Application of ifi27 gene as a target for liver cancer treatment

Abstract

The present application relates to the technical field of biological medicine, and particularly relates to the application of IFI27 gene as a liver cancer treatment target point. The present application first discovers that IFI27 is significantly up-regulated in liver cancer tissues, and the expression level in normal tissues adjacent to cancer is low, showing good tumor specificity, indicating that IFI27 plays an important role in liver cancer progression. Further, through in vitro cell experiments, it is confirmed that the knockdown of IFI27 expression by shRNA can significantly inhibit the proliferation activity, clonogenicity and migration and invasion ability of liver cancer cell lines, providing strong evidence for IFI27 gene as a liver cancer treatment target point. In vivo animal experiments further verify that the knockdown of IFI27 expression can effectively inhibit the growth of liver tumors. In summary, IFI27 can be used as a potential new target for liver cancer targeted therapy, and small molecule inhibitors or biological agents targeting IFI27 have research value and clinical transformation potential for the development of innovative drugs for liver cancer. The present application provides a new theoretical basis and intervention strategy for the precision treatment of liver cancer.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of the IFI27 gene as a therapeutic target for liver cancer. Background Technology

[0002] Hepatocellular carcinoma (HCC), a malignant tumor of the liver, is one of the most common malignant tumors worldwide, with consistently high incidence and mortality rates. The pathogenesis of liver cancer is complex, involving abnormal regulation of multiple genes and signaling pathways. Currently, treatment methods for liver cancer mainly include surgical resection, liver transplantation, radiofrequency ablation, chemotherapy, and targeted therapy. However, due to the highly invasive nature of liver cancer, its high recurrence rate, and tendency to metastasize, most patients are diagnosed at an advanced stage, resulting in poor treatment outcomes and a poor prognosis. Therefore, identifying new therapeutic targets and developing more effective therapeutic drugs is of significant clinical importance.

[0003] Interferon alpha-inducible protein 27 (IFI27) is an interferon-stimulated gene (ISG) that can be induced by interferon during viral infection and immune responses. IFI27 protein is located on the mitochondrial membrane, endoplasmic reticulum membrane, and nuclear membrane, and participates in the regulation of biological processes such as apoptosis, immune responses, and viral replication. Recent studies have found that IFI27 is abnormally expressed in various malignant tumors and is closely related to tumor occurrence, development, invasion, and metastasis. However, the specific role of IFI27 in liver malignancies and its potential as a therapeutic target have not been fully investigated and confirmed.

[0004] Currently, targeted therapy for liver cancer mainly focuses on the following aspects: (1) Anti-angiogenic drugs: such as sorafenib and lenvatinib, which block tumor angiogenesis by inhibiting the activity of vascular endothelial growth factor receptor (VEGFR) and other kinases. These drugs are currently the first-line treatment for advanced liver cancer, but they can only prolong the survival of patients by a few months and there are drug resistance problems. (2) Immune checkpoint inhibitors: such as PD-1 / PD-L1 inhibitors (nivolumab, pembrolizumab, etc.) and CTLA-4 inhibitors, which attack tumor cells by activating the body's immune system. These drugs have shown certain efficacy in some liver cancer patients, but the overall efficacy rate is still low and there are immune-related adverse reactions. (3) Targeted drugs for specific signaling pathways: such as mTOR inhibitors, c-Met inhibitors, etc., these drugs intervene in key signaling pathways in the occurrence and development of liver cancer. However, clinical studies show that the monotherapy efficacy of these drugs is limited and is often accompanied by drug resistance. (4) Gene therapy and RNA interference technology: These technologies inhibit tumor growth by regulating the expression of specific genes, such as gene silencing or overexpression strategies targeting certain oncogenes or tumor suppressor genes. However, these technologies are still in the research stage and have not yet been widely used in clinical practice.

[0005] However, existing liver cancer treatment technologies have many shortcomings and limited efficacy: (1) The overall efficacy of existing targeted drugs and immunotherapy drugs is low. Most patients can only achieve a few months of extended survival and cannot achieve long-term survival or cure. (2) Drug resistance problem: Whether it is chemotherapy drugs or targeted drugs, liver cancer cells are prone to drug resistance, leading to treatment failure. The drug resistance mechanism is complex and involves compensatory activation of multiple signaling pathways and gene mutations. (3) Large toxic side effects: Existing treatment drugs are often accompanied by serious adverse reactions, such as liver and kidney function damage, bone marrow suppression, immune-related adverse reactions, etc., which seriously affect the quality of life of patients. Some patients are forced to terminate treatment because they cannot tolerate it. (4) Lack of specific targets: The pathogenesis of liver cancer is complex and involves abnormalities in multiple genes and signaling pathways. Existing drugs lack targeting of liver cancer specific markers, resulting in insufficient selectivity and precision of treatment. Summary of the Invention

[0006] The purpose of this invention is to provide a new therapeutic target for the treatment of liver cancer.

[0007] To achieve the above objectives, the present invention provides the use of substances that inhibit the activity of IFI27 protein or reduce the expression of its encoding gene in the preparation of drugs for treating liver cancer.

[0008] Specifically, the active ingredient of the drug is any one of the following: a gene editing reagent that specifically knocks out the IFI27 coding gene; a small interfering RNA that specifically inhibits the activity of the IFI27 protein or reduces the expression of its coding gene; a small molecule compound that specifically inhibits the activity of the IFI27 protein or reduces the expression of its coding gene; or an antibody or ligand that specifically binds to the IFI27 protein.

[0009] Specifically, the active ingredient of the drug is an artificially designed functional RNA, such as shRNA, siRNA, dsRNA, miRNA, or sgRNA, that specifically inhibits the activity of the IFI27 protein or reduces the expression of its encoding gene.

[0010] Specifically, the active ingredient of the drug is an RNA that specifically inhibits the activity of the IFI27 protein or reduces the expression of its encoding gene. This RNA is either shRNA that knocks out the IFI27 gene or a vector used to express the shRNA. The vector is any one of a plasmid vector, a lentiviral vector, or an adeno-associated virus vector.

[0011] Specifically, the nucleotide sequence of the shRNA that knocks out the IFI27 gene is shown in SEQ ID NO: 1, specifically: shRNA-1:5'-GGATCCGAGTTCATCCTGGGCTCCATTGCTCGAGCAATGGAGCCCAGGATGAACTTTTTTTGAATTC-3'; further, the shRNA is packaged into a lentiviral vector.

[0012] Specifically, the nucleotide sequence of the sgRNA that knocks out the IFI27 gene is shown in SEQ ID NO: 2, specifically: 5′-GGCACCATTCTAGCTGGTTCG-3′; further, the sgRNA is packaged into an adeno-associated virus vector.

[0013] Furthermore, this invention also provides the application of the IFI27 protein and its encoding gene as biomarkers in the preparation of liver cancer diagnostic kits.

[0014] This invention is the first to discover that IFI27 is significantly upregulated in hepatocellular carcinoma (HCC) tissues, while its expression level is low in adjacent normal tissues, exhibiting good tumor specificity and indicating that IFI27 plays an important role in HCC progression. Furthermore, in vitro cell experiments confirmed that knocking down IFI27 expression using shRNA significantly inhibits the proliferation, colony formation, and migration / invasive abilities of HCC cell lines, providing strong evidence for IFI27 as a therapeutic target for HCC. In vivo animal studies further validated that knocking down IFI27 expression effectively inhibits liver tumor growth. In conclusion, IFI27 can serve as a potential new target for targeted therapy of HCC, and small molecule inhibitors or biologics targeting IFI27 have research value and clinical translational potential for developing innovative drugs for HCC. This invention provides new theoretical basis and intervention strategies for precision treatment of HCC. Attached Figure Description

[0015] Figure 1 The discovery of high specific expression of the IFI27 gene in hepatocellular carcinoma (HCC) tissues is shown in Figure A, where UMAP dimensionality reduction clustering analysis illustrates the distribution of different cell types in HCC and adjacent normal tissues; Figure B shows the differential expression of the IFI27 gene between HCC cells and normal hepatocytes; Figure C analyzes the differential expression of IFI27 in HCC tissues (Tumor) and adjacent normal tissues (PT) using the Cancer Genome Atlas (TCGA) liver cancer database (TCGA-LIHC). Note: **p<0.01, ****p<0.0001.

[0016] Figure 2 Immunohistochemical staining and quantitative analysis of liver cancer tissue and adjacent tissue sections after partial hepatectomy in 10 patients with hepatocellular carcinoma were performed using IFI27 specific antibody; Note: **p<0.01.

[0017] Figure 3 To verify the knockdown efficiency of IFI27 using qRT-PCR (A) and Western Blot (B); Note: *p<0.05.

[0018] Figure 4 To investigate the effect of knocking down the IFI27 gene on the proliferation of Huh7 cells; Note: ****p<0.0001.

[0019] Figure 5 To investigate the effect of knocking down the IFI27 gene on the cloning ability of Huh7 cells; Note: *p<0.05.

[0020] Figure 6 To investigate the effect of knocking down the IFI27 gene on the migration ability of Huh7 cells; Note: ****p<0.0001.

[0021] Figure 7 To investigate the effect of knocking down the IFI27 gene on the number and diameter of tumors in a mouse model of liver cancer; Note: ****p<0.0001. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] Example 1

[0024] The IFI27 gene is specifically highly expressed in liver cancer tissues.

[0025] We first collected tissue samples from six liver cancer patients who underwent partial hepatectomy at the Department of Hepatobiliary and Pancreatic Surgery, First Affiliated Hospital of Zhengzhou University. These samples included liver cancer tissue and adjacent tissue (1 cm from the tumor margin). The tissue samples were prepared into single-cell suspensions using standard methods, and single-cell RNA sequencing analysis was performed using the GEXSCOPE® microfluidic chip single-cell RNA sequencing (scRNA-seq) technology platform.

[0026] UMAP dimensionality reduction clustering analysis revealed the distribution of different cell types in hepatocellular carcinoma and adjacent normal tissues. Through integrated analysis and unsupervised clustering of single-cell transcriptome data from six patients, multiple cell types were identified, laying the foundation for subsequent analysis of IFI27 gene expression patterns in different cell types. Figure 1 A). Furthermore, differential expression of the IFI27 gene was found between hepatocellular carcinoma cells and adjacent normal cells. IFI27 gene expression was significantly upregulated in hepatocellular carcinoma cells, while expression was low in adjacent normal hepatocellular carcinoma cells (P>0.0001). The results are as follows: Figure 1 As shown in B.

[0027] We then used the Cancer Genome Atlas (TCGA) liver cancer database (TCGA-LIHC) to analyze the expression differences of IFI27 in liver cancer tissues (Tumor) and adjacent normal tissues (PT). The results showed that in the vast majority of samples, the IFI27 expression level in liver cancer tissues was significantly higher than that in the corresponding adjacent normal tissues (p>0.001), consistent with the single-cell sequencing results mentioned above. Figure 1 As shown in C.

[0028] Furthermore, we collected tissue samples from 10 hepatocellular carcinoma patients who underwent partial hepatectomy at the Department of Hepatobiliary and Pancreatic Surgery, First Affiliated Hospital of Zhengzhou University. These samples included hepatocellular carcinoma tissue and adjacent tissue (1 cm from the tumor margin). The tissue samples were fixed in 4% paraformaldehyde, dehydrated, embedded in paraffin, sectioned, and then immunohistochemically stained with IFI27 specific antibody, followed by quantitative analysis. The results are as follows: Figure 2 As shown, significant differences were observed under both low magnification (20x) and high magnification (40x). The proportion of IFI27-positive cells was high and the staining intensity was strong in hepatocellular carcinoma tissue, while the staining intensity was weak in the control group, showing a significant difference between the two groups (p<0.01). This result indicates that the expression level of IFI27 protein in hepatocellular carcinoma tissue is significantly higher than that in the control group (non-hepatocellular carcinoma tissue).

[0029] In summary, IFI27 is specifically highly expressed in liver cancer, and it may be involved in the development and progression of liver cancer, showing potential as a diagnostic biomarker and therapeutic target for liver cancer.

[0030] To further illustrate the potential of the IFI27 gene as a diagnostic biomarker and therapeutic target for liver cancer, the following examples demonstrate the therapeutic effect of the IFI27 gene in liver cancer at both in vivo and in vitro levels by constructing an IFI27 knockdown virus.

[0031] Example 2

[0032] 1. Construction of IFI27 knockdown slow virus vector

[0033] Designed shRNA sequence targeting the human IFI27 gene, with the following target sequence: shRNA-1: 5'-GGATCCGAGTTCATCCTGGGCTCCATTGCTCGAGCAATGGAGCCCAGGATGAACTTTTTTTGAATTC-3' (SEQ ID NO: 1, named shIFI27-1).

[0034] Negative control shRNA: ACTACCGTTGTTATAGGTG (SEQ ID NO: 2, named shNC)

[0035] The sequences of the negative control shRNA (shNC) and the knockdown experimental group shIFI27-1 were amplified by PCR. The amplified target fragment was bound to a linearized vector, and the binding product was transformed into DH5α / Stbl3 competent cells. The bacterial cultures of shNC and shIFI27-1 were plated onto solid culture plates containing the corresponding antibiotics. Two positive clones were selected for sequencing. If the sequencing results matched the target sequence, the target plasmid was successfully constructed. Viral packaging was performed using a four-plasmid lentiviral system: the lentiviral transfer plasmid carrying the target fragment, the viral packaging helper plasmid LV003-VSVG, the viral packaging helper plasmid LV003-REV, and the viral packaging helper plasmid LV003-GP were extracted with high purity and free of endotoxin. The four plasmids were then co-transfected into 293T cells using Easyfectin transfection reagent. 72 hours after transfection, the supernatants of shNC and shIFI27-1 were collected, concentrated, and their titers were determined for later use.

[0036] 2. Lentiviral infection of human liver cancer cells Huh7

[0037] Huh7 cells were seeded in 6-well plates. When the cell confluence reached 30-40%, shNC and shIFI27-1 lentiviruses containing polybrene (final concentration 5 μg / ml) were added for infection, with the MOI (multiple of infection) set at 10-20. After 24 hours of infection, the medium was replaced with fresh complete medium, and the cells were cultured for another 24 hours.

[0038] Subsequently, puromycin (5ug / ml) was added for positive cell selection, and selection continued for 7-10 days, with the puromycin-containing medium being changed every 2-3 days until all uninfected control cells died. The selected stable cell lines were then analyzed using qRT-PCR and Western Blot to verify the IFI27 knockdown efficiency. Results are as follows... Figure 3 As shown, the mRNA of IFI27 in Huh7 cells of the shIFI27 group (IFI27 knockdown group) Figure 3 A) and protein ( Figure 3 The expression levels of B) were significantly lower than those of the negative control group (shNC), indicating that the present invention successfully constructed Huh7 cells with knockdown of the IFI27 gene.

[0039] 3. Effects of IFI27 gene knockdown on Huh7 cell proliferation

[0040] The effect of IFI27 gene knockdown on the proliferation of Huh7 cells was investigated using a CCK-8 proliferation assay. Specifically, Huh7 cells from the negative control group (shNC) and the IFI27 knockdown group (shIFI27) were seeded into 96-well plates, with 2000 cells per well and 5 replicates per group. After cell attachment, 10 μL of CCK8 reagent was added to each well on days 1, 2, 3, 4, 5, and 6. The cells were incubated at 37°C for 1 hour. The absorbance (OD value) at 450 nm was measured using a microplate reader.

[0041] The results are as follows Figure 4 As shown, the proliferation rate of cells in the shIFI27 group was significantly slower than that in the shNC group over time (p<0.0001), indicating that knocking down the IFI27 gene significantly inhibited the proliferation ability of Huh7 cells.

[0042] 4. Effect of IFI27 gene knockdown on the cloning ability of Huh7 cells

[0043] Huh7 cells from the negative control group (shNC) and the IFI27 knockdown group (shIFI27) were seeded into 6-well plates, 1000 cells per well, with 3 replicates per group. After culturing in complete culture medium for 14 days, the culture medium was discarded, and the cells were washed three times with physiological saline. After removing the residual physiological saline, 2 ml of 4% paraformaldehyde was added to each well for fixation for 30 min. After washing three times with physiological saline, the physiological saline was removed, and 2 ml of crystal violet dye was added to each well for staining for 1-2 hours. After staining three times with physiological saline, the cells were washed once with sterile, enzyme-free water. After air-drying upside down, the cells were photographed, and the images were processed using ImageJ software to calculate the number of cell clones.

[0044] The results are as follows Figure 5 As shown, the number of cell clones in the shIFI27 group was significantly lower than that in the shNC group (p<0.05), indicating that knocking down IFI27 significantly reduced the cloning ability of liver cancer cells.

[0045] 5. Effects of IFI27 gene knockdown on Huh7 cell migration ability

[0046] Using 24-well Transwell chambers (8 μm pores), Huh7 cells from the negative control group (shNC) and the IFI27 knockdown group (shIFI27) were resuspended in serum-free DMEM medium and the cell density was adjusted to 5 × 10⁶ cells / mL. 4Cells / mL were collected, and 200 μL of cell suspension was added to the upper chamber of a Transwell cell culture medium. 600 μL of DMEM medium containing 10% FBS was added to the lower chamber as a chemokine. Three replicates were made for each group. After incubation at 37°C and 5% CO2 for 24 hours, the Transwell chambers were removed, and unmigrated cells on the surface of the upper chamber were gently wiped away with a cotton swab. Cells were washed with PBS, fixed with 4% paraformaldehyde for 15 minutes, stained with 0.1% crystal violet for 30 minutes, washed with PBS, and air-dried. Five fields of view (100× or 200× magnification) were randomly selected and photographed under an inverted microscope.

[0047] The results are as follows Figure 6 As shown, the number of cells that migrated to the lower chamber in the shIFI27 group was significantly lower than that in the shNC group (p<0.0001), indicating that knocking down IFI27 significantly reduced the migration ability of liver cancer cells.

[0048] In summary, we investigated the effect of the IFI27 gene on liver cancer at the in vitro cellular level by constructing Huh7 cells with IFI27 gene knockdown. The experimental results showed that knockdown of the IFI27 gene can significantly inhibit the proliferation activity, colony formation ability, and migration and invasion ability of liver cancer cell lines, providing strong evidence for the IFI27 gene as a therapeutic target for liver cancer.

[0049] Example 3

[0050] Effect of knockdown of IFI27 gene on tumorigenesis in mice

[0051] Experimental Methods: Male C57BL / 6J mice, aged 6-8 weeks and weighing 18-22g, were purchased from SPF (Beijing) Biotechnology Co., Ltd. and housed in an SPF-grade animal facility with free access to food and water, under a 12-hour light / dark cycle. Mice were randomly divided into three groups: control group, model group, and knockout group, with 8 mice in each group. Lentiviral Injection: After mild anesthesia with isoflurane, mice in the knockout group were injected via tail vein with a high-titer shIFI27-1 lentivirus (4 × 10⁻⁶ viral titers per mouse). 11 TU, 200 μL), and tail vein injection of high-titer shNC lentivirus (2 × 10⁻⁶ virus titers per mouse) was administered to the model group and control group. 8TU (200 μL). Due to the liver's filtration function, lentivirus injected via the tail vein mainly accumulates in the liver and infects hepatocytes. The model group and knockdown group used diethylnitrosamine (DEN) to induce a liver cancer model. Specifically, mice were given a single intraperitoneal injection of DEN (25 mg / kg, dissolved in physiological saline), followed by normal feeding. Starting from week 4 after DEN injection, carbon tetrachloride (CCl4, dissolved in olive oil, 2 mL / kg) was injected intraperitoneally twice weekly for 8 weeks to induce chronic liver injury and fibrosis, promoting liver cancer development. Control group mice were injected with the same dose of physiological saline. Mice were sacrificed 16 weeks after lentivirus injection. Liver samples were harvested, and the number and average diameter of liver tumors were recorded.

[0052] The results are as follows Figure 7 As shown in the figure, the model group mice exhibited multiple tumors in their livers compared to the control group, indicating the successful establishment of the mouse hepatocellular carcinoma model. Compared to the model group, the number of liver tumors in mice with IFI27 knockdown was significantly reduced (p<0.001), and the tumor diameter was also significantly smaller. These results indicate that inhibiting or reducing IFI27 gene expression can significantly suppress the growth of hepatocellular carcinoma tumors, and the IFI27 gene holds promise for further research and development as a therapeutic target for hepatocellular carcinoma.

[0053] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of substances that inhibit IFI27 protein activity or reduce the expression of its encoding gene in the preparation of drugs for treating liver cancer, characterized in that, The substance that inhibits the activity of IFI27 protein or reduces the expression of its encoding gene is a knockdown shRNA of the IFI27 gene. The nucleotide sequence of the shRNA is SEQ ID NO: 1, specifically: 5'-GGATCCGAGTTCATCCTGGGCTCCATTGCTCGAGCAATGGAGCCCAGGATGAACTTTTTTTGAATTC-3'.

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