Application of oxysterol-binding protein OSBPL2 as a molecular marker and therapeutic target for colorectal cancer

Abstract

The application provides application of oxysterol binding protein OSBPL2 as a colorectal cancer molecular marker and a treatment target, relates to the technical field of colorectal cancer, and provides application of oxysterol binding protein OSBPL2 as a colorectal cancer molecular marker, application of oxysterol binding protein OSBPL2 in preparation of reagents for detecting colorectal cancer, tissue chips and patient serum, and use of an activator of OSBPL2 gene or OSBPL2 protein in preparation of a drug for preventing and / or treating colorectal cancer. The discovery of OSBPL2 as the colorectal cancer molecular marker can be used for predicting, diagnosing and detecting colorectal cancer, provides a new experimental theoretical basis and a new direction for further studying the pathogenesis of colorectal cancer and exploring the treatment target of colorectal cancer, and can be applied in the field of preparing reagents for predicting, diagnosing and detecting colorectal cancer patients or colorectal cancer treatment drugs, and enriches the diagnosis, detection and treatment means of colorectal cancer.

Description

Technical Field

[0001] The present invention relates to the technical field of colorectal cancer, and in particular to the application of oxysterol-binding protein OSBPL2 as a molecular marker of colorectal cancer and a therapeutic target thereof. Background Art

[0002] Colorectal cancer is a cancer with a high morbidity and mortality rate. Clinically, fecal occult blood test and colonoscopy are mainly used as screening and diagnostic methods for colorectal cancer, but this method cannot fully and effectively confirm the actual condition of the patient and the coverage population is limited. The treatment of colorectal cancer is currently based on surgical resection, supplemented by radiotherapy and chemotherapy. Existing technical means can double the survival rate of colorectal cancer patients, but due to the long incubation period of the disease, usually once the cancer lesions are discovered, they have already metastasized to distant sites. To date, colorectal cancer is still exploring effective tumor diagnostic markers and therapeutic targets.

[0003] Currently, a Chinese patent with publication number CN111411155B discloses the use of lncRNA IGFL2-AS1 as a diagnostic marker for colon cancer. It proposes that knocking down lncRNA IGFL2-AS1 in colon cancer cells can significantly inhibit the proliferation, migration, invasion, and malignancy of SW620 cells, indicating that lncRNA IGFL2-AS1 plays an important role in the pathogenesis of colon cancer and can serve as a new molecular marker and drug target for the diagnosis and treatment of colorectal cancer. Another example is Chinese patent publication number CN111592487B, which discloses a class of diarylethenes containing hydroxamic acid groups as dual-target inhibitors of LSD1 and HDACs, their preparation methods, and applications. The proposed LSD1 / HDACs dual-target inhibitor exhibits significant in vitro antitumor activity against the human colon cancer HCT-116 cell line and the human gastric cancer MGC-803 cell line, providing a foundation for the development of LSD1 / HDACs dual-target inhibitor drugs.

[0004] Tumor metastasis is a major cause of mortality in colorectal cancer, and epithelial–mesenchymal transition (EMT) is particularly critical in this process. EMT is a process in which cells lose epithelial characteristics, such as cytoskeletal remodeling, loss of cell bonding, and polarity, and acquire mesenchymal properties, enhancing their migration and invasion capabilities. Oxysterol binding protein like 2 (OSBPL2) is an intracellular transporter responsible for the transport of sterols and phospholipids. Studies have reported that OSBPL2 deficiency remodels the cytoskeleton of liver cancer cells and inhibits cell migration, adhesion, and growth. However, a function of OSBPL2 in colorectal cancer metastasis has not yet been proposed.

[0005] In view of the above, the present application provides an application of oxysterol binding protein-like 2 (OSBPL2) as a molecular marker and therapeutic target of colorectal cancer. SUMMARY

[0006] In view of the deficiencies in the prior art, the present application aims to provide an application of oxysterol binding protein-like 2 (OSBPL2) as a molecular marker and therapeutic target of colorectal cancer, and the specific solutions are as follows:

[0007] The application of oxysterol binding protein-like 2 (OSBPL2) as a molecular marker of colorectal cancer.

[0008] The application of oxysterol binding protein-like 2 (OSBPL2) in the preparation of reagents, tissue chips, and patient serum for predicting, diagnosing, or detecting colorectal cancer.

[0009] The use of an activator of OSBPL2 gene or OSBPL2 protein in the preparation of a drug for preventing and / or treating colorectal cancer, wherein the activator of OSBPL2 gene includes an expression vector of OSBPL2 gene and / or an expression vector of a transcription factor binding to the promoter region of OSBPL2 gene, and / or an activator or inhibitor of epigenetic modification of OSBPL2 gene, and / or an activator or inhibitor of OSBPL2 gene mutant, and the activator of OSBPL2 protein includes an activator or inhibitor of post-translational modification, protein stability promotion, and activity of OSBPL2 protein, and / or an activator or inhibitor of OSBPL2 protein mutant.

[0010] A drug for preventing and / or treating colorectal cancer, comprising a carrier and an active ingredient, wherein the active ingredient includes an activator of OSBPL2 gene and / or an activator of OSBPL2 protein.

[0011] Further, the activator of OSBPL2 gene includes an expression vector of OSBPL2 gene and / or an expression vector of a transcription factor binding to the promoter region of OSBPL2 gene, and / or an activator or inhibitor of epigenetic modification of OSBPL2 gene, and / or an activator or inhibitor of OSBPL2 gene mutant.

[0012] The activator of OSBPL2 gene refers to a substance that can promote the expression of OSBPL2 gene. These activators include:

[0013] Expression vector of OSBPL2 gene: This is a vector that can introduce the OSBPL2 gene into cells and promote its expression, similar to a "gene syringe". This kind of expression vector is a DNA molecule that contains the complete sequence of the OSBPL2 gene and has promoter, terminator and regulatory elements, etc. regions, which can be transcribed into mRNA molecules in cells, and then translated into OSBPL2 protein. Therefore, using the expression vector of the OSBPL2 gene can achieve the purpose of expressing a large amount of OSBPL2 protein in cells.

[0014] Transcription factor expression vector binding to the promoter region of the OSBPL2 gene: This is a vector that can bind to the promoter region of the OSBPL2 gene and promote gene expression, similar to a "transcription regulator". Transcription factors are a class of proteins that can bind to the promoter region of a gene and can initiate or inhibit the transcription process of the gene. When a transcription factor binds to the promoter region of a gene, it can guide RNA polymerase to transcribe the corresponding mRNA molecule, thereby promoting the expression of the gene.

[0015] Activator or inhibitor of OSBPL2 gene epigenetic modification: This is a substance that can change the epigenetic modification state (such as DNA methylation) of the OSBPL2 gene, thereby promoting or inhibiting gene expression. Epigenetic modification of the OSBPL2 gene refers to chemical modification that affects gene expression without changing the DNA sequence. Epigenetic modifications include DNA methylation, histone modification, non-coding RNA, etc. The activator or inhibitor of the epigenetic modification of the OSBPL2 gene refers to a compound or substance that can affect the epigenetic modification state of the OSBPL2 gene to achieve the regulation of the expression of the OSBPL2 gene. For example, DNA methyltransferase inhibitors or histone deacetylase activators can be used to reduce DNA methylation of the OSBPL2 gene or increase histone deacetylation, thereby promoting the expression of the OSBPL2 gene. Conversely, DNA methylase or histone acetylase inhibitors can increase DNA methylation of the OSBPL2 gene or histone acetylation, thereby inhibiting the expression of the OSBPL2 gene.

[0016] Activator or inhibitor of OSBPL2 gene mutant: This is a substance that can activate or inhibit the expression of OSBPL2 gene mutants (such as mutations that cause abnormal activation or inactivation of gene function). The activator or inhibitor of the OSBPL2 gene mutant is not directly promoting the expression of the OSBPL2 gene, but it actually activates or inhibits the activity of the mutant. Mutant refers to a gene that has undergone some abnormal changes, resulting in its function being affected. Some mutants may cause the expression of the OSBPL2 gene to increase, abnormally activate or decrease, and function to be out of order, so the use of activators or inhibitors of mutants can restore or improve the expression of the OSBPL2 gene.

[0017] In general, these activators or inhibitors can promote the expression and function of the OSBPL2 gene through different pathways.

[0018] Further, the activators of OSBPL2 protein include activators or inhibitors of post-translational modification of OSBPL2 protein, activators or inhibitors of protein stability and activity, and / or activators or inhibitors of OSBPL2 protein mutants.

[0019] The activators of OSBPL2 protein refer to compounds or factors that can promote the function of OSBPL2 protein. These activators include:

[0020] Activators or inhibitors of post-translational modification of OSBPL2 protein: The activators of post-translational modification of OSBPL2 protein refer to substances that can promote the chemical modification of OSBPL2 protein, thereby changing the structure and function of OSBPL2 protein, and further promoting its activity and stability. Chemical modification refers to the process of combining biological molecules under certain conditions with other biological molecules, thereby changing their physical properties and functions, including phosphorylation, methylation, acetylation, ubiquitination, etc. The activators of post-translational modification of OSBPL2 protein can improve the activity and stability of OSBPL2 protein by promoting its phosphorylation, acetylation and other chemical modifications, thereby exerting therapeutic effects. Inhibitors of post-translational modification of OSBPL2 protein refer to a class of compounds or drugs that can inhibit the post-translational modification of OSBPL2 protein. Protein post-translational modification includes phosphorylation, methylation, acetylation, ubiquitination and other types, which can affect the function, stability and interaction of proteins. The post-translational modification of OSBPL2 protein can be inhibited, thereby affecting the function and stability of OSBPL2 protein.

[0021] Activators or inhibitors of OSBPL2 protein mutants: This is a compound or substance that can inhibit the formation of certain mutants or reduce their activity. In some cases, OSBPL2 protein may undergo mutations, leading to changes in its structure or function, thereby affecting its role in cells. The specific effects of activators or inhibitors of OSBPL2 protein mutants depend on the impact caused by the mutants. If the mutant leads to a decrease in the stability of OSBPL2 protein or an acceleration of its degradation, the inhibitor may promote the expression of OSBPL2 protein by preventing such degradation or accelerating the stabilization of the protein. If the mutant causes changes in the function of OSBPL2 protein, the inhibitor may promote the expression of OSBPL2 protein through other pathways. Vice versa.

[0022] Further, the carrier is a combination of one or more of water, saline, buffer, glycerol, ethanol, liposome, lipid, protein, protein-antibody conjugate, peptide, cellulose, nanogel.

[0023] The active ingredient is a pharmaceutically acceptable ingredient, and the "pharmaceutically acceptable" ingredient is a substance suitable for use in humans and / or mammals without excessive adverse side effects (such as toxicity, irritation, and allergy), i.e., with a reasonable benefit / risk ratio. The active ingredient is in an effective amount or effective dose, which means an amount or dose that can produce a function or activity on humans and / or animals and can be accepted by humans and / or animals.

[0024] The effective amount of the active ingredient described in the present application can vary depending on the mode of administration and the severity of the disease to be treated, etc. The selection of the preferred effective amount can be determined by a person skilled in the art according to various factors (for example, through clinical trials). The factors include, but are not limited to, the pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated in the patient, the weight of the patient, the immune status of the patient, the route of administration, etc.

[0025] The carrier is a pharmaceutically acceptable carrier, and the "pharmaceutically acceptable carrier" refers to a carrier for the administration of a therapeutic agent, including various excipients and diluents. The selection of the carrier should be matched with the mode of administration, which is well known to a person skilled in the art.

[0026] Generally, the pharmaceutical preparation should be matched with the mode of administration, and the dosage form of the pharmaceutical composition of the present application is injection, oral preparation (tablet, capsule, oral liquid), transdermal preparation, sustained-release preparation. For example, it is prepared by a conventional method using normal saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition is preferably manufactured under sterile conditions.

[0027] Compared with the prior art, the present application has the following beneficial effects:

[0028] (1) Oxysterol binding protein is a glycoprotein containing acidic phosphate residues, which plays an important biological role in the extracellular matrix, and is closely related to cell adhesion, migration, proliferation, and bone metabolism processes. The discovery of oxysterol binding protein OSBPL2 as a molecular marker of colorectal cancer can be used for prediction, diagnosis and detection of colorectal cancer, providing a new experimental theoretical basis and new direction for further study of the pathogenesis of colorectal cancer, and can be applied in the field of preparing reagents for predicting, diagnosing or detecting colorectal cancer patients, enriching the prediction, diagnosis and detection means of colorectal cancer.

[0029] (2) The present application provides a new therapeutic target for colorectal cancer, and a drug developed for the target can intervene or regulate the activity thereof, so as to achieve the purpose of treating diseases. The target can be effectively used for judging the development of colorectal cancer, selecting a treatment scheme and / or evaluating a prognosis, thereby providing a novel diagnostic agent and / or therapeutic agent for colorectal cancer in the art, which has a clinical application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 A is an immunohistochemical representative graph of OSBPL2 expression in colorectal cancer patients in stage IV;

[0031] Figure 1 B is a Kaplan-Meier survival curve graph of OSBPL2 expression in colorectal cancer patients in stage IV;

[0032] Figure 2 A is a graph of mRNA knockdown of OSBPL2 in colorectal cancer cells detected by real-time PCR;

[0033] Figure 2 B is a graph of protein knockdown of OSBPL2 in colorectal cancer cells detected by western blot;

[0034] Figure 3 A is a graph of the effect of tumors removed from mice injected with shcontrol and OSBPL2 knockdown (KD) tumor cells;

[0035] Figure 3 B is a statistical graph of the weight of the removed tumors;

[0036] Figure 3 C is a statistical graph of the tumor volume after subcutaneous inoculation of tumor cells in nude mice;

[0037] Figure 4 A is a graph of cell staining in transwell cell migration (transwell chamber without Matrigel) and invasion (transwell chamber without Matrigel) experiments;

[0038] Figure 4 B is a statistical graph of the number of migrated cells;

[0039] Figure 4 C is a statistical graph of the number of invasive cells;

[0040] Figure 5 A is a contrast graph of the liver of a mouse liver metastasis model after knockdown of OSBPL2;

[0041] Figure 5 B is a graph of HE staining of the liver of a mouse liver metastasis model after knockdown of OSBPL2;

[0042] Figure 6 Figure A is a western blot detecting the protein elevation of OSBPL2 in colorectal cancer cells;

[0043] Figure 6 Figure B is a contrast graph of the liver of a mouse colorectal cancer liver metastasis treated after OSBPL2 overexpression;

[0044] Figure 6 Figure C is a liver HE staining graph of a mouse colorectal cancer liver metastasis treated after OSBPL2 overexpression. DETAILED DESCRIPTION

[0045] The application will be further described in conjunction with the embodiments and the accompanying drawings, but the embodiments of the application are not limited thereto.

[0046] In this embodiment, the concept of a P value is used. The P value is a statistical index for measuring the difference between the observation result and the hypothesis, and the smaller the P value, the more significant the difference. Generally, a P value less than 0.05 is considered as a threshold value of significant difference, wherein, * represents a P value less than 0.05, ** represents a P value less than 0.01, *** represents a P value less than 0.001, *P<0.05 indicates significant difference, **P<0.01 indicates very significant difference, ***P<0.001 indicates extremely significant difference, *P<0.05, **P<0.01, ***P<0.001 are common statistical symbols used to represent the significant difference between data.

[0047] (1) Colorectal cancer patient samples and tissue chips

[0048] The colorectal cancer patient samples collected in the study were from Yangpu Hospital Affiliated to Tongji University, and the collection and use of the samples were agreed by the patients.

[0049] After the paraffin-embedded colorectal cancer patient tissues were made into tissue chips, immunohistochemical staining experiments were performed, as follows:

[0050] (2) Immunohistochemical staining experiment

[0051] The tissue chips in the above (1) were fixed in 4% paraformaldehyde overnight and then washed with PBS. Then, paraffin embedding and sectioning were performed. After the sections were hydrated and antigen repaired, primary and secondary antibody staining was performed. After hematoxylin staining, dehydration and mounting were performed. The immunohistochemical scoring standard of the sample was the degree of antibody coloring (0-3) and the proportion of positive cells (0-4).

[0052] Experimental results: Figure 1 Figure A is a representative graph of the immunohistochemical staining of OSBPL2 expression in colorectal cancer patients in stage IV, and the coloring degree is shown for easy viewing, Figure 1The color effect is adopted, the coloring degree of the OSBPL2 low expression antibody is 1, the positive cell proportion is 4, and the comprehensive score is 4; the coloring degree of the OSBPL2 high expression antibody is 3, the positive cell proportion is 4, and the comprehensive score is 12; Figure 1 B is the Kaplan-Meier survival curve of OSBPL2 expression in patients with stage IV colorectal cancer, and the curve of OSBPL2 Low is shown below. Figure 1 B, the tissue chip result shows that the less the OSBPL2 expression in stage IV colorectal cancer, the lower the survival rate of the patient, the worse the prognosis, the higher the OSBPL2 expression, the higher the survival rate of the patient, and P<0.05, indicating that the survival rates of patients are significantly different when the two OSBPL2 expression levels are different. It can be known that the prognosis of the stage IV colorectal cancer patient with high expression of OSBPL2 will be better.

[0053] Therefore, it is found that the low expression of OSBPL2 is related to the malignant prognosis of the stage IV colorectal cancer patient, and OSBPL2 can be used as a benign colorectal cancer tumor marker for distant metastasis of colorectal cancer.

[0054] (3) Construction of plasmid and verification of stable cell strain

[0055] In order to verify the biological function of OSBPL2 in colorectal cancer, the present application uses shRNA lentivirus to construct a stable cell strain of OSBPL2 knockdown (KD) in colorectal cancer cells.

[0056] It should be noted that in the gene knockdown experiment, the expression level of the target gene can be reduced by RNA interference technology (such as using shRNA), and then the biological behavior of the cell, such as proliferation, apoptosis, differentiation, etc. Changes are observed. By detecting the change of the mRNA level of the target gene after knockdown, it can be determined whether the expression of the target gene is successfully inhibited. Therefore, in the gene knockdown experiment, detecting the mRNA level of the target gene is a commonly used index.

[0057] In the present application, detecting the change of OSBPL2 mRNA level can be used as one of the indicators in the gene knockdown experiment, and the OSBPL2 mRNA level refers to the mRNA level of the OSBPL2 gene in cells or tissues, also known as the mRNA expression level of OSBPL2. mRNA is the abbreviation of messenger RNA, which is a kind of RNA molecule transcribed from DNA, and has the function of guiding protein synthesis. OSBPL2 mRNA level represents the number of OSBPL2 gene transcribed into mRNA under certain conditions, which is usually detected and analyzed by techniques such as real-time fluorescent quantitative PCR (RT-qPCR). By detecting the change of OSBPL2 mRNA level, biological problems related to OSBPL2 gene expression can be studied, such as gene regulation, signal transduction, cell proliferation, apoptosis, differentiation, etc. Therefore, the detection of OSBPL2 mRNA level is also one of the indicators for confirming that the osbpl2 gene has been successfully knocked down.

[0058] The specific verification process is as follows:

[0059] (a) Culture HT29 and LoVo cells with DMEM sterile culture solution (Invitrogen, 11995065) or 1640 culture medium (Gibco, 11875093) containing 10% fetal bovine serum FBS (Gibco, 10091148), and add 1% penicillin and streptomycin (Gibco, 15140122). The culture dishes or flasks are placed in a 37°C cell culture incubator containing 5% CO2 for culture.

[0060] (b) First, construct the short hairpin RNA (shRNA) of OSBPL2 into the pLKO.1-PURO vector. The shRNA sequence is as follows:

[0061] OSBPL2: 5'-GGATTACTTTGAGCGGAATTT-3';

[0062] OSBPL2: 5'-GGGAGAAACGTATGAATTAAT-3';

[0063] OSBPL2: 5'-GAAGATTTAGGATTCAGATTT-3';

[0064] Scramble: 5'-CCTAAGGTTAAGTCGCCCTCG-3'.

[0065] Secondly, the lentivirus packaging plasmid (psPAX2 / pMD2.G) and the viral vector were transfected into HEK293T cells by transfection reagent Lipofectamine 3000 (Invitrogen). After 48 hours, the supernatant of the cells was collected and filtered with a 0.45 μM filter. The viral liquid was directly infected into colorectal cancer cells for 48 hours, and the viral liquid was removed and screening drug puromycin (Invivogene) was added to screen stable cell strains.

[0066] (c) RNA extraction and qPCR detection, western blot detection

[0067] RNA extraction and qPCR detection refer to extracting RNA in the cell strain in (4) above by Trizol (Novagen). The extracted RNA is reverse transcribed and qPCR experiment is performed by detecting SYBR green fluorescence. The primers for qPCR are as follows:

[0068] OSBPL2:

[0069] 5'-AGAGGTGACCACCTGAGAAAGG-3';

[0070] 5'-GTTGATCCTCCAGAGCAGCTTG-3'.

[0071] β-actin:

[0072] 5'-CACCATTGGCAATGAGCGGTTC-3';

[0073] 5'-AGGTCTTTGCGGATGTCCACGT-3'.

[0074] Experimental results: Figure 2 Using shRNA, stable knockdown cell strains of HCT116, HT29, LoVo and SW620 were constructed by lentivirus infection.

[0075] It should be noted that the stable knockdown cell lines of HCT116, HT29, LoVo and SW620 are constructed by using shRNA (short hairpin RNA) and lentivirus infection, which refers to a gene knockdown experiment in the laboratory. Specifically, by designing and synthesizing shRNA, it is introduced into a lentivirus plasmid, and the plasmid is transfected into HCT116, HT29, LoVo and SW620 and other intestinal cancer cell lines to make them express shRNA in cells. shRNA can specifically bind to the mRNA of the target gene and induce its degradation, thereby inhibiting the expression of the target gene. By using lentivirus as a carrier, stable knockdown of the target gene is achieved. HCT116, HT29, LoVo and SW620 are commonly used intestinal cancer cell lines, so this experiment is used to study the role of the target gene OSBPL2 in the occurrence and development of intestinal cancer. By detecting the expression level of the target gene OSBPL2 in the cells, and observing the changes in biological behaviors such as cell growth, proliferation and apoptosis, the function and mechanism of action of the target gene OSBPL2 can be further revealed.

[0076] Specifically, Figure 2 A is the real-time PCR detection of the mRNA knockdown of OSBPL2 in colorectal cancer cells, and shcontrol is used as a control group to compare with groups 1, 2 and 3 of OSBPL2 shRNA. From the four cases of HCT116, HT29, LoVo and SW620, it can be seen that the OSBPL2 mRNA level values of groups 1, 2 and 3 of OSBPL2 shRNA are much smaller than those of the shcontrol control group. It can be seen that when the mRNA level of the target gene OSBPL2 is reduced, it can be explained that the transcription of the target gene OSBPL2 is inhibited, thereby reducing the expression amount of the protein of the gene.

[0077] Western blot detection refers to a protein immunoblotting experiment. After collecting the cells, they are washed with PBS and lysed with RIPA lysis buffer (Bi Yun Tian). Proteinase inhibitors and phosphatase inhibitors are added. After lysis, loading buffer is added and the sample is boiled. After the sample is added to the SDS-PAGE gel, it is transferred to the membrane. After incubation of the primary and secondary antibodies, ECL color development is performed.

[0078] It should be noted that ECL is a biomolecular detection technique that mainly uses radiolabeled antibodies to bind to the protein to be detected, and then uses enzymatic fluorescent substrate to convert the protein detection result into a fluorescent signal. The intensity of the fluorescent signal can be quantitatively measured by a specific detection device, but it does not have color expression itself. Usually, the results of ECL development are presented in the form of numbers, intensity values or grayscale images. The antibodies in the western blot detection of the present application: OSBPL2 (from Proteintech), β-actin (from Abclonal).

[0079] Figure 2 B is the western blot detection of the protein knockdown of OSBPL2 in colorectal cancer cells. By detecting the expression levels of OSBPL2 and Actin at the same time, shcontrol is used as a control group to compare with groups 1, 2 and 3 of OSBPL2 shRNA. From the four cases of HCT116, HT29, LoVo and SW620, it can be seen that the protein expression level of OSBPL2 is significantly decreased, while the protein expression level of Actin does not change significantly. It can be known that the expression of the target gene OSBPL2 is successfully inhibited.

[0080] It should be noted that Actin is a cytoskeletal protein, which is one of the most important protein components in cells. Actin protein has various biological functions in cells, such as maintaining cell morphology, participating in cell movement, cell division, cell adhesion, etc. Actin protein also regulates various signal transduction pathways in cells, including cell proliferation, apoptosis, differentiation, etc. In cells, actin protein assembles into microfilaments (micro-fiber-like structures) and participates in the formation of cytoskeleton, thereby maintaining the stability and morphology of cells. Actin protein plays an important role in many biological researches, including cancer, muscle disease, nervous system disease, etc.

[0081] Actin is used as an internal control in western blot analysis to correct the differences in protein content between samples and ensure the accuracy of the analysis results. Therefore, in the above western blot detection, Actin is detected as a control in addition to the detection of the target protein OSBPL2. When analyzing the experimental results, the experimenter usually compares the expression level of the target protein with the expression level of Actin, calculates the relative expression amount, and evaluates the change in the expression level of the target protein.

[0082] Moreover, in the above shRNA knockdown experiment, Mean ± SEM represents the mean and standard error of each group of data. Among them, Mean represents the mean, and SEM represents the standard error, which is a statistical measure of the accuracy of the mean, indicating the error range between the mean and the true value. Combined Figure 2 , Mean ± SEM, LoVo and SW620 correspond to **P <0.01, HCT116, HT29 correspond to ***P <0.001. Therefore, **P <0.01 represents a very significant difference, and ***P <0.001 represents an extremely significant difference. It can be seen that in the real-time PCR detection and Western blot detection, the difference in the expression of the target gene OSBPL2 represented by the shcontrol control group and the 1, 2, 3 groups of OSBPL2 shRNA is indeed great.

[0083] Figure 2 A The knockdown efficiency of OSBPL2 mRNA was detected by real-time PCR; Figure 2 B The knockdown efficiency of OSBPL2 protein was detected by western blot.

[0084] (4) Nude mouse tumor formation experiment

[0085] In order to verify the function of OSBPL2 in the growth of colorectal tumors, the shcontrol control group and the OSBPL2 knockdown stable strain were subjected to nude mouse tumor formation experiment, as follows:

[0086] Order 6-week-old male nude mice (BALB / cA-nu / nu) and raise them in a sterile environment. The stable cell line is injected subcutaneously into the back of the nude mouse. After one week, measure the tumor size (every 3-5 days) and observe the tumor growth. The tumor volume is calculated according to the following formula: V (mm 3 ) = 0.5 x length x width 2 After a period of time, the nude mice are sacrificed and the tumors are collected, weighed, and photographed.

[0087] Experimental results: Figure 3 A The same number of shcontrol and OSBPL2 knockdown (KD) tumor cells were injected subcutaneously into nude mice. After 22 days, the HCT116 and HT29 group mice were sacrificed, and after 29 days, the LoVo and SW620 group mice were sacrificed. After the tumors were removed, they were photographed;

[0088] Figure 3B Statistics of tumor weight were taken out, and Mean±SEM was obtained. *P<0.05 was obtained for HCT116, HT29, and LoVo, and ns was obtained for SW620. *P<0.05 indicates a significant difference, and ns indicates no statistically significant difference.

[0089] Figure 3 C. After subcutaneous inoculation of nude mice with tumor cells, tumor size was measured starting on day 8 and then every 3-4 days. Tumor volume was calculated. The shcontrol control group is the lower line, and the OSBPL2 KD group is the upper line. Mean ± SEM is shown. **P < 0.01 for the HCT116 and HT29 groups, ***P < 0.001 for the LoVo group, and ns for the SW620 group. *P < 0.05 indicates a significant difference, **P < 0.01 indicates a very significant difference, ***P < 0.001 indicates an extremely significant difference, and ns indicates no statistically significant difference.

[0090] The results showed that knocking down OSBPL2 significantly accelerated the growth of colorectal tumors, except for SW620 cells, which showed no difference ( Figure 3 ).

[0091] (5) Transwell cell migration and invasion assay

[0092] To verify the function of OSBPL2 in colorectal cancer metastasis, we first used transwell cell migration and invasion assays to detect the effect of OSBPL2 on the migration and invasion of colorectal cancer cells.

[0093] Stable cell lines (5×10 5 Cells were seeded in the upper chamber of a transwell chamber (Corning) using 100 μL of culture medium (100 μL / well, serum-free) and 800 μL of complete culture medium was added to the lower chamber. For invasion assays, the chambers were first coated with Matrigel basement membrane matrix. After 48-72 hours of culture, the chambers were removed and fixed with paraformaldehyde (Seville), then stained with crystal violet (Biyuntian) and photographed.

[0094] Figure 4 A transwell chamber was plated with equal numbers of shcontrol and OSBPL2 KD HCT116, HT29, LoVo, and SW620 cells. After culturing for 2-3 days, the cells were harvested, stained with crystal violet, and photographed under a light microscope.

[0095] Figure 4B. Counting the number of migrating cells. Comparison of the shcontrol control group with OSBPL2 shRNA groups 1, 2, and 3 shows that (mean ± SEM) for the HT29 group, *P < 0.05 indicates a significant difference; for the HCT116, LoVo, and SW620 groups, **P < 0.01 indicates a very significant difference. Experimental results in HCT116, HT29, LoVo, and SW620 cells indicate that knockdown of OSBPL2 significantly promotes the migration of colorectal cancer cells.

[0096] Figure 4 C. Counting the number of invasive cells. Comparison of the shcontrol control group with OSBPL2 shRNA groups 1, 2, and 3 reveals that *P<0.05 for the HT29 group, where *P<0.05 indicates a significant difference; **P<0.01 for the LoVo and SW620 groups; and **P<0.01 for the HCT116 group, where ***P<0.001 indicates an extremely significant difference. The experimental results in HCT116, HT29, LoVo, and SW620 cells indicate that knockdown of OSBPL2 significantly promotes the invasive ability of colorectal cancer cells.

[0097] (6) Mouse liver metastasis model

[0098] To verify the in vivo function of OSBPL2 in colorectal cancer metastasis, we used the shcontrol control group and OSBPL2 knockdown stable strain to construct a mouse liver metastasis model.

[0099] It should be noted that the mouse liver metastasis model refers to the process of transplanting tumor cells into the mouse liver to observe whether the tumor cells can successfully grow, invade, and metastasize. This model is suitable for studying the metastasis and invasion mechanisms of tumor cells in the liver, such as liver cancer, pancreatic cancer, and colorectal cancer. The details are as follows:

[0100] Anesthetize the mouse in an anesthesia box. After the mouse's righting reflex disappears, surgery can be performed. After transferring the mouse to a table, maintain anesthetic inhalation with a mask. Use ophthalmic scissors or tissue scissors to cut the skin to expose the muscular layer. Use ophthalmic scissors to cut the muscle to expose the spleen. Pull the spleen slightly out, fix the spleen with forceps, and aspirate a certain amount of cells (2x10 6=Individual / only, 50ul), push the spleen with an insulin needle. After withdrawing the needle, press the injection site quickly with an alcohol cotton swab, press under pressure to prevent cell suspension from seeping and bleeding. After timing for 5 minutes, the mouse spleen is lifted, and the spleen is separated along the lower edge of the spleen. There are small bleeding points and electrocoagulation to stop bleeding. When separating to the splenic artery and splenic vein, the blood vessels are ligated until the spleen is completely severed. Observe whether there is active bleeding, return the abdominal contents, and use 1-2 interrupted sutures of the muscular layer depending on the size of the incision with a round needle. The skin is sutured with several interrupted / continuous sutures of a triangular needle depending on the size of the incision. The mouse is placed on a heating pad and returned to the mouse cage after its recovery.

[0101] Figure 5 A is a comparison of the liver in the mouse liver metastasis model after OSBPL2 knockdown; Figure 5 B is a HE staining image of the liver of a mouse liver metastasis model after OSBPL2 knockdown. The results showed that the reduction of OSBPL2 expression significantly promoted liver metastasis in mice, and the size and number of liver metastases increased significantly ( Figure 5 A). Representative livers from each group were selected for HE staining. The results showed that the area of ​​tumor infiltration in the liver increased significantly after OSBPL2 knockdown. Figure 5 B.

[0102] Figure 6 A is a graph showing the protein elevation of OSBPL2 in colorectal cancer cells detected by western blot; Figure 6 B is a comparison of the livers of mice treated with colorectal cancer liver metastasis after OSBPL2 overexpression; Figure 6 C is a HE staining image of the liver of colorectal cancer mice treated with liver metastasis after OSBPL2 overexpression. The full-length CDS sequence of OSBPL2 was cloned into the pCDH vector and OSBPL2 overexpression (OE) stable cells were constructed by lentivirus. The expression of OSBPL2 was detected by western blot ( Figure 6 A). Overexpression of OSBPL2 can significantly inhibit liver metastasis caused by colorectal cancer cells, as evidenced by a decrease in the size and number of liver metastases ( Figure 6 B). Representative livers from each group were selected for HE staining, and the results showed that the area of ​​tumor infiltration in the liver was significantly reduced after OSBPL2 overexpression ( Figure 6 C).

[0103] By knocking down OSBPL2 and inhibiting OSBPL2 protein expression, they found that inhibiting OSBPL2 protein expression induced colorectal tumor growth, promoted colorectal cancer cell migration, invasion, and tumor metastasis, while overexpressing OSBPL2 significantly treated colorectal cancer liver metastasis in mice. Therefore, OSBPL2 protein is a potential new target for colorectal cancer treatment. Based on this, studying its therapeutic targets in colorectal cancer can provide treatment ideas and theoretical basis for colorectal cancer-related diseases and provide new targets for screening and preparing drugs for colorectal cancer treatment.

[0104] The above description is merely a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiment. All technical solutions based on the concept of the present invention are within the scope of protection of the present invention. It should be noted that for those skilled in the art, various improvements and modifications that do not depart from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. Use of an activator of the OSBPL2 gene or OSBPL2 protein in the preparation of a medicament for preventing and / or treating colorectal cancer, characterized in that: The activator of the OSBPL2 gene includes an expression vector of the OSBPL2 gene and / or an expression vector of a transcription factor that binds to the promoter region of the OSBPL2 gene.

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