Application of CGREF1 inhibitor in preparation of medicine for preventing and treating colorectal cancer metastasis

Abstract

The invention discloses an application of a CGREF1 inhibitor in preparation of a medicine for preventing and / or treating colorectal cancer metastasis and an application of CGREF1 in prognosis prediction of a colorectal cancer patient. It is found that inhibition of CGREF1 expression has no significant influence on proliferation of colorectal cancer cells, but migration and invasion of the colorectal cancer cells can be significantly inhibited. Besides, it is found for the first time that high expression of the CGREF1 is related to remarkable shortening of the survival time of the colorectal cancer patient, univariate analysis and multivariate Cox regression analysis both show that the expression of the CGREF1 is an independent prognosis factor for survival of the colorectal cancer patient, and the above discovery prompts that the CGREF1 is potentially applied to prognosis prediction of the colorectal cancer patient as a biomarker, and the application prospect is wide. Clinical doctors can be assisted in judging the prognosis of the patient, so that a personalized treatment scheme is formulated.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to the use of CGREF1 inhibitor in the preparation of a drug for preventing and treating colorectal cancer metastasis. BACKGROUND

[0002] Colorectal cancer (CRC) is a major global health challenge. Despite the significant improvement in patient prognosis by surgery, chemotherapy (e.g. FOLFOX regimen), targeted therapy (e.g. anti-EGFR, anti-VEGF antibodies) and immunotherapy (e.g. PD-1 inhibitors), early diagnosis difficulty (about 20% patients have distant metastasis at the time of diagnosis), easy recurrence and metastasis (5-year recurrence rate is about 30% after surgery) and drug resistance are still the main reasons leading to patient death. Therefore, finding precise prognostic biomarkers and effective therapeutic targets is the core requirement of colorectal cancer research.

[0003] CGREF1, also known as CGR11 (Cell Growth Regulatory Gene 11 Protein) or Hydrophobestin, is encoded by the gene (Gene ID: 10669) on human chromosome 2q37.3. CGREF1 protein consists of 301 amino acids, with a molecular weight of about 31 kDa (partially cleaved body can present 11 kDa or 47 kDa bands), and its most significant structural feature is two highly conserved EF-hand calcium binding domains. EF-hand domain is a typical structure of calcium-dependent proteins (consisting of helix-loop-helix motif), responsible for binding calcium ions and mediating downstream functions, which is also the structural basis for CGREF1 to play a role in cell adhesion. In addition, the subcellular localization of CGREF1 is mainly in the cytoplasm (Swiss Prot: Q99674), suggesting that it may be involved in signal transduction or protein-protein interaction in the cytoplasm. SUMMARY

[0004] The present application explores the role of CGREF1 in the proliferation and metastasis of colorectal cancer cells by traditional molecular biology methods, and unexpectedly finds that inhibiting the expression of CGREF1 has no significant effect on the proliferation of colorectal cancer cells, but can significantly inhibit the migration and invasion of colorectal cancer cells. This finding suggests the potential use of inhibiting CGREF1 in preventing and treating colorectal cancer metastasis. In addition, the present application first finds that high expression of CGREF1 is significantly associated with a significantly shortened survival period of colorectal cancer patients, wherein univariate analysis shows that advanced stage (p=0.001), lymph node metastasis (p=0.008), venous invasion (p=0.000) and CGREF1 expression (p=0.001) are significant prognostic factors of colorectal cancer, and multivariate Cox regression analysis shows that CGREF1 expression (p=0.014) and venous invasion (p=0.034) are independent prognostic factors for the survival of colorectal cancer patients. The above findings suggest that CGREF1 as a biomarker has potential application in the prognosis prediction of colorectal cancer patients.

[0005] The first aspect of the present application aims to provide the use of a CGREF1 inhibitor in the preparation of a drug for preventing and / or treating colorectal cancer metastasis.

[0006] The second aspect of the present application aims to provide the use of CGREF1 in the prognosis prediction of colorectal cancer patients.

[0007] The third aspect of the present application aims to provide a pharmaceutical composition comprising a CGREF1 inhibitor and a pharmaceutically acceptable excipient.

[0008] The fourth aspect of the present application aims to provide the use of the pharmaceutical composition according to the third aspect of the present application in the preparation of a drug for preventing and / or treating colorectal cancer metastasis.

[0009] The technical solution adopted by the present application is:

[0010] The first aspect of the present application provides the use of a CGREF1 inhibitor in the preparation of a drug for preventing and / or treating colorectal cancer metastasis.

[0011] In some embodiments of the present application, the CGREF1 inhibitor comprises at least one of the following:

[0012] A1) a substance that inhibits the activity of CGREF1 protein;

[0013] A2) a substance that reduces the expression of CGREF1 protein;

[0014] A3) a substance that silences the CGREF1 gene;

[0015] A4) a substance that knocks out the CGREF1 gene;

[0016] A5) a substance that inhibits the expression of CGREF1 gene.

[0017] In some embodiments of the present application, the CGREF1 inhibitor comprises a nucleic acid molecule, a protein molecule and a small molecule compound.

[0018] In some embodiments of the present application, the nucleic acid molecule comprises microRNA, siRNA, shRNA, dsRNA, sgRNA and antisense oligonucleotide.

[0019] In some embodiments of the present application, the protein molecule is a specific antibody of CGREF1, and the compound is a small molecule or polypeptide compound that binds to CGREF1.

[0020] In some embodiments of the present application, the siRNA sequence is shown in SEQ ID NO: 5.

[0021] In some embodiments of the present application, the colorectal cancer metastasis comprises, but is not limited to, lymphatic metastasis, liver metastasis, lung metastasis, peritoneal metastasis, bone metastasis and brain metastasis of colorectal cancer.

[0022] In some embodiments of the present application, the prevention and / or treatment of colorectal cancer metastasis comprises inhibition of migration and invasion of tumor cells.

[0023] In some embodiments of the present application, the inhibition of migration and invasion of tumor cells comprises inhibition of F-actin expression and pseudopod formation.

[0024] In a second aspect of the present application, there is provided a use of CGREF1 in prognosis prediction of colorectal cancer patients.

[0025] In some embodiments of the present application, the prognosis of colorectal cancer patients is predicted based on a threshold of the Immunoreactive Score Analysis (IRS) of CGREF1 in colorectal cancer patients.

[0026] In some embodiments of the present application, the criteria for predicting the prognosis of colorectal cancer patients based on the threshold of the IRS of CGREF1 in colorectal cancer patients are:

[0027] comparing the IRS score of the colorectal cancer patient with the IRS score threshold,

[0028] if the IRS score of the colorectal cancer patient is greater than the IRS score threshold, the prognosis of the colorectal cancer patient is determined to be poor,

[0029] if the IRS score of the colorectal cancer patient is less than or equal to the IRS score threshold, the prognosis of the colorectal cancer patient is determined to be good.

[0030] In some embodiments of the present application, the prognosis prediction comprises 5-year survival prediction.

[0031] In some embodiments of the present application, the IRS score threshold = 6.

[0032] The inventors found that high expression of CGREF1 is significantly associated with shorter survival of colorectal cancer patients, wherein univariate analysis showed that advanced stage (p = 0.001), lymph node metastasis (p = 0.008), venous invasion (p = 0.000) and CGREF1 expression (p = 0.001) are significant prognostic factors of colorectal cancer, and multivariate Cox regression analysis showed that CGREF1 expression (p = 0.014) and venous invasion (p = 0.034) are independent prognostic factors for survival of colorectal cancer patients.

[0033] In a third aspect of the present application, a pharmaceutical composition comprising a CGREF1 inhibitor and a pharmaceutically acceptable excipient is provided.

[0034] In some embodiments of the present application, the CGREF1 inhibitor comprises a nucleic acid molecule, a protein molecule and a small molecule compound.

[0035] In some embodiments of the present application, the nucleic acid molecule comprises microRNA, siRNA, shRNA, dsRNA, sgRNA and antisense oligonucleotide.

[0036] In some embodiments of the present application, the protein molecule is a specific antibody of CGREF1, and the compound is a small molecule or polypeptide compound that binds to CGREF1.

[0037] In some embodiments of the present application, the siRNA sequence is shown in SEQ ID NO: 5.

[0038] In an embodiment of the present application, the pharmaceutically acceptable excipient comprises at least one of solvent, propellant, solubilizer, co-solvent, emulsifier, coloring agent, binding agent, disintegrating agent, filling agent, lubricant, wetting agent, osmotic pressure regulator, stabilizer, glidant, flavoring agent, preservative, suspending agent, coating material, fragrance, anti-adhesion agent, integrating agent, penetration enhancer, pH regulator, buffer, plasticizer, surfactant, foaming agent, antifoaming agent, thickening agent, inclusion agent, humectant, absorbent, diluent, flocculating agent and deflocculating agent, filter aid, release retardant, carrier.

[0039] The above pharmaceutically acceptable excipients are generally recognized as suitable for this purpose and as non-active ingredients of medicaments. A compilation of pharmaceutically acceptable excipients can be found in the "Handbook of Pharmaceutical Excipients", 2ndEdition, Edited by A. Wade and P. J. Weller; Published by the American Pharmaceutical Association, Washington and The Pharmaceutical Press, London, 1994; "Chinese Pharmacopoeia - Name and Index of Pharmaceutical Excipients", and the like reference books.

[0040] In some embodiments of the present application, the dosage form of the pharmaceutical composition comprises a gastrointestinal administration dosage form, or a non-gastrointestinal administration dosage form.

[0041] In some embodiments of the present application, the gastrointestinal administration dosage form comprises at least one of a powder, a tablet, a granule, a capsule, a sustained release agent, a solution, a dry suspension, an effervescent tablet, an emulsion, a suspension, a syrup, a drop, a chewable tablet.

[0042] In some embodiments of the present application, the non-gastrointestinal administration dosage form comprises at least one of an injection administration dosage form, a respiratory administration dosage form, a skin administration dosage form, a mucosa administration dosage form, a cavity administration dosage form.

[0043] In some embodiments of the present application, the pharmaceutical composition is administered to a subject in need thereof in an effective dose.

[0044] In some embodiments of the present application, the subject in need thereof comprises a human and an animal.

[0045] In some embodiments of the present application, when the subject in need thereof is a human, the effective dose is in the range of 20-100 mg / day.

[0046] In a fourth aspect of the present application, there is provided use of the pharmaceutical composition according to the third aspect of the present application in the preparation of a medicament for preventing and / or treating metastasis of colorectal cancer.

[0047] In some embodiments of the present application, the metastasis of colorectal cancer comprises, but is not limited to, lymphatic metastasis, liver metastasis, lung metastasis, peritoneal metastasis, bone metastasis, and brain metastasis of colorectal cancer.

[0048] In some embodiments of the present application, the preventing and / or treating metastasis of colorectal cancer comprises inhibiting migration and invasion of tumor cells.

[0049] In some embodiments of the application, the inhibition of migration and invasion of tumor cells comprises inhibition of F-actin expression and pseudopod formation.

[0050] The present application has the following advantages:

[0051] The present application explores the role of CGREF1 in colorectal cancer cell proliferation and metastasis by traditional molecular biology methods, and unexpectedly finds that inhibiting CGREF1 expression has no significant effect on the proliferation of colorectal cancer cells, but can significantly inhibit the migration and invasion of colorectal cancer cells. This finding suggests the potential use of inhibiting CGREF1 in preventing and treating colorectal cancer metastasis. In addition, the present application first finds that high expression of CGREF1 is significantly associated with a significantly shortened survival period of colorectal cancer patients, wherein both univariate analysis and multivariate Cox regression analysis show that CGREF1 expression is an independent prognostic factor for the survival of colorectal cancer patients. The above findings suggest that CGREF1 as a biomarker has potential application in the prognosis prediction of colorectal cancer patients, which can assist clinicians in judging the prognosis of patients and thus developing personalized treatment plans. BRIEF DESCRIPTION OF DRAWINGS

[0052] The above and / or additional aspects and advantages of the present application will become apparent and more readily appreciated from the following description of the embodiments, taken in conjunction with the following drawings of which:

[0053] Figure 1 shows the expression of CGREF1 in normal tissues and colorectal cancer tissues; wherein Figure 1 A shows the expression of CGREF1 in 32 kinds of malignant tumors in the TIMER2.0 database, Figure 1 B shows the expression of CGREF1 in normal colon tissue and CRC clinical samples, Figure 1 C and Figure 1 D respectively shows the expression of CGREF1 in the whole research population Figure 1 C) and the paired CRC and normal colorectal samples Figure 1 D) in the TCGA database, Figure 1 E shows the mRNA expression level of CGREF1 in CRC samples and paired normal tissues in clinical samples.

[0054] Figure 2 shows the relationship between CGREF1 expression and the clinical prognosis of CRC; wherein Figure 2 A-2B respectively shows the IRS score distribution of CGREF1 expression in CRC samples and paired normal tissues Figure 2 A) and H&E staining results Figure 2 B), Figure 2 C shows the results of correlation analysis between CGREF1 expression and clinical pathology (advanced stage), Figure 2D shows the results of CGREF1 expression correlation analysis with clinicopathology (lymph node metastasis, venous invasion), Figure 2 E shows the results of CGREF1 expression correlation analysis with clinicopathology (tumor budding), Figure 2 F shows the results of Kaplan-Meier survival analysis, Figure 2 G shows the ROC curve of CGREF1 expression level for 5-year survival prediction of colorectal cancer patients.

[0055] Figure 3 shows the effect of CGREF1 on CRC cell proliferation, wherein Figure 3 A-3B shows that CGREF1 siRNA can effectively knock down the mRNA Figure 3 A) and protein Figure 3 B) expression levels in SW480 cells and RKO cells, Figure 3 C-3E shows the effect of knocking down CGREF1 on tumor cell proliferation, respectively using CCK8 detection, colony formation experiment and EdU cell proliferation experiment.

[0056] Figure 4 shows the effect of CGREF1 on the invasion and metastasis ability of CRC, wherein Figure 4 A shows the results of Transwell migration experiment, Figure 4 B shows the results of scratch experiment, Figure 4 C shows the effect of CGREF1 knockdown on the growth of primary colorectal tumor, Figure 4 D-4E shows the effect of CGREF1 knockdown on the number of liver metastasis, Figure 4 F shows the effect of CGREF1 knockdown on the expression of F-actin and pseudopod formation in CRC cells.

[0057] In Figures 1-4 , "ns" is no significant difference, "*" is p<0.05, "**" is p<0.01, and "***" is p<0.001. DETAILED DESCRIPTION

[0058] The concept and technical effects of the present application will be described below in conjunction with the embodiments to fully understand the purpose, features and effects of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, not all embodiments. Based on the embodiments of the present application, other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0059] In addition, for a better understanding of the present application, numerous specific details are given in the following detailed description. It will be understood by those skilled in the art that the present application can be practiced without certain specific details. In some embodiments, well-known ingredients, methods, means, etc. are not described in detail because they are frequently deemed to be insignificant to an understanding of the present application.

[0060] Example 1: CGREF1 is highly expressed in colorectal cancer tissues

[0061] This example first detected the expression of CGREF1 in 32 kinds of malignant tumors using the TIMER2.0 database (https: / / timer.cistrome.org / ). The "32 kinds of malignant tumors" module in the TIMER2.0 database was selected to obtain the expression data of CGREF1 in colon adenocarcinoma (COAD) and rectal adenocarcinoma (READ) (compared with normal tissues). The results showed that CGREF1 was significantly up-regulated in 13 kinds of tumors including colon adenocarcinoma and rectal adenocarcinoma compared with normal tissues Figure 1 A).

[0062] To further explore the expression of CGREF1 in colorectal cancer (CRC), the RNA-seq data of 275 CRC samples in the TCGA database (https: / / portal.gdc.cancer.gov / ) and 349 normal colon tissues in the GEPIA2 database (http: / / gepia2.cancer-pku.cn / ) were analyzed. The R language (ggplot2 package) was used to draw box plots to compare the expression differences of CGREF1 between tumor and normal tissues (Wilcoxon rank sum test); paired t-test was used to analyze the expression differences between 275 CRC samples and 345 normal intestinal epithelial tissues in the TCGA database, as well as 19 pairs of paired CRC tissues and normal intestinal epithelial tissues from Shenzhen Bao'an District People's Hospital. The results showed that the expression of CGREF1 in CRC samples was significantly higher than that in normal colorectal epithelial tissues Figure 1 B), and whether in the whole study population Figure 1 C) or in paired tissues Figure 1 D), CGREF1 was significantly highly expressed in CRC.

[0063] To verify this result, the expression of CGREF1 in 19 pairs of primary CRC tumors (T) and adjacent normal colorectal tissues (N) from the People's Hospital of Bao'an District, Shenzhen City was detected by qRT-PCR. The experimental steps are as follows: 1) sample collection: 19 pairs of primary CRC tumors (T) and adjacent normal colorectal tissues (N) were collected, frozen in liquid nitrogen and stored at -80°C; 2) RNA extraction: total RNA was extracted using the RNA simple total RNA extraction kit (Tiangen, Cat No: DP419), and the RNA concentration was detected using NanoDrop (OD260 / 280 = 1.8-2.0); 3) reverse transcription: RNA was reverse transcribed into cDNA using the kit All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (Quangen, Cat No: AU341) (reaction system: 1 μg RNA, 5x All-in-One Reaction Mix for qPCR (4 μL), TransScript Uni All-in-One Enzyme Mix (1 μL), RNase-free Water (make up to 20 ul)), the system was incubated at 50°C for 5 minutes; 4) qPCR: qPCR reaction was performed according to the method and steps provided in the instructions using Green qPCR SuperMix (Quangen, Cat No: AQ601-01-V2); 5) result calculation: the relative expression of CGREF1 was calculated according to the 2 (-ΔΔCt) method (GAPDH as internal reference, primer sequences as follows: CGREF1: 5'-ACGATGACAGTGTTAATCCTGC-3' (forward primer, SEQ ID NO: 1) and 5'-CCTAGTCCCTTTAGGTAGCTCTG-3' (reverse primer, SEQ ID NO: 2); GAPDH: 5'-ACAGTCAGCCGCATCTTCTT-3' (forward primer, SEQ ID NO: 3) and 5'-GGATGCCACAGGACTCCAT-3' (reverse primer, SEQ ID NO: 4)), and the expression difference between tumor and normal tissue was compared using paired t test. The results showed that the CGREF1 mRNA level in 84.2% (16 / 19) of CRC samples was higher than that in the paired normal tissue Figure 1 E). The above results show that CGREF1 is highly expressed in CRC.

[0064] Example 2: CGREF1 high expression is related to CRC progression and poor prognosis

[0065] The present embodiment performed immunohistochemical staining (IHC) analysis on 80 pairs of paraffin-embedded CRC samples and paired normal tissues from Hunan Cancer Hospital. The specific steps include: deparaffinization and hydration of tissue sections, immersion in xylene I, II, III for 10 minutes each, followed by gradient ethanol (100%, 95%, 85%, 75%) rehydration for 5 minutes each, and washing with PBS for 3 times, 5 minutes each. Then, antigen retrieval was performed by placing the sections in sodium citrate antigen retrieval solution (Biogenics, Cat No: AR0024) for high pressure retrieval for 5 minutes, and then naturally cooling to room temperature. Then, 3% BSA was used for room temperature blocking for 30 minutes, followed by primary antibody incubation, adding CGREF1 antibody (Proteintech, Cat No: 13323-1-AP) diluted at a dilution ratio of 1:100, 4°C incubation overnight, PBS washing (washing 3 times, 5 minutes each), and then adding immunohistochemical mouse / rabbit secondary antibody (Sivabio, Cat No: G1303-10ML), 37°C incubation for 20 minutes. In the color development step, the color developing solution was prepared according to the instructions of the DAB kit (Solarbio, Cat No: DA1010), added to the sections, and then observed under a microscope for color development (1-5 minutes), and then washed with distilled water to terminate the reaction. Finally, hematoxylin counterstaining was performed, and then the sections were sealed with neutral resin, and observed under an optical microscope. According to the immunoreaction score (IRS), the interpretation is as follows: IRS = staining intensity (0-3 points: none, weak, moderate, strong, respectively) x positive cell proportion (0-4 points: 0%, 1-25%, 26-50%, 51-75%, 76-100%, respectively), total score 0-12 points (≤6 points for low expression, >6 points for high expression).

[0066] The results show that CGREF1 is overexpressed in 61.25% (49 / 80) of CRC samples; in contrast, only 25% of normal colorectal tissues show weak expression of CGREF1 (immunoreaction score, IRS 1-3), and none of the 80 normal tissues shows moderate (IRS 4-6) or strong expression (IRS 7-12) Figure 2 A-2B, Table 1). Clinical pathological correlation analysis using chi-square test shows that CGREF1 high expression is associated with more invasive tumor phenotypes, including advanced stage (p=0.006), lymph node metastasis (p=0.007), tumor cell budding (p=0.017), and vein invasion (p=0.043) Figure 2 C-2D, Table 2). In addition, the expression of CGREF1 is significantly up-regulated in high-level budding areas (bd3) Figure 2 E, Table 2).

[0067] In addition, the results of Kaplan-Meier survival analysis on the above-mentioned 80 cases from Hunan Cancer Hospital show that CGREF1 high expression is significantly associated with a significantly shortened survival period of patients (Figure 2 F) Univariate analysis showed that advanced stage (p=0.001), lymph node metastasis (p=0.008), venous invasion (p=0.000) and CGREF1 expression (p=0.001) were significant prognostic factors; multivariate Cox regression analysis showed that CGREF1 expression (p=0.014) and venous invasion (p=0.034) were independent prognostic factors for patient survival (Table 3). The above results suggest that CGREF1 can be used as a prognostic biomarker indicating CRC progression and invasiveness.

[0068] In addition, by time-dependent ROC curve, Figure 2 G clearly shows the analysis of CGREF1 expression level for 5-year survival prediction of colorectal cancer patients, and the AUC value (0.728) and curve shape (high true positive rate, low false positive rate) directly prove the effectiveness of CGREF1 as a long-term survival prediction biomarker.

[0069] Table 1: Expression of CGREF1 in normal colon tissue and CRC tissue

[0070]

[0071] The p value is the result of analyzing the relationship between normal colon tissue and tumor tissue and the strength of CGREF1 expression using the chi-square test.

[0072] Table 2: Relationship between CGREF1 expression and CRC clinicopathological parameters

[0073]

[0074]

[0075] The p value is the result of analyzing the relationship between CGREF1 expression and gender, histopathological grade, microsatellite stability status, etc. using the chi-square test.

[0076] Table 3: Univariate and multivariate Cox regression calculation of disease-free survival

[0077]

[0078]

[0079] The T-test analysis method was used for univariate analysis, and the Cox regression analysis method was used for multivariate analysis.

[0080] Example 3: Effect of CGREF1 on CRC cell proliferation and invasion and metastasis ability

[0081] This example aims to explore the effect of CGREF1 on CRC cell proliferation and invasion and metastasis ability.

[0082] (1) Constructing CGREF1 knockdown expression of CRC cell lines

[0083] Cell culture: SW480, RKO cells (CRC cell lines) were cultured with Gibco DMEM medium (Cat No: 11965-092) containing 10% Gibco FBS (Cat No: 10099-141) and 1% penicillin-streptomycin, and incubated in a 37°C, 5% CO2 incubator.

[0084] siRNA transfection: CGREF1 siRNA (5'-GCACATTGTTCAAGTGGAGAA-3' (SEQ ID NO: 5)) and nonsense control sequence (5'-TTCTCCGAACGTGTCACGTTT-3' (SEQ ID NO: 6)) were designed and synthesized by Shenguo Company. One day before transfection, SW480 cells and RKO cells were seeded in 6-well plates (5 x 10 5 cells / well) and cultured to 70% confluence. Then, according to the operation steps of Thermo Fisher Lipofectamine TM 3000 (Cat No: L3000015) instructions, mix siRNA and its control group with Lipofectamine TM 3000 at a final concentration of 50 nM, incubate at room temperature for 15 minutes, then add to cell wells, and detect the knockdown efficiency after 48 hours of culture.

[0085] Knockdown efficiency verification: First, extract the RNA of the transfected cells, then detect the CGREF1 mRNA level of the transfected cells by qRT-PCR method, and the experimental method and steps are the same as in Example 1. Western blot was used to detect the CGREF1 protein expression of the transfected cells, and the steps were as follows: total protein was extracted from cells using RIPA protein lysis buffer containing phenylmethylsulfonyl fluoride (PMSF), and lysed on ice for 30 minutes, then centrifuged to obtain the supernatant, then the protein concentration was quantified by BCA kit (Thermo Fisher, Cat No: 23225), then 30 μg of protein sample was loaded according to the protein concentration for SDS-PAGE electrophoresis (10% separating gel), PVDF membrane was transferred after blocking, then CGREF1 primary antibody (Proteintech, Cat No: 13323-1-AP, 1:1000 dilution) was used for 4°C incubation overnight. After washing the membrane, incubate the secondary antibody (Rabbit anti-RM3002, Mouse anti-RM3001) at room temperature for 1 hour, wash the membrane again, then use ECL chemiluminescence method to detect the signal intensity on the membrane. Finally, analyze the protein band gray value with ImageJ software.

[0086] The experimental results show that siRNA can effectively knock down CGREF1 expression in SW480 and RKO cells, and qRT-PCR and Western blot verify the knockdown efficiency of siRNA Figure 3 A-3B).

[0087] (2) Proliferation experiment

[0088] CCK8 detection: After siRNA transfection, the cells were inoculated in 96-well plates (1 x 10 3 cells / well), 5 replicates per group. After 0, 24, 48, and 72 hours of culture, 10 μL of CCK-8 reagent was added to each well, and incubated at 37°C for 2 hours. Then the 450 nm absorbance (OD value) of the sample was detected by a microplate reader (Bio-Rad), and a growth curve was drawn.

[0089] Colony formation experiment: After siRNA transfection, the cells were inoculated in 6-well plates (5 x 10 2 cells / well), 3 replicates per group. After 14 days of culture, the medium was changed every 3 days. After colony formation, the cells were washed with PBS twice, then fixed with 4% paraformaldehyde for 30 minutes, and then stained with 0.1% crystal violet (Sigma, Catalog No. C3886) for 10 minutes. After washing the residual crystal violet with tap water, the colonies were counted.

[0090] EdU cell proliferation experiment: After siRNA transfection, the cells were inoculated on coverslips in 6-well plates and cultured for 24 hours. EdU reagent (final concentration 10 μM) in the EdU-488 cell proliferation detection kit (Bi Yun Tian, Catalog No. C0071S) was added and incubated at 37°C for 2 hours. The cells were fixed with 4% paraformaldehyde, and then permeabilized with 0.5% Triton X-100 for 10 minutes. Then the Click reaction was performed according to the kit instructions (add Click reaction solution, incubate at room temperature for 30 minutes in the dark), and the nuclei were stained with DAPI (Sigma, Catalog No. D9542, 1:1000 dilution) for 10 minutes before mounting. Observed under a fluorescence microscope (Zeiss), and the proportion of EdU positive cells was counted.

[0091] The results of the proliferation experiment show that CGREF1 knockdown has no significant effect on CRC cell proliferation Figure 3 C-3E).

[0092] (3) Migration and invasion experiment

[0093] Scratch assay: siRNA-transfected cells were seeded in 6-well plates and cultured to 100% confluence. Then, a 200-μL gun tip was used to make a scratch perpendicular to the bottom of the well. After washing twice with PBS to remove floating cells, serum-free medium was added. At different time points, photographs were taken using a microscope (x100) and the scratch width was measured using ImageJ software. The migration rate was calculated as follows: migration rate = (0-hour scratch width - 24-hour scratch width) ÷ 0-hour scratch width x 100.

[0094] Transwell migration assay: Transwell chambers (Corning, item number: 3422) were used. DMEM medium containing 20% FBS (500 μL) was added to the lower chamber, and siRNA-transfected cells (1 x 105 cells / well) were added to the upper chamber. The cells were diluted with serum-free medium to 200 μL. After incubation at 37°C and 5% CO2 for 24 hours, the chambers were removed, and the cells in the upper and lower chambers were fixed with 4% paraformaldehyde for 15 minutes. Finally, the cells were stained with 0.1% crystal violet for 10 minutes. The cells that did not migrate in the upper chamber were removed with a cotton swab, and the number of cells that migrated to the lower chamber was counted under a microscope (3 replicate wells per group, 5 fields per well). 5 Transwell migration assay: Transwell chambers (Corning, item number: 3422) were used. DMEM medium containing 20% FBS (500 μL) was added to the lower chamber, and siRNA-transfected cells (1 x 105 cells / well) were added to the upper chamber. The cells were diluted with serum-free medium to 200 μL. After incubation at 37°C and 5% CO2 for 24 hours, the chambers were removed, and the cells in the upper and lower chambers were fixed with 4% paraformaldehyde for 15 minutes. Finally, the cells were stained with 0.1% crystal violet for 10 minutes. The cells that did not migrate in the upper chamber were removed with a cotton swab, and the number of cells that migrated to the lower chamber was counted under a microscope (3 replicate wells per group, 5 fields per well).

[0095] The results of the migration and invasion assay showed that knocking down CGREF1 significantly inhibited the migration and invasion of CRC cells (A-4B). Figure 4 The results of the migration and invasion assay showed that knocking down CGREF1 significantly inhibited the migration and invasion of CRC cells (A-4B).

[0096] (4) F-actin immunofluorescence staining

[0097] siRNA-transfected cells were seeded in a confocal dish and incubated at 37°C and 5% CO2 for 24 hours. Then, the cells were fixed with 4% paraformaldehyde for 15 minutes, washed with PBS, and permeabilized with 0.5% Triton X-100 for 10 minutes. Next, the cells were blocked with 3% BSA at room temperature for 1 hour. Alexa Fluor 594-labeled phalloidin (UElandy, item number: YP0052L, diluted 1:200) was added for F-actin staining, and the cells were incubated at room temperature in the dark for 1 hour to bind F-actin. The cells were stained with DAPI for 5 minutes, washed with PBS, and mounted with an anti-fluorescence quenching mounting medium. The distribution of F-actin (pseudopod formation) was observed and photographed using a Zeiss LSM 880 laser confocal microscope.

[0098] The results of F-actin immunofluorescence staining showed that knocking down CGREF1 inhibited the expression of F-actin (fibrous actin) and pseudopod formation in CRC cells (F), suggesting that CGREF1 promotes the migration and invasion of CRC cells by regulating the cytoskeleton. Figure 4 The results of F-actin immunofluorescence staining showed that knocking down CGREF1 inhibited the expression of F-actin (fibrous actin) and pseudopod formation in CRC cells (F), suggesting that CGREF1 promotes the migration and invasion of CRC cells by regulating the cytoskeleton.

[0099] (5) In vivo metastasis experiment

[0100] The role of CGREF1 in tumor metastasis in vivo was explored by tumor orthotopic implantation and liver metastasis detection.

[0101] The animal model was selected as 6-8 week old female BALB / c nude mice (Charles River, item number: 025), and the adaptive feeding lasted for 1 week (room temperature 22-25℃, 12 hours light and dark cycle, free drinking and drinking water).

[0102] The 6-8 week old female BALB / c nude mice were selected to receive RKO cell orthotopic transplantation surgery under anesthesia, and the CGREF1 knockdown RKO cells or negative control group (nonsense control sequence transfection) RKO cells prepared in Example 3 were resuspended with PBS (2x10 6 The mice were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg / kg), and after laparotomy, the cell suspension was injected into the cecum wall, and the incision was sutured. After the operation, penicillin (100,000 U / each) was injected to prevent infection. The body weight of the mice was measured every week, and the health status of the mice was observed, and the mice were sacrificed 6 weeks after implantation. The primary colon tumor and liver were removed, washed with PBS, and the liver tissue was fixed with 4% paraformaldehyde for 24 hours, paraffin-embedded section (4 μm thick), and then H&E staining was performed to observe the liver metastasis foci.

[0103] The results of the in vivo metastasis experiment showed that CGREF1 knockdown did not affect the growth of primary colorectal tumors ( Figure 4 C), but could significantly reduce the number of liver metastasis foci ( Figure 4 D-4E).

[0104] The embodiments of the application are described in detail above in combination with the drawings, but the application is not limited to the above embodiments, and various changes can be made within the knowledge range of those skilled in the art without departing from the purpose of the application. In addition, the embodiments of the application and the features in the embodiments can be combined with each other without conflict.

Claims

1. Use of CGREF1 inhibitors in the preparation of drugs for the prevention and / or treatment of colorectal cancer metastasis.

2. The use according to claim 1, characterized in that, The CGREF1 inhibitors include nucleic acid molecules, protein molecules, and small molecule compounds.

3. The use according to claim 2, characterized in that, The nucleic acid molecules include microRNA, siRNA, shRNA, dsRNA, sgRNA, and antisense oligonucleotides.

4. The use according to claim 3, characterized in that, The siRNA sequence is shown in SEQ ID NO:

5.

5. The use according to claim 1, characterized in that, The colorectal cancer metastases include lymph node metastases, liver metastases, lung metastases, peritoneal metastases, bone metastases, and brain metastases.

6. The use according to claim 1, characterized in that, The prevention and / or treatment of colorectal cancer metastasis includes inhibiting the migration and invasion of tumor cells, and the inhibition of tumor cell migration and invasion includes inhibiting F-actin expression and pseudopodia formation.

7. Use of a pharmaceutical composition in the preparation of a medicament for the prevention and / or treatment of colorectal cancer metastasis, said pharmaceutical composition comprising a CGREF1 inhibitor and pharmaceutically acceptable excipients.

8. The use of CGREF1 in prognostic prediction of colorectal cancer patients.

9. The use according to claim 8, characterized in that, Predicting the prognosis of colorectal cancer patients based on the CGREF1 immune response score (IRS) threshold, wherein the criteria for predicting the prognosis of colorectal cancer patients based on the CGREF1 IRS score threshold are as follows: Compare IRS scores with IRS score thresholds in colorectal cancer patients. If a colorectal cancer patient's IRS score is greater than the IRS score threshold, the prognosis is considered poor; if the IRS score is less than or equal to the IRS score threshold, the prognosis is considered good.

10. The use according to claim 8, characterized in that, The prognostic predictions include 5-year survival predictions.

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