CN115612745A - Application of IPO7 in colorectal cancer treatment and prognosis prediction - Google Patents

Abstract

The invention discloses application of IPO7 in colorectal cancer treatment and prognosis prediction, provides a product for diagnosing colorectal cancer/diagnosing colorectal cancer metastasis/predicting colorectal cancer prognosis, and also provides a pharmaceutical composition for treating colorectal cancer. According to the invention, extensive and in-depth experiments prove that IPO7 can effectively diagnose colorectal cancer and the metastasis of the colorectal cancer and can effectively predict the prognosis of the colorectal cancer, and proliferation, migration, invasion, apoptosis and division experiments prove that the effect of treating the colorectal cancer can be achieved by inhibiting the expression of IPO7.

Description

Application of IPO7 in colorectal cancer treatment and prognosis prediction

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to application of IPO7 in colorectal cancer treatment and prognosis prediction.

Background

Colorectal Cancer (CRC) has become the third largest malignancy worldwide, second only to lung and female breast Cancer, and has become the second leading cause of Cancer-related death in 2020. Because colorectal cancer is hidden, specific symptoms and signs are not always present in the early stage, most patients are in the middle and late stage when visiting the doctor, even have multiple metastases of the whole body, the chance of surgical treatment is lost, and the colorectal cancer is the main reason for death. Even after surgical treatment, the patient's prognosis varies significantly. Therefore, in order to develop a comprehensive and individual treatment scheme for patients, improve the prognosis level of patients, and find effective molecular markers and specific drug targets for colorectal cancer diagnosis and prognosis.

Disclosure of Invention

In order to make up the defects of the prior art, the invention provides the molecular marker IPO7 which can realize the diagnosis, the metastasis diagnosis, the treatment and the prognosis prediction of colorectal cancer.

In order to realize the purpose, the invention adopts the following technical scheme:

a first aspect of the invention provides a product for diagnosing colorectal cancer/diagnosing colorectal cancer metastasis/predicting colorectal cancer prognosis, the product comprising an agent capable of detecting the expression level of IPO7.

Further, the reagent is selected from an oligonucleotide probe that specifically recognizes the IPO7 gene, a primer that specifically amplifies the IPO7 gene, or a binding agent that specifically binds to a protein encoded by the IPO7 gene.

Further, the product comprises a chip, a kit or a nucleic acid membrane strip.

A second aspect of the invention provides a pharmaceutical composition for the treatment of colorectal cancer, comprising an inhibitor of IPO7.

Further, the inhibitor inhibits proliferation, migration, invasion, apoptosis and/or division of colorectal cancer.

Further, the inhibitor comprises a nucleic acid inhibitor and a protein inhibitor.

A third aspect of the present invention provides a method of screening for a candidate drug for the treatment of colorectal cancer, the method comprising: treating the culture system expressing or containing the IPO7 gene or its encoded protein with a substance to be screened; and detecting expression or activity of the IPO7 gene or protein encoded thereby in said system; wherein, when the substance to be screened inhibits the expression level or activity of the IPO7 gene or a protein encoded by the same, the substance to be screened is a candidate drug for the treatment of colorectal cancer.

A fourth aspect of the invention provides a system/device for diagnosing/diagnosing metastasis/predicting prognosis of colorectal cancer, the system/device comprising:

an acquisition unit: for obtaining the expression level of IPO7 in the sample;

a processing unit: and obtaining a diagnosis/diagnosis metastasis/prognosis prediction result of the colorectal cancer according to the expression condition of the IPO7.

A fifth aspect of the present invention provides a computer readable storage medium having stored thereon a computer program which, when executed by a processor, implements the system/apparatus of the fourth aspect of the present invention.

A sixth aspect of the invention provides the use of any one of:

(1) Use of an inhibitor of IPO7 or a pharmaceutical composition according to the second aspect of the invention in the manufacture of a product for the treatment of colorectal cancer;

(2) Use of a reagent for detecting the expression level of IPO7 or a product according to the first aspect of the invention for the manufacture of a tool for diagnosing colorectal cancer/diagnosing colorectal cancer metastasis/predicting colorectal cancer prognosis;

(3) Use of IPO7 for screening a candidate drug for the treatment of colorectal cancer;

(4) Use of IPO7 for promoting macrophage differentiation;

(5) Use of IPO7 in the construction of a system/device for diagnosing colorectal cancer/diagnosing colorectal cancer metastasis/predicting colorectal cancer prognosis;

(6) Use of IPO7 in the construction of a computer readable storage medium for diagnosing colorectal cancer/diagnosing colorectal cancer metastasis/predicting colorectal cancer prognosis.

The invention has the advantages and beneficial effects that:

the molecular marker IPO7 provided by the invention can effectively diagnose colorectal cancer and metastasis of the colorectal cancer, and can achieve the purpose of treating the colorectal cancer by inhibiting proliferation, migration, invasion, apoptosis and division of colorectal cancer cells, and in addition, the IPO7 can well predict the prognosis of the colorectal cancer.

Drawings

FIG. 1 is a protein expression difference volcano diagram, wherein 1A is a protein expression difference diagram of a colorectal cancer primary focus and a normal tissue, and 1B is a protein expression difference diagram of a colorectal cancer liver metastasis and a primary focus;

FIG. 2 is a Wien diagram of groups of co-up/down regulated proteins;

FIG. 3 is a graph showing the difference in protein expression of IPO7 in colorectal normal tissue, primary foci and liver metastasis, wherein 3A is a graph showing the result of immunohistochemical staining and 3B is a graph showing the statistics of immunohistochemical scores;

FIG. 4 is a graph showing the difference in protein expression of IPO7 in colorectal cancer and paracarcinoma, wherein 4A is a graph showing the result of immunohistochemical staining and 4B is a graph showing the statistics of immunohistochemical score;

FIG. 5 is a graph of the relationship between IPO7 and overall survival of colorectal cancer patients;

FIG. 6 is a diagram of the detection of the knockdown efficiency of IPO7 and the construction of stable transgenic cell lines, wherein 6A is a diagram of the knockdown efficiency of siRNA to IPO7 in DLD1, 6B is a diagram of the mRNA expression level of IPO7 in stable transgenic cell lines, 6C is a Western immunoblot diagram of IPO7 in stable transgenic cell lines, and 6D is a diagram of lentivirus-transfected stable transgenic cell lines with knockdown of IPO 7;

FIG. 7 is a graph of the proliferative effect of knockdown of IPO7 on DLD1 cells;

FIG. 8 is a graph of the clonality impact of knockdown of IPO7 on DLD1 cells, where 8A is a clonality map and 8B is a clonality statistic;

FIG. 9 is a graph of the effect of knockdown of IPO7 on the migratory capacity of DLD1 cells, where 9A is a score healing profile, 9B is a 24h wound healing rate statistic, and 9C is a 48h wound healing rate statistic;

fig. 10 is a graph showing the effect of knocking down IPO7 on the migration and invasion abilities of DLD1 cells, wherein 10A is a DLD1 cell migration ability map, 10B is a DLD1 cell migration ability statistical map, 10C is an invasion ability map of DLD1 cells, and 10D is an invasion ability statistical map of DLD1 cells;

fig. 11 is a graph showing the effect of knocking-down IPO7 on DLD1 cell cycle, in which 11A is a graph showing the distribution ratio of the cells in different stages of the shmc, 11B is a graph showing the distribution ratio of the cells in different stages of the shmpo 7-1, 11C is a graph showing the distribution ratio of the cells in different stages of the shmpo 7-2, 11D is a graph showing the distribution statistics of the cells in G1 stage, 11E is a graph showing the distribution statistics of the cells in G2 stage, and 11F is a graph showing the distribution statistics of the cells in S stage;

fig. 12 is a graph of the apoptotic effect of knockdown of IPO7 on DLD1 cells, where 12A is a graph of distribution ratio of the shmc at different apoptotic stages, 12B is a graph of distribution ratio of the ship IPO7-1 at different apoptotic stages, 12C is a graph of distribution ratio of the ship IPO7-2 at different apoptotic stages, 12D is a graph of early apoptotic cell distribution, and 12E is a graph of late apoptotic cell distribution;

FIG. 13 is a graph showing the correlation between IPO7 and macrophage infiltration, in which 13A is a graph showing the correlation between low expression of IPO7 and macrophage infiltration, and 13B is a graph showing the correlation between high expression of IPO7 and macrophage infiltration.

Detailed Description

The following provides definitions of some terms used in this specification. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The invention provides a product for diagnosing colorectal cancer/diagnosing colorectal cancer metastasis/predicting colorectal cancer prognosis, the product comprising an agent capable of detecting the expression level of IPO7.

IPO7 includes wild type, mutant or fragments thereof. The term encompasses full-length, unprocessed IPO7, as well as any form of IPO7 that results from processing in a cell. The term encompasses naturally occurring variants (e.g., splice variants or allelic variants) of IPO7. The term encompasses for example human IPO7 as well as IPO7 from any other vertebrate source, including IPO7 of mammals such as primates and rodents (e.g. mice and rats), gene ID:10527.

In the present invention, diagnosis and variations of the term thereof refer to the discovery, judgment or cognition of an individual's health state or condition based on one or more signs, symptoms, data or other information associated with the individual. The health status of an individual may be diagnosed as healthy/normal (i.e., absence of a disease or condition), or may be diagnosed as unhealthy/abnormal (i.e., presence of an assessment of a disease or condition or characteristic). The above terms diagnosis and variants of the terms include early detection of disease associated with a particular disease or condition; the nature or classification of the disease; discovery of progression, cure or recurrence of disease; discovery of response to disease after treatment or therapy of an individual.

In the present invention, metastasis refers to a process in which cancer cells originating from one organ or part of the body migrate to another part of the body with or without transport of body fluids and continue to repeat. The metastasized cells can subsequently form tumors that can metastasize further. Metastasis is therefore called the spread of cancer, from the part of the body where it originally occurred to other parts of the body.

In an embodiment of the invention, metastasis refers to hepatic metastasis of colorectal cancer.

In the present invention, prognosis refers to anticipation with respect to medical development (e.g., long-term survival likelihood, disease-free survival rate, etc.), including positive prognosis including disease progression such as relapse, colorectal cancer growth, metastasis, and drug-resistant mortality, or negative prognosis including disease remission such as disease-free state, disease improvement such as colorectal cancer regression or stabilization.

In the present invention, expression level or level of expression generally refers to the amount of a biomarker in a biological sample. Expression generally refers to the process by which information (e.g., gene coding and/or epigenetic information) is converted into structures present and operating in a cell. Thus, as used herein, expression may refer to transcription into a polynucleotide, translation into a polypeptide, or even polynucleotide and/or polypeptide modification (e.g., post-translational modification of a polypeptide). Transcribed polynucleotide, translated polypeptide or polynucleotide and/or polypeptide modified (e.g., post-translational modification of a polypeptide) fragments should also be considered expressed, whether they are derived from a transcript produced by alternative splicing or a degraded transcript, or from post-translational processing of a polypeptide (e.g., by proteolysis). Expressed genes include genes that are transcribed into a polynucleotide (e.g., mRNA) and then translated into a polypeptide, as well as genes that are transcribed into RNA but not translated into a polypeptide (e.g., transport and ribosomal RNA).

Further, the reagent for detecting the expression level of IPO7 is selected from an oligonucleotide probe specifically recognizing the IPO7 gene, a primer specifically amplifying the IPO7 gene, or a binding agent specifically binding to a protein encoded by the IPO7 gene.

In the present invention, a probe refers to a molecule capable of binding to a specific sequence or subsequence or other portion of another molecule. Unless otherwise indicated, a probe generally refers to a polynucleotide probe that is capable of binding to another polynucleotide (often referred to as a target polynucleotide) by complementary base pairing. Depending on the stringency of the hybridization conditions, a probe can bind to a target polynucleotide that lacks complete sequence complementarity to the probe. The probe may be directly or indirectly labeled, and includes within its scope a primer. Hybridization modes include, but are not limited to: solution phase, solid phase, mixed phase or in situ hybridization assays.

In the present invention, a primer refers to a short nucleic acid sequence, which can form a base pair (basepair) with a complementary template (template) and serves as an origin of replication template, as a nucleic acid sequence having a short free 3 'terminal hydroxyl group (free 3' hydroxyl group).

In the present invention, a binding agent refers to a naturally occurring or non-naturally occurring molecule that specifically binds a target. Examples of specific binding agents include, but are not limited to, proteins, peptides, nucleic acids, carbohydrates, and lipids.

In the present invention, the binding agent that specifically binds to the protein encoded by the IPO7 gene is, for example, a receptor for the protein IPO7, a lectin that binds to the protein IPO7, an antibody against the protein IPO7, a peptide antibody (peptidebody) against the protein IPO7, a bispecific dual binding agent or a bispecific antibody format. Specific examples of specific binding agents are peptides, peptidomimetics, aptamers, spiegelmers, dappin, ankyrin repeat proteins, kunitz-type domains, antibodies, single domain antibodies and monovalent antibody fragments.

Further, the product comprises a chip, a kit or a nucleic acid membrane strip.

In the invention, the chip comprises a gene chip and a protein chip, wherein the gene chip comprises an oligonucleotide probe aiming at an IPO7 gene and used for detecting the transcription level of the IPO7 gene, and the protein chip comprises a specific binding agent of the IPO7 protein; the kit comprises a gene detection kit and a protein detection kit, wherein the gene detection kit comprises a reagent or a chip for detecting the IPO7 gene transcription level, and the protein detection kit comprises a reagent or a chip for detecting the IPO7 protein expression level.

In the present invention, a kit refers to a set of components provided in the context of a system for sequencing nucleotides and/or isolating nucleotide sequences and/or diagnosing a subject with a disease or infection based on the presence, absence and/or amount of expressed nucleotide sequences from a sample or cell.

The kit comprises a gene detection kit and a protein detection kit, wherein the gene detection kit comprises a reagent or a chip for detecting the IPO7 gene transcription level, and the protein detection kit comprises a reagent or a chip for detecting the IPO7 protein expression level.

The kit of the invention comprises one or more substances from the following group: container, instructions for use, positive control, negative control, buffer, adjuvant or solvent.

The kit can be also attached with an instruction book of the kit, wherein the instruction book describes how to adopt the kit for detection, how to judge the tumor development by using the detection result and how to select a treatment scheme.

The components of the kit may be packaged in the form of an aqueous medium or in lyophilized form. Suitable containers in the kit generally include at least one vial, test tube, flask, pet bottle, syringe, or other container in which a component may be placed and, preferably, suitably aliquoted. Where more than one component is present in the kit, the kit will also typically comprise a second, third or other additional container in which the additional components are separately disposed. However, different combinations of components may be contained in one vial. The kits of the invention also typically include a container for holding the reactants, sealed for commercial sale. Such containers may include injection molded or blow molded plastic containers in which the desired vials may be retained.

The chip, kit or membrane strip of the present invention can be used for detecting the expression level of a plurality of genes or proteins including IPO7 gene or protein and expression products thereof (e.g., a plurality of genes or proteins associated with colorectal cancer). The multiple markers of the colorectal cancer are detected simultaneously, so that the accuracy of colorectal cancer diagnosis or prognosis prediction can be greatly improved.

The present invention provides a pharmaceutical composition for the treatment of colorectal cancer, comprising an inhibitor of IPO7.

In the present invention, the inhibitor refers to any substance that can reduce the activity of the IPO7 protein, reduce the stability of the IPO7 gene or protein, down-regulate the expression of the IPO7 protein, reduce the effective acting time of the IPO7 protein, or inhibit the transcription and translation of the IPO7 gene, and these substances can be used in the present invention as a substance useful for down-regulating IPO7, and thus can be used for preventing or treating colorectal cancer.

In the present invention, the inhibitor includes a nucleic acid inhibitor and a protein inhibitor. Wherein the nucleic acid inhibitor is selected from: an interfering molecule capable of inhibiting the expression or gene transcription of IPO7 gene, which targets IPO7 or its transcript, comprising: shRNA (small hairpin RNA), small interfering RNA (siRNA), dsRNA, microrna, antisense nucleic acid, or a construct capable of expressing or forming said shRNA, small interfering RNA, dsRNA, microrna, antisense nucleic acid. The protein inhibitor is selected from substances that specifically bind to the IPO7 protein, such as antibodies or ligands that are capable of inhibiting the activity of the IPO7 protein.

In an embodiment of the invention, the inhibitor is selected from nucleic acid inhibitors.

In a particular embodiment of the invention, the nucleic acid inhibitor is selected from the group consisting of shRNA (small hairpin RNA) and small interfering RNA (siRNA).

The pharmaceutical compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. Oral administration or injection administration is preferred. The pharmaceutical composition of the present invention may contain any of the usual non-toxic pharmaceutically acceptable carriers, adjuvants or excipients.

The medicaments of the invention can also be combined with other medicaments for the treatment of colorectal cancer, and the other therapeutic compounds can be administered simultaneously with the main active ingredient (e.g. an inhibitor of IPO 7), even in the same composition. Other therapeutic compounds may also be administered alone in a composition or dosage form different from the main active ingredient. A partial dose of the main ingredient (e.g. an inhibitor of IPO 7) may be administered simultaneously with the other therapeutic compound, while the other dose may be administered separately.

The pharmaceutical compositions of the present invention also include a pharmaceutically acceptable carrier.

Pharmaceutically acceptable carriers include, but are not limited to, diluents, binders, surfactants, humectants, adsorbent carriers, lubricants, fillers, disintegrants.

Wherein the diluent is lactose, sodium chloride, glucose, urea, starch, water, etc.; binders such as starch, pregelatinized starch, dextrin, maltodextrin, sucrose, acacia, gelatin, methyl cellulose, carboxymethyl cellulose, ethyl cellulose, polyvinyl alcohol, polyethylene glycol, polyvinyl pyrrolidone, alginic acid and alginates, xanthan gum, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, and the like; surfactants such as polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, glyceryl monostearate, cetyl alcohol, etc.; humectants such as glycerin, starch, etc.; adsorption carriers such as starch, lactose, bentonite, silica gel, kaolin, and bentonite, etc.; lubricants such as zinc stearate, glyceryl monostearate, polyethylene glycol, talc, calcium stearate and magnesium stearate, polyethylene glycol, boric acid powder, hydrogenated vegetable oil, sodium stearyl fumarate, polyoxyethylene monostearate, monolaurocyanate, sodium lauryl sulfate, magnesium lauryl sulfate, etc.; fillers such as mannitol (granular or powder), xylitol, sorbitol, maltose, erythrose, microcrystalline cellulose, polymeric sugar, coupling sugar, glucose, lactose, sucrose, dextrin, starch, sodium alginate, laminarin powder, agar powder, calcium carbonate, sodium bicarbonate, etc.; disintegrating agent such as crosslinked vinylpyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl methyl, crosslinked sodium carboxymethyl cellulose, and soybean polysaccharide.

The present invention provides a system/apparatus for diagnosing colorectal cancer/diagnosing colorectal cancer metastasis/predicting colorectal cancer prognosis, the system/apparatus comprising:

an acquisition unit: for obtaining the expression level of IPO7 in the sample;

a processing unit: obtaining the diagnosis/diagnosis metastasis/prognosis prediction result of the colorectal cancer according to the expression condition of the IPO7.

If the expression level of the IPO7 is significantly up-regulated compared with a normal sample, the diagnosis result is colorectal cancer;

compared with the primary focus of colorectal cancer, if the expression level of IPO7 is remarkably up-regulated, the diagnosis result is colorectal cancer liver metastasis;

a high protein expression level of IPO7 indicates a poor prognosis for colorectal cancer.

The invention is further illustrated with reference to the following specific examples. It should be understood that the particular embodiments described herein are presented by way of example and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention.

Example 1 expression of IPO7 in colorectal cancer

1.1 Experimental materials

Tissue specimen information for proteomics: collecting paired paracancer normal tissues (more than 10cm away from the edge of a tumor), a primary colorectal cancer focus and a liver metastasis focus of colorectal cancer patients who are subjected to gastrointestinal surgical operation excision in Beijing university Hospital and do not receive preoperative neoadjuvant radiotherapy and chemotherapy and are combined with simultaneous liver metastasis in 2019-2020-7 months (3 patients have obvious clinical symptoms such as combined bleeding and obstruction, and the primary colorectal cancer focus and the liver metastasis focus can be excised, so that right hemicolectomy and liver partial excision are adopted, and preoperative neoadjuvant therapy is not received). Dividing all the obtained tissue specimens into two parts, quickly freezing one part in liquid nitrogen 30 minutes after the tissue specimens are separated, and then storing in an ultra-low temperature refrigerator at minus 80 ℃; the other part was fixed in 4% tissue cell fixative and then paraffin embedded. The final proteomics study was included in a total of 3 tissue specimens from colorectal cancer patients, the detailed clinical pathology data of which are shown in table 1. Extracting protein in corresponding tissues, and performing TMT (TMT-labeled quantitative proteomics) research after quality control is qualified.

Organizing a chip: 10 cases of paraffin specimens of colorectal cancer tissues, liver metastasis tissues and paracancer normal tissues of patients subjected to gastrointestinal surgery simultaneous colorectal cancer resection and liver resection in Beijing university Hospital in 1 month to 2021 month in 2019 are selected, and all the patients do not receive chemoradiotherapy before operation.

Large cohort paraffin specimens of colorectal cancer and paired paracancerous normal tissues: collecting colorectal cancer specimens which are surgically removed in the Beijing university Hospital during the 12 th 2014-2016 12 th month of gastrointestinal surgery, and taking the specimens into the standard that the specimens are pathologically diagnosed as colorectal cancer; before operation, new auxiliary radiotherapy and chemotherapy and targeted therapy are not received; contains complete clinical pathological data (including sex, age, tumor size, histological type, differentiation degree, vascular cancer embolus, TNM stage, AJCC stage, etc.) and follow-up information (survival time and survival status). Patients with combined other tumors were excluded when diagnosing colorectal cancer. Together, 128 pairs of normal tissue specimens were included for colorectal cancer and its paired paracarcinoma. All specimens were prepared into tissue chips and stored in the surgery tumor research laboratory of people's hospital of Beijing university.

1.2 Experimental methods

1.2.1 proteomics analysis

The samples were standardized and subjected to omics analysis by Hangzhou Jingjie Biotechnology GmbH.

1.2.2 immunohistochemical staining

(1) Dewaxing and hydrating: baking the paraffin sections at 60 to 70 ℃ for 2 hours, putting the paraffin sections into fresh xylene, and soaking the paraffin sections for 10 minutes and multiplying the times by 3; after removing the redundant liquid, placing the mixture in absolute ethyl alcohol, and soaking for 3 minutes and 3 times; removing redundant liquid, and soaking in 95% ethanol for 3 min × 2 times; removing redundant liquid, placing in 75% ethanol, soaking for 3 minutes and multiplying 2 times; washing with distilled water for 1 min, and placing in PBS buffer solution;

(2) Antigen retrieval: preheating an antigen repairing solution (1 x) to 95-100 ℃ before use, soaking the slices in the antigen repairing solution, heating at 95 ℃ for 15 minutes, cooling to room temperature within about 20-30 minutes, washing with a PBS (phosphate buffer solution) for 1-2 times, and washing for 3-5 minutes each time;

(3) Blocking endogenous peroxidase: according to the size of the tissue, adding a proper amount of endogenous peroxidase blocker (covering all the tissue), incubating for 10 minutes at room temperature, and washing for 3 minutes by 3 times by using PBS buffer solution;

(4) Dropping primary antibody: diluting the primary antibody with an antibody diluent according to a proper proportion according to a primary antibody specification, dropwise adding a proper amount of the primary antibody according to the tissue size, and standing overnight at 4 ℃; washing with PBS buffer for 3 min × 3 times;

(5) Dropwise adding a reaction enhancing solution: dripping a proper amount of reaction enhancing solution (only covering the tissue) and incubating for 20 minutes at room temperature; washing with PBS buffer for 3 min × 3 times;

(6) Dropwise adding an enhanced enzyme-labeled goat anti-rabbit IgG polymer: dripping a proper amount of an enhanced enzyme-labeled goat anti-rabbit IgG polymer, and incubating for 20 minutes at room temperature; washing with PBS buffer for 3 min × 3 times;

(7) Preparing DAB color development liquid: adding 1 drop (about 50 microliter) of reagent 1 (DAB concentrated solution) into 1ml of reagent 2 (DAB substrate solution), and uniformly mixing to prepare DAB working solution;

(8) Counterdyeing: washing with tap water, incubating with hematoxylin staining solution for 20 s, washing with tap water to remove floating color, differentiating with 0.5% hydrochloric acid and ethanol, and washing with tap water to turn blue;

(9) Dehydrating and transparency: soaking in 75% ethanol for 3 min × 2 times; after removing the redundant liquid, placing the mixture in 95 percent ethanol, and soaking for 3 minutes and 2 times; after removing the redundant liquid, placing the mixture in absolute ethyl alcohol, and soaking for 3 minutes and 3 times; after removing the redundant liquid, placing the mixture in fresh dimethylbenzene, and soaking the mixture for 10 minutes and 3 times;

(10) Sealing: after neutral gum is dripped, a cover glass is covered and a slide is sealed;

(11) And (4) interpretation of results: the tissue staining proportion and intensity are observed, two pathologists with abundant experience assist and double-blind film reading is carried out, and the experimental result interpretation is evaluated by adopting a semi-quantitative scoring standard: according to the proportion of the positive staining cells of the corresponding index (< 10% to be judged as 0 point, 10% -40% to be judged as 1 point, 40% -70% to be judged as 2 points and more than 70% to be judged as 3 points) and the positive staining intensity of the corresponding index (0 point represents no staining: 1 point represents staining yellow: 2 point represents staining yellow: 3 point represents staining yellow), the staining indexes are obtained by adding the scores of the positive staining cells and the positive staining intensity of the corresponding index, wherein 0-3 points represent weak expression, and 4-6 points represent strong expression.

2.2.2 statistical analysis

Statistical analysis and mapping were performed using SPSS22.0 and GraphPad Prism 8. And (4) after the homogeneity of variance test conforms to normal distribution, performing difference comparison between groups by adopting an independent sample t test.

1.3 results of the experiment

Proteomics results show that in the samples of the primary focus of colorectal cancer and normal tissues beside the cancer, the expression of 171 proteins is obviously up-regulated, and the expression of 100 proteins is obviously down-regulated; among liver metastasis tissues and primary foci of colorectal cancer, 92 proteins were significantly up-regulated, and 99 proteins were significantly down-regulated (fig. 1).

And screening the differential expression protein of the normal tissue from the proteomic result into tumor-associated protein, and further analyzing the expression of the tumor-associated protein in the liver metastasis of the colorectal cancer. It was found by plotting a wien graph for two groups of differentially expressed proteins that there were 1 co-upregulated protein (IPO 7) in the two groups of differentially expressed proteins, which may be a key tumor-associated protein affecting liver metastasis in colorectal cancer (fig. 2).

In 10 paired colorectal cancer primary foci, liver metastases, and normal colorectal epithelial tissues, IPO7 was expressed higher in colorectal cancer primary foci and liver metastases than in normal tissues, and in liver metastases than in tumor tissues, consistent with proprotein results (fig. 3).

128 colorectal cancer patients matched with the colorectal cancer and paracancerous normal tissues, and IPO7 in the tumor tissue of the colorectal cancer patients was significantly higher than that in the normal tissue (p < 0.001), further verifying the preliminary experimental results (FIG. 4).

Example 2 use of IPO7 in prognosis prediction of colorectal cancer

2.1 Experimental materials

Patients and tissues: the same experimental materials as in example 1,1.1, colorectal cancer and paracarcinoma normal tissue matched large-queue paraffin specimens.

2.2 Experimental methods

2.2.1 statistical analysis

The Kaplan-Meier method is adopted to draw a survival curve of the colorectal cancer patient, the significance test is carried out through a log-rank test, the relation of the clinical pathological indexes of the protein expression colorectal cancer patient is analyzed through a chi-square test, and the difference P <0.05 has statistical significance.

2.3 results of the experiment

Expression of IPO7 was clearly correlated with lymph node metastasis, distant metastasis and tumor stage in colorectal cancer patients (Table 2, p-straw 0.01). Simultaneous survival analysis showed that the overall survival rate of colorectal cancer patients with high IPO7 expression was significantly lower than that of low-expression patients (FIG. 5, p- <0.05)

Example 3 use of IPO7 in the treatment of colorectal cancer

3.1 Experimental materials

Cell line: the human colon cancer cell line DLD1 and the human embryonic kidney cell HEK293T are purchased from a national experimental cell resource sharing service platform.

3.2 Experimental methods

3.2.1 siRNA transfection

(1) Designing small interfering RNA aiming at the sequence of IPO7, wherein the sequence is shown in a table 3;

(2) Placing the siRNA dry powder in a refrigerator at the temperature of 20 ℃ below zero for storage, centrifuging for 5 minutes at 5000rpm before use, centrifuging RNA powder on the tube wall to the tube bottom, carefully opening the tube in an ultra-clean workbench, adding a proper amount of enzyme-free water to prepare a 10 mu M solution, and subpackaging for storage;

(3) Inoculating cells to be transfected into a 6-well plate one day before transfection, wherein the density is based on the cell fusion rate of 50% in the next day of transfection;

(4) Preparing a transfection mixed solution in an ultra-clean workbench, preparing two sterile enzyme-free centrifuge tubes (a tube and a tube B) for each group of siRNA, adding 5 mu l of siRNA and 200 mu l of Opti-MEM into the tube A, adding 9 mu l of RNAiMAX and 200 mu l of Opti-MEM into the tube B, then slowly adding the liquid in the tube B into the tube A, gently mixing uniformly, and standing for 10 to 15 minutes at room temperature;

(5) And (3) replacing a fresh culture medium in a 6-hole plate, slowly adding the transfection mixed solution, shaking uniformly, placing in an incubator for continuous culture, replacing the fresh culture medium after 24 hours, and carrying out subsequent experiments after 48 to 72 hours.

3.2.2 Lentiviral packaging and transfection

(1) Inoculating HEK293T cells into a 10cm cell culture dish, wherein the density is based on the cell fusion degree of 70-80% on the next day;

(2) Preparing a transfection mixed solution in an ultra-clean workbench, preparing two sterile enzyme-free centrifuge tubes (a tube and a tube B) for each plasmid to be transfected, adding 2.5 mu g shRNA plasmid and 5.0 mu l Lenti-Pac-HIV mixture into the tube A, adding 200 mu l Opti-MEM, uniformly mixing, adding 15 mu l Endofetin and 200 mu l OptiMEM into the tube B, slowly adding the liquid in the tube B into the tube A, slightly mixing, and standing at room temperature for 10 to 15 minutes;

(3) Replacing the fresh culture medium for the cells, adding the transfection mixed solution into the corresponding cells for continuous culture, and replacing the fresh culture medium after 8 hours;

(4) 48 hours after transfection, the virus-containing medium was collected in a 15ml centrifuge tube, centrifuged at 4 ℃ at 2000g for 10 minutes, and after centrifugation, the supernatant was filtered through a 0.45 μm filter to remove cell debris;

(5) According to the slow virus liquid: concentration reagent =5 volume of 1 lentiviral supernatant and concentration reagent were mixed and incubated overnight at 4 ℃;

(6) 3500g of the centrifugal virus liquid is centrifuged at 4 ℃ for 25 minutes, the supernatant is discarded after centrifugation, 1ml of DMEM is added into the sediment to regenerate the suspended slow virus sediment, and the slow virus liquid is subpackaged and stored at-80 ℃;

(7) Before the slow virus transfects the cells, the cells are paved in a culture dish of 6cm, and the density is based on 70-80% of the fusion degree in the next day of virus transfection;

(8) On the infection day, replacing the fresh culture medium, adding a proper amount of virus solution into the culture solution, adding Polybrene according to the proportion of 1/1000, continuously culturing for 24 hours, and then replacing the fresh culture medium for continuous culture;

(9) And observing the fluorescence expression intensity of the infected cells after infection for 48 to 72 hours, and screening the cells by using puromycin with proper concentration to obtain a stably transfected cell strain.

3.2.3 cell proliferation assay

(1) After the cells of the experimental group and the control group are digested, the cell density is adjusted to 10 ⁴ /ml；

(2) Each group of cells of the experimental group and the control group are provided with 6 repeated holes, 100 mul of single cell suspension is added into each hole of a 96-hole cell culture plate respectively, the cells are required to be uniformly mixed before the single cell suspension is sucked each time, the same amount of PBS buffer solution is added into blank holes around the cells to reduce the influence of the evaporation of the culture medium on the experimental result, 5 96-hole plates are continuously added, and all 96-hole plates are placed in a cell culture box for continuous culture;

(3) After the cells are adhered to the wall by paving the plate for 8 to 10 hours, replacing the culture medium in the culture holes with a fresh culture medium, adding 10 mu l of CCK8 reagent into each hole, uniformly mixing the CCK8 reagent and the culture medium in a ratio of 1;

(4) Taking out the culture plate after 2 hours, and detecting the absorbance of the culture hole at the wavelength of 450nm by using an enzyme-labeling instrument;

(5) The above operations were repeated after 24 hours, 48 hours, 72 hours, and 96 hours from the first measurement, and the absorbance of the cells of the experimental group and the control group was obtained at different time points.

3.2.4 clonogenic experiments

(1) After the cells of the experimental group and the control group are digested, the cell density is adjusted to 10 ³ Adding 500 mu l of cell suspension into each 6-well plate, and then supplementing the culture medium for continuous culture;

(2) Changing a fresh culture medium every 3 days, continuously culturing the cells for 8 to 10 days, observing the cells, and finishing culturing when the number of cells of each cell colony is 80 to 100;

(3) Discarding the culture medium, carefully cleaning the cells for 1-2 times by using PBS at room temperature, and adding 2ml of 4% tissue cell fixing solution into each hole to fix the cells for 15 minutes;

(4) And (3) discarding the fixing solution, carefully cleaning the solution for 1 to 2 times by using PBS, adding 2ml of crystal violet into each hole, dyeing the solution for 15 minutes, slowly washing the crystal violet dye solution by using ultrapure water, taking a picture after the culture plate is air-dried, and analyzing the result by using ImageJ.

3.2.5 scratch repair experiment

(1) Carrying out a scratch repair test by using a wound healing insert produced by Ibidi company, and placing the wound healing insert and tweezers required by the test under ultraviolet light for disinfection for 1 to 2 hours for later use before the test;

(2) Placing the wound healing inserts into a 12-hole plate, wherein each hole is provided with one wound healing insert, and after the wound healing inserts are placed, checking the adhesion degree of the inserts and a culture plate to avoid the inserts from loosening in the cell culture process;

(3) Digesting and centrifuging cells of an experimental group and a control group, and adjusting the cell density to 3 to 5 multiplied by 10 ⁵ Shaking up, adding 80 μ l of cell suspension into each well of the insert, and gently shaking the culture plate to distribute the cells uniformly;

(4) Removing the plug-in unit after the cells are completely attached to the wall, carefully washing the cells which are not attached to the wall by using PBS, observing under an inverted microscope, photographing and recording, and recording the result as 0 hour;

(5) Continuously culturing the cells by using a culture medium containing 3% fetal calf serum, observing the healing condition of the scratch after 24 hours and 48 hours, and photographing and recording;

(6) The results were analyzed using ImageJ and the wound healing rate was calculated, wound healing rate = (24 hours or 48 hours blank area-0 hours area part area)/0 hours blank area.

3.2.6 Transwell experiment

The Transwell experiment is divided into a migration experiment and an invasion experiment.

Migration experiment:

(1) Hydrated basement membrane: add 70. Mu.l RPMI1640 serum free medium to each chamber, incubate 30 minutes at 37 ℃ in cell incubator and carefully aspirate off the residual medium;

(2) Paving a plate: the cells of the experimental group and the control group were digested, and the cell density was adjusted to 5X 10 using serum-free RPMI1640 medium ⁵ Adding 200 mu l of uniformly mixed single cell suspension into the upper layer of a Transwell chamber, adding 750 mu l of fresh culture medium containing 10% serum into the lower layer of the chamber, carefully shaking the culture plate to ensure that the cells in the upper chamber are uniformly distributed, and placing the culture plate in an incubator to continue culturing for 24-48 hours;

(3) Cell fixation and staining: culturing for a proper time, taking out the small chamber, washing the small chamber for 1-2 times by using normal-temperature PBS (phosphate buffer solution), placing the small chamber in 4% tissue cell fixing solution for fixing for 15 minutes, then washing the small chamber for 1-2 times by using PBS (phosphate buffer solution) to wash away residual fixing solution, placing the small chamber in crystal violet dye solution for dyeing for 15 minutes, washing away residual crystal violet by using PBS after dyeing is finished, carefully wiping the upper layer of a basement membrane by using a cotton swab, and wiping off cells which do not migrate;

(4) Recording: after the liquid in the chamber is dried in the air, the chamber is placed under a microscope for observation, 3 visual fields are randomly selected for photographing and recording, and the number of the migrated cells is counted by using ImageJ.

Invasion experiment:

(1) Spreading glue: melting Matrigel in a refrigerator at 4 ℃ one day before the experiment, placing a gun head and a centrifuge tube which are needed to be used in the experiment in a refrigerator at-20 ℃ for pre-cooling, and using serum-free RPMI1640 to perform the steps of 1: diluting the matrigel according to the proportion of 10, then adding 70 mu l of matrigel into the upper layer of each small chamber, and putting the culture plate in an incubator at 37 ℃ for incubation for 5 to 6 hours to solidify the matrigel;

(2) Invasion assay the rest of the procedure was identical to the migration assay.

3.2.7 cell cycle assays

(1) Collecting cells: digesting and centrifuging the cells of the experimental group and the control group, and collecting 10 ⁶ Centrifuging the cells, removing the supernatant, re-suspending the cells by using PBS, centrifuging and removing the supernatant;

(2) Fixing: adding 1ml PBS at room temperature for resuspension of cells, slowly adding the cells into 3ml absolute ethyl alcohol pre-cooled at-20 deg.C, and standing overnight at-20 deg.C;

(3) Hydration: centrifuging the cells, removing ethanol, adding 5ml of room-temperature PBS, standing for 15 minutes to hydrate the cells, centrifuging and removing supernatant;

(4) Dyeing: adding 1ml DNA staining reagent, mixing, incubating at 37 deg.C in dark for 30 min, and detecting on flow meter.

3.2.8 apoptosis assay

(1) Digesting and centrifuging the cells of the experimental group and the control group, counting and collecting 10 ⁶ Washing the cells 1 to 2 times by using precooled PBS (phosphate Buffer solution) in a centrifugal manner, and adding 500 mu l of 1 × Binding Buffer into the cell sediment for resuspending the cells;

(2) Add 5. Mu.l Annexin V-FITC and 10. Mu.l PI into each tube, shake and incubate for 5 minutes at room temperature in the dark;

(3) Annexin V-FITC detection by a FITC channel and PI detection by a PI channel on a cell flow instrument.

3.2.9 statistical analysis

Data analysis and mapping were performed using GraphPad Prism 8 and SPSS22.0, with results expressed as mean ± SD, and differences between the two groups were compared using independent sample t-test, with P <0.05 being statistically significant.

3.3 results of the experiment

4 different siRNAs (siRNA-1, siRNA-2, siRNA-3 and siRNA-4) can obviously down-regulate the expression of IPO7 mRNA, wherein the effect of low knock-out of siRNA-1 and siRNA-2 is most obvious; selecting sequences of siRNA-1 and siRNA-2 to construct shRNA, and further packaging lentivirus-transformed DLD1 cells to construct stable transgenic cell strains for knocking down IPO 7; after the stable transgenic cell strain expressing green fluorescence is obtained by puromycin screening, the knocking efficiency of IPO7 in the stable transgenic cell strain at the mRNA level and the protein level is detected again, and the result shows that the expression of IPO7 can be obviously reduced by two different interference sequences, so that the construction success of the stable transgenic cell strain with the knocked-down IPO7 is proved, and the stable transgenic cell strain can be used for the next experiment (figure 6).

The results of the clonogenic experiments showed that the proliferative capacity of DLD1 cells was significantly inhibited after downregulation of IPO7 expression compared to control cells (FIG. 7, p- <0.05). Meanwhile, the results of colony formation experiments showed that inhibition of IPO7 expression significantly decreased the clonogenic capacity of DLD1 cells (number of cell clones: shNC: 121.7. + -. 13.43, shIPO7-1: 87.67. + -. 11.02, shIPO7-2: 89.67. + -. 13.32, shIPO7-1vs. shNC, P < -0.05 shIPO7-2vs. shNC, p < -0.05; FIG. 8).

Scratch repair experiments showed that the wound healing rate of the knockdown IPO7 group was significantly lower than that of the control group (24-hour wound healing rate: shNC:17.4% ± 0.9045%; shIPO 7-1.

Migration experiments showed that the migration ability of DLD1 cells was significantly inhibited after interfering with IPO7 expression (number of cells reaching the lower surface of the chamber: shNC: 452.3. + -. 22.01: shIPO7-1: 157.0. + -. 1.73: shIPO7-2: 161.7. + -. 10.50: shIPO7-1vs. shNC, P < -0.01: shIPO7-2vs. shNC, p <0.05; FIG. 10A, 10B); the results of the invasion experiments showed that interfering with IPO7 expression significantly reduced the invasive capacity of DLD1 cells (number of cells reaching the lower surface of the chamber: shNC: 490.0. + -. 25.24, shIPO7-1: 206.7. + -. 4.04 shIPO7-2: 197.7. + -. 24.50 shIPO7-1vs. shNC, P <0.01 shIPO7-2vs. shNC, P <0.01; FIG. 110C, 10D).

Cell cycle assay results show that after down-regulating IPO7 expression, the number of cells in S phase of colon cancer cells is significantly increased (shp: 31.16% ± 0.6929%; shp o 7-1.

The apoptosis detection results show that the IPO7 knockdown group and the control group have obvious difference in early apoptosis (the percentage of early apoptotic cells: shNC:3.687% + -0.4283%; shIPO 7-1. These results indicate that interfering IPO7 significantly promotes apoptosis of colorectal cancer, and it can be seen that IPO7 promotes tumor cell survival by inhibiting apoptosis of tumor cells (fig. 12).

Example 4 relationship between IPO7 and colorectal cancer immune cell infiltration

4.1 Experimental materials

Rabbit anti-IPO 7 monoclonal antibody, abcam; rabbit anti-CD 68 polyclonal antibody, CST; rabbit anti-CD 163 polyclonal antibody, CST; rabbit HLA-DR polyclonal antibody, CST; multi-marker immunohistochemical kit, baino biotech, beijing.

4.2 Experimental methods

4.2.1 tissue chip immunohistochemistry

Immunohistochemical staining was performed as in example 1, 1.2 of the experimental procedure 1.2.

4.2.2 Paraffin section Multi-target immunofluorescence

(1) In the multi-target immunofluorescence dyeing, horseradish peroxidase (HRP) marked secondary antibodies are selected to activate different fluorescent dyes, and the multi-target dyeing is realized through multiple dyeing cycles by means of marking of different fluorescent dyes;

(2) After the primary antibody incubation is finished, using a washing solution to soak and wash the glass slide for 3 times, each time for 5 minutes, removing residual liquid on the glass slide, dropwise adding an HRP secondary antibody working solution, immersing the sample area, and incubating for 15 minutes at room temperature;

(3) Using washing liquid to dip and wash the glass slide for 3 times, each time for 5 minutes, removing residual liquid, then dropwise adding 100 mu l of 1 Xdye working solution on the glass slide, immersing the sample area, and incubating for 15 minutes at room temperature;

(4) Soaking and washing the glass slide for 3 times and 5 minutes each time by using a washing solution, repairing by using microwaves after washing is finished, naturally cooling the room temperature to the room temperature, and soaking and washing the glass slide for 3 times and 5 minutes each time by using the washing solution;

(5) Dyeing the next target according to the steps;

(6) DAPI was used to counterstain nuclei and incubated for 15 minutes at room temperature in the dark;

(9) Sealing: using the anti-fluorescence quenching mounting agent, mount, observe and collect the image under the fluorescence microscope.

4.2.3 statistical analysis

Download abundance data of different tumor infiltrating immune cells in the TIMER database, including B cells, CD4+ T cells, CD8+ T cells, neutrophils, macrophages and dendritic cells, and assess the correlation between IPO7 expression and six types of immune cell infiltration in colorectal cancer.

Data analysis and mapping were performed using software GraphPad Prism 8 and SPSS22.0, differences between the two groups were compared using t-test, relation between IPO7 expression and macrophage infiltration in paraffin specimens was analyzed using chi-square test, P <0.05 is statistically significant for differences.

4.3 results of the experiment

The results showed a strong correlation between IPO7 expression and macrophage infiltration in either colon or rectal cancer (Table 4)

Expression of IPO7 was positively correlated with macrophage infiltration in colorectal cancer, with M1-type macrophage infiltration being down-regulated and M2-type macrophage infiltration being up-regulated in colorectal cancer tissues with high expression of IPO7, and M2-type macrophage infiltration being down-regulated and M1-type macrophage infiltration being up-regulated in colorectal cancer tissues with low expression of IPO7 (fig. 13).

The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.

Claims (10)

1. A product for diagnosing colorectal cancer/diagnosing colorectal cancer metastasis/predicting colorectal cancer prognosis, comprising an agent capable of detecting the expression level of IPO7.

2. The product according to claim 1, wherein the reagent is selected from an oligonucleotide probe specifically recognizing the IPO7 gene, a primer specifically amplifying the IPO7 gene, or a binding agent specifically binding to a protein encoded by the IPO7 gene.

3. The product of claim 1, wherein the product comprises a chip, a kit, or a nucleic acid membrane strip.

4. A pharmaceutical composition for the treatment of colorectal cancer, characterized in that it comprises an inhibitor of IPO7.

5. The pharmaceutical composition of claim 4, wherein the inhibitor inhibits proliferation, migration, invasion, apoptosis and/or division of colorectal cancer.

6. The pharmaceutical composition of claim 5, wherein the inhibitor comprises a nucleic acid inhibitor or a protein inhibitor.

7. A method of screening for a candidate drug for the treatment of colorectal cancer, the method comprising: treating the culture system expressing or containing the IPO7 gene or its encoded protein with a substance to be screened; and detecting expression or activity of the IPO7 gene or protein encoded thereby in said system; wherein, when the substance to be screened inhibits the expression level or activity of the IPO7 gene or the protein encoded by the gene, the substance to be screened is a candidate drug for treating colorectal cancer.

8. A system/apparatus for diagnosing colorectal cancer/diagnosing colorectal cancer metastasis/predicting colorectal cancer prognosis, the system/apparatus comprising:

an acquisition unit: for obtaining the expression level of IPO7 in the sample;

a processing unit: and obtaining a diagnosis/diagnosis metastasis/prognosis prediction result of the colorectal cancer according to the expression condition of the IPO7.

9. A computer-readable storage medium, having stored thereon a computer program which, when executed by a processor, implements the system/apparatus of claim 8.

10. Any one of the following applications:

(1) Use of an inhibitor of IPO7 or a pharmaceutical composition according to any of claims 4-6 for the manufacture of a product for the treatment of colorectal cancer;

(2) Use of a reagent for detecting the expression level of IPO7 or a product according to any one of claims 1 to 3 for the preparation of a tool for the diagnosis of colorectal cancer/the diagnosis of colorectal cancer metastasis/the prediction of the prognosis of colorectal cancer;

(3) Use of IPO7 for screening a candidate drug for the treatment of colorectal cancer;

(4) Use of IPO7 for promoting macrophage differentiation;

(5) Use of IPO7 in the construction of a system/device for diagnosing colorectal cancer/diagnosing colorectal cancer metastasis/predicting colorectal cancer prognosis;

(6) Use of IPO7 in the construction of a computer readable storage medium for diagnosing colorectal cancer/diagnosing colorectal cancer metastasis/predicting colorectal cancer prognosis.

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