Application of C6orf120 inhibitor in preparation of medicine for treating hepatocellular carcinoma

Abstract

The invention belongs to the field of antitumor drugs, and provides application of a C6orf120 inhibitor in preparation of a drug for treating hepatocellular carcinoma. According to the application, a key target C6orf120 for connecting a tumor cell EGFR cancer promoting pathway and an ECM mechanical microenvironment is found, and a nucleic acid inhibition form capable of being directly medicated is given, so that multi-pathway and multi-cell link synergistic inhibition on the hepatocellular carcinoma is realized, and a new choice is provided for treatment of the hepatocellular carcinoma.

Description

Technical Field

[0001] This application belongs to the field of antitumor drugs. Specifically, this application provides the use of C6orf120 inhibitors in the preparation of drugs for treating hepatocellular carcinoma. Background Technology

[0002] Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer, accounting for approximately 90% of all liver cancer cases. Current epidemiological data shows that HCC ranks among the most common malignant tumors worldwide and is a significant cause of cancer-related death. HCC often develops based on long-term chronic liver disease. In my country, HCC associated with hepatitis B virus (HBV) infection is predominant, but HCC caused by non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), and alcoholic liver disease is also increasing annually.

[0003] Currently, treatment options for HCC include local therapies such as surgical resection, ablation, and transarterial chemoembolization (TACE), as well as systemic therapies such as small molecule targeted drugs, anti-angiogenic drugs, and immune checkpoint inhibitors. In recent years, explorations have also emerged in immunotherapy such as CAR-T, TCR-T, and tumor-infiltrating lymphocytes (TILs), and oncolytic virus therapy. However, overall, most of these treatments still target single molecular targets or pathways within a single cell population: small molecule targeted drugs primarily act on receptor tyrosine kinases such as VEGFR, FGFR, PDGFR, and EGFR, or their downstream signaling pathways; immune checkpoint inhibitors primarily act on molecules such as PD-1 and CTLA-4 on T cells; and immunotherapy enhances the recognition and killing of tumor cells by specific effector cells (such as CAR-T cells). However, solid tumors, including HCC, exhibit significant spatial and temporal heterogeneity: signaling pathway dependencies and drug sensitivity vary considerably among different clones within the tumor, and the tumor microenvironment contains multiple components such as tumor cells, hepatocytes, hepatic stellate cells, immune cells, and endothelial cells. These factors collectively lead to the fact that interventions targeting only a single cell or signaling pathway often fail to achieve stable and durable therapeutic effects, easily resulting in partial remission, rapid drug resistance, or immune escape.

[0004] On the other hand, HCC is a typical progressive disease, and its development usually follows a continuous process of "chronic hepatitis—liver fibrosis—cirrhosis—liver cancer." During this process, the deposition and remodeling of the extracellular matrix (ECM) in the liver persists for a long time. Collagen fibers gradually accumulate and undergo enhanced alignment and cross-linking, leading to a transformation of the liver and tumor-specific ECM from soft to rigid. This quantitative and qualitative change in the ECM promotes tumor cell proliferation, invasion, and activation of pro-cancer signaling pathways through the hardened matrix; on the other hand, it limits immune cell infiltration and affects angiogenesis by forming a dense and rigid matrix barrier. Therefore, starting with improving the ECM microenvironment—a common upstream physical stimulus—it is hoped that multiple cell types can be simultaneously modulated at the same intervention point, mitigating the impact of tumor heterogeneity on drug efficacy to some extent.

[0005] C6orf120 is an N-glycosylated extracellular matrix-associated protein (ECM-associated protein) that has been shown to be expressed in liver tissue and involved in immune regulation, inflammatory responses, and liver diseases. Existing research primarily focuses on expression profiles and overall function. The precise mechanisms of action of C6orf120 within the ECM microenvironment, its direct involvement in collagen fiber rearrangement and ECM sclerosis, and its specific role in the development and progression of hepatocellular carcinoma (HCC) remain unclear. Particularly in the field of HCC, there is currently no systematic literature reporting on whether C6orf120 affects tumor growth, immune invasion, and angiogenesis by regulating tumor cell oncogenic signals and the ECM microenvironment. Furthermore, no technical solutions have been found that clearly define C6orf120 as a key molecule regulating the ECM mechanical microenvironment and for its use in the prevention and treatment of HCC.

[0006] It is worth noting that previous studies have observed that high expression of C6orf120 in some solid tumors with significant fibrosis is associated with malignant tumor progression and poor prognosis, suggesting that C6orf120 may play a pro-cancer role in the "fibrosis-tumor progression" axis, thus providing indirect evidence for its use as a therapeutic target for solid tumors (including HCC). Summary of the Invention

[0007] In summary, the existing technology has the following main shortcomings: (1) Existing HCC treatments mostly target single cells or single molecules, which are difficult to address the significant tumor heterogeneity and complex tumor microenvironment as a whole, and are prone to unstable drug efficacy and drug resistance. (2) There is a lack of specific and druggable molecular targets for the common upstream physical stimulation of ECM deposition and hardening throughout the entire course of HCC development. It is still difficult to improve the function of multiple cells such as tumor cells, immune cells and vascular endothelial cells by "softening or reshaping the ECM microenvironment" to inhibit tumor progression in an overall way. (3) The relationship between C6orf120 and tumor cell pro-cancer pathways is still unclear, and there is no technical solution for comprehensive intervention using nucleic acid drugs (such as siRNA) that target C6orf120.

[0008] In response to the above situation, this application provides the use of C6orf120 inhibitors in the preparation of drugs for treating hepatocellular carcinoma.

[0009] On the other hand, this application provides the use of C6orf120 inhibitors in the preparation of drugs to prevent hepatocellular carcinoma metastasis.

[0010] Furthermore, the drug has one or more of the following effects: (1) Inhibit the EGFR tumor-promoting signaling pathway in hepatocellular carcinoma tumor cells; (2) Inhibits the proliferation of hepatocellular carcinoma tumor cells; (3) Downregulate the expression of EGFR downstream extracellular matrix-related genes and remodel the extracellular matrix microenvironment of hepatocellular carcinoma tumor cells; (4) Promotes the infiltration of immune cells; (5) Inhibits angiogenesis in hepatocellular carcinoma tumors.

[0011] Furthermore, the inhibition of the EGFR-promoting signaling pathway in hepatocellular carcinoma tumor cells includes a significant reduction in EGFR phosphorylation levels and / or total EGFR levels.

[0012] Furthermore, the inhibition of hepatocellular carcinoma tumor cell proliferation includes reducing the proportion of Ki67-positive cells and weakening cell proliferation activity.

[0013] Furthermore, the immune cells are NK cells.

[0014] Furthermore, the EGFR downstream extracellular matrix-related genes include Col1A1, COL4A1, LAMA2, and ELN.

[0015] Furthermore, the C6orf120 inhibitor is a nucleic acid molecule that inhibits the expression of the C6orf120 gene, an antibody or functional polypeptide that inhibits the function of the C6orf120 protein, or a small molecule compound that interferes with the key structural domain of C6orf120 or its binding interface with the ECM / receptor.

[0016] This invention demonstrates, through a C6orf120 gene knockout mouse hepatocellular carcinoma model and in vitro intervention experiments using C6orf120-targeting siRNA, that intervention with C6orf120 can inhibit the EGFR pro-cancer pathway in tumor cells, reduce the expression of downstream ECM-related genes, improve the ECM biomechanical microenvironment, enhance immune infiltration, and inhibit angiogenesis. Those skilled in the art can design and obtain various forms of C6orf120 inhibitors based on this, and equivalent variations are considered to fall within the scope of protection of this invention, with siRNA-based nucleic acid inhibitors being the preferred embodiment.

[0017] Furthermore, the nucleic acid molecules that inhibit C6orf120 gene expression are siRNA, dsRNA, shRNA, or ASO.

[0018] Furthermore, the C6orf120 inhibitor is a nucleic acid molecule, specifically siRNA, that inhibits the expression of the C6orf120 gene.

[0019] Furthermore, the nucleotide sequence of the siRNA is SEQ ID NO.1, SEQ ID NO.2 and / or SEQ ID NO.3.

[0020] Furthermore, the drug also includes a pharmaceutically acceptable carrier.

[0021] Furthermore, the drug is an injectable form.

[0022] The nucleic acid molecules described herein may be modified using methods that are not permitted in the art, including but not limited to ribose modification (such as LNA, 2'-OME, 2'-F, etc.), phosphate backbone modification, base methylation, uracil and adenine substitution, N-acetylgalactosamine coupling, conjugates, etc., to improve in vivo stability and reduce immunogenicity.

[0023] Pharmaceutically acceptable carriers can be selected from, but are not limited to, carriers / excipients known to be available for small nucleic acid drugs and antibody drugs, such as liposomes, exosomes, polymer carriers, solvents, cosolvents, pH adjusters, osmotic pressure adjusters, antioxidants, preservative coatings, capsules, fillers, binders, sustained-release agents, controlled-release agents, lubricants, etc.

[0024] On the other hand, this application provides a pharmaceutical composition for treating hepatocellular carcinoma or preventing hepatocellular carcinoma metastasis, said pharmaceutical composition comprising a C6orf120 inhibitor.

[0025] The administration routes of the drug / drug composition include, but are not limited to, intravenous administration, portal vein administration, or local administration; the drug / drug composition may also be used in combination with EGFR inhibitors, immune checkpoint inhibitors, antifibrotic drugs, or antiangiogenic drugs to achieve synergistic intervention on tumor proliferation, ECM mechanical status, immune infiltration, and angiogenesis in the same patient.

[0026] The drug / drug composition is preferably used for hepatocellular carcinoma patients with high C6orf120 expression and / or high EGFR signaling activation, accompanied by significant ECM deposition and increased tumor tissue stiffness.

[0027] Compared with the prior art, the present invention has the following beneficial effects: (1) A key target, C6orf120, connecting the EGFR pro-cancer pathway in tumor cells with the ECM biomechanical microenvironment was proposed, and a directly druggable nucleic acid inhibition form was given: This invention, combining database analysis, animal models, and in vitro siRNA intervention experiments, reveals that C6orf120 is highly expressed in HCC tumor tissues and is associated with poor prognosis. It can activate the EGFR pro-cancer pathway in tumor cells and upregulate ECM-related genes, thereby promoting ECM deposition and sclerosis. Intervention with siRNA targeting C6orf120 can simultaneously inhibit EGFR activation, downregulate ECM gene expression, and alleviate ECM sclerosis, providing a new target and feasible nucleic acid drug form for pharmacological intervention along the "tumor cell pro-cancer pathway—ECM microenvironment" axis in solid tumors.

[0028] (2) By inhibiting C6orf120, tumor progression is synergistically inhibited through multiple pathways and cellular processes: C6orf120 deficiency not only restricts the growth of hepatocellular carcinoma in mice, but also reduces intratumoral collagen fiber deposition and ECM sclerosis, decreases tumor stiffness, inhibits angiogenesis, and simultaneously promotes deep NK cell infiltration and enhances the production of effector molecules such as IFN-γ. Compared with traditional treatments that target only a single cell or a single signaling pathway, this invention achieves simultaneous intervention on multiple pathways, including tumor cells, ECM, immune cells, and vascular endothelial cells, through the inhibition of a single target.

[0029] (3) It has the potential for prognostic assessment and patient stratification, which is conducive to achieving precision medicine: High expression of C6orf120 is associated with decreased survival rates in HCC patients, making it a promising candidate as an adjunct biomarker for implementing the treatment strategy of this invention. It could be used to identify patient populations more likely to benefit from C6orf120 inhibition, thereby providing a basis for personalized and precision treatment. Attached Figure Description

[0030] Figure 1A Images of C6orf120 immunohistochemical staining of liver tissues from different donors in the THE HUMAN PROTEIN ATLAS public database.

[0031] Figure 1B The overall survival Kaplan-Meier curves are based on the TCGA-LIHC cohort and grouped by C6orf120 expression levels.

[0032] Figure 1C Survival curves for high and low C6orf120 expression groups within each clinical stage (from top to bottom: stage I, stage II, stage III).

[0033] Figure 2 A schematic diagram illustrating the construction and identification of C6orf120 gene knockout mice: Part A illustrates the CRISPR / Cas9 gene editing strategy targeting the exons of the C6orf120 coding region; Part B shows the PCR amplification and sequencing results of F0 and subtype representative morphologies, used to distinguish C6orf120. WT With C6 KO Mouse; Part C shows the immunofluorescence staining image of C6orf120 in mouse liver tissue, which shows a significant reduction in C6orf120 protein deposition in the liver tissue of knockout mice.

[0034] Figure 3 Results of C6orf120 gene knockout mouse subcutaneous hepatocellular carcinoma tumor model and tumor size determination: Part A shows the timeline of Hepa1-6 cell seeding and sampling; Part B, the left image, shows representative C6 cells. WT With C6 KO Photographs of the appearance of subcutaneous tumors in mice. The middle and right images are bar charts showing the statistical values ​​of tumor volume and weight in the two groups of mice, respectively.

[0035] Figure 4 Schematic diagram illustrating the effect of inhibiting C6orf120 on the structure and mechanical properties of the extracellular matrix within tumor cells: Part A represents C6. WT With C6 KO Second harmonic imaging (SHG) collagen fiber images of mouse tumor tissue and quantitative results of collagen signal intensity per unit field of view; Part B presents shear wave ultrasound elastography images of tumors and statistical results of shear wave velocity, used to reflect differences in tumor tissue stiffness.

[0036] Figure 5 Schematic diagram illustrating the effect of C6orf120 on tumor intratumoral immune cell infiltration and immune effector molecule expression: Part A shows multiplex immunofluorescence images of frozen sections of tumor, displaying CD8. + T cells and NCR1 +NK cells in C6 WT With C6 KO Spatial distribution within the tumor; Part B shows flow cytometry analysis of tumor-infiltrating immune cells and CD45. + IFN-γ + Statistical results of cell proportions.

[0037] Figure 6 A schematic diagram illustrating the effect of C6orf120 on tumor angiogenesis: Part A shows a color Doppler ultrasound blood flow image of a mouse tumor, displaying C6... WT With C6 KO The differences in blood flow signals within the tumors were observed in group A; Part B shows CD31 immunofluorescence staining of tumor tissue, reflecting the distribution of vascular endothelial cells; Part C shows flow cytometry analysis of single-cell suspensions of tumor cells. Scatter plot of cell proportions and statistical results.

[0038] Figure 7A Western blot analysis results of p-EGFR and t-EGFR in HepG2 cells treated with recombinant C6orf120 protein rCF at different time points.

[0039] Figure 7B Immunofluorescence images of EGFR subcellular localization in the control group and rCF-treated group, showing EGFR clustering and endocytosis.

[0040] Figure 7C A statistical graph showing the qPCR results of mRNA expression levels of downstream genes in the EGFR pathway, such as Cyclin D1, c-FOS, EGR1, and extracellular matrix.

[0041] Figure 8A A bar chart showing the relative expression levels of ECM-related genes such as C6orf120, Cyclin D1, Col1A1, and LAMA2 after transfection with negative control siRNA (NC) or siRNA targeting C6orf120 (siC6) by qPCR.

[0042] Figure 8B The images show Ki67 / DAPI immunofluorescence staining patterns of liver cancer cells in the NC and siC6 groups, along with the statistical results of the average Ki67 fluorescence intensity per field, reflecting changes in cell proliferation activity.

[0043] Figure 8C The Western blot results and corresponding quantitative analysis diagrams for the expression of Ki67, COL1A2, MMP9 and C6orf120 proteins in NC and siC6 group cells show that downregulation of C6orf120 inhibits the proliferation and expression of ECM-related proteins.

[0044] Figure 9 A schematic diagram illustrating the effect of C6orf120 on the activation of the EGFR signaling pathway in tumor cells: Part A shows the statistical results of p-EGFR and t-EGFR protein expression and their relative levels after transfection with negative control siRNA (NC) or C6orf120-targeting siRNA (siC6) by Western blot. Part B shows the EGFR / DAPI immunofluorescence images of the NC group and the siC6 group under the conditions of treatment with the control reagent DMSO or the EGFR activator EGF, and the statistical results of the total EGFR fluorescence intensity / field of view for each group. Detailed Implementation

[0045] Example 1: Analysis of C6orf120 and HCC survival using TCGA and immunohistochemical databases This embodiment uses publicly available tumor databases to analyze the expression of C6orf120 in human liver tissue and its relationship with the prognosis of HCC patients.

[0046] First, immunohistochemical results of human liver tissue were retrieved from the HUMAN PROTEIN ATLAS public database. Tissue microarray images were created from three representative cases (aged 54–63 years, including male and female volunteers). Figure 1A As shown, C6orf120 protein was generally expressed at a medium to high level in the liver tissues of volunteers of different genders.

[0047] Subsequently, gene expression matrices and corresponding survival follow-up data were downloaded from the TCGA Liver hepatocellular carcinoma (TCGA-LIHC) cohort, and the samples were processed using statistical software (R 4.1.2). Patients were divided into high-expression and low-expression groups based on the median C6orf120 mRNA expression level, and overall survival was analyzed using the Kaplan-Meier method and log-rank test. Results are as follows: Figure 1B As shown, among all HCC patients, the survival rate of the high C6orf120 expression group (red curve in the figure) was significantly lower than that of the low expression group (black curve in the figure), with a hazard ratio (HR) greater than 1 and a statistically significant p-value in the log-rank test, suggesting that high C6orf120 expression is associated with poor prognosis of HCC.

[0048] Furthermore, the TCGA-LIHC cohort was divided into stage I, II, and III subgroups according to clinical stage, and survival analysis was performed within each stage based on C6orf120 expression levels. The results are as follows: Figure 1CAs shown, in HCC patients at different clinical stages, the survival rate of the high C6orf120 expression group showed a significant downward trend, and statistically significant survival differences were observed in each stage.

[0049] In summary, the results of this embodiment indicate that C6orf120 is expressed in liver tissue, and the high expression of this protein is closely related to the reduced survival rate of HCC patients. This suggests that C6orf120 can serve as a potential molecular marker for prognostic assessment of hepatocellular carcinoma, providing a basis for subsequent intervention targeting C6orf120.

[0050] Example 2: Construction of C6orf120 gene knockout mice This embodiment uses CRISPR / Cas9 technology to introduce mutations through non-homologous recombination repair, causing a frameshift and loss of function in the mouse C6orf120 gene protein. Figure 2 Part A of the study). The specific construction method was as follows: Cas9 mRNA and gRNA were obtained through in vitro transcription; Cas9 mRNA and gRNA were microinjected into the fertilized eggs of C57BL / 6J mice to obtain F0 generation mice. PCR amplification and sequencing confirmed that the F0 generation mice were positive for gene knockout. These mice were then mated with C57BL / 6J mice, and the offspring were bred through heterozygous mating to obtain homozygous mice. Wild-type mice born in the same littermate served as experimental controls. The gene knockout effect in the offspring mice was identified by PCR amplification and sequencing. Figure 2 Part B, C6 WT Representing wild-type mice, C6 KO Representative gene knockout mice), and immunofluorescence staining of liver tissue confirmed that, compared with wild-type mice, C6orf120 protein deposition in the liver tissue of knockout mice was significantly reduced ( ). Figure 2 Part C).

[0051] Example 3: Construction of a mouse model of hepatocellular carcinoma and determination of tumor size To evaluate the effect of C6orf120 deletion on tumor growth, C6orf120 gene knockout mice (C6) were selected. KO ) and wild-type control mice with the same background (C6) WT Mice were matched for sex and age, and routinely housed under SPF conditions. Hepa1-6 mouse hepatocellular carcinoma cells were used as the seed cells, and the experimental procedure was as follows: Figure 3 As shown in Part A. Specifically, Hepa1-6 cells were cultured in standard medium containing 10% fetal bovine serum to the logarithmic growth phase, digested, and then collected. The cells were resuspended in sterile PBS, and each mouse was inoculated with 1×10⁶ cells. 6Hepa1-6 cell suspension was subcutaneously inoculated into one side of the back of mice using a sterile syringe to establish a hepatocellular carcinoma tumor model. Mice were sacrificed at least four weeks after inoculation, and tumor tissue was extracted. The longest diameter (L) and shortest vertical diameter (W) of the tumor were measured, and the result was calculated using the common formula V = (L × W) / (L × W). 2 Calculate the tumor volume using a 1 / 2 ratio and weigh the tumor using an electronic balance. Record representative tumor morphologies by photograph, such as... Figure 3 As shown in Figure B (left); the statistical results of tumor volume and tumor weight are as follows: Figure 3 As shown in Part B.

[0052] The results show that, compared with C6 WT Compared to the previous group, C6 KO The tumor volume and tumor weight of the mice in the group were significantly reduced, and the difference was statistically significant. Figure 3 (Part B of the study). The results above suggest that in the Hepa1-6 hepatocellular carcinoma tumor model, the deletion of C6orf120 can significantly inhibit tumor growth, providing in vivo functional evidence for the application of C6orf120 as a therapeutic target and inhibitor of hepatocellular carcinoma.

[0053] Example 4: Evaluation of the effects of C6orf120 inhibition on the structure and mechanical properties of the extracellular matrix within tumor cells. To evaluate the effects of C6orf120 inhibition on the extracellular matrix structure and mechanical properties of tumors, the C6orf120 inhibitor established in Example 3 was selected. WT With C6 KO Hepatocellular carcinoma-bearing mice were examined at both histological and in vivo imaging levels.

[0054] First, frozen sections of tumor tissue were prepared from euthanized mice and subjected to second harmonic generation (SHG) imaging using a two-photon microscope to observe and analyze the spatial arrangement and relative content of collagen fibers. In this embodiment, the two-photon mode of the Leica DIVE system was used, with a fixed excitation wavelength, to acquire multi-field images of the tumor parenchyma. Then, the collagen SHG signal intensity within each field of view was quantified using ImageJ image analysis software, and the collagen intensity per unit field of view was statistically obtained. The results are as follows: Figure 4 As shown in part A, C6 WT In the C6^KO group, relatively dense and coarse collagen fiber bundles with high SHG signal intensity were observed in the tumor tissue; while in the C6^KO group, collagen fibers were significantly sparse and weakened in arrangement. Quantitative analysis showed that C6^KO tumors... KO The collagen intensity per unit field of view of group C6 was significantly lower than that of group C6. WT The study suggests that C6orf120 deficiency may reduce the degree of collagen deposition and fibrosis within tumors.

[0055] Secondly, to evaluate the mechanical properties of tumor tissue at the in vivo level, shear wave elastography was performed on live mice to measure the propagation velocity of shear waves within the tumor. Specifically, after anesthetizing and fixing the mice, a small animal ultrasound imaging system was used to locate the subcutaneous tumor in B-mode, and then switched to shear wave imaging mode. A region of interest (ROI) was delineated in the tumor area, and the average shear wave velocity value within the tumor was automatically acquired. The results are as follows: Figure 4 As shown in Part B, tumors in group C6^WT exhibited higher shear wave velocities in shear wave imaging, suggesting relatively stiff tissue; while C6 KO The shear wave velocity of the tumors in the two groups was significantly reduced, and the statistical results showed that the difference between the two groups was statistically significant. Combined with the results of the decrease in tumor volume and tumor weight in Example 3, it is shown that the absence of C6orf120 not only reduces the deposition of collagen fibers and their dense rearrangement in the extracellular matrix of tumor cells, but also reduces the overall stiffness of tumor tissue at the in vivo level, transforming the tumor ECM from a "hardened" state to a relatively "softened" state.

[0056] In summary, this embodiment demonstrates that inhibiting C6orf120 can improve the tumor microenvironment (ECM) at both the extracellular matrix structure and mechanical properties levels, providing direct experimental evidence that C6orf120 inhibitors exert their anti-tumor effects by "softening the tumor ECM and improving the tumor microenvironment."

[0057] Example 5: Evaluation of the effects of C6orf120 inhibition on tumor intratumoral immune cell infiltration and immune effector molecules To further evaluate the effects of inhibiting C6orf120 on tumor intratumoral immune cell infiltration and immune effector molecules, the C6orf120 inhibitor established in Example 3 was selected. WT With C6 KO Immunological tests were performed on hepatocellular carcinoma-bearing mice at both the tissue and cellular levels, and the results are shown below. Figure 5 .

[0058] First, the extracted tumor tissue was frozen sectioned, and multiplex immunofluorescence staining was used to analyze the spatial distribution of immune cells within the tumor tissue. In this embodiment, DAPI was used to label cell nuclei, and CD8 antibody was used to label CD8. + T cells (shown as blue) and NK cells labeled with NCR1 antibody (shown as red) were stained. After staining, whole-tissue images of the tumor were obtained under a confocal microscope, with magnified observation of the tumor margins and internal regions, such as... Figure 5 As shown in Part A.

[0059] The results showed that in C6 WT In the group of tumors, CD8 + T cells are mainly distributed at the tumor margin or in the surrounding stroma, and CD8 cells enter the tumor's internal parenchyma. + Relatively few T cells; NCR1 + Overall NK cell infiltration was low, with a limited number of NK cells visible within the tumor. In contrast, C6... KO CD8 in the group of tumors + T cells were distributed in both the peripheral zone and the internal parenchyma, and the overall degree of infiltration was similar to that of C6. WT There was no significant decrease compared to the previous group; while NCR1 + NK cells were significantly enriched within the tumor, and a large number of K cells were observed to aggregate in the tumor parenchyma, suggesting that the absence of C6orf120 facilitates the penetration of NK cells through the tumor extracellular matrix barrier and into the tumor.

[0060] Secondly, to evaluate changes in local tumor immune effector molecules, C6 was analyzed. WT With C6 KO Tumor tissue from tumor-bearing mice was digested into single cells to prepare a suspension of tumor-infiltrating immune cells. The proportion of interferon-γ (IFN-γ) positive cells was detected by flow cytometry. Specifically, hematopoietic immune cells were first labeled with CD45 antibody, and dead cells were excluded by combining live cell dyes. + Intracellular staining was used to detect IFN-γ expression levels in the Live cell population.

[0061] Flow cytometry images Figure 5 As shown in Part B, C6 WT CD45 in the group of tumors + IFN-γ + Cells account for approximately 34%, while C6 KO The proportion of this group rose to approximately 49%, and quantitative statistical results showed that C6 KO Group CD45 + IFN-γ + The percentage of cells was significantly higher than that of C6. WT Group( Figure 5 (Part B, right figure). This result indicates that in the context of C6orf120 deletion, the levels of effector molecules such as IFN-γ produced by tumor-infiltrating immune cells are increased, thereby enhancing anti-tumor immune activity.

[0062] The combined results of immunofluorescence spatial localization and flow cytometry analysis show that inhibiting C6orf120 can promote the infiltration and aggregation of NK cells in the tumor, and increase the expression levels of effector molecules such as IFN-γ in tumor-infiltrating immune cells, thereby improving the tumor immune microenvironment. This provides experimental evidence that the C6orf120 inhibitor proposed in this invention exerts its anti-tumor effect by promoting immune cell infiltration and enhancing its effector function.

[0063] Example 6: Evaluation of the effect of C6orf120 inhibition on tumor angiogenesis To evaluate the effect of inhibiting C6orf120 on tumor angiogenesis, the C6-based inhibitor established in Example 3 was used again. WT With C6 KO Hepatocellular carcinoma-bearing mice were examined using in vivo blood flow imaging, histological immunofluorescence, and flow cytometry. Results are shown below. Figure 6 .

[0064] First, Doppler ultrasound blood flow imaging was performed on mice. After anesthetizing and fixing the mice, subcutaneous tumors were located using a small animal ultrasound imaging system in B-mode. Then, the system was switched to color Doppler mode, and regions of interest (ROIs) were delineated within the tumor area. The distribution of blood flow signals within and around the tumor was recorded. Figure 6 As shown in part A, C6 WT Abundant color Doppler flow signals were observed within the tumor group, suggesting numerous blood vessels and active blood flow within the tumor; while C6 KO Under the same conditions, the color Doppler flow signal in the tumor area was significantly reduced, indicating reduced tumor blood supply and restricted angiogenesis.

[0065] Secondly, frozen sections of tumor tissue were prepared from sacrificed mice, and immunofluorescence staining was used to detect the vascular endothelial marker CD31. DAPI was used to label cell nuclei, and anti-CD31 antibody was used to label vascular endothelial cells (shown in red). Images of the tumor parenchyma were acquired under a confocal microscope, such as... Figure 6 As shown in Part B. The results show that C6 WT CD31 in group of tumors + The number of cells is relatively large and their distribution is relatively dense; while C6 KO CD31 in group of tumors + The cells were significantly sparse, suggesting that C6orf120 deficiency may reduce angiogenesis in tumor tissue.

[0066] Furthermore, to quantitatively analyze the proportion of vascular endothelial cells within tumors at the cellular level, C6... WT With C6 KOTumor tissue from tumor-bearing mice was enzymatically digested to prepare single-cell suspensions, and the proportion of CD31-positive cells was detected by flow cytometry. Specifically, hematopoietic immune cells were labeled with CD45 antibody, and CD31 expression was further detected in the CD45-negative (CD45-) cell population (non-immune cells). CD45-CD31+ cells were used as the tumor intracellular vascular endothelial cell population for statistical analysis.

[0067] Flow cytometry and statistical results are as follows: Figure 6 As shown in section C, in C6 WT In the tumor group, CD45-CD31+ cells accounted for a relatively high proportion of the live cell population; while in C6... KO In group C6, the proportion of this group decreased significantly, and quantitative comparison showed that the proportion of this group decreased significantly. KO The percentage of CD45-CD31+ cells in group C45-CD31+ was significantly lower than that in group C6. WT The differences between the groups were statistically significant.

[0068] The combined results of ultrasound blood flow imaging, CD31 immunofluorescence, and flow cytometry showed that C6orf120 deficiency led to a reduction in tumor blood flow signals, a decrease in CD31+ blood vessel density, and a reduction in the proportion of CD45-CD31+ vascular endothelial cells. This indicates that inhibiting C6orf120 can effectively inhibit tumor angiogenesis, thereby weakening the blood supply conditions of tumor tissue. This provides experimental evidence for the anti-tumor effect of the C6orf120 inhibitor proposed in this invention by limiting angiogenesis.

[0069] Example 7: Activation of the EGFR pro-cancer signaling pathway in liver cancer cells by C6orf120 To investigate the regulatory role of C6orf120 on tumor cell-mediated oncogenic signals, this study added recombinant C6orf120 protein (rCF) to an in vitro hepatocellular carcinoma model and observed its effects on epidermal growth factor receptor (EGFR) activation and downstream signal transcription. Human hepatocellular carcinoma cell line (HepG2) was starved of serum and divided into a control group (Control, Ctl) and a recombinant C6orf120 treatment group (rCF). Samples were collected at different time points, and the results are shown below. Figures 7A-7C .

[0070] First, the phosphorylation level of EGFR was detected. A predetermined concentration of recombinant C6orf120 protein was added to the treatment group. Cells were collected at 0, 0.5, 1, 6, and 9 h time points. Total protein levels were measured by Western blot to detect phosphorylated EGFR (p-EGFR) and total EGFR (t-EGFR), using GAPDH as an internal control. Figure 7AAs shown in the figure. The results showed that, compared with 0 h or the control group, the p-EGFR signal was significantly enhanced from 0.5 h after rCF treatment and continued to increase in subsequent time, while the t-EGFR expression did not change significantly, suggesting that C6orf120 can promote the activation and phosphorylation of EGFR without changing the total amount of EGFR.

[0071] To further observe the subcellular localization changes of EGFR, immunofluorescence staining was performed on cells in the control group and the rCF-treated group. DAPI was used to label the cell nuclei, and EGFR antibody was used to label the receptor (shown as magenta). The cells were then observed under a confocal microscope. Figure 7B As shown in the figure. The results showed that in the control group cells, EGFR was mainly distributed on the cell membrane surface and was in a relatively "resting" state; while after rCF treatment, EGFR formed obvious clusters in the local cell membrane and showed endocytosis into the cell, suggesting that C6orf120 stimulation can induce EGFR clustering and endocytosis, which is a morphological manifestation of its receptor activation and signal transduction process.

[0072] Based on EGFR activation, real-time quantitative PCR was used to detect the expression levels of downstream proliferative / early response genes, including Cyclin D1, c-FOS, and EGR1. The results are as follows: Figure 7C As shown in the figure above, compared with the control group, the mRNA levels of the above genes in the rCF treatment group were significantly increased, indicating that the activation of the C6orf120-EGFR axis can drive the enhancement of related pro-cancer signals, pro-proliferation pathways and transcriptional programs in liver cancer cells. Figure 7C The figure below shows that, compared with the control group, the extracellular matrix genes PAPLN, ELN, COL4A1, and COL1A1 were significantly upregulated in the rCF treatment group, indicating that activation of the C6orf120-EGFR axis can drive the transcription of extracellular matrix genes in liver cancer cells.

[0073] This embodiment demonstrates that in liver cancer cells, C6orf120 directly enhances pro-cancer signals within tumor cells by promoting EGFR receptor phosphorylation, activation, clustering, and endocytosis, thereby upregulating the transcription of multiple downstream pro-proliferative / early response genes and extracellular matrix genes. This indicates that inhibitors of C6orf120 possess a dual effect of blocking EGFR pro-cancer signals and inhibiting extracellular matrix synthesis.

[0074] Example 8: Evaluation of the effects of C6orf120-targeting siRNA on hepatocellular carcinoma cell proliferation and extracellular matrix synthesis. To evaluate the effects of inhibiting C6orf120 on tumor cell proliferation and extracellular matrix (ECM) synthesis at the cellular level, this study transfected cells with C6orf120-targeting siRNAs (siRNA1: UCUDUUCUCUGGACGAUAUUATT, SEQ ID NO.1; siRNA2: UGAACCACGAGGGCAAGAUAGTT, SEQ ID NO.2; siRNA3: CAAGGGAGAUGCGGAUCUGUATT, SEQ ID NO.3; all experimental results were obtained after treating cells with an equal mixture of the three siRNAs) in vitro. Changes in proliferation-related molecules and ECM-related genes / proteins were detected, and the results are shown in Figure 8.

[0075] Human hepatocellular carcinoma cell line (HepG2) was selected and cultured to the logarithmic growth phase before being divided into two groups: a negative control group (NC) transfected with non-specific control siRNA, and an experimental group (siC6) transfected with specific siRNA targeting human C6orf120. After a certain period of transfection, total RNA was extracted, and the mRNA expression levels of C6orf120, Cyclin D1, and ECM-related genes Col1A1 and LAMA2 were detected using real-time quantitative PCR (qPCR), with GAPDH as an internal control. Results are as follows: Figure 8A As shown, compared with the NC group, the expression of C6orf120 mRNA in the siC6 group was significantly decreased, and the relative expression levels of Cyclin D1, Col1A1, and LAMA2 were also significantly reduced.

[0076] Ki67 is a recognized nuclear antigen associated with cell proliferation. It is expressed only in cells at active phases of the cell cycle (G1, S, G2, M) and not in quiescent cells. By statistically analyzing the proportion of Ki67-positive cells, the number of cells in a proliferative state can be directly reflected. Compared to simply counting the total number of cells or OD450 values, this method provides a more sensitive and reliable assessment of whether cell proliferation is inhibited. To further observe changes in cell proliferation levels, Ki67 immunofluorescence staining was performed on both groups of cells, and DAPI was used to label cell nuclei. Images were acquired under a confocal microscope, and the average fluorescence intensity of Ki67 in each field of view was quantitatively analyzed. Representative images and statistical results after 48 hours are shown below. Figure 8B As shown, Ki67 staining was significantly weakened in the siC6 group cells, and the average fluorescence intensity / field of Ki67 was significantly lower than that in the NC group, suggesting that C6orf120 knockdown can inhibit the proliferation activity of liver cancer cells.

[0077] In addition, total protein was extracted from cells in the NC and siC6 groups, and the expression of C6orf120, Ki67, and ECM-related proteins COL1A2 and MMP9 was detected by Western blot, with GAPDH as an internal control. Representative bands and quantitative results are shown below. Figure 8C As shown, the expression of C6orf120 protein was significantly downregulated in the siC6 group, and the expression levels of Ki67, COL1A2 and MMP9 were also reduced accordingly.

[0078] In summary, this embodiment demonstrates that siRNA targeting C6orf120 can effectively downregulate the expression of C6orf120, while simultaneously inhibiting the expression of proliferation-related molecules and multiple ECM-related genes / proteins in liver cancer cells. This demonstrates at the cellular level that C6orf120 inhibitors possess both anti-proliferation and extracellular matrix synthesis-inhibiting effects.

[0079] Example 9 Evaluation of the inhibitory effect of C6orf120-targeting siRNA on EGFR signaling pathway activation in liver cancer cells To further verify whether inhibiting C6orf120 can block the EGFR pro-cancer signaling pathway in tumor cells, this embodiment transfected C6orf120-targeting siRNA into liver cancer cells. The expression and activation of EGFR were detected by Western blot and immunofluorescence, respectively. The results are shown in [Figure number missing]. Figure 9 .

[0080] Hepatocellular carcinoma cells were still used, divided into NC and siC6 groups for siRNA transfection. After transfection, total cellular protein was collected, and Western blot was used to detect phosphorylated EGFR (p-EGFR) and total EGFR (t-EGFR) levels, with GAPDH as an internal control. Representative bands and quantitative results are shown below. Figure 9 As shown in Part A, compared with the NC group, the relative expression levels of t-EGFR and p-EGFR in the siC6 group cells were significantly decreased, suggesting that the overall expression and activation state of EGFR were inhibited after C6orf120 was knocked down.

[0081] To observe the effect of C6orf120 downregulation on EGFR protein localization and overall fluorescence intensity, transfected NC and siC6 cells were treated with DMSO (solvent control) or exogenous EGF (EGFR activator), respectively. EGFR immunofluorescence staining was then performed, and cell nuclei were labeled with DAPI. Images were acquired under a confocal microscope, and the average fluorescence intensity of EGFR in each field of view was quantitatively analyzed. Results are as follows: Figure 9As shown in Part B: Under DMSO conditions, EGFR fluorescence in the cell membrane and cytoplasm of the siC6 group was significantly reduced; under EGF stimulation, EGFR fluorescence intensity increased in the NC group, indicating that the EGFR pathway was activated, while the EGFR fluorescence signal in the siC6 group was still significantly lower than that in the NC group. Statistical results showed that the EGFR fluorescence intensity in the siC6 group decreased significantly under both basal conditions and EGF stimulation.

[0082] In summary, this embodiment demonstrates that siRNA targeting C6orf120 can reduce the total expression of EGFR in liver cancer cells and inhibit its phosphorylation activation, while weakening the overall fluorescence signal of EGFR under basal and ligand stimulation conditions. This suggests that C6orf120 is an important positive regulator of the EGFR oncogenic pathway, and its inhibitors can effectively block the EGFR signaling pathway in tumor cells.

Claims

1. Application of C6orf120 inhibitors in the preparation of drugs for treating hepatocellular carcinoma.

2. Application of C6orf120 inhibitors in the preparation of drugs to prevent hepatocellular carcinoma metastasis.

3. In the application according to claim 1 or 2, the drug has one or more of the following effects: (1) Inhibit the EGFR tumor-promoting signaling pathway in hepatocellular carcinoma tumor cells; (2) Inhibits the proliferation of hepatocellular carcinoma tumor cells; (3) Downregulate the expression of EGFR downstream extracellular matrix-related genes and remodel the extracellular matrix microenvironment of hepatocellular carcinoma tumor cells; (4) Promotes the infiltration of immune cells; (5) Inhibits angiogenesis in hepatocellular carcinoma tumors.

4. The application according to any one of claims 1-3, wherein the C6orf120 inhibitor is a nucleic acid molecule that inhibits the expression of the C6orf120 gene, an antibody or functional polypeptide that inhibits the function of the C6orf120 protein, a small molecule compound that interferes with the key structural domain of C6orf120 or its binding interface with the ECM / receptor.

5. In the application according to claim 4, the nucleic acid molecule that inhibits the expression of the C6orf120 gene is siRNA, dsRNA, shRNA, or ASO.

6. In the application according to claim 4, the C6orf120 inhibitor is a nucleic acid molecule that inhibits the expression of the C6orf120 gene, and is siRNA.

7. The application according to claim 6, wherein the nucleotide sequence of the siRNA is SEQ ID NO.1, SEQ ID NO.2 and / or SEQ ID NO.

3.

8. The application according to any one of claims 1-7, wherein the medicament further comprises a pharmaceutically acceptable carrier.

9. The application according to claim 8, wherein the drug is an injectable preparation.

10. A pharmaceutical composition for treating hepatocellular carcinoma or preventing metastasis of hepatocellular carcinoma, characterized in that, The pharmaceutical composition contains a C6orf120 inhibitor.

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