Use of inhibitors targeting c6orf223 protein in the preparation of a medicament for treating a tumor

Abstract

The application discloses an application of an inhibitor targeting C6orf223 protein in preparation of a tumor treatment drug, and belongs to the technical field of biological medicines. The application finds that the C6orf223 protein can be combined with PRMT5, promotes assembly of a PRMT5 / MEP50 hetero-octamer compound, and thus improves PRMT5 activity and enrichment of H4R3me2s in a series of genes. The application finds that the C6orf223 protein is highly expressed in various tumors; a mouse in-situ model shows that the C6orf223 protein significantly promotes tumor growth and metastasis by improving PRMT5 activity. The application effectively inhibits growth and metastasis of colorectal cancer by using a ferritin shell to wrap siRNA specifically targeting the C6orf223 and shRNA specifically targeting the C6orf223. Therefore, the application provides a new treatment target and treatment strategy for various tumors.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to application of an inhibitor targeting C6orf223 protein in preparation of a tumor treatment drug. BACKGROUND

[0002] In recent years, RNA interference technology is developing rapidly, and also brings new dawn and revolutionary changes to cancer treatment. As a new cancer treatment method, RNA interference technology has great potential and development prospects. RNA interference (RNAi) is a small interfering RNA (siRNA) mediated and specific gene silencing phenomenon involving specific enzymes, which is found in biological organisms in recent years. siRNA is a double-stranded RNA molecule composed of 20-25 nucleotides. In the RNAi pathway, siRNA interferes with gene expression by hybridizing with complementary mRNA molecules. This interference triggers mRNA degradation, thereby inhibiting the expression of specific genes. RNAi can theoretically silence all genes, so siRNA can be used as a targeted therapeutic drug to specifically regulate the expression of disease-related genes. Short hairpin RNA (shRNA) is a kind of artificially synthesized RNA with a tight hairpin turn structure, which can silence the expression of targeted genes through the principle of RNA interference technology.

[0003] Tumors are common and frequently-occurring diseases, among which malignant tumors are the most serious diseases that endanger human health. The etiology, pathogenesis and prevention and treatment of these tumors are the focus of research in this field. Tumor targeted therapy is designed to develop targeted inhibitors for tumor-specific functional molecules, including antibodies, small molecules, biological carriers, etc. These targeted molecules can specifically inhibit the growth, migration and chemotherapy resistance of tumor cells, but have no destructive effect on normal tissue cells. Therefore, targeted therapy can greatly prolong the survival time and quality of life of patients. However, the available targeted therapy targets are limited, and the available targeted drugs are scarce. Therefore, it is urgent to develop a targeted drug that can effectively inhibit tumor growth and metastasis. SUMMARY

[0004] In order to solve the above-mentioned deficiencies existing in the prior art, the purpose of the present application is to provide application of an inhibitor targeting C6orf223 protein in preparation of a tumor treatment drug, so as to effectively inhibit the growth and metastasis of tumors.

[0005] The technical scheme for solving the above technical problems of the present application is as follows: providing application of an inhibitor targeting C6orf223 protein in preparation of a tumor treatment drug.

[0006] Further, the amino acid sequence of the C6orf223 protein is shown as SEQ ID NO. 1.

[0007] The present application provides an inhibitor targeting the C6orf223 protein, which can reduce the expression amount of the C6orf223 protein in tumor cells; the inhibitor comprises siRNA targeting the C6orf223 protein.

[0008] Further, the siRNA targeting the C6orf223 protein comprises siC6orf233-F and siC6orf233-R; wherein the nucleotide sequences of siC6orf233-F and siC6orf233-R are shown as SEQ ID NO. 8 and SEQ ID NO. 9 respectively.

[0009] Further, the inhibitor further comprises shRNA targeting the C6orf223 protein.

[0010] Further, the nucleotide sequence of the shRNA targeting the C6orf223 protein is shown as SEQ ID NO. 2.

[0011] The present application provides a recombinant vector comprising the above-mentioned siRNA targeting the C6orf223 protein or the above-mentioned shRNA targeting the C6orf223 protein.

[0012] The present application provides the use of the above-mentioned inhibitor in the preparation of a drug for treating tumors.

[0013] Further, the tumors comprise colorectal cancer, bladder urothelial carcinoma, breast invasive carcinoma, cholangiocarcinoma, esophageal carcinoma, renal clear cell carcinoma, gastric adenocarcinoma and endometrial carcinoma.

[0014] The present application provides the use of the above-mentioned recombinant vector in the preparation of a drug for treating tumors.

[0015] Further, the tumors comprise colorectal cancer, bladder urothelial carcinoma, breast invasive carcinoma, cholangiocarcinoma, esophageal carcinoma, renal clear cell carcinoma, gastric adenocarcinoma and endometrial carcinoma.

[0016] The present application further provides a drug for treating tumors, which comprises at least one of the above-mentioned inhibitor and the above-mentioned recombinant vector.

[0017] Further, the tumors comprise colorectal cancer, bladder urothelial carcinoma, breast invasive carcinoma, cholangiocarcinoma, esophageal carcinoma, renal clear cell carcinoma, gastric adenocarcinoma and endometrial carcinoma.

[0018] The application has the following beneficial effects: the application finds that C6orf223 protein can be combined with PRMT5, promote the assembly of PRMT5 / MEP50 hetero-octamer complex, thereby improving the activity of PRMT5 and the enrichment of H4R3me2s in a series of genes. The application analyzes the expression of C6orf223 protein in normal tissues and tumor tissues, finds that it is negative in most normal tissues, and is low in lung and kidney; but is highly expressed in a variety of tumors. The in situ mouse model shows that C6orf223 protein significantly promotes the growth and metastasis of colorectal cancer by improving the activity of PRMT5. The application effectively inhibits the growth and metastasis of colorectal cancer by using ferritin shell to encapsulate siRNA specifically targeting C6orf223 and shRNA specifically targeting C6orf223. Therefore, the application provides a new treatment target and treatment strategy for a variety of tumors. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 A diagram of the molecular mechanism of C6orf223-mediated PRMT5 / MEP50 hetero-octamer assembly;

[0020] Figure 2 A diagram of the amino acid sequence of the arginine-rich region of C6orf223; arginine is marked in red;

[0021] Figure 3 A diagram of the expression level of C6orf223 in paired or unpaired normal peritumoral tissues and primary tumors of CRC patients;

[0022] Figure 4 A diagram of the expression level of C6orf223 protein in primary colorectal cancer P and liver metastasis samples M, NCG mice injected with HT29 cells in situ; wherein the HT29 cells carry an empty vector or overexpress PRMT5;

[0023] Figure 5 A diagram of the immunohistochemical image and quantitative analysis of C6orf223 in the tissue chip;

[0024] Figure 6 A diagram of the Kaplan-Meier analysis of the survival curve of colorectal cancer patients with low and high expression of C6orf223;

[0025] Figure 7 A diagram of the expression level of C6orf223 and PRMT5 in HT29 cells transfected with full-length or different C6orf223 mutations or C6orf223 and PRMT5 knockdown, and the expression level of C6orf223 in C6orf223 knockdown HCT116 cells;

[0026] Figure 8 Migration assay plot for HT29 cells with full length or different C6orf223 mutations or C6orf223 and PRMT5 knockdown;

[0027] Figure 9 Clonogenic plot for HT29 cells with full length or different C6orf223 mutations or C6orf223 and PRMT5 gene knockdown;

[0028] Figure 10 Migration assay plot for HCT116 cells after C6orf223 knockdown, followed by rescue with full length C6orf223, C6orf223 Δ1 or C6orf223 C178S;

[0029] Figure 11 Clonogenic plot for HCT116 cells after C6orf223 knockdown, followed by rescue with full length C6orf223, C6orf223 Δ1 or C6orf223 C178S;

[0030] Figure 12 Cell proliferation plot for HCT116 cells after C6orf223 knockdown, followed by rescue with full length C6orf223, C6orf223 Δ1 or C6orf223 C178S;

[0031] Figure 13 Image and weight plot for cecal tumors of NCG mice injected with HCT116 cells in situ;

[0032] Figure 14 Image and quantification plot for liver metastasis of NCG mice injected with HCT116 cells in situ;

[0033] Figure 15 HE staining and quantification plot for liver metastasis foci of NCG mice injected with HCT116 cells in situ;

[0034] Figure 16 Survival curve analysis plot for NCG mice injected with HCT116 cells in cecum;

[0035] Figure 17 Expression level plot of C6orf223 in cancer and normal tissues;

[0036] Figure 18 Process schematic and detection plot for tHFn(+) protein shell encapsulated C6orf223 siRNA;

[0037] Figure 19 Expression level plot of TfR1 in cancer cells;

[0038] Figure 20Figure for expression level of C6orf223 in HCT116 cells after siGFP@tHFn(+), siC6orf223@tHFn(+), or siC6orf223@Lipo2000 treatment;

[0039] Figure 21 Figure for expression level of C6orf223, GATA5, WWTR1, FGFR1, and CLU in HCT116 cells after PBS, tFHn(+), siGFP@tHFn(+), siC6orf223@tHFn(+), or siC6orf223@Lipo2000 treatment;

[0040] Figure 22 Figure for images and quantification of HCT116 cell migration assay after PBS, tFHn(+), siGFP@tHFn(+), siC6orf223@tHFn(+), or siC6orf223@Lipo2000 treatment;

[0041] Figure 23 Figure for images and quantification of HCT116 cell colony formation after PBS, tFHn(+), siGFP@tHFn(+), siC6orf223@tHFn(+), or siC6orf223@Lipo2000 treatment;

[0042] Figure 24 Figure for ex vivo fluorescence images of mouse tumors and major organs after PBS, tFHn(+), siGFP@tHFn(+), siC6orf223@tHFn(+), treatment;

[0043] Figure 25 Figure for HE staining images of major organs after different treatments;

[0044] Figure 26 Figure for images and weight of cecal tumors of NCG mice orthotopically injected with HCT116 cells;

[0045] Figure 27 Figure for bright field images and quantification of metastatic foci of NCG mice after cecal injection of HCT116 cells;

[0046] Figure 28 Figure for HE staining and quantification of metastatic foci of NCG mice after cecal injection of HCT116 cells;

[0047] Figure 29 Figure for immunohistochemistry images and quantification of C6orf223, GATA5, WWTR1, FGFR1, and CLU expression levels in primary tumors of mice orthotopically injected with HCT116 cells;

[0048] Figure 30 Survival curve analysis plot of NCG mice injected with HCT116 cells in cecum. DETAILED DESCRIPTION

[0049] The following examples are intended to illustrate but not limit the present application. Unless otherwise indicated, conventional conditions or manufacturer's recommended conditions were used in the examples. Unless otherwise indicated, reagents and instruments used were conventional products available commercially.

[0050] Primary and liver metastatic colorectal cancer specimens involved in the embodiments of the present application were obtained from the Seventh Medical Center of General Hospital of People's Liberation Army, and the informed consent of all donors was obtained; NOD / ShiLtJ Gpt-Prkdcem26Cd52Il2rgem26Cd22 / Gptc (NCG) mice were purchased from Gem Pharmatech Co., Ltd., and all animal experiments were approved by the Biomedical Research Ethics Committee.

[0051] Experimental methods:

[0052] (1) Co-IP-MS experiment: HEK293T cells were transfected with Vigofect (T001, Vigorous) corresponding plasmids, and cells were collected after 48 h. After cell lysis, the cell lysate was pre-cleaned with Pierce Protein A / G magnetic beads (88803, Thermo). When immunoprecipitating, equal amounts of cell lysate and primary antibody were incubated at 4°C overnight, then protein G magnetic beads were added and incubated at 4°C for 4 h. Then the magnetic beads were boiled with 1×SDS-PAGE sample buffer. Cells or homogenized tissues were lysed in RIPA lysis buffer containing protease inhibitors (04693132001, Roche), centrifuged at 13000 rpm for 10 min, and the supernatant was collected and 5×SDS-PAGE sample buffer (E153-01, GenStar) was added. Protein samples were separated by 10wt% SDS-PAGE and then transferred to PVDF membranes. The membrane was blocked with 5wt% skim milk in PBS at room temperature for 1 h, and incubated with primary antibody at 4°C overnight. After washing three times with 1×PBST (0.05wt% Tween 20 in 1×PBS), the secondary antibody conjugated with horseradish peroxidase (HRP) was incubated at room temperature for 1 h. The target protein was detected by western Dura Extended Duration Substrate (34076, Thermo) chemiluminescence imaging system.

[0053] (2) Western Blot: Cells or homogenized tissues were lysed in RIPA lysis buffer (04693132001, Roche) with protease inhibitors, then centrifuged at 13000 rpm for 10 min, the supernatant was collected, and 5x SDS-PAGE sample buffer (E153-01, GenStar) was added. Protein samples were separated by 10wt% SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5wt% skim milk in 1x PBS at room temperature for 1 h, and incubated with primary antibodies on a shaker at 4°C overnight. After washing with 1x PBST three times, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 1 h. The target proteins were detected by western Dura Extended Duration Substrate (34076, Thermo) chemiluminescence imaging system.

[0054] (3) Blue Native-PAGE (BN-PAGE) detection method: adapted from the user guide of Native PAGE Novex Bis-Tris Gel System (Thermo). Cells or homogenized tissues were lysed in 1x Native PAGE sample buffer (BN2003, Thermo) with 1wt% diantherin and protease inhibitors. After centrifugation at 13000 rpm for 30 min at 4°C, the supernatant was collected, and UltraNuclease (20156ES25, YEASEN) was added to remove nucleic acids. The sample was added with MgCl2 to a final concentration of 2mM and incubated at 37°C for 30 min. The protein concentration was determined by Pierce BCA Protein Assay Kit (23225, Thermo). Before gel loading, 5wt% Coomassie Brilliant Blue G-250 buffer was added to the sample to a final concentration of 0.25wt%. BN-PAGE was performed in a 3-12wt% gradient Native Page precast gel (BN1003, Thermo). Unless otherwise specified, all the above procedures should be performed on ice or at 4°C.

[0055] For Western Blot, proteins were transferred to PVDF membranes in lx NuPAGE Transfer Buffer (NP0006-1, Thermo Fisher Scientific) after electrophoresis. The transfer was performed at 4 °C, 25 V constant voltage for 3 h. After transfer, the membranes were incubated with 8 wt% acetic acid for 15 min, then rinsed with deionized water and air-dried. The air-dried membranes were wetted with methanol, then rinsed with deionized water before immunodetection. Coomassie staining, after electrophoresis, the gel was placed in fixation solution (40 wt% methanol, 10 wt% acetic acid) and microwaved for 45 s. After discarding the solution, Coomassie Brilliant Blue R-250 solution (DE0702, BioDee) was poured in and microwaved for 2 min. After staining, destaining solution (30 wt% ethanol, 10 wt% acetic acid) was used to shake the gel on a shaker until the desired background was achieved.

[0056] (4) Preparation of siRNA@tHFn (+): prepared according to the preparation method of the prior art (reference: ① Jin Y, Zhang B, Li J, Guo Z, Zhang C, Chen X, et al. Bioengineered protein nanocarrier facilitating siRNA escape from lysosomes for targeted RNAi therapy in glioblastoma. Sci Adv. 2025; 11(8): eadr9266.; ② Zhang B, Chen X, Tang G, Zhang R, Li J, Sun G, et al. Constructing a nanocage-based universal carrier for delivering TLR-activating nucleic acids to enhance antitumor immunotherapy. Nano Today. 2022; 46: 101564.), then the protein and siRNA concentrations were determined by BCA protein assay kit and Qubit 4 fluorometer, respectively.

[0057] (5) In vivo biological safety of siC6orf223@tHFn(+): The potential toxic effects of siC6orf223@tHFn(+) on normal tissues were evaluated in healthy BALB / c mice. The mice were injected with PBS, tHFn(+), siGFP@tHFn(+) or siC6orf223@tHFn(+) via the tail vein twice a week for 8 consecutive weeks. To evaluate histopathological changes, the major organs of the mice in each group, such as the heart, liver, lung, spleen, kidney, colon, etc., were embedded and sectioned, and hematoxylin-eosin staining (HE staining) was performed to detect the effects of siC6orf223@tHFn(+) on normal tissues.

[0058] (6) Immunohistochemistry: Human or mouse tissues were fixed in a 4wt% paraformaldehyde solution overnight, then transferred to 70wt% ethanol and processed into paraffin-embedded. The slides were deparaffinized, rehydrated and boiled in 10mM pH 6.0 citric acid buffer (ZLI-9064, Zsbio) for 3min. At room temperature, the slides were blocked with 5wt% goat serum for 1h, then incubated with the primary antibody overnight at 4°C. The next day, incubate with the appropriate anti-species secondary antibody (ZSGB-BIO) for 40min, then stain with DAB Kit (ZLI-9017, ZSCB-BIO) and hematoxylin. Finally, the slides were dehydrated and mounted with neutral resin.

[0059] (7) Colony formation experiment and treatment: 1000 HCT116, HT29 or SW480 cells were resuspended in 1mL growth medium and inoculated in a 12-well plate. The medium was replaced every 3 days. Then, the colonies were fixed with 4wt% paraformaldehyde solution (PFA) at 4°C for 1h and stained with 0.5wt% crystal violet. The colonies were counted directly under a microscope. The cells were treated with 50μg / mL tHFn(+), siGFP@tHFn(+), siC6orf223@tHFn(+), PBS or lipo2000 (cell transfection reagent) wrapped with siC6orf223, respectively.

[0060] (8) In situ injection mouse model and treatment: 6-week-old female NCG mice were selected, and 2×10 6 HCT116 cells were injected into the cecum wall, and randomly divided into groups. One week after inoculation, 1×PBS, tHFn(+), siGFP@tHFn(+) or siC6orf223@tHFn(+) were administered intravenously, once every other day.

[0061] (9) Spleen injection mouse model: 6-week-old female NCG mice were selected. The mice were anesthetized with isoflurane, and the surgical site was disinfected with 75wt% ethanol. After being wrapped with gauze, a small surgical incision was made at the skin and peritoneum to expose the spleen. 50 μL of 1x PBS solution containing 5x10 5 HCT116 or 1x10 5 HT29 cells was injected into the spleen. After transplantation, the peritoneal muscle and skin were closed.

[0062] (10) Cell culture, lentiviral vector and infection: Human colorectal cancer cell lines HCT116, HT29, SW480, SW620 and DLD1 were derived from the American Type Culture Collection (ATCC). Patient-derived xenograft CRC cell lines CRC57 and CRC119 were obtained. All CRC cell lines were cultured in RPMI Medium 1640 basic (C11875500BT, Gibco) supplemented with 10wt% fetal bovine serum (ST30-3302, PAN-Seratech) and 1wt% penicillin-streptomycin solution (SV30010, Cytiva). HEK293T cells were cultured in DMEM basic medium (C11965500BT, Gibco) supplemented with 10wt% bovine serum and 1wt% penicillin-streptomycin solution. HEK293F cells were cultured in OPM-293 CD05 medium (81075-001, OPM) supplemented with 1wt% penicillin-streptomycin solution. All adherent cell lines were cultured in a cell culture incubator at 37°C, 5% CO2; HEK293F cells were cultured on an orbital shaker platform at 135 rpm in a humidified atmosphere at 37°C, 8% CO2.

[0063] (11) Targeting C6orf223: shRNAs targeting PRMT5 and C6orf223 were cloned into lentiviral vector pLKO.1 (purchased from Beijing Zhuangmeng International Biological Gene Technology Co., Ltd.). Wild-type and different truncated PRMT5 and C6orf223 were cloned into lentiviral vector pCDH (purchased from Beijing Zhuangmeng International Biological Gene Technology Co., Ltd.). The above lentiviral constructs were co-transfected with helper plasmids into 293T cells, and the virus was collected 48h after transfection to infect cells and construct stable knockdown or overexpression cell lines. siRNAs against GFP and C6orf223 were synthesized using the T7 RNAi Transcription Kit (Vazyme Biotech, Nanjing). The shRNA and siRNA sequences are shown in Table 1.

[0064] Table 1 shRNA and siRNA sequences

[0065]

[0066] (12) Real-time fluorescence quantitative (RT-qPCR): RNA was purified by TRIzol reagent (15596, Thermo Fisher Scientific) or Rapure total RNA kit (R4011-03, Magen), and cDNA was synthesized using HiScript II Q Select RT SuperMix (R223, Vazyme). Real-time quantitative reverse transcription PCR was performed on a real-time PCR instrument using Fast Sybr Green PCR master mix (Q311-02, Vazyme). β-actin was amplified as a control. All primers were verified by a series of template cDNA dilution amplification, and the primer sequences are shown in Table 2.

[0067] Table 2 Primer sequences

[0068]

[0069] Example 1: C6orf223 protein promotes the assembly of PRMT5 / MEP50 multi-protein complex

[0070] To study how C6orf223 promotes the assembly of PRMT5 / MEP50 multi-protein complex, the interaction region of PRMT5 was first determined. PRMT5 was truncated into three different domains, i.e. PRMT5-N (37-290 amino acids), PRMT5-M (294-464 enzyme-containing amino acids) and PRMT5-C (467-635 amino acids) (see Figure 1 A). C6orf223 with HA tag was expressed in HEK293T cells with three Myc-tagged PRMT5 truncations, respectively. Co-immunoprecipitation with anti-Myc antibody showed that only PRMT5-C retained the interaction with C6orf223 (see Figure 1 B). PRMT5-C has a groove with negatively charged amino acid residues on the surface, while C6orf223 has many positively charged amino acid residues on the surface, especially arginine content as high as 16.9% (see Figure 1 C and Figure 2 ; Figure 2 Bold and underlined are the amino acid sequences of the arginine-rich region of C6orf223). It is therefore speculated that the interaction between C6orf223 and PRMT5 may depend on electrostatic attraction. Then the 6 arginine-rich regions in C6orf223 (about 12 amino acids in each region) were focused on, and each region was deleted, and HA-tagged C6orf223 mutants and His-tagged PRMT5 were co-expressed in HEK293T cells. From Figure 1D and 1G, only the mutant lacking the N-terminal arginine-rich region failed to interact with PRMT5; after deletion of the N-terminal arginine-rich region, C6orf223 could not increase the PRMT5 / MEP50 multiprotein complex and SDMA levels. The above results show that the N-terminal arginine-rich region of C6orf223 interacts with the C-terminal negatively charged groove region of PRMT5.

[0071] C6orf223 protein contains two cysteines, C23 and C178, respectively. Mutating these two cysteines or mutating both of them at the same time (see Figure 1 E). These mutants are labeled as HA (HA1, HA2 and HA1+2) or His (His1, His2 and His1+2) and co-expressed in HEK293T cells. It can be seen from Figure 1 F that C23 mutation (HA1 or His1) does not affect the formation of C6orf223 dimer; in contrast, once C178 is mutated (HA2 or His2), C6orf223-HA and C6orf223-His cannot interact with each other. Therefore, C6orf223 mediates its dimerization through disulfide bonds between C178. It can be seen from Figure 1 G that C178 mutation fails to increase the PRMT5 / MEP50 multiprotein complex and SDMA levels, indicating that C6orf223 promotes the formation of PRMT5 / MEP50 multiprotein complex depends on C6orf223 dimerization.

[0072] Example 2: C6orf223 protein promotes colorectal cancer metastasis

[0073] The present application studies the expression of C6orf223 protein in normal colon, primary colorectal cancer and colorectal cancer liver metastasis. It can be seen from Figure 3 that the expression level of C6orf223 protein in tumor tissue is significantly higher than that in normal colon tissue. In addition, from the liver metastases collected from CRC patients and in situ mouse models, the expression of C6orf223 protein is significantly increased compared with in situ tumors, which is consistent with the observation that C6orf223 promotes the formation of PRMT5 / MEP50 (see Figure 4 ). Further detection of the expression level of C6orf223 protein on the tissue chip of 25 pairs of matched primary colorectal cancer and liver metastasis samples found that the expression level of C6orf223 protein in liver metastasis was much higher than that in primary CRC (see Figure 5 ). In addition, high expression level of C6orf223 protein is also associated with poor prognosis of CRC patients (see Figure 6 ).

[0074] This invention then investigates the impact of C6orf223 on CRC progression. Figures 7-9 It was found that ectopic expression of C6orf223 in HT29 cells enhanced CRC cell migration and colony formation. Deletion of the N-terminal arginine-rich region of C6orf223, the C6orf223C178 mutation, or decreased PRMT5 expression levels did not promote CRC cell migration and colony formation. Further, in HCT116 cells with C6orf223 knockdown, C6orf223 lacking the N-terminal arginine-rich region (C6orf223 Δ1) and the C178 mutant C6orf223 (C6orf223 C178S) were reintroduced. Figures 10-12 It was found that reducing the expression level of C6orf223 significantly inhibited cell migration, colony formation, and proliferation. Further analysis in an orthotopic mouse model showed that C6orf223 protein promotes CRC growth and liver metastasis through its N-terminal arginine-rich region and C178 (see...). Figures 13-15 Furthermore, in a spleen injection model, knockdown of C6orf223 significantly inhibited CRC liver metastasis, and reintroduction of full-length C6orf223 restored liver metastasis levels, while reintroduction of C6orf223 Δ1 and C6orf223 C178S mutants did not restore CRC liver metastasis (see [link to original text]). Figure 15 Lowering the expression level of C6orf223 protein prolongs the survival of tumor-bearing mice, while overexpression of C6orf223 protein shortens the survival of tumor-bearing mice (see...). Figure 16 ).

[0075] Example 3: Targeting C6orf223 can inhibit colorectal cancer tumor growth and metastasis.

[0076] First, the expression of C6orf223 in normal tissues was analyzed using the GEPIA2 database. Figure 17 It is known that C6orf223 is negative in most normal tissues and expressed at low levels in the lungs and kidneys, but it is highly expressed in a variety of tumors (such as bladder urothelial carcinoma (BLCA), invasive breast carcinoma (BRCA), cholangiocarcinoma (CHOL), esophageal carcinoma (ESCA), clear cell renal cell carcinoma (KIRC), gastric adenocarcinoma (STAD), and endometrial carcinoma (UCEC)), especially colorectal cancer (see [link to relevant documentation]). Figure 17 Then, C6orf223-specific siRNAs (SEQ ID NO. 8-9) were designed and encapsulated in a ferritin shell (tHFn(+)) by adjusting the pH (see [link to siRNA]). Figure 18Ferritin is an important iron storage protein with a hollow interior cavity suitable for drug delivery. Ferritin nanocage has obvious advantages such as low toxicity and immunogenicity because it naturally exists in the human body. In addition, the receptor transferrin receptor 1 (TfR1) of ferritin is highly expressed in cancer cells.

[0077] The present application proves that TfR1 is indeed highly expressed in cancer cells (see Figure 19 ), 50 μg / mL siC6orf223@tHFn(+) significantly reduces the expression of C6orf223 (see Figure 20 ), and siC6orf223@tHFn(+) reduces WWTR1, FGFR1 and CLU, and increases the expression of GATA5 (see Figure 21 ). Functionally, siC6orf223@tHFn(+) significantly inhibits the migration and clonogenicity of CRC cells in vitro (see Figures 22-23 ).

[0078] The biodistribution and biosafety of siC6orf223@tHFn(+) were then studied. PBS, tHFn(+), cy5-siGFP@tHFn(+) and cy5-siC6orf223@tHFn(+) were intravenously injected into HCT116 mice, respectively. Ex vivo fluorescence images showed that there were obvious fluorescence signals in the tumors of cy5-siGFP@tHFn(+) and cy5-siC6orf223@tHFn(+) treated mice, suggesting that siC6orf223@tHFn(+) accumulated in the tumor (see Figure 24 ). In addition, the potential toxic effects of siC6orf223@tHFn(+) on normal tissues were also evaluated in healthy BALB / c mice. The main organs of mice treated with siGFP@tHFn(+) and siC6orf223@tHFn(+) maintained typical physiological morphology and no obvious histopathological abnormalities or damage were observed (see Figure 25 ). In summary, the siC6orf223@tHFn(+) nanocarrier has tumor enrichment and good biosafety. Orthotopic mouse models showed that siC6orf223@tHFn(+) significantly inhibited the growth and liver metastasis of CRC in vivo (see Figures 26-28 ). Immunohistochemical staining showed that knocking down C6orf223 can up-regulate the expression of GATA5 and down-regulate the expression of WWTR1, FGFR1 and CLU in tumor tissues (see Figure 29 ). In addition, siC6orf223@tHFn(+) treatment prolonged the survival of tumor mice (see Figure 30 ).

[0079] The above merely describes preferred embodiments of the present application and is not used to limit the present application, and any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. The application of an inhibitor targeting C6orf223 protein in the preparation of a drug for treating colorectal cancer, characterized in that, The inhibitor can reduce the expression level of C6orf223 protein in tumor cells; the inhibitor is a siRNA targeting C6orf223 protein; the siRNA targeting C6orf223 protein includes siC6orf233-F and siC6orf233-R; wherein the nucleotide sequences of siC6orf233-F and siC6orf233-R are shown in SEQ ID NO.8 and SEQ ID NO.9, respectively.

2. The application according to claim 1, characterized in that, The amino acid sequence of the C6orf223 protein is shown in SEQ ID NO.

1.

3. The application according to claim 1, characterized in that, The inhibitor may also be an shRNA targeting the C6orf223 protein; the nucleotide sequence of the shRNA targeting the C6orf223 protein is shown in SEQ ID NO.

2.

4. A recombinant vector, characterized in that, This includes the siRNA targeting the C6orf223 protein as described in claim 1 or the shRNA targeting the C6orf223 protein as described in claim 3.

5. The use of the recombinant vector according to claim 4 in the preparation of a drug for treating colorectal cancer.

6. A drug for treating colorectal cancer, characterized in that, The drug comprises the inhibitor of claim 1 or 3 or the recombinant vector of claim 4.

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