Application of NAT9 expression inhibitor in preparation of medicine for preventing, relieving and / or treating liver cancer

Abstract

The invention provides application of an NAT9 expression inhibitor in preparation of a medicine for preventing, relieving and / or treating liver cancer. It is found for the first time that the mRNA and protein expression levels of NAT9 in hepatocellular carcinoma (HCC) tissue are obviously higher than those in para-carcinoma and normal liver tissue, and the high expression of NAT9 is closely related to prognosis indexes such as poor overall lifetime and progression-free lifetime of a patient, so that the value of NAT9 serving as a liver cancer diagnosis marker and a new treatment target is determined. On the basis, the invention provides the application of the NAT9 inhibitor in preparing the medicine for preventing, relieving and / or treating the liver cancer. NAT9 expression is down-regulated through siRNA, and the proliferation activity, the clone forming ability, the migration ability and the invasion ability of liver cancer cells HLF can be effectively inhibited. The invention further provides a pharmaceutical composition containing the inhibitor, a brand new target spot and an effective intervention strategy are provided for liver cancer treatment, and the pharmaceutical composition has great clinical development prospects and application value.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the use of NAT9 expression inhibitors in the preparation of drugs for the prevention, relief and / or treatment of liver cancer. Background Technology

[0002] Hepatocellular carcinoma (HCC) is the most prevalent histological type of primary liver cancer, accounting for 70%-85% of all cases. In China, liver cancer ranks among the top malignant tumors in terms of incidence, being the fourth most common cancer, and its mortality rate ranks third. Currently, the preferred treatment for liver cancer is early surgical resection. However, due to the insidious nature of early symptoms, most patients are diagnosed at an advanced stage, thus losing the opportunity for radical surgery. The natural course of the disease in these patients is usually only 3-6 months, and they can only receive palliative treatment or systemic chemotherapy, but the overall efficacy is limited and the prognosis is poor. Therefore, there is an urgent need to develop new treatment strategies and targets.

[0003] The NAT9 (N-Acetyltransferase 9) gene is a protein-coding gene belonging to the N-acetyltransferase family. Proteins in this family are widely involved in the acetylation modification of various small molecule substrates and are important components of intracellular phase I and II biotransformation metabolic pathways. The NAT9 protein possesses acetyltransferase activity, catalyzing the transfer of acetyl groups to non-aminoacyl receptor molecules, and also exhibits N-acetyltransferase activity. These enzymatic properties indicate that NAT9 plays a crucial role in maintaining cellular metabolic homeostasis and regulating the biotransformation of endogenous small molecules or exogenous compounds.

[0004] Existing research suggests that NAT9 is associated with various disease states, including acquired color blindness and psoriasis, indicating its potential involvement in cellular stress responses, metabolic homeostasis regulation, and immune-related processes. Although the literature reports on the fundamental functions of NAT9 in cellular metabolic regulation and signaling pathways, its role in tumors, particularly hepatocellular carcinoma, remains unclear. Currently, there is no research or application regarding NAT9's involvement in the development and progression of liver cancer or its use as a therapeutic target; therefore, further exploration in this area has significant scientific importance and potential clinical value.

[0005] Therefore, it is necessary to develop a target and drugs for screening drugs to prevent, alleviate and / or treat liver cancer. Summary of the Invention

[0006] The purpose of this invention is to provide the application of NAT9 expression inhibitors in the preparation of drugs for the prevention, alleviation, and / or treatment of liver cancer. It provides a novel target and effective intervention strategy for the treatment of liver cancer, and has significant clinical development prospects and application value.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect of the invention, the use of the NAT9 gene as a target gene in screening drugs for the prevention, relief and / or treatment of liver cancer is provided, wherein the screening method includes screening for substances that can inhibit NAT9 expression.

[0008] In a second aspect of the invention, the use of NAT9 expression inhibitors in the preparation of medicaments for the prevention, relief and / or treatment of liver cancer is provided.

[0009] Furthermore, the NAT9 expression inhibitor can suppress the transcriptional expression of the NAT9 gene or inhibit the biological function of the NAT9 protein.

[0010] Furthermore, the NAT9 expression inhibitor is siRNA, shRNA, antisense nucleic acid, ribozyme, or an antibody or antigen-binding fragment thereof that specifically binds to and inhibits the function of the NAT9 protein.

[0011] Furthermore, the sequence of the siRNA is shown in SEQ ID NO.1-2.

[0012] In a third aspect of the invention, a medicament for preventing, alleviating and / or treating liver cancer is provided, characterized in that the medicament comprises a NAT9 expression inhibitor.

[0013] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0014] Furthermore, the excipients are selected from one of the following: fillers, disintegrants, binders, lubricants, sweeteners, or colorants.

[0015] Furthermore, the dosage form of the drug includes one of granules, tablets, pills, capsules, injections, and dispersants.

[0016] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages: 1. Innovation and Clinical Relevance of Target Discovery: This invention, for the first time, through large-scale bioinformatics analysis (covering 15 public datasets and 3993 samples) combined with clinical sample validation, confirms that NAT9 is specifically highly expressed in hepatocellular carcinoma (HCC) tissues, and that its high expression is significantly correlated with poor overall survival (OS), progression-free survival (PFS), disease-free survival (DFS), and disease-specific survival (DSS). This discovery not only reveals that NAT9 is a key factor driving HCC progression but also establishes it as a novel therapeutic target for HCC with significant prognostic value, overcoming the technological gap in the prior art regarding the unknown role of NAT9 in HCC.

[0017] 2. Effectiveness and Multidimensional Inhibitory Effects of the Treatment Strategy: This invention is the first to propose and validate through functional experiments that "inhibiting NAT9" can be an effective strategy for treating liver cancer. Specifically, by specifically downregulating NAT9 expression through siRNA or lentiviruses carrying shRNA, significant anti-cancer effects can be produced in multiple aspects in in vitro models: (1) Significantly inhibits cell proliferation: The experimental results of CCK-8 and EdU show that inhibiting NAT9 can effectively reduce the activity of liver cancer cells and significantly inhibit their DNA synthesis, thereby curbing tumor growth.

[0018] (2) Effectively inhibiting colony formation: Colony formation experiments have shown that inhibiting NAT9 can significantly weaken the ability of liver cancer cells to form colonies, indicating that it has a profound impact on the long-term proliferation and self-renewal ability of tumor cells.

[0019] (3) Strong inhibition of migration and invasion: The scratch healing experiment and the Transwell invasion experiment jointly confirmed that inhibiting NAT9 can significantly reduce the migration ability and invasion ability of liver cancer cells across the matrix membrane, which is crucial for inhibiting liver cancer metastasis.

[0020] 3. Broad Application Prospects: This invention not only provides the theoretical basis for NAT9 as a target, but also offers specific technical means to achieve intervention at this target (such as specific siRNA and shRNA lentiviruses) and a complete scheme for developing it into drugs (multiple dosage forms). These technical solutions have clear effects and strong operability, laying a solid foundation for the development of a new class of anti-liver cancer drugs with a clear mechanism of action, and have good prospects for clinical translation and market application potential. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This is a graph showing the results of analyzing the differences in NAT9 expression in liver cancer tissue, adjacent non-cancerous tissue, cirrhotic tissue, and normal liver tissue using multiple publicly available datasets (including GSE22058, GSE25097, GSE36376, GSE14520, GSE10143, GSE54236, GSE63898, TCGA-LIHC, ICGC-LIRI-JP, GSE87630, GSE76427, OEP000321, GSE121248, GSE89377, and GSE148355) in Embodiment 1 of the present invention.

[0023] Figure 2 Example 1 of this invention analyzes the relationship between NAT9 expression levels and patient survival outcomes based on the TCGA-LIHC and GSE54236 datasets, including overall survival (OS), progression-free survival (PFS), disease-free survival (DFS), and disease-specific survival (DSS).

[0024] Figure 3 The figure shows the results of detecting the expression level of NAT9 protein in liver cancer tissue and corresponding adjacent tissue of 6 liver cancer patients in Example 2 of this invention using the Western Blot method.

[0025] Figure 4 The figure shows the experimental results of using Western blot to verify the effect of the NAT9 expression inhibitor on the NAT9 protein expression level in liver cancer cells in Example 3 of this invention.

[0026] Figure 5 The figure shows the experimental results of the effect of NAT9 expression inhibitor on the proliferation ability of liver cancer cells in Example 4 of the present invention. The left figure shows the experimental results of CCK-8, and the right figure shows the experimental results of EdU probe labeling.

[0027] Figure 6 This is a graph showing the experimental results of the effect of NAT9 expression inhibitor on the clonogenic ability of liver cancer cells in Example 5 of the present invention.

[0028] Figure 7 This is a graph showing the experimental results of the effect of NAT9 expression inhibitor on the invasive ability of liver cancer cells in Example 6 of the present invention.

[0029] Figure 8 This is a graph showing the experimental results of the effect of NAT9 expression inhibitor on the migration ability of liver cancer cells in Example 4 of the present invention. Detailed Implementation

[0030] The present invention will be described in detail below with reference to specific embodiments and examples, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific embodiments and examples are for illustrative purposes only and are not intended to limit the present invention.

[0031] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict, this specification shall prevail.

[0032] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be obtained by purchasing them from the market or by existing methods.

[0033] The application of the NAT9 gene in this application in the preparation of drugs for the prevention and / or treatment of liver cancer will be described in detail below with reference to embodiments and experimental data.

[0034] Example 1: Bioinformatics Analysis of NAT9 Expression and Prognostic Value in Hepatocellular Carcinoma 1. Objective To systematically analyze the expression differences of NAT9 mRNA in hepatocellular carcinoma (HCC) tissues, adjacent normal tissues, cirrhotic tissues, and normal liver tissues using public gene expression databases, and to evaluate the correlation between NAT9 expression levels and clinical prognosis of HCC patients.

[0035] 2. Methods 2.1 Data Collection: Fifteen liver cancer-related datasets were downloaded from the Gene Expression Omnibus (GEO), The Cancer Genome Atlas (TCGA), and International Cancer Genome Consortium (ICGC) databases, including GSE22058, GSE25097, GSE36376, GSE14520, GSE10143, GSE54236, GSE63898, TCGA-LIHC, ICGC-LIRI-JP, GSE87630, GSE76427, OEP000321, GSE121248, GSE89377, and GSE148355, totaling 3993 samples.

[0036] 2.2 Data Standardization: (1) Microarray data: Download the original CEL file, use the R language affy package to perform RMA algorithm standardization, and obtain the expression value after log2 transformation. For multiple probes of the same gene, take the median as the expression value of the gene.

[0037] (2) RNA-seq data: The normalized read counts data provided by the TCGA-LIHC and ICGC-LIRI-JP projects were used directly and transformed by log2(x+1).

[0038] 2.3 Differential Expression Analysis: The expression levels of NAT9 in hepatocellular carcinoma (HCC) tissues and adjacent / normal liver tissues were compared using the limma or DESeq2 packages in R. A statistically significant difference was defined as log2(Fold Change) > 1 and an adjusted P-value (adj. P. Val) < 0.05.

[0039] 2.4 Survival Analysis: In the TCGA-LIHC and GSE54236 datasets, which contain complete survival information, patients were divided into high-expression and low-expression groups using the median NAT9 expression level as the cutoff value. Survival curves for overall survival (OS), progression-free survival (PFS), disease-free survival (DFS), and disease-specific survival (DSS) were plotted using the Kaplan-Meier method. The Log-rank test was used to compare survival differences between groups, with P < 0.05 considered statistically significant.

[0040] 3. Results like Figure 1As shown, in all 15 included datasets, the mRNA expression level of NAT9 in hepatocellular carcinoma tissues was significantly higher than that in the corresponding adjacent normal tissues, cirrhotic tissues, and normal liver tissues (P<0.05).

[0041] like Figure 2 As shown, in the TCGA-LIHC and GSE54236 datasets, the OS, PFS, DFS and DSS of patients in the NAT9 high expression group were significantly shorter than those in the low expression group (Log-rank P<0.05).

[0042] 4. Conclusion NAT9 is specifically highly expressed in liver cancer tissues, and its high expression is an independent risk factor for the prognosis of liver cancer patients, suggesting that NAT9 may serve as a potential diagnostic biomarker and therapeutic target for liver cancer.

[0043] Example 2: Western blot analysis was used to analyze the expression differences of NAT9 in hepatocellular carcinoma tissues and adjacent normal tissues, and the inhibitory efficiency of NAT9 in HLF cell lines was evaluated. 1. Purpose To validate the expression of NAT9 in clinical liver cancer samples at the protein level.

[0044] 2. Methods 2.1 Sample Source: Cancer tissue and paired adjacent tissue (>5cm from the cancer margin) were collected from 6 patients with pathologically confirmed hepatocellular carcinoma. None of the patients had received radiotherapy or chemotherapy before surgery. Sample acquisition was approved by the ethics committee and with the informed consent of the patients.

[0045] 2.2 Preparation of protein extraction solution Prepare the protein extraction buffer according to the following ratio: RIPA lysis buffer: 1 mL; 50× protease inhibitor: 20 μL; 100× phosphatase inhibitor A: 10 μL; 100× phosphatase inhibitor B: 10 μL; 100× PMSF: 10 μL. Mix well and use at low temperature.

[0046] 2.3 Methods for extracting cell proteins Discard the culture medium and wash with PBS; add protein extraction buffer to lyse cells for 5 min and collect with a scraper; then sonicate to disrupt the cells, and incubate the lysate on ice for 10 min. Centrifuge at 12,000 rpm and 4°C for 10 min, and collect the supernatant as total cell protein.

[0047] 2.4 Methods for extracting tissue proteins Take 30-50 mg of tissue and add N×10 μL of protein extraction buffer according to tissue mass (N, mg); add grinding beads and perform mechanical grinding (e.g., 120s, 70Hz). After lysis, incubate on ice for 15 min, and centrifuge at 12,000 rpm, 4℃ for 10 min. Collect the supernatant as total tissue protein.

[0048] 2.5 Denaturation and Preservation of Protein Samples Mix the protein supernatant with 5× loading buffer at a ratio of 4:1, heat at 100℃ for 5 min to denature the protein; after cooling to room temperature, it can be used directly for electrophoresis or for cryopreservation.

[0049] 2.6 Western Blot Experimental Method Place the pre-prepared SDS-PAGE gel into the electrophoresis tank and add electrophoresis buffer; add protein samples and pre-stained markers to the upper sample wells. Electrophoresis is performed at 80 V, and after the samples enter the separating gel, the voltage is adjusted to 120 V. Electrophoresis is stopped when the bromophenol blue reaches near the bottom of the gel.

[0050] The PVDF membrane was cut and activated with methanol before being placed in transfer buffer. The gel and PVDF membrane were assembled in a sponge-filter paper-gel-membrane-filter paper-sponge configuration and placed in a wet transfer apparatus. The membrane was transferred at 4°C and 200mA for 60 min.

[0051] After transfer, the membrane was washed with TBST and blocked with TBST containing 10% skim milk powder for 2 hours. Then, diluted primary antibody was added and incubated overnight at 4°C (NAT9, Abclonal, A7204; GAPDH, proteintech, 60004-1-Ig). After washing, HRP-labeled secondary antibody was added and incubated at room temperature for 1 hour, followed by another wash.

[0052] The membrane was placed in an ECL chemiluminescent substrate for color development and then exposed in an imaging system to acquire a strip image.

[0053] 3. Results like Figure 3 As shown, in the paired samples of 6 patients, the gray value of the NAT9 protein band in cancer tissue (T) was significantly higher than that in the corresponding adjacent normal tissue (N). After calibration with the internal reference GAPDH, the difference was statistically significant (P<0.01). 4. Conclusion The embodiments of the present invention confirmed at the protein level that NAT9 is highly expressed in liver cancer tissues, consistent with the results at the mRNA level.

[0054] Example 3: Construction of NAT9-targeting shRNA lentiviral vector and establishment of stable knockdown cell lines 1. Purpose We constructed a lentiviral vector that could stably knock down the NAT9 gene and established a human hepatocellular carcinoma (HLF) cell model with downregulated NAT9 expression.

[0055] 2. Methods 2.1 shRNA sequence design and synthesis: A specific shRNA interference fragment was designed targeting the human NAT9 (Gene ID: 26151) mRNA sequence. Its template sequence is as follows: Chain of Justice (SEQ ID NO.1): 5'-GATGTTCCAGAAACTTCACTTCTCGAGAAGTGAAGTTTCTGGAACATC-3' Antisense chain (SEQ ID NO.2): 3'-CTACAAGGTCTTTGAAGTGAAGAGCTCTTCACTTCAAAGACCTTGTAG-5'.

[0056] 2.2 Lentiviral vector construction: After annealing the synthesized double-stranded oligonucleotides to form double strands, they were cloned into the pLV2-U6-EGFP-Puro lentiviral vector, which was double-digested with AgeI and EcoRI, to construct the recombinant plasmid pLV2-U6-shNAT9-EGFP-Puro. The correctness of the inserted sequence was verified by colony PCR and sequencing.

[0057] 2.3 Lentiviral Packaging: The virus was packaged using a three-plasmid system. Healthy 293T cells were seeded in 10 cm culture dishes. When the cell density reached 70%-80%, the following plasmids were co-transfected using liposome transfection: 8 μg of pLV2-U6-shNAT9-EGFP-Puro (or the empty control plasmid pLV2-U6-EGFP-Puro), 6 μg of the packaging plasmid psPAX2, and 2 μg of the envelope plasmid pMD2.G. Six hours after transfection, the medium was replaced with complete medium (DMEM + 10% FBS). The virus-containing supernatant was collected after 48 and 72 hours of culture, filtered through a 0.45 μm filter, concentrated by ultracentrifugation, and stored at -80°C.

[0058] 2.4 Cell infection and screening: Human hepatocellular carcinoma (HLC-HF) cells were seeded in 6-well plates (5 × 10⁵ cells / well) and cultured to a density of approximately 30%. Viral supernatant (MOI = 10) and polybrene at a final concentration of 5 μg / mL were added. After 24 h of infection, the medium was replaced with fresh complete medium and cultured for another 48 h. Puromycin at a final concentration of 2 μg / mL was then added for selection, continuing for approximately one week until all untransfected control cells died. Monoclonal cell lines were obtained using limiting dilution, and EGFP green fluorescence expression was observed using fluorescence microscopy to assess infection efficiency. Finally, a stably knocked-down NAT9 HLF-shNAT9 cell line and an empty vector control HLF-NC cell line were obtained.

[0059] 2.5 Knockout efficiency verification: Total protein was extracted from HLF-shNAT9 and HLF-NC cells according to the method in Example 2, and Western blotting was performed to verify the knockdown effect of NAT9 protein.

[0060] 3. Results like Figure 4 As shown, compared with the HLF-NC group, the expression level of NAT9 protein in HLF-shNAT9 cells was significantly inhibited, with a knockdown efficiency of over 70%.

[0061] 4. Conclusion This invention successfully constructed a shRNA lentivirus targeting NAT9 and established a stable NAT9 knockdown liver cancer cell model, providing a reliable tool for subsequent functional experiments.

[0062] Example 4: The ability of NAT9 expression inhibitors to suppress the proliferation of liver cancer cells 1. Purpose To assess the effect of downregulating NAT9 expression on the proliferation ability of hepatocellular carcinoma cells.

[0063] 2. Methods 2.1 CCK-8 Experiment: HLF-NC and HLF-shNAT9 cells in logarithmic growth phase were digested and resuspended, and seeded at a density of 3000 cells per well in 96-well plates, with 5 replicates per group. At 24 h, 48 h, and 72 h post-seeding, 10 μL of CCK-8 reagent (Dojin Chemical, Japan) was added to each well, and the cells were incubated at 37°C for 2 h. The absorbance (OD value) of each well was measured using a microplate reader at 450 nm. Cell proliferation curves were plotted with time on the x-axis and OD value on the y-axis.

[0064] 2.2 EdU cell proliferation detection: Cells were seeded in 24-well plates and cultured to approximately 50% density. Following the BeyoClick™ EdU-555 kit instructions, medium containing 10 μM EdU was added to each well and incubated for 2 h. The medium was discarded, and cells were washed with PBS, fixed with 4% paraformaldehyde for 15 min, and permeabilized with 0.3% Triton X-100 for 15 min. Click reaction solution was added and incubated (in the dark), followed by Hoechst 33342 staining of the cell nuclei. After washing with PBS, cells were observed under a fluorescence microscope, and multiple fields of view were randomly photographed. The percentage of EdU-positive cells (red fluorescence) out of all Hoechst-positive cells (blue fluorescence) was counted.

[0065] 3. Results like Figure 5 As shown in the left figure, the CCK-8 experiment results showed that at 48 h and 72 h time points, the OD values ​​of the HLF-shNAT9 group were significantly lower than those of the HLF-NC group (P<0.01), indicating that NAT9 knockdown significantly inhibited cell proliferation.

[0066] like Figure 5 As shown in the right figure, the EdU experiment results showed that the EdU-positive cell rate in the HLF-shNAT9 group was significantly lower than that in the HLF-NC group (P<0.001), further confirming that NAT9 knockdown can inhibit the DNA replication activity of liver cancer cells.

[0067] 4. Conclusion The embodiments of the present invention demonstrate that knocking down NAT9 can significantly inhibit the in vitro proliferation ability of liver cancer cells.

[0068] Example 5: NAT9 expression inhibitors weaken the ability of hepatocellular carcinoma cells to form clones. 1. Purpose To assess the effect of downregulating NAT9 expression on the monoclonal formation ability of hepatocellular carcinoma cells.

[0069] 2. Methods HLF-NC and HLF-shNAT9 cells were digested to prepare single-cell suspensions and counted. They were seeded at a low density (500 cells / well) in 6-well plates, with 3 replicates per group. The plates were incubated statically at 37°C with 5% CO2, with the medium replaced every 3-4 days. Culture was stopped after approximately 10-14 days when cell colonies were visibly formed. The medium was discarded, and the cells were carefully washed with PBS, fixed with 4% paraformaldehyde for 15 min, and stained with 0.1% crystal violet solution for 20 min. Residual staining was slowly washed away with running water, and the cells were air-dried at room temperature. Cells were scanned and photographed, and the number of cell colonies larger than 50 μm in diameter was counted.

[0070] 3. Results like Figure 6 As shown, the number of cell clones formed in the HLF-shNAT9 group was significantly less than that in the HLF-NC group (P<0.001).

[0071] 4. Conclusion The embodiments of the present invention demonstrate that knocking down NAT9 can significantly weaken the long-term proliferation and self-renewal ability of liver cancer cells.

[0072] Example 6: NAT9 expression inhibitors reduce the invasive ability of liver cancer cells. 1. Purpose To assess the effect of downregulating NAT9 expression on the invasive ability of hepatocellular carcinoma cells.

[0073] 2. Methods (Transwell invasion assay) Thaw the Transwell chamber (8 μm pore size, Corning) Matrigel (BD Biosciences) overnight at 4°C, and dilute it 1:8 with serum-free, pre-chilled DMEM medium. Spread 100 μL of the diluted Matrigel evenly onto the upper chamber membrane surface and incubate at 37°C for 4-5 h to solidify. Resuspend HLF-NC and HLF-shNAT9 cells in serum-free medium and adjust the cell density to 5 × 10⁶ cells / year. 5 / mL. Add 100 μL of cell suspension to the upper chamber and 600 μL of culture medium containing 20% ​​FBS as a chemical inducer to the lower chamber. After incubation at 37°C for 36 h, remove the chamber and gently wipe away the cells and matrix gel in the upper chamber with a cotton swab. Fix the chamber membrane with 4% paraformaldehyde for 15 min and stain with 0.1% crystal violet for 20 min. After washing with PBS, randomly select 5 fields of view under a microscope and count the number of cells that have passed through the matrix gel to reach the lower chamber surface.

[0074] The results are as follows Figure 7 As shown, the number of cells invading the lower chamber in the HLF-shNAT9 group was significantly less than that in the HLF-NC group (P<0.001). 4. Conclusion The embodiments of the present invention demonstrate that knocking down NAT9 can significantly inhibit the in vitro invasive ability of liver cancer cells.

[0075] Example 7: NAT9 expression inhibitors reduce the migration ability of liver cancer cells. 1. Purpose To assess the effect of downregulating NAT9 expression on the migration ability of hepatocellular carcinoma cells.

[0076] 2. Method (Scratch Healing Experiment) HLF-NC and HLF-shNAT9 cells were seeded at high density in 6-well plates and cultured until cell confluence reached over 90%. Using a 200 μL sterile pipette tip, three straight scratches were made perpendicular to the bottom of the wells on each cell monolayer. The cells were gently washed 2-3 times with PBS to remove cell debris. The medium was then replaced with serum-free medium to inhibit cell proliferation. The scratches at 0 h were photographed and marked under a microscope (100x). After culturing the plates for another 48 h, the same locations were photographed again. The width of the scratches was measured using ImageJ software, and the scratch healing rate was calculated: Healing rate (%) = [(0 h scratch width - 48 h scratch width) / 0 h scratch width] × 100%.

[0077] 3. Results like Figure 8 As shown, after 48 h of culture, the scratch healing rate of the HLF-shNAT9 group was significantly lower than that of the HLF-NC group (P<0.01). 4. Conclusion The embodiments of the present invention demonstrate that knocking down NAT9 can significantly inhibit the in vitro migration ability of liver cancer cells.

[0078] Example 8: Formulation of the pharmaceutical composition The pharmaceutical compositions of the present invention can be prepared into various dosage forms according to clinical needs. Several exemplary formulations are listed below, but the scope of the invention is not limited thereto.

[0079] 1. Injectable: Take the lentivirus particles prepared in Example 3 (titer ≥ 1 × 10⁻⁶) 8 Add 1 mL of IFU / mL to PBS buffer (pH 7.4) containing 5% sucrose and 1% human serum albumin to a final volume of 10 mL. Filter the solution aseptically through a 0.22 μm microporous membrane, dispense into vials, and freeze to obtain the injection.

[0080] 2. Lyophilized powder for injection: The above-mentioned injection solution was dispensed into 1 mL vials, placed in a freeze dryer, and pre-frozen, dried once, dried twice, and then capped and sealed to prepare lyophilized powder for injection. It was reconstituted with physiological saline before use.

[0081] 3. Lipid nanoparticle (LNP) formulations: The siRNA (SEQ ID NO.1-2) described in Example 5 was mixed with ionizable lipids, phospholipids, cholesterol and PEG-lipids in a certain proportion, and LNP formulations loaded with siRNA were prepared using microfluidic technology. These formulations can be used for intravenous injection to improve in vivo delivery efficiency.

[0082] The above examples fully demonstrate that targeting NAT9 and downregulating its expression through inhibitors (such as shRNA lentivirus and siRNA) can effectively inhibit the malignant biological behavior of liver cancer cells, exhibiting significant anti-liver cancer effects and broad prospects for clinical application.

[0083] Finally, it should be noted that the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0084] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0085] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. Use of the NAT9 gene as a target gene in screening of drugs for preventing, alleviating and / or treating liver cancer, characterized in that, The screening method comprises screening a substance capable of inhibiting expression of NAT9.

2. Use of a NAT9 expression inhibitor in the preparation of a medicament for preventing, alleviating and / or treating liver cancer.

3. Use according to claim 2, characterized in that, The NAT9 expression inhibitor can inhibit transcriptional expression of the NAT9 gene or inhibit the biological function of the NAT9 protein.

4. Use according to claim 3, characterized in that, The NAT9 expression inhibitor is siRNA, shRNA, antisense nucleic acid, ribozyme, or antibody or antigen-binding fragment thereof that specifically binds to and inhibits the function of the NAT9 protein.

5. Use according to claim 4, characterized in that, The sequence of the siRNA is shown in SEQ ID NO. 1-2.

6. Use according to claim 3, characterized in that, The NAT9 expression inhibitor is a lentivirus capable of stably expressing a NAT9 RNA interference fragment.

7. A medicament for preventing, alleviating and / or treating liver cancer, characterized by, The medicament comprises a NAT9 expression inhibitor.

8. The medicament according to claim 7, characterized in that, The medicament further comprises a pharmaceutically acceptable excipient.

9. The medicament according to claim 8, characterized in that, The pharmaceutically acceptable excipient is selected from one of a filler, a disintegrant, a binder, a lubricant, a sweetener or a colorant.

10. The medicament according to claim 7, characterized in that, The dosage form of the medicament comprises one of a tablet, a powder, a granule, a capsule, an oral solution, an injection or a sustained-release preparation.

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