as an active ingredient, and [2] an FAM83H protein expression inhibitor is an FAM83H siRNA. The therapeutic agent for colorectal cancer according to [1] above, or [3] siRNA consisting of a double-stranded RNA sequence comprising the RNA sequence shown in SEQ ID NO: 1 and its complementary sequence, or SEQ ID NO: 3 The therapeutic agent for colorectal cancer according to [2] above, which is a siRNA comprising a double-stranded RNA sequence comprising the RNA sequence shown in FIG. 5 and a complementary sequence thereof, and [4] siRNA of FAM83H are incorporated into a vector It is related with the colon cancer therapeutic agent as described in said [2] or [3] characterized by the above-mentioned.

  In addition, the present invention provides [5] (a) a step of quantifying FAM83H protein in a biological sample derived from a subject; (b) the amount of FAM83H protein in the biological sample, a colon cancer non-affected person or colon cancer Comparing and evaluating the amount of FAM83H protein in a biological sample derived from an affected person, the present invention relates to a method for collecting data for evaluating the risk of colorectal cancer onset and recurrence.

  According to the colorectal cancer therapeutic agent of the present invention, colorectal cancer can be treated without side effects such as tooth enamel formation failure by devising the administration method.

(A) is a graph showing the results of plotting the mRNA expression level by microarray analysis of FAM83H in non-cancerous tissue and colorectal cancer tissue. (B) is a graph showing the results of plotting mRNA expression levels by qPCR of FAM83H in non-cancerous tissue and colon cancer tissue. It is a graph which shows the number of living cells of S1 cell, S2 cell, and control cell every 24, 48, and 72 hours after siRNA introduction | transduction. It is a figure which shows the result of the Western blot analysis of S1 cell and control cell at the time of 2 days after siRNA introduction | transduction. (A) shows an image observed using a confocal microscope using an anti-keratin 18 antibody and an anti-α-tubulin antibody as primary antibodies. The scale in the image is 10 μm. (B) is a graph which shows the normal formation rate of the spindle body in the cell division phase in S1 cell, S2 cell, and those control cells, and D-S1 cell and D-control cell. (A) shows an image of S1 cells observed using a confocal microscope using an anti-lamin B2 antibody and an anti-α-tubulin antibody as primary antibodies. The scale in the image is 500 μm. (B) is a graph showing the normal formation rate of the Lamin B spindle matrix during cell division in S1 cells, S2 cells and control cells. (A) is a figure which shows the image of the cell sheet immediately after the wound and 24 hours after in wound healing analysis. (B) is a graph which shows the movement distance of the cell located in the front-end | tip of a cell-free surface. (A) is a figure which shows the image of the cell sheet immediately after wounding and 6 hours after in wound healing analysis. The scale in the image is 10 μm. (B) is a graph which shows the length of the cell projection of the cell located in the front-end | tip of a cell-free surface. It is a figure which shows the image of the cell sheet immediately after the wound in wound healing analysis, and 6 hours after.

  The therapeutic agent for colorectal cancer of the present invention is not particularly limited as long as it contains an FAM83H protein expression inhibitor or function inhibitor as an active ingredient, and data for evaluating the risk of colorectal cancer onset and recurrence according to the present invention is not limited. The collection method includes: (a) a step of quantifying FAM83H protein in a biological sample derived from a subject; and (b) the amount of FAM83H protein in the biological sample, a biological sample derived from a person not affected by colorectal cancer, and And / or the step of comparing / evaluating the amount of FAM83H protein in a biological sample derived from a colorectal cancer-affected person; and the ascending colon cancer and transverse colon cancer are not particularly limited. , Descending colon cancer, sigmoid colon cancer, rectal cancer, or cecal cancer, and the administration target of the colon cancer therapeutic agent of the present invention is human, monkey, mouse, rat Hamsters can guinea pig, cattle, pigs, horses, rabbits, sheep, goats, cats, be mentioned mammals dogs like.

  The FAM83H protein expression-suppressing substance in the present invention may be any substance that completely or significantly suppresses FAM83H protein expression at the FAM83H gene transcription level or translation level. Inhibitors include FAM83H siRNA, antisense RNA, antisense DNA, and the like, as well as ribozymes having FAM83H mRNA cleavage activity and FAM83H miRNA.

  The FAM83H siRNA includes a sense RNA having a sequence homologous to a partial sequence of the FAM83H mRNA, which can be used in RNAi (RNA interference) that suppresses expression in a sequence-specific manner by degrading FAM83H mRNA. A double-stranded RNA having 18 to 26 bases, preferably 19 to 25 bases, more preferably 20 to 24 bases, and particularly preferably 21 to 23 bases, including antisense RNA having a complementary sequence to the corresponding sense RNA. Specifically, it consists of a double-stranded RNA sequence containing 5'-ACACGAAGGCCAUUCUGGAGCAGAU-3 '(SEQ ID NO: 1) and its complementary sequence 3'-UGUGCUUCCGGUAAGACCUCGUCUA-5' (SEQ ID NO: 2). FAM83H siRNA, 5'-CAACGCCUUGUACAGCAGCAACCUU-3 '(SEQ ID NO: 3) and its complementary sequence 3'-GUUGCGGAAC An FAM83H siRNA consisting of a double-stranded RNA sequence containing AUGUCGUCGUUGGAA-5 ′ (SEQ ID NO: 4) can be mentioned, preferably 5′-ACACGAAGGCCAUUCUGGAGCAGAU-3 ′ (SEQ ID NO: 1) and its complementary sequence 3 From FAM83H siRNAS1 consisting of '-UGUGCUUCCGGUAAGACCUCGUCUA-5' (SEQ ID NO: 2), 5'-CAACGCCUUGUACAGCAGCAACCUU-3 '(SEQ ID NO: 3) and its complementary sequence 3'-GUUGCGGAACAUGUCGUCGUUGGAA-5' (SEQ ID NO: 4) SiRNAS2 of FAM83H can be mentioned, and siRNAS1 of FAM83H can be particularly preferably exemplified. Moreover, the said siRNA can also be used 1 type, or 2 or more types.

  The FAM83H siRNA may include a FAM83H siRNA derivative or a FAM83H siRNA precursor, and the FAM83H siRNA precursor may include a FAM83H gene shRNA (short hairpin RNA).

  As the FAM83H shRNA, a single-stranded RNA having a double-stranded structure in the molecule by partially including a palindromic base sequence and a structure like a short hairpin having a protruding portion at the 3 ′ end After such shRNA is introduced into a cell, it is decomposed into a double-stranded RNA having a length of, for example, 21 to 23 bases by Dicer, and can cause RNAi similarly to the above siRNA. As a double-stranded RNA having a length of 21 to 23 bases, 5′-ACACGAAGGCCAUUCUGGAGCAGAU-3 ′ (SEQ ID NO: 1) and its complementary sequence 3′-UGUGCUUCCGGUAAGACCUCGUCUA-5 ′ (SEQ ID NO: 2) or 5 ′ Examples include '-CAACGCCUUGUACAGCAGCAACCUU-3' (SEQ ID NO: 3) and its complementary sequence, 3'-GUUGCGGAACAUGUCGUCGUUGGAA-5 '(SEQ ID NO: 4).

  Examples of the FAM83H siRNA derivatives include derivatives that have been modified to enhance the RNAi effect and improve the stability of RNA so that they are not easily degraded by intracellular RNase. 3 ′ overhang type derivatives in which several U, preferably 2 to 4, more preferably 2 or 3, most preferably 2 are added on the 3 ′ side, polyethylene glycol, cholesterol, or 2 Derivatives appropriately modified by anti-RNase treatment such as methylation treatment or thiophosphorylation treatment such as '-o-methyl' can be mentioned.

  The siRNA of FAM83H can be designed based on known sequence information about FAM83H (for example, NCBI Reference Sequence: NM_198488.3). Specifically, it can be designed based on the sequence shown in SEQ ID NO: 9. It can be produced by using a known production method such as a synthetic production method or a production method using a gene recombination technique.

  Examples of the production method by the synthesis include a method of synthesizing double-stranded RNA by a conventional method based on the sequence information. That is, a sense strand oligoribonucleotide and an antisense strand oligoribonucleotide are protected with a protecting group. SiRNA, which is a double-stranded oligoribonucleotide, can be synthesized by an organic synthesis method using a synthesizer using four kinds of ribonucleotides with a (adenosine, guanosine, cytidine, uridine).

  Examples of the production method using the above-described gene recombination technique include a method of expressing FAM83H siRNA by incorporating it into an expression vector. For example, inserting a FAM83H siRNA expression cassette downstream of an appropriate promoter in the vector. Thus, an siRNA expression vector can be constructed. As such siRNA expression vectors, known ones including commercially available vectors for expression of double-stranded RNA can be used, but when they are introduced into mammals, they are preferably viral vectors. For example, a mouse leukemia retrovirus vector, an adeno-associated virus vector, an adenovirus vector, a lentivirus vector, and the like can be mentioned. Adenovirus vectors are preferred, and expression is performed when constructing an expression system using a vector. In addition to causing it, regulatory sequences that regulate expression, such as the U6 promoter, can also be included.

  FAM83H siRNA can be introduced into colon cancer cells by infecting the cells with the above-described recombinant viral vector. By directly infecting a cancer cell with a recombinant viral vector, the period in which it can be present in the cell becomes longer and the efficiency of gene expression suppression is higher than when double-stranded siRNA is directly introduced into the cell.

  FAM83H siRNA can also be introduced into colon cancer cells by lipofection or the like. Specifically, a liposome composed of phosphatidylserine (PS) having a negative charge can be used, but N- [1- (2,3-dioleyloxy) which is a cationic lipid that forms a more stable liposome. ) Propyl] -N, N, N-triethylammonium chloride (DOTMA), 2,3-dioleyloxy-N- [2 (sperminecarboxamido) ethyl] -N, N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), medium It can be introduced using artificial liposomes and the like, which have intracellular transferability and nuclear transferability mixed with functional lipid dioleoylphosphatidyl ethanolamine (DOPE).

  Examples of the antisense RNA and antisense DNA include RNA and DNA having a base sequence complementary to the sequence of FAM83H mRNA, which suppresses the synthesis of FAM83H protein by forming a hybrid with FAM83H mRNA. In addition, examples of the ribozyme include RNA molecules having catalytic activity, which exhibit an effect as a therapeutic agent for colorectal cancer by cleaving FAM83H mRNA.

  Examples of the miRNA include RNA having a base sequence complementary to the sequence of FAM83H mRNA, which causes transcription and translation inhibition and suppresses the expression of FAM83H protein.

  Examples of the function inhibitor of FAM83H protein in the present invention include anti-FAM83H protein antibodies and peptide aptamers for FAM83H protein.

Examples of the anti-FAM83H protein antibody include an antibody that specifically binds to the FAM83H protein and can inhibit the function of the FAM83H protein, and may be either a polyclonal antibody or a monoclonal antibody, and an antibody fragment (for example, , Fab, F (ab ′) ₂ ) or a recombinant antibody (eg, scFv).

  The anti-FAM83H protein antibody can be prepared by an appropriate method known to those skilled in the art using the FAM83H protein or an appropriate peptide fragment thereof, or various derivatives or complexes thereof as an immunogen. For example, polyclonal antibodies can be prepared by administering to appropriate animals such as mice, rats, rabbits, goats, chickens, etc., and preparing them from the antiserum. Monoclonal antibodies can be prepared according to "Clone antibodies", Kasuna Chomune, Hiroaki Terada, Yodogawa Shoten, 1990; "Monoclonal Antibody" James W. Goding, third edition, Academic Press, 1996)).

  According to the FAM83H protein expression inhibitor or function inhibitor, the growth of colon cancer cells can be suppressed, apoptosis in colon cancer cells can be induced, or the normal formation rate of spindles during the cell division phase of colon cancer cells can be reduced. In this sense, the colorectal cancer therapeutic agent of the present invention is a colon cancer cell growth inhibitor, an apoptosis inducer in colorectal cancer cells, a cell of colon cancer cells. It can be said that it is an agent for reducing the normal formation rate of spindles at the mitotic stage, or a cell motility function inhibitor for colon cancer cells.

  When the colon cancer therapeutic agent of the present invention is administered as an agent for lowering the normal formation rate of the spindle in the cell division phase of the colon cancer cells, lamin B2 aggregates at the cell periphery during the cell division phase, resulting in colon cancer cells. Alternatively, spindle matrix formation is inhibited during cell division in colorectal cancer cell lines. Thus, when spindle formation is inhibited, it is considered that the growth of colon cancer cells is suppressed.

  When the therapeutic agent for colorectal cancer of the present invention is administered as an apoptosis inducer in the above-mentioned colorectal cancer cells, the apoptosis marker is significantly expressed in the colorectal cancer cells or colorectal cancer cell lines, resulting in an apoptosis-inducing action. Examples of the apoptosis marker include fragmented PARP and fragmented caspase-3. Thus, when apoptosis is induced, colon cancer cells are thought to die.

  When the therapeutic agent for colorectal cancer of the present invention is administered as the above-mentioned inhibitor of cell motility function of colon cancer cells, the motility function of cells in colorectal cancer cells or colorectal cancer cell lines is inhibited. Thus, when the motor function of colon cancer cells is inhibited, it is considered that colon cancer metastasis is suppressed.

  The therapeutic agent for colorectal cancer of the present invention can be made into an appropriate preparation by a conventional method, and is an auxiliary component consisting of a known pharmacologically acceptable carrier or diluent, and other agents for the treatment and prevention of cancer. A physiologically active substance can be further contained.

  The therapeutic agent for colorectal cancer of the present invention is not particularly limited as long as the desired therapeutic effect for colorectal cancer is obtained, and examples thereof include intravenous administration, subcutaneous administration, transdermal administration, enema administration, and the like. Is preferred.

  As the dose of the therapeutic agent for colorectal cancer of the present invention, depending on the type of active ingredient, the severity of the disease state, the treatment policy, the patient's age, weight, sex, general health condition, and the genetic background of the patient, Those skilled in the art can appropriately select, for example, when administering FAM83H siRNAS1, 0.1 to 100 mg / day / kg bw, preferably 0.5 to 50 mg / day / kg bw, Preferably, 1 to 10 mg / day / kgbw, more preferably 3 to 7 mg / day / kgbw.

  In the method for collecting data for evaluating the risk of the onset and recurrence of colorectal cancer according to the present invention, the biological sample derived from a person without colorectal cancer is a negative control, and the biological sample derived from a person with colorectal cancer is a positive control. . The biological sample derived from the subject is preferably stool, blood, or a biopsy specimen, and FAM83H can be quantified by any method known to those skilled in the art. For example, ELISA using an anti-FAM83H protein antibody And methods utilizing various immunological specific reactions such as Western blotting and immunostaining can be used.

  In the method of collecting the data, for example, when the amount of FAM83H protein in the biological sample derived from the subject is equal to or greater than the amount of FAM83H protein in the biological sample derived from a colorectal cancer patient, It can be judged that the risk of cancer onset / recurrence is high, and the amount of FAM83H protein in the biological sample derived from the subject is significant compared to the amount of FAM83H protein in the biological sample derived from a person not affected by colorectal cancer If the risk is high, it can be determined that the risk of colorectal cancer onset / recurrence is high.

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, the technical scope of this invention is not limited to these illustrations.

(Colon cancer cell line)
As colon cancer cell lines, HCT116 cells and DLD-1 cells purchased from ATCC were used. Each cell was cultured in IMDM medium (Invitrogen) supplemented with 10% fetal calf serum (Invitrogen) in a 5% CO ₂ atmosphere.

[Example 1]
[Gene expression analysis of human colon cancer tissue]
At the Chiba University Hospital, a colon cancer tissue extracted from 13 colorectal cancer patients and the surrounding non-cancerous tissue were collected as specimens. Total RNA was extracted from each tissue using RNeasy Plus kit (Qiagen).

Of the collected samples, 12 samples were used to comprehensively analyze the difference in gene expression level between colon cancer tissue and non-cancerous tissue by microarray analysis. ARNA was prepared from TotalRNA using Gene Chip (R) 3 'IVT Express Kit (Affymetrix), all-genotype microarray (Gene Chip Human Genome U133 Plus 2.0 Array) (Affimetrix) and probe set Were used for microarray analysis. The expression values of all genes obtained by such microarray analysis were normalized by the median value using GeneSpringGX software (Agilent Technology), and the expression ratio of FAM83H having a high expression level was determined for each patient in colorectal cancer. Tissue: Calculated as a non-cancerous tissue and the log ₂ values plotted are shown in FIG. Significant difference test was performed by Student's t test (corresponding). The results are shown in FIG.

The expression of FAM83H mRNA was identified by microarray analysis using the 226129_at probe set. In order to examine the reproducibility, the FAM83H gene was newly added to 10 samples among the samples used in the microarray analysis. Eleven colorectal cancer tissues plus one specimen and the surrounding non-cancerous tissues were used as specimens, and Power CYBR (R) Green reagent and Applied Biosystems 7900HT Fast Real Time PCR System (manufactured by Applied Biosystems) Was analyzed by qPCR. The primer sequences used were forward primer 5′-CGACAAGTGCCGTGTCAACC-3 ′ (SEQ ID NO: 5) and reverse primer 5′-ACTTCCCAGTGCGGCAGTAG-3 ′ (SEQ ID NO: 6). As primers for actin gene for expression level correction, forward primer 5′-AGAAAATCTGGCACCACACC-3 ′ (SEQ ID NO: 7) and reverse primer 5′-GGGGTGTTGAAGGTCTCAAA-3 ′ (SEQ ID NO: 8) were used. The expression ratio of FAM83H for each patient was calculated as colon cancer tissue: non-cancerous tissue, and the log ₂ values were plotted. FIG. 1 (b) shows the result.

(result)
As is clear from FIG. 1 (a), it was suggested by microarray analysis that the FAM83H mRNA expression level was significantly increased in the colon cancer tissue as compared with the non-cancerous tissue. Further, as is clear from FIG. 1 (b), it is shown by qPCR analysis that the FAM83H mRNA expression level is significantly increased in the colon cancer tissue as compared with the non-cancerous tissue, and the expression of the FAM83H gene is It was confirmed to be specific.

[Example 2]
[Colon cancer cell proliferation test]
In order to examine the effect of suppressing the transcription of the FAM83H gene, examination was performed using FAM83H siRNA. Details of the sequence of the FAM83H siRNA used are as follows.
FAM83H siRNAS1
5'-ACACGAAGGCCAUUCUGGAGCAGAU-3 '(SEQ ID NO: 1)
3'-UGUGCUUCCGGUAAGACCUCGUCUA-5 '(SEQ ID NO: 2)
FAM83H siRNAS2
5'-CAACGCCUUGUACAGCAGCAACCUU-3 '(SEQ ID NO: 3)
3'-GUUGCGGAACAUGUCGUCGUUGGAA-5 '(SEQ ID NO: 4)

  A cell proliferation test of FAM83H knockdown colon cancer cells (hereinafter sometimes referred to as “FAM83H knockdown HCT116 cells”) was performed by introducing FAM83H siRNA into the HCT116 cells. Ten thousand HCT116 cells were seeded in a 12-well dish and cultured for 2 days. The two types of FAM83H siRNAS1 and siRNAS2 were transfected into HCT116 cells using lipofectamine RNAiMAX (Invitrogen), respectively, and FAM83H knockdown HCT116 cells into which FAM83H siRNAS1 was introduced (hereinafter referred to as “S1 cells”). And FAM83H knockdown HCT116 cells into which siRNA S2 of FAM83H was introduced (hereinafter also referred to as “S2 cells”). As control siRNA, Medium GC Duplex # 2 (manufactured by Invitrogen) was used and similarly introduced into HCT116 cells as control cells.

  The viable cell numbers of S1, S2 and control cells were counted every 24, 48 and 72 hours after introduction of siRNA, respectively, using trypan blue staining. The same experiment was performed three times, and the average number of live cells and the standard deviation were calculated from the results of the three experiments and plotted. The results are shown in FIG.

(result)
As is clear from FIG. 2, it was confirmed that the numbers of S1 cells and S2 cells were significantly decreased by Student's t-test (no correspondence) compared to control cells (*, p <0.005). **, p <0.00005; ***, p <0.0005). Specifically, the number of S1 cells 72 hours after introduction of siRNA is about 5% of the control cells, the number of S2 cells is 30% of the control cells, and the introduction of FAM83H siRNA. There was a significant cell number reduction effect. Therefore, it was suggested that FAM83H is necessary for the growth and survival of colon cancer cells.

[Example 3]
[Western blot analysis]
Western blot analysis of the above S1 cells was carried out 2 days after siRNA introduction. Extraction of HCT116 cell total protein was performed by the Laemmli method using an SDS sample buffer. Proteins were separated by 5-20% gradient polyacrylamide gel electrophoresis (DRC) (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Milipore, Bedford, Mass.). The membrane was blocked using Blocking One (Nacalai Tesque). Anti-PARP degradation product antibody (# 5625, manufactured by Cell Signaling Technology), anti-caspase-3 degradation product antibody (# 9664, manufactured by Cell Signaling Technology), and anti-FAM83H antibody (HPA024604, manufactured by Sigma) As a control, an anti-actin antibody (sc-1615, manufactured by Santa Cruz Biotechnology) was added as a primary antibody, incubated, washed, and then HRP-labeled anti-goat IgG antibody (sc-2020, manufactured by Santa Cruz Biotechnology) ) And HRP-labeled anti-rabbit IgG antibody (NA934, manufactured by GE Healthcare) were added as a labeled secondary antibody, incubated, and then washed. Light was emitted by a chemiluminescence method using an ECL reagent (manufactured by GE Healthcare) and detected with a LAS-4000 image analyzer (manufactured by GE Healthcare). The results are shown in FIG.

(result)
As apparent from FIG. 3, fragmented PARP (cleaved PARP) and fragmented caspase-3 (cleaved caspase 3), which are known as apoptosis markers, were detected in S1 cells, and the production of FAM83H protein was suppressed. Therefore, it was shown that FAM83H is necessary for the growth and survival of colon cancer cells, and that deletion of FAM83H leads colon cancer cells to apoptosis.

[Example 4]
[Immunostaining 1]
Cells (hereinafter also referred to as “D-S1 cells”) in which siRNA S1 of FAM83H is introduced into the S1 cells and S2 cells, and DLD1 cells using Lipofectamine RNAiMAX (manufactured by Invitrogen), and Medium to DLD1 cells. The function of FAM83H was further examined by introducing GC Duplex # 2 and immunostaining the cells (hereinafter also referred to as “D-control cells”). In the immunostaining, each of the above cells was fixed by immersing in 100% methanol cooled to −20 ° C. for 1 minute, blocked with 3% bovine serum albumin, then reacted in the order of primary antibody and secondary antibody. The next antibody solution was mixed with DAPI (4 ′, 6-diamidino-2-phenylindole) to co-stain DNA. The cells were observed using a confocal microscope (LSM710, manufactured by Zeiss). As a primary antibody, an anti-keratin 18 antibody (MS-142-P0, manufactured by Thermo Scientific) and an anti-α-tubulin antibody (YOL1 / 34, manufactured by Santa Cruz Biotechnology) were used. Alexa488-labeled anti-mouse IgG antibody and Alexa594-labeled anti-rat IgG antibody (both manufactured by Invitrogen) were used. A similar experiment was performed three times, and the average value and standard deviation of the proportion of mitotic cells forming normal spindles were calculated from the results of the three experiments and plotted. The results are shown in FIGS. 4 (a) and (b).

(result)
As apparent from FIG. 4 (b), according to Student's t-test (no correspondence), the normal formation rate of the spindle in the cell division phase in S1 cells, S2 cells, and D-S1 cells was found to be in the control cells (D- (*, P <1 × 10 ⁻⁶ ; **, p <0.0005). Further, as is clear from FIG. 4 (a), in the image obtained by observing cells in which siRNAS1 of FAM83H was introduced using a confocal microscope, it is observed that the spindle is normally formed in the control cells. However, abnormalities in keratin cytoskeleton and spindle formation were observed in S1 cells, as is apparent from the staining of keratin 18 and α-tubulin.

[Example 5]
[Immunostaining 2]
In the same manner as in Example 4 except that anti-lamin B2 antibody (10895-1-AP, manufactured by ProteinTech Group) and anti-α-tubulin antibody (DM1A, manufactured by Sigma) were used as primary antibodies. Immunostaining was performed on S1, S2 and control cells. The cells were observed using a confocal microscope. Two days after siRNA introduction, the same experiment was performed three times, and the average value and standard deviation of the proportion of mitotic cells in which lamin B2 formed a normal matrix were calculated from the results of the three experiments and graphed. . The results are shown in FIGS. 5 (a) and (b).

(result)
As apparent from FIG. 5 (b), the normal formation rate of the spindle matrix of lamin in the cell division phase of S1 cells and S2 cells is significantly different from the control cells by Student's t test (no correspondence). Low (*, p <1 × 10 ⁻⁵ ; **, p <0.0005) was confirmed. Specifically, the number of normal spindle-forming cells in the cell division phase of S1 cells 2 days after introduction of siRNA is about 5% of the control cells, and the number of normal spindle-forming cells in the cell division phase of S2 cells is It was about 22% of the control cells, and there was a remarkable spindle formation inhibitory effect by introduction of FAM83H siRNA. As is clear from FIG. 5 (a), in the control cells, it is observed that lamin B2 forms a matrix outside the spindle and the spindle is normally formed. In S1 cells, lamin B2 Were observed to aggregate at the periphery of the cell, inhibiting the formation of the spindle matrix of lamin. As described above, FAM83H is thought to promote cancer cell division through the formation of a lamin spindle matrix. Spindle dysplasia induces cell death through the cessation of cell division and abnormal chromosome distribution, and is considered to be one of the mechanisms that induce cell growth inhibition.

[Example 6]
[Wound healing analysis 1]
In order to examine the functions related to the movement of the S1 cells and S2 cells, Wound healing analysis was performed. S1 cells and S2 cells were wounded on the cell sheet 24 hours after the introduction of siRNA, and the cells migrated and filled in the formed cell-free surface, immediately after the wound and 24 hours later Observed below. The same experiment was performed three times, and the average value and standard deviation of the movement distance of the cells located at the tip of the cell-free surface of the cell sheet were calculated from the results of the three experiments and graphed. The results are shown in FIGS. 6 (a) and (b).

(result)
As is clear from FIG. 6 (b), it was shown that the moving distance of the cell sheet tip decreased significantly with respect to S1 cells and S2 cells compared to control cells (*, p <0.001; **, p <0.005). Specifically, the moving distance of S1 cells was about one-fourth that of control cells, and the moving distance of S2 cells was about one-half that of control cells. In addition, as is clear from FIG. 6 (a), the control cells migrated substantially on the cell-free surface after 24 hours, whereas in S1 cells, the movement speed of the cell sheet tip decreased significantly. It was confirmed that the cell motility function was suppressed.

[Example 7]
[Wound healing analysis 2]
The S1 cells were wound on the cell sheet after 36 hours from the introduction of siRNA, and 6 hours later, they were immunostained with an antibody against α-tubulin and observed with a confocal microscope. About 100 cells per experiment, the length of the leaf-like temporary foot located at the tip of the cell sheet was measured from the staining of α-tubulin, and the average value and standard deviation were obtained from the results of three experiments. And was graphed. The results are shown in FIGS. 7 (a) and (b).

(result)
As is clear from FIG. 7 (b), it was shown that the length of the leaf-like temporary foot of the cell sheet tip decreased significantly with respect to the S1 cell as compared with the control cell (*, p <0. 05). Further, as apparent from FIG. 7 (a), when the tip of the cell sheet was observed under the confocal microscope, it was shown that the formation of leaf-like temporary feet of cells located at the tip of the cell sheet was suppressed in S1 cells. It was. The lamellipodia is formed when cells move, and the suppression of the formation of lamellipodia suggests that the movement of colon cancer cells is suppressed.

[Example 8]
[Wound healing analysis 3]
The S1 cells were wound on the cell sheet after 36 hours from the introduction of siRNA, and 6 hours later, the cells were immunostained with an antibody against α-tubulin and an antibody against keratin 8, and observed with the confocal microscope. The results are shown in FIG.

(result)
As is clear from FIG. 8, keratin filament depolymerization was induced with cell migration in the control tip cells, but it was observed that S1 cells promoted bundling of the keratin skeleton and suppressed migration. It was. It is considered that one of the mechanisms for suppressing the migration ability of cancer cells is to suppress the reconstruction of the keratin skeleton necessary for cell migration.

[Example 9]
As the FAM83H siRNAS3, the same experiment as in each of the above examples was performed using the following sequences. As a result, a colon cancer cell growth inhibitory effect, an apoptosis inducing effect, and a motor function inhibitory effect were observed. The sequence of the double-stranded DNA composing FAM83H siRNAS3 is as follows.
FAM83H siRNA S3
5'-UCCACUCGAAGAGGAAGUCCAACUA-3 '(SEQ ID NO: 10)
3'-AGGUGAGCUUCUCCUUCAGGUUGAU-5 '(SEQ ID NO: 11)

Claims (5)

A therapeutic agent for colorectal cancer comprising a FAM83H protein expression inhibitor or function inhibitor as an active ingredient. The therapeutic agent for colorectal cancer according to claim 1, wherein the FAM83H protein expression inhibitor is FAM83H siRNA. SiRNA consisting of a double-stranded RNA sequence containing the RNA sequence shown in SEQ ID NO: 1 and its complementary sequence, or a double-stranded RNA sequence containing the RNA sequence shown in SEQ ID NO: 3 and its complementary sequence The therapeutic agent for colorectal cancer according to claim 2, which is an siRNA comprising: The agent for treating colorectal cancer according to claim 2 or 3, wherein FAM83H siRNA is incorporated into a vector. A method for collecting data for evaluating the risk of colorectal cancer onset / relapse comprising the following steps (a) and (b).
(A) quantifying FAM83H protein in a biological sample derived from a subject;
(B) a step of comparing and evaluating the amount of FAM83H protein in the biological sample and the amount of FAM83H protein in a biological sample derived from a colon cancer non-affected person or a colon cancer affected person;