Application of inhibitor of targeted C6orf223 protein in preparation of medicine for treating tumors

Abstract

The invention discloses application of an inhibitor targeting C6orf223 protein in preparation of a medicine for treating tumors, and belongs to the technical field of biological medicine. The invention finds that the C6orf223 protein can be combined with the PRMT5 to promote the assembly of a PRMT5 / MEP50 heterooctamer compound, so that the activity of the PRMT5 and the enrichment of H4R3me2s in a series of genes are improved. The invention discovers that the C6orf223 protein is highly expressed in various tumors; an in-situ mouse model shows that the C6orf223 protein significantly promotes the growth and metastasis of tumors by improving the activity of PRMT5. According to the invention, the growth and metastasis of colorectal cancer are effectively inhibited by respectively wrapping siRNA (small interfering Ribonucleic Acid) of specific targeting C6orf223 and shRNA (short hairpin Ribonucleic Acid) of specific targeting C6orf223 with a ferritin shell. Therefore, the invention provides new therapeutic targets and therapeutic strategies for various tumors.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to the application of an inhibitor targeting the C6orf223 protein in the preparation of anti-tumor drugs. Background Art

[0002] In recent years, RNA interference technology has experienced rapid development, bringing new hope and revolutionary changes to cancer treatment. As an emerging cancer treatment method, RNA interference technology has great potential and development prospects. RNA interference (RNAi) is a phenomenon of specific gene silencing mediated by small interfering RNA (siRNA) and involving specific enzymes, which is commonly present in organisms. 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, which triggers mRNA degradation and thus inhibits the gene expression of specific genes. In theory, RNAi can silence all genes. Therefore, siRNA can be used as a targeted therapeutic drug to specifically regulate the expression of disease-related genes. Short hairpin RNA (shRNA) is a type of artificially synthesized RNA with a tight hairpin turn structure, which can silence the expression of target genes based on the principle of RNA interference technology.

[0003] Tumors are common and frequently-occurring diseases, among which malignant tumors are currently the most serious diseases endangering human health. The etiology, pathogenesis, prevention and treatment of these tumors are the focus of research in this field. Tumor targeted therapy is to design and develop targeted inhibitors, including antibodies, small molecules, biological carriers, etc., against functional molecules specifically expressed in tumors. Such targeted molecules can specifically inhibit the growth, migration and chemotherapy resistance of tumor cells without damaging normal tissue cells. Therefore, targeted therapy can greatly extend the survival time and quality of life of patients. However, at the present stage, 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 of the Invention

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

[0005] The technical solution for the present invention to solve the above technical problems is as follows: providing the application of an inhibitor targeting the C6orf223 protein in the preparation of anti-tumor drugs.

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

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

[0008] Furthermore, the siRNA targeting the C6orf223 protein includes siC6orf233-F and siC6orf233-R; the nucleotide sequences of siC6orf233-F and siC6orf233-R are shown in SEQ ID NO.8 and SEQ ID NO.9 respectively.

[0009] Furthermore, the inhibitor also includes shRNA targeting the C6orf223 protein.

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

[0011] The present invention provides a recombinant vector, which includes the above-mentioned siRNA targeting the C6orf223 protein or the above-mentioned shRNA targeting the C6orf223 protein.

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

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

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

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

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

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

[0018] The present invention has the following beneficial effects: The present invention discovers that the C6orf223 protein can bind to PRMT5, promoting the assembly of the PRMT5 / MEP50 heterooctameric complex, thereby enhancing the activity of PRMT5 and the enrichment of H4R3me2s in a series of genes. The present invention analyzes the expression of the C6orf223 protein in normal tissues and tumor tissues and finds that it is negative in most normal tissues, with low expression in the lung and kidney; however, it is highly expressed in various tumors. The in situ mouse model shows that the C6orf223 protein significantly promotes the growth and metastasis of colorectal cancer by enhancing the activity of PRMT5. The present invention respectively uses ferritin shells to encapsulate siRNA specifically targeting C6orf223 and shRNA specifically targeting C6orf223 to effectively inhibit the growth and metastasis of colorectal cancer. Therefore, the present invention provides new therapeutic targets and treatment strategies for various tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a molecular mechanism diagram of C6orf223-mediated PRMT5 / MEP50 heterooctamer assembly; Figure 2 It is an amino acid sequence diagram of the arginine-rich region of C6orf223; among them, arginine is marked in red; Figure 3 It is a diagram of the expression levels of C6orf223 in paired or unpaired normal peritumoral tissues and primary tumors of CRC patients; Figure 4 It is a diagram of the expression levels of the C6orf223 protein in primary colorectal cancer P and liver metastasis samples M of NCG mice injected with HT29 cells in situ, and primary colorectal cancer P and liver metastasis samples M; among them, the HT29 cells carry a blank vector or overexpress PRMT5; Figure 5 It is an immunohistochemical image and quantitative analysis diagram of C6orf223 in a tissue chip; Figure 6 It is a Kaplan-Meier analysis diagram of the survival curves of colorectal cancer patients with low and high expression of C6orf223; Figure 7 It is a diagram of the expression levels of C6orf223 and PRMT5 in HT29 cells transfected with full-length or different C6orf223 mutants or knocked down with C6orf223 and PRMT5, and the expression level of C6orf223 in HCT116 cells knocked down with C6orf223; Figure 8 It is a migration experiment diagram of HT29 cells transfected with full-length or different C6orf223 mutants or knocked down with C6orf223 and PRMT5; Figure 9Colony formation diagrams of HT29 cells with full-length or different C6orf223 mutations or knockdown of C6orf223 and PRMT5 genes; Figure 10 Migration assay diagrams of HCT116 cells after knockdown of C6orf223 and reconstitution with full-length C6orf223, C6orf223 Δ1, or C6orf223 C178S; Figure 11 Colony formation diagrams of HCT116 cells after knockdown of C6orf223 and reconstitution with full-length C6orf223, C6orf223 Δ1, or C6orf223 C178S; Figure 12 Cell proliferation diagrams of HCT116 cells after knockdown of C6orf223 and reconstitution with full-length C6orf223, C6orf223 Δ1, or C6orf223 C178S; Figure 13 Images and weight diagrams of cecal tumors in NCG mice injected orthotopically with HCT116 cells; Figure 14 Images and quantification diagrams of liver metastases in NCG mice injected orthotopically with HCT116 cells; Figure 15 HE staining and quantification diagrams of liver metastases in NCG mice injected orthotopically with HCT116 cells; Figure 16 Survival curve analysis diagrams of NCG mice injected with HCT116 cells into the cecum; Figure 17 Expression level diagrams of C6orf223 in cancer tissues and normal tissues; Figure 18 Schematic diagrams and detection diagrams of the process of C6orf223 siRNA encapsulated by tHFn(+) protein shells; Figure 19 Expression level diagrams of TfR1 in cancer cells; Figure 20 Expression level diagrams of C6orf223 in HCT116 cells treated with siGFP@tHFn(+), siC6orf223@tHFn(+), or siC6orf223@Lipo2000; Figure 21 Expression level diagrams of C6orf223, GATA5, WWTR1, FGFR1, and CLU in HCT116 cells treated with PBS, tFHn(+), siGFP@tHFn(+), siC6orf223@tHFn(+), or siC6orf223@Lipo2000; Figure 22Images and quantitative graphs of HCT116 cell migration assays after treatment with PBS, tFHn(+), siGFP@tHFn(+), siC6orf223@tHFn(+), or siC6orf223@Lipo2000; Figure 23 Images and quantitative graphs of HCT116 cell colony formation after treatment with PBS, tFHn(+), siGFP@tHFn(+), siC6orf223@tHFn(+), or siC6orf223@Lipo2000; Figure 24 Ex vivo fluorescence images of tumors and major organs in mice after treatment with PBS, tHFn(+), siGFP@tHFn(+), siC6orf223@tHFn(+); Figure 25 HE staining images of major organs after different treatments; Figure 26 Images and weight graphs of cecal tumors in NCG mice injected with HCT116 cells in situ; Figure 27 Bright-field images and quantitative graphs of metastatic foci in NCG mice after cecal injection of HCT116 cells; Figure 28 HE staining and quantitative graphs of metastatic foci in NCG mice after cecal injection of HCT116 cells; Figure 29 Immunohistochemical images and quantitative graphs of the expression levels of C6orf223, GATA5, WWTR1, FGFR1, and CLU in primary tumors of mice injected with HCT116 cells in situ; Figure 30 Survival curve analysis graphs of NCG mice after cecal injection of HCT116 cells. Detailed implementation manners

[0020] The following examples are only used to explain the present invention and are not intended to limit the scope of the present invention. For those not specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. For reagents or instruments not specified for the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0021] The primary and liver metastatic colorectal cancer specimens involved in the examples of the present invention were obtained from the Seventh Medical Center of the Chinese PLA General Hospital, and the informed consent of all donors has been obtained; NOD / ShiLtJGpt-Prkdcem26Cd52Il2rgem26Cd22 / Gptc (NCG) mice were purchased from Gem Pharmatech Company, and all animal experiments have obtained the approval of the Biomedical Research Ethics Committee.

[0022] Experimental methods: (1)Co-Immunoprecipitation / Mass Spectrometry (Co-IP-MS) experiment: HEK293T cells were transfected with the corresponding plasmids using Vigofect (T001, Vigorous), and the cells were harvested 48 h later. After cell lysis, the cell lysate was pre-cleared using Pierce Protein A / G magnetic beads (88803, Thermo). During immunoprecipitation, an equal volume of cell lysate and the primary antibody were added and incubated overnight at 4 °C. Then, protein G magnetic beads were added and incubated for 4 h at 4 °C. Subsequently, the magnetic beads were boiled with 1× SDS-PAGE sample buffer. Cells or homogenized tissues were lysed in RIPA lysis buffer supplemented with protease inhibitors (04693132001, Roche). After centrifugation at 13,000 rpm for 10 min, the supernatant was collected and 5× SDS-PAGE sample buffer (E153-01, GenStar) was added. The protein samples were separated by 10 wt% SDS-PAGE and then transferred to a PVDF membrane. The membrane was blocked with 5 wt% non-fat milk in PBS for 1 h at room temperature and incubated overnight at 4 °C with the primary antibody. After washing three times with 1× PBST (containing 0.05 wt% Tween 20 in 1× PBS), the membrane was incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at room temperature. The target protein was detected using a western Dura Extended Duration Substrate (34076, Thermo Fisher Scientific) chemiluminescent imaging system.

[0023] (2)Western Blot: Cells or homogenized tissues were lysed in RIPA lysis buffer supplemented with protease inhibitors (04693132001, Roche). After centrifugation at 13,000 rpm for 10 min, the supernatant was collected and 5× SDS-PAGE sample buffer (E153-01, GenStar) was added. The protein samples were separated by 10 wt% SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked with 5 wt% non-fat milk in 1× PBS for 1 h at room temperature and incubated overnight at 4 °C with the primary antibody on a shaker. After washing three times with 1× PBST, the membrane was incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at room temperature. The target protein was detected using a western Dura Extended Duration Substrate (34076, Thermo Fisher Scientific) chemiluminescent imaging system.

[0024] (3)Blue Native-PAGE (BN-PAGE) detection method: Adapted from the user guide of the Native PAGE Novex Bis-Tris gel system (Thermo Fisher Scientific). Cells or homogenized tissues were lysed in 1× NativePAGE sample buffer (BN2003, Thermo Fisher Scientific), with 1 wt% digitonin and protease inhibitors added to the buffer. After centrifugation at 13,000 rpm for 30 min at 4 °C, the supernatant was collected and UltraNuclease (20156ES25, YEASEN) was added to remove nucleic acids. MgCl2 was added to the sample to a final concentration of 2 mM, and the sample was incubated at 37 °C for 30 min. The protein concentration was determined using the Pierce BCA Protein Assay Kit (23225, Thermo Fisher Scientific). Before loading, 5 wt% Coomassie Brilliant Blue G-250 buffer was added to the sample to a final concentration of 0.25 wt%. BN-PAGE was performed in a 3-12 wt% gradient Native Page precast gel (BN1003, Thermo Fisher Scientific). Unless otherwise specified, all of the above procedures should be carried out on ice or at 4 °C.

[0025] For Western Blot, after electrophoresis, the proteins were transferred to a PVDF membrane in 1× NuPAGE Transfer Buffer (NP0006-1, Thermo Fisher Scientific). Transfer was carried out at a constant voltage of 25 V at 4 °C for 3 h. After transfer, the membrane was incubated in 8 wt% acetic acid for 15 min, then rinsed with deionized water and air-dried. The air-dried membrane was moistened with methanol, then rinsed with deionized water and subjected to immunodetection. For Coomassie staining, after electrophoresis, the gel was placed in a fixing solution (40 wt% methanol, 10 wt% acetic acid) and microwaved for 45 s. After discarding the liquid, the Coomassie Brilliant Blue R-250 solution (DE0702, BioDee) was poured in and microwaved for 2 min. After staining, the gel was shaken on a shaker using a destaining solution (30 wt% ethanol, 10 wt% acetic acid) until the desired background was obtained.

[0026] (4)Preparation of siRNA@tHFn(+): It was prepared according to the preparation method of the prior art (references: ① Jin Y, Zhang B, Li J, Guo Z, Zhang C, Chen X, et al. Bioengineered proteinnanocarrier facilitating siRNA escape from lysosomes for targeted RNAitherapy 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 universalcarrier for delivering TLR-activating nucleic acids to enhance antitumorimmunotherapy. Nano Today. 2022;46:101564.), and then the protein and siRNA concentrations were measured using a BCA protein assay kit and a Qubit 4 fluorometer, respectively.

[0027] (5)In vivo biosafety of siC6orf223@tHFn(+): The potential toxic effects of siC6orf223@tHFn(+) on normal tissues were evaluated in healthy BALB / c mice. Mice were injected via the tail vein with PBS, tHFn(+), siGFP@tHFn(+), or siC6orf223@tHFn(+) twice a week for 8 consecutive weeks. To evaluate histopathological changes, the main organs of mice in each group, such as the heart, liver, lung, spleen, kidney, and colon, were embedded in paraffin sections and stained with hematoxylin and eosin (HE staining) to detect the effects of siC6orf223@tHFn(+) on normal tissues.

[0028] (6)Immunohistochemistry: Human or mouse tissues were fixed in 4 wt% paraformaldehyde solution overnight and then transferred to 70 wt% ethanol and processed into paraffin-embedded blocks. The slides were deparaffinized, rehydrated, and boiled in 10 mM citrate buffer at pH 6.0 (ZLI-9064, Zsbio) for 3 min. At room temperature, the slides were blocked with 5 wt% goat serum for 1 h and then incubated with the primary antibody overnight at 4 °C. The next day, the slides were incubated with the appropriate anti-species enzyme-labeled secondary antibody (ZSGB-BIO) for 40 min, and then stained with DAB Kit (ZLI-9017, ZSCB-BIO) and hematoxylin. Finally, the slides were dehydrated and sealed with neutral resin.

[0029] (7)Colony formation assay and treatment: 1000 HCT116, HT29, or SW480 cells were resuspended in 1 mL of growth medium and seeded in a 12-well plate. The medium was changed every 3 days. Subsequently, the colonies were fixed with 4 wt% paraformaldehyde solution (PFA) at 4 °C for 1 h and stained with 0.5 wt% crystal violet. The colonies were directly counted under a microscope. The cells were treated with 50 μg / mL tHFn(+), siGFP@tHFn(+), siC6orf223@tHFn(+), PBS, or lipo2000 (a cell transfection reagent) encapsulating siC6orf223, respectively.

[0030] (8)Orthotopic injection mouse model and treatment: Six-week-old female NCG mice were selected, and 2×10 6 HCT116 cells were carefully injected into the cecal wall using an insulin syringe. The mice were randomly grouped and treated with 1×PBS, tHFn(+), siGFP@tHFn(+), or siC6orf223@tHFn(+) by intravenous injection at one week after inoculation, once every other day.

[0031] (9)Spleen injection mouse model: Six-week-old female NCG mice were selected. The mice were anesthetized with isoflurane, and the surgical site was disinfected with 75 wt% ethanol. After wrapping with gauze, a small surgical incision was made in the skin and peritoneum to expose the spleen. 50 μL of 1×PBS solution containing 5×10 5 HCT116 or 1×10 5 HT29 cells was injected into the spleen. After transplantation, the peritoneum muscle and skin were surgically closed.

[0032] (10)Cell culture, lentiviral vectors, and infection: Human colorectal cancer cell lines HCT116, HT29, SW480, SW620, and DLD1 were obtained 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 10 wt% fetal bovine serum (ST30-3302, PAN-Seratech) and 1 wt% penicillin-streptomycin solution (SV30010, Cytiva). HEK293T cells were cultured in DMEM basic medium (C11965500BT, Gibco) supplemented with 10 wt% bovine serum and 1 wt% penicillin-streptomycin solution. HEK293F cells were cultured in OPM-293 CD05 medium (81075-001, OPM) supplemented with 1 wt% penicillin-streptomycin solution. All adherent cell lines were cultured in a cell culture incubator at 37 °C and 5% CO2; HEK293F cells were cultured on an orbital shaker platform rotating at 135 rpm in a humidified atmosphere at 37 °C and 8% CO2.

[0033] (11)Targeting C6orf223: shRNAs targeting PRMT5 and C6orf223 were cloned into the lentiviral vector pLKO.1 (purchased from Beijing Zhuangmeng International Biotechnology Co., Ltd.). Wild-type and different truncated PRMT5 and C6orf223 were cloned into the lentiviral vector pCDH (purchased from Beijing Zhuangmeng International Biotechnology Co., Ltd.). The above lentiviral constructs were co-transfected with the helper plasmid into 293T cells. After 48 h of transfection, the virus was collected and used to infect cells to construct stable knockdown or overexpression cell lines. siRNAs against GFP and C6orf223 were synthesized using the T7 RNAi transcription kit (Vazyme Biotech, Nanjing). Among them, the shRNA and siRNA sequences are shown in Table 1.

[0034] Table 1 shRNA and siRNA sequences

[0035] (12) Real-time fluorescence quantitative (RT-qPCR): RNA was purified using TRIzol reagent (15596, Thermo Fisher Scientific) or Rapure total RNA kit (R4011-03, Magen), and cDNA was synthesized using HiScriptⅡQ Select RTSuperMix (R223, Vazyme). Real-time quantitative reverse transcription PCR was performed on a real-time PCR instrument using Fast SybrGreen PCR Master Mix (Q311-02, Vazyme). β-actin was amplified as a control. All primers were verified by amplification of a series of template cDNA dilutions, and the primer sequences are shown in Table 2.

[0036] Table 2 Primer sequences

[0037] Example 1: C6orf223 protein promotes the assembly of the PRMT5 / MEP50 multiprotein complex To study how C6orf223 promotes the assembly of the PRMT5 / MEP50 multiprotein complex, the interaction region of PRMT5 was first determined. PRMT5 was truncated into three different domains, namely PRMT5-N (amino acids 37-290), PRMT5-M (amino acids 294-464 containing the enzyme), and PRMT5-C (amino acids 467-635) (see Figure 1 A). C6orf223 carrying an HA tag was expressed in HEK293T cells together with three Myc-tagged PRMT5 truncations respectively. Co-immunoprecipitation with an anti-Myc antibody showed that only PRMT5-C retained the interaction with C6orf223 (see Figure 1 B). There is a groove with negatively charged amino acid residues on the surface of PRMT5-C, while there are many positively charged amino acid residues on the surface of C6orf223, especially with an 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 regions of C6orf223). Therefore, it is speculated that the interaction between C6orf223 and PRMT5 may depend on electrostatic attraction. Then, six arginine-rich regions in C6orf223 (each region has about 12 amino acids) were focused on, each region was deleted respectively, and the HA-tagged C6orf223 mutant and His-tagged PRMT5 were co-expressed in HEK293T cells. By Figure 1As can be seen from D and 1G, only the mutant lacking the N-terminal arginine-rich region fails to interact with PRMT5; after deleting the N-terminal arginine-rich region, C6orf223 cannot increase the levels of the PRMT5 / MEP50 multi-protein complex and SDMA. The above results indicate that the N-terminal arginine-rich region of C6orf223 interacts with the negatively charged groove region at the C-terminus of PRMT5.

[0038] The C6orf223 protein contains two cysteines, C23 and C178 respectively. Mutate these two cysteines separately or simultaneously (see Figure 1 E). These mutants were labeled as HA (HA1, HA2 and HA1+2) or His (His1, His2 and His1+2), and co-expressed in HEK293T cells. As Figure 1 shown in F, the mutation of C23 (HA1 or His1) does not affect the formation of the 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 a disulfide bond between C178. As Figure 1 shown in G, the mutation of C178 fails to increase the levels of the PRMT5 / MEP50 multi-protein complex and SDMA, indicating that the promotion of the formation of the PRMT5 / MEP50 multi-protein complex by C6orf223 depends on C6orf223 dimerization.

[0039] Example 2: C6orf223 protein promotes colorectal cancer metastasis The present invention studied the expression of C6orf223 protein in normal colon, primary colorectal cancer and liver metastases of colorectal cancer. As Figure 3 shown, the expression level of C6orf223 protein in tumor tissues is significantly higher than that in normal colon tissues. In addition, in liver metastases collected from CRC patients and orthotopic mouse models, the expression of C6orf223 protein is significantly increased compared with that in orthotopic tumors, which is consistent with the observation of higher levels of the PRMT5 / MEP50 multi-protein complex and the promotion of PRMT5 / MEP50 formation by C6orf223 in liver metastases (see Figure 4 ). Further, the expression level of C6orf223 protein was detected on the tissue microarray of 25 pairs of matched primary colorectal cancer and liver metastasis samples, and it was found that the expression level of C6orf223 protein in liver metastases is much higher than that in primary CRC (see Figure 5 ). In addition, the high expression level of C6orf223 protein is also associated with poor prognosis of CRC patients (see Figure 6 ).

[0040] Then, the present invention studied the effect of C6orf223 on CRC progression. As Figures 7 - 9 shown, the ectopic expression of C6orf223 in HT29 cells enhanced the migration and colony formation of CRC cells. When the arginine-rich region at the N-terminus of C6orf223 was deleted, C6orf223 C178 was mutated, or the expression level of PRMT5 was decreased, the migration and colony formation of CRC cells could not be promoted. Further, C6orf223, C6orf223 lacking the N-terminal arginine-rich region (C6orf223 Δ1), and C6orf223 C178 mutant (C6orf223 C178S) were re-supplemented in HCT116 cells with knocked-down C6orf223. As Figures 10 - 12 shown, reducing the expression level of C6orf223 could significantly inhibit cell migration, colony formation, and proliferation. The in situ mouse model further showed that C6orf223 protein promoted CRC growth and liver metastasis through its N-terminal arginine-rich region and C178 (see Figures 13 - 15 ). In addition, the spleen injection model showed that the knockdown of C6orf223 significantly inhibited CRC liver metastasis. Re-supplementation of full-length C6orf223 could restore its liver metastasis level, while re-supplementation of C6orf223 Δ1 and C6orf223 C178S mutants could not restore CRC liver metastasis (see Figure 15 ). Reducing the expression level of C6orf223 protein could prolong the survival period of tumor-bearing mice, and the overexpression of C6orf223 protein could shorten the survival period of tumor-bearing mice (see Figure 16 ).

[0041] Example 3: Targeting C6orf223 can inhibit the growth and metastasis of colorectal cancer tumors First, the expression of C6orf223 in normal tissues was analyzed in the GEPIA2 database. As Figure 17 shown, C6orf223 was negative in most normal tissues, with low expression in the lung and kidney, but C6orf223 was highly expressed in various tumors (such as bladder urothelial carcinoma (BLCA), breast invasive carcinoma (BRCA), cholangiocarcinoma (CHOL), esophageal carcinoma (ESCA), kidney clear cell carcinoma (KIRC), gastric adenocarcinoma (STAD), and endometrial carcinoma (UCEC)), especially colorectal cancer (see Figure 17 ). Then, C6orf223-specific siRNA (SEQ ID NO.8-9) was designed and encapsulated in the ferritin shell (tHFn(+)) by adjusting the pH (see Figure 18). Ferritin is an important iron storage protein with a hollow internal cavity suitable for drug delivery. Ferritin nanocages have obvious advantages such as low toxicity and immunogenicity because they naturally exist in the human body. In addition, the receptor of ferritin, transferrin receptor 1 (TfR1), is highly expressed in cancer cells.

[0042] This invention confirmed that TfR1 was indeed highly expressed in cancer cells (see Figure 19 ), 50 μg / mL siC6orf223@tHFn(+) significantly reduced the expression of C6orf223 (see Figure 20 ), and siC6orf223@tHFn(+) decreased WWTR1, FGFR1, and CLU and increased the expression of GATA5 (see Figure 21 ). Functionally, siC6orf223@tHFn(+) significantly inhibited the migration and colony formation of CRC cells in vitro (see Figures 22 - 23 ).

[0043] Then, the biodistribution and biosafety of siC6orf223@tHFn(+) were studied. PBS, tHFn(+), cy5-siGFP@tHFn(+), and cy5-siC6orf223@tHFn(+) were intravenously injected into HCT116 mice, respectively. Ex vivo fluorescence images showed obvious fluorescence signals in the tumors of mice treated with cy5-siGFP@tHFn(+) and cy5-siC6orf223@tHFn(+), indicating that siC6orf223@tHFn(+) accumulated in the tumors (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 injuries were observed (see Figure 25 ). In summary, the siC6orf223@tHFn(+) nanocarrier has tumor enrichment and good biosafety. The orthotopic mouse model showed that siC6orf223@tHFn(+) significantly inhibited CRC growth and liver metastasis in vivo (see Figures 26 - 28 ). Immunohistochemical staining showed that knockdown of C6orf223 upregulated the expression of GATA5 and downregulated the expression of WWTR1, FGFR1, and CLU in tumor tissues (see Figure 29 ). In addition, treatment with siC6orf223@tHFn(+) prolonged the survival time of tumor-bearing mice (see Figure 30 ).

[0044] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Application of an inhibitor targeting C6orf223 protein in the preparation of drugs for treating tumors.

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

1.

3. An inhibitor targeting C6orf223 protein, characterized in that: The inhibitor can reduce the expression level of C6orf223 protein in tumor cells; the inhibitor includes siRNA targeting C6orf223 protein.

4. The inhibitor according to claim 3, characterized in that 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.

5. The inhibitor according to claim 3, characterized in that The inhibitors also include shRNA targeting C6orf223 protein.

6. The inhibitor according to claim 5, characterized in that The nucleotide sequence of the shRNA targeting C6orf223 protein is shown in SEQ ID NO.

2.

7. A recombinant vector, characterized in that: The method comprises the siRNA targeting C6orf223 protein according to claim 3 or the shRNA targeting C6orf223 protein according to claim 5.

8. Use of the inhibitor according to any one of claims 3 to 6 in the preparation of drugs for treating tumors.

9. Use of the recombinant vector according to claim 7 in the preparation of drugs for treating tumors.

10. A drug for treating tumors, characterized in that: The drug comprises at least one of the inhibitor according to any one of claims 3 to 6 and the recombinant vector according to claim 7.

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