Application of TROAP in the Diagnosis and Treatment of Colorectal Cancer

Abstract

The present invention discloses the application of TROAP in the diagnosis and treatment of colorectal cancer. The biomarker TROAP provided by the present invention can effectively diagnose colorectal cancer, diagnose the stage / metastasis of colorectal cancer, predict the prognosis of colorectal cancer, and at the same time can inhibit the proliferation, invasion and lipid droplet formation of colorectal cancer by inhibiting TROAP, so as to achieve the effect of treating colorectal cancer, and can also enhance the sensitivity to chemotherapy drugs. The TROAP biomarker provided by the present invention provides new ideas for the diagnosis, treatment and drug resistance of colorectal cancer, and has broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to the application of TROAP in the diagnosis and treatment of colorectal cancer. Background Art

[0002] Colorectal cancer (CRC) is one of the most common malignant tumors, with both its incidence and mortality ranking among the top, and there is a great heterogeneity in the prognosis of patients. The 5-year survival rate of early-stage CRC patients is as high as 90%, while that of late-stage patients is only 10%. Among patients in the advanced stage, the outcomes vary greatly, and half of the patients experience recurrence or metastasis. In-depth understanding of the occurrence and progression mechanisms of CRC is crucial for improving the diagnosis and prognosis of this disease. Therefore, finding diagnostic biomarkers with high sensitivity and specificity is of great significance for the early diagnosis and treatment of CRC. Summary of the Invention

[0003] To make up for the deficiencies of the prior art, the present invention provides the application of TROAP in the diagnosis and treatment of colorectal cancer.

[0004] To achieve the above object, the present invention adopts the following technical solutions:

[0005] The first aspect of the present invention provides any one of the following applications of a reagent for detecting the expression level of TROAP:

[0006] (1) Preparing a product for diagnosing colorectal cancer;

[0007] (2) Preparing a product for diagnosing the stage / metastasis of colorectal cancer;

[0008] (3) Preparing a product for predicting the prognosis of colorectal cancer.

[0009] Furthermore, the reagent includes primers for amplifying TROAP, probes for recognizing TROAP, or binders for binding to the protein encoded by the TROAP gene.

[0010] Furthermore, the sequences of the primers for amplifying TROAP are as shown in SEQ ID NO: 6-7.

[0011] Furthermore, the reagent further includes a detectable label.

[0012] Furthermore, the product includes a kit, a test strip.

[0013] The second aspect of the present invention provides the application of an inhibitor of TROAP in preparing a pharmaceutical composition for treating colorectal cancer / increasing the sensitivity of colorectal cancer to chemotherapeutic agents / inhibiting the formation of lipid droplets in colorectal cancer.

[0014] Furthermore, the inhibitor includes nucleic acid inhibitors, protein inhibitors, compounds.

[0015] Furthermore, the nucleic acid inhibitor includes siRNA, shRNA, ribozyme.

[0016] Furthermore, the nucleic acid inhibitor is selected from shRNA.

[0017] Furthermore, the sequence of the shRNA is as shown in SEQ ID NO:1.

[0018] Furthermore, the chemotherapeutic agent includes platinum drugs and / or pyrimidine drugs.

[0019] Furthermore, the platinum drug is selected from cisplatin.

[0020] Furthermore, the pyrimidine drug is selected from uracil analogs.

[0021] Furthermore, the uracil analog is selected from 5-FU.

[0022] The third aspect of the present invention provides a pharmaceutical composition, and the pharmaceutical composition includes an inhibitor of TROAP.

[0023] Furthermore, the pharmaceutical composition further includes other drugs.

[0024] Furthermore, the other drugs include other drugs for treating colorectal cancer / increasing the sensitivity of colorectal cancer to chemotherapeutic agents / inhibiting the formation of lipid droplets in colorectal cancer.

[0025] Furthermore, the other drugs for treating colorectal cancer include one or more of antiviral drugs, chemotherapeutic drugs, targeted therapy drugs, immunotherapy drugs, and traditional Chinese medicine drugs.

[0026] Furthermore, the other drugs for increasing the sensitivity of colorectal cancer to chemotherapeutic agents / inhibiting the formation of lipid droplets in colorectal cancer include fatty acid synthesis inhibitors and / or Akt inhibitors.

[0027] Furthermore, the fatty acid synthesis inhibitor includes an inhibitor of one or more of ACLY, ACC1, and MOGAT2.

[0028] Furthermore, the inhibitor includes a nucleic acid inhibitor, a protein inhibitor, and a compound.

[0029] Furthermore, the nucleic acid inhibitor includes siRNA, shRNA, ribozyme.

[0030] Furthermore, the nucleic acid inhibitor is selected from shRNA.

[0031] Furthermore, the shRNA sequence of ACLY is as shown in SEQ ID NO:2.

[0032] Further, the shRNA sequence of ACC1 is as shown in SEQ ID NO:3.

[0033] Further, the compound inhibitor of ACLY is selected from SB-204990.

[0034] Further, the compound inhibitor of ACC1 is selected from PF-05175157.

[0035] Further, the Akt inhibitor is selected from AZD5363.

[0036] Further, the pharmaceutical composition further comprises a pharmaceutically compatible carrier.

[0037] The fourth aspect of the present invention provides a method for screening a candidate drug for treating colorectal cancer, the method comprising: testing the effect of a candidate drug on the level of TROAP in a sample, wherein, after using the candidate drug, a decrease in the level of TROAP indicates that the candidate drug has an effect in treating colorectal cancer.

[0038] The fifth aspect of the present invention provides the use of TROAP as a target in screening candidate drugs for treating colorectal cancer.

[0039] Further, the method comprises: testing the effect of a candidate drug on the level of TROAP in a sample, wherein, after using the candidate drug, a decrease in the level of TROAP indicates that the candidate drug has an effect in treating colorectal cancer.

[0040] The sixth aspect of the present invention provides a method for regulating the sensitivity of colorectal cancer to chemotherapeutic agents / the formation of lipid droplets in colorectal cancer, the method comprising administering an inhibitor of TROAP.

[0041] Further, the chemotherapeutic agent comprises a platinum-based drug and / or a pyrimidine-based drug.

[0042] Further, the platinum-based drug is selected from cisplatin.

[0043] Further, the pyrimidine-based drug is selected from uracil analogs.

[0044] Further, the uracil analog is selected from 5-FU.

[0045] Further, the method further comprises administering other drugs for regulating the sensitivity of colorectal cancer to chemotherapeutic agents / the formation of lipid droplets in colorectal cancer.

[0046] Further, the other drugs for regulating the sensitivity of colorectal cancer to chemotherapeutic agents / the formation of lipid droplets in colorectal cancer comprise fatty acid synthesis inhibitors and / or Akt inhibitors.

[0047] Furthermore, the fatty acid synthesis inhibitor includes an inhibitor of one or more of ACLY, ACC1, and MOGAT2.

[0048] Furthermore, the inhibitor includes a nucleic acid inhibitor, a protein inhibitor, and a compound.

[0049] Furthermore, the nucleic acid inhibitor includes siRNA, shRNA, and ribozyme.

[0050] Furthermore, the nucleic acid inhibitor is selected from shRNA.

[0051] Furthermore, the shRNA sequence of ACLY is as shown in SEQ ID NO:2.

[0052] Furthermore, the shRNA sequence of ACC1 is as shown in SEQ ID NO:3.

[0053] Furthermore, the compound inhibitor of ACLY is selected from SB-204990.

[0054] Furthermore, the compound inhibitor of ACC1 is selected from PF-05175157.

[0055] Furthermore, the Akt inhibitor is selected from AZD5363.

[0056] Furthermore, the method is a method for non-therapeutic purposes.

[0057] The seventh aspect of the present invention provides a method for regulating apoptosis / migration / adhesion of colorectal cancer cells, the method comprising administering an inhibitor of TROAP.

[0058] Furthermore, the method is a method for non-therapeutic purposes.

[0059] The eighth aspect of the present invention provides a method for regulating the expression of any one of the following substances, the method comprising administering an inhibitor of TROAP, the substances including one or more of Ki67, PI3K, AKT, p-AKT, p-mTor, CyclinD1, CDK4, CDC-25c, NF-κB, p-STAT3, β-catenin, Bcl-2, XIAP, Zeb1, sluge, MMP9, P27, p-p38, LC-3α / β, Caspase1, Gasdemin D, ING5, P300, HDAC1, MOGAT2, ACLY, ACC1, AC-H3, and AC-H4.

[0060] Furthermore, the method is a method for non-therapeutic purposes.

[0061] The ninth aspect of the present invention provides the application of TROAP in constructing a computational model for diagnosing colorectal cancer / diagnosing colorectal cancer metastasis / staging / predicting the prognosis of colorectal cancer.

[0062] The tenth aspect of the present invention provides the application of TROAP in constructing a system / device for diagnosing colorectal cancer / diagnosing colorectal cancer metastasis / staging / predicting the prognosis of colorectal cancer.

[0063] Advantages and beneficial effects of the present invention:

[0064] The biomarker TROAP provided by the present invention can effectively diagnose colorectal cancer, diagnose the stage / metastasis of colorectal cancer, predict the prognosis of colorectal cancer, and at the same time, by inhibiting TROAP, it can inhibit the proliferation, invasion, and lipid droplet formation of colorectal cancer, achieving the effect of treating colorectal cancer. At the same time, it can also enhance the sensitivity of colorectal cancer to chemotherapeutic drugs. The TROAP biomarker provided by the present invention provides new ideas for the diagnosis, treatment, and drug resistance of colorectal cancer and has broad application prospects. Description of the drawings

[0065] Figure 1 It is the differential expression map of TROAP in the Xiantao database;

[0066] Figure 2 It is the differential expression map of TROAP in the UALCAN database;

[0067] Figure 3 It is the differential expression map of TROAP in the GEO database;

[0068] Figure 4 It is the overall survival and progression-free survival map of colorectal cancer patients;

[0069] Figure 5 It is the differential expression and survival map of TROAP in colorectal cancer tissues;

[0070] Figure 6 It is the effect of overexpressing or knocking down TROAP on cell proliferation in an animal model;

[0071] Figure 7 It is the effect of overexpressing or knocking down TROAP on cell apoptosis, migration, invasion, and adhesion;

[0072] Figure 8 It is the effect of overexpressing or knocking down TROAP on related factors;

[0073] Figure 9 It is the sequencing result map;

[0074] Figure 10 It is the drug sensitivity map of overexpressing or knocking down TROAP;

[0075] Figure 11 It is a diagram showing the effects of overexpression or knockdown of TROAP on fatty acid synthesis-related factors;

[0076] Figure 12 It is a diagram showing the effects of overexpression or knockdown of TROAP, ACC1, ACLY, PA, or HG on lipid droplet formation;

[0077] Figure 13 It is a diagram showing the drug sensitivity of overexpression or knockdown of TROAP, ACC1, or ACLY;

[0078] Figure 14 It is a diagram showing the drug sensitivity, the effect on lipid droplet formation, and the effect on signal pathway-related factors of overexpression or knockdown of TROAP, Akt agonist, or Akt inhibitor;

[0079] Figure 15 It is a diagram showing the effects of overexpression or knockdown of TROAP and HDAC inhibitor on drug sensitivity and lipid droplet formation. Detailed implementation manners

[0080] The following provides definitions of some terms used in this specification. Unless otherwise specified, all technical and scientific terms used herein generally have the same meaning as commonly understood by those of ordinary skill in the art to which this invention pertains.

[0081] The present invention provides any of the following applications of a reagent for detecting the expression level of TROAP:

[0082] (1) Preparing a product for diagnosing colorectal cancer;

[0083] (2) Preparing a product for diagnosing the stage / metastasis of colorectal cancer;

[0084] (3) Preparing a product for predicting the prognosis of colorectal cancer.

[0085] In one embodiment, TROAP includes wild-type, mutant, or a fragment thereof. This term encompasses full-length, unprocessed TROAP, any form of TROAP resulting from processing in cells, and naturally occurring variants of TROAP (such as splice variants or allelic variants). This term encompasses, for example, human TROAP and TROAP from any other vertebrate source, including mammals such as primates and rodents (e.g., mice and rats), Gene ID: 10024.

[0086] In one embodiment, diagnosing the stage of colorectal cancer refers to diagnosing the TNM stage of colorectal cancer, and the specific stages include Stage1, Stage2, Stage3, and Stage4.

[0087] In one embodiment, diagnosing colorectal cancer metastasis refers to colorectal cancer lymph node metastasis. When there is no lymph node metastasis, it is denoted as N0. As the degree and scope of lymph node metastasis increase, they are sequentially denoted as N1 to N2. The larger the number, the more lymph node metastases there are.

[0088] In one embodiment, prognosis refers to the expectation regarding medical development (e.g., long-term survival probability, disease-free survival rate, etc.), including positive prognosis or negative prognosis. The negative prognosis includes disease progression such as recurrence, colorectal cancer growth, metastasis, and drug-resistant mortality, and the positive prognosis includes disease remission such as disease-free status, and disease improvement such as regression or stability of colorectal cancer.

[0089] The reagent further includes a detectable label.

[0090] In one embodiment, a label refers to a composition capable of generating a detectable signal indicating the presence of a target polynucleotide in a test sample. Suitable labels include but are not limited to radioisotopes, nucleotide chromophores, enzymes, substrates, fluorescent molecules, chemiluminescent moieties, magnetic particles, bioluminescent moieties. Thus, a label is any composition that can be detected by a device or method, including but not limited to spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, chemical detection devices, or any other suitable device. In some embodiments, the label can be visually detected without the aid of a device. A label is used to refer to any chemical group or moiety having a detectable physical property or any compound capable of causing a chemical group or moiety to exhibit a detectable physical property, such as an enzyme that catalyzes the conversion of a substrate into a detectable product. A label also encompasses compounds that inhibit the manifestation of a specific physical property. A label can also be a compound that is a member of a binding pair, and the other member of which has a detectable physical property.

[0091] Among them, radioisotopes include but are not limited to 3 H, 14 C, 35 S, 125 I, 131 I.

[0092] Enzymes include but are not limited to horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, acetylcholinesterase.

[0093] Fluorescent molecules include but are not limited to FITC, rhodamine, lanthanide phosphors.

[0094] The product includes a kit, a chip, a test strip, a nucleic acid membrane strip.

[0095] The product includes a kit, a test strip.

[0096] In one embodiment, the kit further includes a buffer, a preservative, or a protein stabilizer. The kit may also contain the necessary components for detecting the detectable reagent (e.g., a substrate). The kit may also contain a control sample or a series of control samples, which can be assayed and compared with the test samples contained therein. Each component of the kit is usually encapsulated in a separate container, and all the different containers are packed in a package and accompanied by instructions for observing whether the test subject has a disease related to abnormal expression of the target gene or is at risk of developing the disease.

[0097] Other components of the kit include, but are not limited to: tools for collecting biological samples, tools for labeling the detection reagent (binding agent), a membrane for immobilizing the TROAP protein or TROAP nucleic acid in the biological sample, tools for loading the biological sample onto the membrane, tools for binding the reagent to TROAP in the subject's biological sample, a secondary antibody, tools for isolating total RNA from the subject's biological fluid, tools for performing gel electrophoresis, tools for generating cDNA from the isolated total RNA, tools for performing hybridization assays, and tools for performing PCR.

[0098] The present invention provides the use of an inhibitor of TROAP in the preparation of a pharmaceutical composition for treating colorectal cancer / increasing the sensitivity of colorectal cancer to chemotherapeutic agents / inhibiting the formation of lipid droplets in colorectal cancer.

[0099] In one embodiment, an inhibitor refers to a substance that can specifically bind to TROAP, preferably human TROAP, or bind to the polynucleotide of TROAP or a fragment thereof, and inhibit the activity and / or expression of the TROAP protein or polynucleotide. It includes nucleic acid inhibitors, protein inhibitors, and compounds.

[0100] In a preferred embodiment, the inhibitor of TROAP is selected from nucleic acid inhibitors, and the nucleic acid inhibitors include siRNA, shRNA, and ribozymes.

[0101] In a specific embodiment, the nucleic acid inhibitor of TROAP is selected from shRNA.

[0102] The chemotherapeutic agents include platinum drugs and / or pyrimidine drugs.

[0103] In one embodiment, the platinum drugs include, but are not limited to, cisplatin, carboplatin, nedaplatin, oxaliplatin, and lobaplatin.

[0104] In a specific embodiment, the platinum drug is selected from cisplatin.

[0105] In one embodiment, the pyrimidine drugs include, but are not limited to, cytosine analogs and uracil analogs.

[0106] In a preferred embodiment, the pyrimidine drug is selected from uracil analogs.

[0107] In one embodiment, the uracil analogs include but are not limited to 5-fluorouracil (fluorouracil, 5-FU or f5U), floxuridine, capecitabine.

[0108] In a specific embodiment, the uracil analog is selected from 5-fluorouracil (fluorouracil, 5-FU or f5U).

[0109] The pharmaceutical composition further comprises other drugs.

[0110] The other drugs include other drugs for treating colorectal cancer / increasing the sensitivity of colorectal cancer to chemotherapeutic agents / inhibiting the formation of lipid droplets in colorectal cancer.

[0111] In one embodiment, the other drugs for treating colorectal cancer include antiviral drugs, chemotherapeutic drugs, targeted therapy drugs, immunotherapy drugs, Chinese patent drugs or any combination thereof.

[0112] In one embodiment, the antiviral drugs include entecavir, lamivudine, sofosbuvir, danoprevir, tenofovir disoproxil fumarate, adefovir dipivoxil, oseltamivir, telbivudine, ritonavir.

[0113] The chemotherapeutic drugs include fluorouracil, cyclophosphamide, doxorubicin, cisplatin, carboplatin, mitomycin, daunorubicin, epirubicin, gemcitabine, irinotecan, oxaliplatin, mitoxantrone.

[0114] The targeted therapy drugs include sorafenib, regorafenib, lenvatinib, donafenib, regorafenib, apatinib, cabozantinib.

[0115] The immunotherapy drugs include atezolizumab, sintilimab, camrelizumab, tislelizumab, bevacizumab, nivolumab, pembrolizumab.

[0116] The Chinese patent drugs include Compound Cantharidin Capsules, Kang'ai Ping Pills, Cinobufacini Capsules, Zhenxiang Capsules, Zhenqi Fuzheng Granules.

[0117] The pharmaceutical composition further comprises a pharmaceutically compatible carrier.

[0118] In one embodiment, pharmaceutical compatibility refers to a non-toxic material that does not interact with the action of the active components of the pharmaceutical composition. The pharmaceutical compatibility carrier refers to a natural or synthetic, organic or inorganic component that is used in combination with the active component to facilitate application. In one embodiment, the pharmaceutical compatibility carrier includes one or more compatible solid or liquid fillers, diluents or encapsulating substances, and the carrier is suitable for administration to a patient. The components of the pharmaceutical composition of the present invention generally do not interact significantly to affect the desired therapeutic effect of the drug.

[0119] The present invention provides the application of TROAP in constructing a system / device for diagnosing colorectal cancer / diagnosing colorectal cancer metastasis / staging / predicting the prognosis of colorectal cancer.

[0120] The system / device includes:

[0121] An acquisition unit: used to acquire the expression level of TROAP in a sample;

[0122] A processing unit: obtaining a diagnosis / diagnosis of metastasis / staging / prognosis prediction result of colorectal cancer according to the expression of TROAP.

[0123] Compared with a normal sample, if the expression level of TROAP shows a significant up-regulation, the diagnosis result is colorectal cancer.

[0124] A high protein expression level of TROAP indicates a poor prognosis of colorectal cancer.

[0125] The present invention will be further described below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are presented by way of example and are not intended to limit the present invention. Without departing from the scope of the present invention, the main features of the present invention can be used in various embodiments.

[0126] Examples

[0127] 1. Experimental materials and methods

[0128] 1) Cell culture and transfection

[0129] Colorectal cancer cell lines (HCT15, RKO) were from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences. The cells were maintained in RPMI-1640 medium (BD Bioscience) containing 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 μg / mL streptomycin at 37 °C with 5% carbon dioxide in a humidified atmosphere. TROAP was ectopically overexpressed in HCT15 cells using the pcDNA3.1-TROAP-3×Flag plasmid (NM_005480), and TROAP was knocked down in RKO cells using pGPU6 / GFP-shTROAP. The shTROAP target sequence: 5'-GCCCTGTGTTTCATTCCAGTT-3' (NM_005480, SEQ ID NO:1). The plasmids pcDNA3.1-ACC1-3×Flag, pcDNA3.1-ATP citrate lyase (ACLY)-3×Flag, shRNA-ACC1 (acetyl-CoA carboxylase 1), and shACLY plasmids were purchased from HonorGene. The shACLY target sequence: 5′-CCTATGACTATGCCAAGACTA-3′ (SEQ ID NO:2), and the shACC1 target sequence: 5′-TACAAGGGATACAGGTATTTA-3′ (SEQ ID NO:3). The ACC1 inhibitor (PF-05175157) and ACLY inhibitor (SB-204990) were purchased from AbMole. The HDAC inhibitor SAHA was purchased from MCE. All plasmids were transfected using Lipofectamine 3000 (Thermo Fisher Scientific). They were treated with cisplatin (DDP) or 5-fluorouracil (5-FU). The cells were treated with the Akt agonist SC79 (Beyotime) and the Akt inhibitor AZD5363 (Beyotime), and the changes in cell phenotypes and the expression of their related proteins were observed.

[0130] 2) Cell proliferation assay

[0131] Cell Counting Kit-8 (CCK-8) was used to evaluate the number of viable cells. Initially, 5.0×10 3 cells / well were seeded in 96-well plates. After cell adhesion, according to the experimental time points, CCK-8 solution (10 μL) was added to each well. After incubation for 3 hours at 37 °C in a humidified atmosphere with 5% CO2, the absorbance was measured at 450 nm using a microplate reader (Multiskan FC microplate photometer). The data were carefully analyzed and visualized using Prism 8.

[0132] 3) Flow cytometry for apoptosis detection

[0133] For the apoptosis assay, 1×106 Cells. Apoptotic cells were detected using a flow cytometer FC500 (Beckman Coulter, USA), stained with Annexin V-phycoerythrin (PE) and 7-aminoactinomycin (7-AAD) (BD Pharmingen, USA). The staining method was carried out strictly according to the reagent instructions.

[0134] 4) Scratch assay

[0135] Cells were seeded in 6-well plates at a density of 5×10 5 cells / well. Until the cells were nearly 80%-90% confluent, the monolayer cells were scratched with a 200 μL pipette tip. Then the cell debris in the scratch was rinsed 3 times with PBS. Then the cells were cultured in serum-free medium and photographed at 48 h and 72 h. Finally, the cell migration area was analyzed using ImageJ software.

[0136] 5) Migration and invasion assays

[0137] For the migration assay, 1.0×10 5 cells were seeded in serum-free RPMI 1640, and 200 μL of the cell suspension was cultured in the upper chamber of a transwell plate (BD Bioscience, USA). The lower chamber contained 600 μL of RPMI 1640 supplemented with 10% FBS. After incubation for 24 h, non-migrating cells were scrubbed and washed with PBS. Migrated cells were fixed with 100% methanol and stained with hematoxylin for 10 minutes. For the invasion assay, the procedure was the same as above except that the upper chamber of the transwell plate was coated with matrix.

[0138] 6) Nile red staining

[0139] Cells were cultured overnight in 12-well culture plates. After removing the medium, the cells were washed 3 times with PBS and then fixed with 100% methanol for 20 minutes. After fixation, the cells were washed 3 times with PBS, stained with 1 mg / mL Nile red for 15 minutes. After washing 3 times with PBS, the cells were stained with 0.5 μg / mL DAPI (Solarbio) for 5 minutes and then imaged under an ECHO fluorescence microscope. The images were acquired and analyzed using Image J software (v1.8.0; National Institutes of Health, USA).

[0140] 7) Specimen information

[0141] Tumor and adjacent normal tissue specimens were stored at -80 °C in the Affiliated Hospital of Chengde Medical College from 2020 to 2021. Routine pathological paraffin blocks were prepared by surgical resection, and the frozen samples were used for protein and RNA extraction. There were 78 cases of tumor tissue and normal tissue respectively.

[0142] Inclusion criteria: Pathologically diagnosed with colorectal cancer and not received any form of anti-cancer treatment before surgery.

[0143] Exclusion criteria: Patients with a history of previous tumors or concurrent other malignancies; those who received neoadjuvant chemotherapy and other anti-tumor treatments before surgery.

[0144] Before the start of the clinical study, the patients did not receive chemotherapy, radiotherapy or adjuvant treatment, and all patients provided written consent to use tumor tissues for the clinical study. The Ethics Committee of the Affiliated Hospital of Chengde Medical College approved the conduct of this clinical study. All protocols were carried out in accordance with relevant guidelines and regulations. During or after data collection, information that could identify individual participants was not available.

[0145] 8) Tissue microarray

[0146] The pathological specimens were fixed with 4% paraformaldehyde, dehydrated with alcohol, washed with xylene and then embedded. Sections of 4 μm were cut from the paraffin blocks, stained with hematoxylin and eosin to guide subsequent histological analysis. Representative areas of adjacent normal tissues and solid tumors were identified under a light microscope. Tissue nuclei were extracted from the paraffin blocks and transferred to recipient pathological blocks. The TMA blocks were cut into 4-μm-thick sections for immunohistochemical examination.

[0147] 9) Immunohistochemistry

[0148] After baking at 60 °C for 2 hours, the slides were dewaxed and rehydrated three times in graded alcohol. Antigen retrieval was performed in a microwave oven for 20 minutes. 3% hydrogen peroxide in methanol was applied for 30 minutes to block endogenous peroxidase activity. 5% bovine serum albumin was applied for 30 min to block non-specific binding sites for 30 min. Then, rabbit anti-TROAP antibody (13634-1-AP, Proteintech) was incubated at 37 °C for 3 hours. The slides were washed three times with TBST, and then incubated with goat anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody (1:200; P0399; DAKO, Japan) at 37 °C for 2 h. After washing with TBS, diaminobenzidine staining was performed to observe specific staining, and then hematoxylin staining was carried out. Then, dehydration and mounting were performed. Finally, observation was carried out with a microscope from Nikon (Nikon Corporation, Japan).

[0149] 10) RT-PCR

[0150] According to the instruction manual, total RNA was extracted from colorectal cancer and paired adjacent cancer tissues using the Qiagen RNeasy Mini Kit (74104, QIAGEN, Germany), quantified, and cDNA was obtained by reverse transcription of total RNA (2 μg). The primers used for real-time PCR were: GAPDH (glyceraldehyde-3-phosphate dehydrogenase), forward 5′-GTCTCCTCTGACTTCAACAGCG-3′ (SEQ ID NO:4) and reverse 5′-ACCACCCTGTTGCTGTAGCCAA-3′ (131 bp, SEQ ID NO:5); TROAP, forward 5'-GGACCAGGAGAACCAAGAT-3' (SEQ ID NO:6), reverse 5'-GTTCCGAGGCTGACTGAT-3' (151 bp, SEQ ID NO:7). The BIO-RAD CFX96TM real-time system was used, and iTaqTM Universal Green Supermix (Bio-Rad, Bio-Rad Laboratories, Inc., Singapore) was used for PCR. After briefly centrifuging the cDNA microarray plate, 10 μL of quantitative PCR mixture (2×), 1 μL of each primer (10 μM), and 7 μL of RNase-free ddH2O were added. The experimental procedure was pre-denaturation at 95 °C for 15 min, denaturation at 95 °C for 10 s, annealing at 60 °C for 20 s, extension at 72 °C for 30 s, for 45 cycles. GAPDH was used as an internal reference.

[0151] 11) Western blot assay

[0152] Proteins were extracted from fresh samples and cells using RIPA lysis buffer and quantified using a BCA protein assay kit (NCM; WB6502). Equal amounts of protein were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a PVDF membrane (Millipore). To reduce non-specific antigen binding, the membrane was incubated with 5% skim milk in TBST for 1.5 h at room temperature and then incubated with the primary antibody overnight at 4 °C. After washing three times with TBST, the anti-mouse or anti-rabbit HRP-conjugated secondary antibody (1:5000; #7074; CST) was incubated at room temperature for 2 h, and Western Bright TM ECL chemiluminescence analysis detection kit (K-12045-D50; Advansta, USA) was used, and protein bands were visualized using C300 (Azure Biosystems). Measurement was performed using Image J software (v1.8.0) (National Institutes of Health, USA), with GAPDH as an internal reference.

[0153] 12) Bioinformatics analysis

[0154] Using the XiTao platform (https: / / www.xiantaozi.com / ) and the UALCAN database (http: / / ualcan.path.uab.edu), the expression, related genes and signaling pathways of the TROAP gene in colorectal cancer were analyzed. The prognostic value of TROAP in colorectal cancer was evaluated using Kaplan-Meier (http: / / www.kmplot.com / ). The differentially expressed genes and related genes of TROAP in colorectal cancer were identified using the XiTao database. Protein-protein interaction (PPI) networks were constructed using the differentially expressed genes to determine key hub genes and identify the signaling pathways of colorectal cancer. Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and gene set enrichment analysis (GSEA) were performed on these related genes in colorectal cancer.

[0155] 13) Statistical analysis

[0156] Data analysis was performed using SPSS 23.0 software. The chi-square test was used to compare ratios, and the t-test was used for mean comparison. Survival analysis was performed using the Kaplan-Meier curve and log-rank test, and multivariate analysis was performed using the Cox proportional hazards regression model. A P value less than 0.05 was considered statistically significant.

[0157] 14) Subcutaneous tumorigenesis

[0158] BALB / c mice (6 - 8 weeks old) were purchased and raised according to the ethical guidelines of the Experimental Animal Center of Chengde Medical College Affiliated Hospital. HCT15 cells with stable overexpression of TROAP and RKO cells with TROAP knockdown (1×10 6 cells per 0.1 ml PBS per mouse) were subcutaneously injected into the lower back of BALB / c mice (7 mice per group). The long and short diameters of the tumors were measured using a vernier caliper every 3 days for 24 consecutive days. The tumor volume (mm 3 ) was calculated: volume = length × width2 × 0.5. The tumors were removed and photographed for preservation as paraffin specimens.

[0159] 2. Experimental results

[0160] Compared with normal mucosa, the expression of TROAP mRNA in colorectal cancer was significantly increased in the XiTao ( Figure 1 ), UALCAN ( Figure 2 ) and GEO ( Figure 3 ) databases (p < 0.05).

[0161] The expression of TROAP was negatively correlated with the overall survival of colorectal cancer patients ( Figure 4 ).

[0162] Western blot and RT-PCR analysis showed that the expression of TROAP in colorectal cancer tissues was significantly increased compared with that in normal tissues ( Figure 5 ).

[0163] Through Western blot verification, TROAP (TROAP) was successfully overexpressed in HCT15 cells, and TROAP (shTROAP) was knocked down in RKO cells. In HCT15 cells, CCK8, subcutaneous tumorigenesis assay and histochemical staining showed that overexpression of TROAP increased cell proliferation ( Figure 6 ).

[0164] Flow cytometry and TUNEL staining showed that overexpression of TROAP reduced apoptosis. From scratch and transwell assays, it could be seen that the migration and invasiveness of TROAP-transfected cells were higher. From adhesion assays, it could be seen that the adhesion of TROAP-transfected cells was stronger ( Figure 7 ).

[0165] Western blot experiments showed that overexpression of TROAP in HCT15 cells was accompanied by upregulation of Ki67, PI3K, AKT, p-AKT, p-mTor, CyclinD1, CDK4, CDC-25c, NF-κB, p-STAT3, β-catenin, Bcl-2, XIAP, Zeb1, slug, MMP9, and downregulation of P27, p-p38, LC-3α / β, Caspase1, Gasdemin D expression. Knockdown of TROAP had the opposite effect ( Figure 8 ).

[0166] Sequencing results: The differentially expressed proteins between the two groups were screened using (log2|fold-change|≥1.2 and p<0.05) (upregulation) and (log2|fold-change|≤0.83 and p<0.05) (downregulation) as the criteria. Compared with HCT15 cells, the results showed that 36 proteins were upregulated and 63 proteins were downregulated in the TROAP overexpression group; compared with RKO cells, 133 proteins were upregulated and 60 proteins were downregulated in the TROAP knockdown cells. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed on the differentially expressed proteins. In TROAP vs HCT15, the top three in the Biological Process (BP) category were adenyl ribonucleotide binding, ATP binding and drug binding. The Cellular Component (CC) category included membrane part, integral component of membrane and intrinsic component of membrane. The Molecular Function (MF) category included maintenance of location, cytoskeletal anchoring at nuclear membrane, maintenance of location in cell and maintenance of protein location in cell. Shigellosis, NOD-like receptor signaling pathway, and motor proteins were the top 3 pathways with the most protein enrichment. In shTROAP vs RKO, the top three in the BP category were cation binding, DNA binding and metal ion binding. The CC category included extracellular matrix component, laminin complex and basal lamina. In the MF category, there were positive regulation of angiogenesis, extracellular matrix organization and extracellular structure organization. PI3K-Akt signaling pathway, Pathways in cancer, and Focal adhesion were the top 3 pathways with the most protein enrichment( Figure 9 ).

[0167] TROAP overexpression leads to resistance of HCT15 cells to DDP and 5-FU, while its knockdown increases the sensitivity of RKO cells to these two drugs( Figure 10 ).

[0168] WB experiments showed that TROAP overexpression increased the levels of ING5, P300, HDAC1, MOGAT2 (monoacylglycerol transferase 2), ACLY, and ACC1, and decreased the levels of AC-H3 and AC-H4. TROAP knockdown had the opposite effect( Figure 11 ).

[0169] Nile red staining showed that TROAP overexpression (ov-TROAP) promoted lipid droplet formation in HCT15, while TROAP knockdown (sh-TROAP) inhibited lipid droplet formation in RKO. Overexpression of ACC1 or ACLY (ov-ACC1 or ov-ACLY) or exposure to high glucose (HG) or palmitic acid (PA) restored the effect of TROAP knockdown on lipid droplet formation, and knockdown of ACC1 or ACLY (shACC1 or shACLY) attenuated the effect of TROAP overexpression on lipid droplet formation( Figure 12 ).

[0170] TROAP overexpression promoted chemoresistance to 5-FU and DDP, while TROAP knockdown had the opposite effect. Chemoresistance of HCT15 and RKO cells with overexpressed or knocked-down TROAP was further deteriorated under the action of HG and PA. Overexpression of ACC1 or ACLY restored the effect of TROAP knockdown on chemoresistance, and knockdown of ACC1 or ACLY (shACC1 or shACLY) or inhibitors (ACC1 inhibitor or ACLY inhibitor) attenuated the effect of TROAP overexpression on chemoresistance( Figure 13 ).

[0171] After treatment with the Akt agonist SC79 (5 μM), the drug sensitivity of TROAP-knockdown RKO cells to 5-FU and DDP was reversed. In contrast, in the TROAP-overexpression group, treatment with the Akt inhibitor AZD5363 (1 μM) reversed resistance to 5-FU and DDP. Nile red staining showed that in the TROAP-knockdown group, SC79 treatment could stimulate lipid droplet production, while in the TROAP-overexpression group, AZD5363 treatment inhibited lipid droplet formation. In the TROAP-knockdown group treated with the Akt agonist, the expression of AKT, p-Akt, p-mTor, NF-κB, ACC1, ACLY, and MOGAT2 could be reversed, while in the TROAP-overexpression group treated with the Akt inhibitor, the opposite results were observed( Figure 14 ).

[0172] Fatty acid synthesis largely depends on ACLY, ACC1, and MOGAT2, all of which are associated with chemoresistance. ING5-mediated lipid droplet formation is related to chemotherapy resistance. It was observed that TROAP enhanced the resistance of HCT15 cells to 5-FU and DDP by upregulating ING5, MOGAT2, ACLY, and ACC1.

[0173] Nile red staining showed that the HDAC inhibitor SAHA could promote lipid droplet formation in TROAP-overexpressing cells. SAHA could also enhance the chemoresistance of TROAP-transfected cells to 5-FU and DDP. RT-PCR showed that SAHA could increase the mRNA expression levels of MOGAT2, ACLY, and ACC1 in TROAP-overexpressing cells. WB showed that SAHA could upregulate the levels of AC-H3, AC-H4, ING5, P300, MOGAT2, ACLY, and ACC1 in TROAP-overexpressing cells and decrease the level of HDAC1 ( Figure 15 ).

[0174] Combining these findings, it is speculated that TROAP induces chemoresistance and lipid droplet formation in colorectal cancer cells through the ING5, Akt / mTor pathway, and abnormal deacetylation of histones.

[0175] In summary, the upregulation of TROAP expression is closely related to the occurrence and subsequent progression of colorectal cancer. Its abnormal expression can be used as an indicator of the invasive behavior and poor prognosis of colorectal cancer. TROAP promotes the proliferation, invasion, lipid droplet formation, and chemoresistance of colorectal cancer through the ING5, Akt / mTor pathway, and abnormal deacetylation of histones.

[0176] The description of the above embodiments is only for understanding the method of the present invention and its core idea. It should be noted that for those of ordinary skill 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 within the protection scope of the claims of the present invention.

Claims

1. Use of a TROAP inhibitor in the preparation of a pharmaceutical composition for increasing the sensitivity of colorectal cancer to chemotherapeutic agents / inhibiting the formation of lipid droplets in colorectal cancer, wherein the chemotherapeutic agent is selected from platinum drugs and / or pyrimidine drugs, the platinum drug is selected from cisplatin, the pyrimidine drug is selected from uracil analogs, and the uracil analogs are selected from 5-FU. The shRNA sequence of the TROAP inhibitor is shown in SEQ ID NO:

1.

2. The use according to claim 1, characterized in that: The pharmaceutical composition also includes a pharmaceutically compatible carrier.

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