Use of a cnot9 expression inhibitor in the preparation of a medicament for preventing, alleviating and / or treating liver cancer

Abstract

The application provides an application of a CNOT9 expression inhibitor in preparation of a medicine for preventing, alleviating and / or treating liver cancer. It is found for the first time that the mRNA and protein expression levels of CNOT9 in hepatocellular carcinoma (HCC) tissues are significantly higher than those in paracancerous and normal liver tissues, and the high expression is closely related to the prognosis indexes such as the overall survival and progression-free survival of patients, thereby establishing the value of CNOT9 as a liver cancer diagnosis marker and a new treatment target. Based on this, the application provides an application of a CNOT9 inhibitor in preparation of a medicine for preventing, alleviating and / or treating liver cancer. By down-regulating the expression of CNOT9 through siRNA, the proliferation activity, clonogenicity, migration and invasion ability of liver cancer cells HLF can be effectively inhibited. The application further provides a medicine composition comprising the inhibitor, thereby providing a brand-new target and effective intervention strategy for liver cancer treatment, and having a great clinical development prospect and application value.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the use of CNOT9 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 main type of primary liver cancer, accounting for 70%-85% of all liver cancers. In China, liver cancer ranks among the top malignant tumors in incidence and third in mortality. Currently, early surgical resection is the preferred treatment, but most patients are diagnosed at an advanced stage, losing the opportunity for surgery. The natural course of the disease in advanced patients is only 3-6 months, and existing chemotherapy and palliative treatments have limited effectiveness, resulting in a very poor prognosis. Therefore, developing new therapeutic targets and strategies is an urgent clinical need.

[0003] Current targeted therapies for liver cancer still have limitations, such as insufficient target specificity, high rates of drug resistance, and unsatisfactory inhibitory effects on tumor metastasis. There is an urgent need to discover new molecular targets and develop drugs that can effectively inhibit the progression of liver cancer.

[0004] 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

[0005] The purpose of this invention is to provide the application of CNOT9 expression inhibitors in the preparation of drugs for the prevention, relief and / or treatment of liver cancer, providing a novel target and effective intervention strategy for liver cancer treatment, with significant clinical development prospects and application value.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect of the invention, the use of the CNOT9 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 CNOT9 expression.

[0007] In a second aspect of the invention, the use of a CNOT9 expression inhibitor in the preparation of a medicament for the prevention, relief and / or treatment of liver cancer is provided.

[0008] Furthermore, the CNOT9 expression inhibitor can inhibit the transcriptional expression of the CNOT9 gene or inhibit the biological function of the CNOT9 protein.

[0009] Furthermore, the CNOT9 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 CNOT9 protein.

[0010] Furthermore, the sequence of the siRNA is shown in SEQ ID NO.1-2.

[0011] 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 CNOT9 expression inhibitor.

[0012] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0013] Furthermore, the excipients are selected from one of the following: fillers, disintegrants, binders, lubricants, sweeteners, or colorants.

[0014] Furthermore, the dosage form of the drug includes one of granules, tablets, pills, capsules, injections, and dispersants.

[0015] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages: This invention demonstrates significant effects in the treatment of hepatocellular carcinoma by inhibiting the expression of specific targets: (1) Significantly inhibits malignant tumor proliferation: In vitro experiments have confirmed that target downregulation can effectively reduce the activity of liver cancer cells, inhibit DNA synthesis and clone formation, thereby curbing tumor growth and self-renewal ability.

[0016] (2) Effectively blocks the metastasis process: Target inhibition can significantly weaken the migration and invasion ability of liver cancer cells, which plays a key role in preventing tumor spread and metastasis.

[0017] (3) Outstanding clinical translation potential: This invention provides specific inhibitor regimens (such as siRNA, lentivirus) and various drug formulations, laying a solid foundation for the development of novel anti-liver cancer drugs and having clear clinical application prospects. Attached Figure Description

[0018] 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.

[0019] Figure 1This is a graph showing the results of analyzing the differences in CNOT9 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.

[0020] Figure 2 Example 1 of this invention analyzes the relationship between CNOT9 expression level and patient survival outcomes based on the TCGA-LIHC and GSE76427 datasets, including overall survival (OS), progression-free survival (PFS), and disease-specific survival (DSS).

[0021] Figure 3 The figure shows the results of detecting the expression level of CNOT9 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.

[0022] Figure 4 The figure shows the experimental results of using Western blot to verify the effect of the CNOT9 expression inhibitor on the CNOT9 protein expression level in liver cancer cells in Example 3 of this invention.

[0023] Figure 5 This is a graph showing the experimental results of the effect of CNOT9 expression inhibitor on the migration ability of liver cancer cells in Example 4 of the present invention.

[0024] Figure 6 This is a graph showing the experimental results of the effect of CNOT9 expression inhibitor on the invasive ability of liver cancer cells in Example 5 of the present invention.

[0025] Figure 7 This is a graph showing the experimental results of the effect of CNOT9 expression inhibitor on the clonogenic ability of liver cancer cells in Example 6 of the present invention.

[0026] Figure 8 The figure shows the experimental results of the effect of CNOT9 expression inhibitor on the proliferation ability of liver cancer cells in Example 7 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. Detailed Implementation

[0027] 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.

[0028] 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.

[0029] 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.

[0030] The application of the CNOT9 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.

[0031] Example 1: Bioinformatics Analysis of CNOT9 Expression and Prognostic Value in Hepatocellular Carcinoma 1. Experimental Objective Using public gene expression databases, we systematically analyzed the expression differences of CNOT9 mRNA in hepatocellular carcinoma (HCC) tissues, adjacent normal tissues, cirrhotic tissues, and normal liver tissues, and assessed the correlation between CNOT9 expression levels and clinical prognosis of HCC patients.

[0032] 2. Experimental Methods 2.1 Data Collection: Fifteen liver cancer-related datasets were downloaded from the Gene Expression Omnibus (GEO), The Cancer Genome Atlas (TCGA), and the International Cancer Genome Consortium (ICGC) databases, including GSE22058, GSE25097, GSE36376, GSE14520, GSE10143, GSE54236, GSE63898, TCGA-LIHC, ICGC-LIRI-JP, GSE87630, and GSE76427. OEP000321, GSE121248, GSE89377, GSE148355, totaling 3993 samples.

[0033] 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.

[0034] (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).

[0035] 2.3 Differential Expression Analysis: The expression levels of CNOT9 in hepatocellular carcinoma tissues and adjacent / normal liver tissues were compared using the limma or DESeq2 packages in R. The statistical significance was defined as log2(Fold Change) > 1 and adjusted P-value (adj. P. Val) < 0.05.

[0036] 2.4 Survival Analysis: In the TCGA-LIHC and GSE76427 datasets, which contain complete survival information, patients were divided into high-expression and low-expression groups using the median CNOT9 expression level as the cutoff value. Kaplan-Meier survival curves for overall survival (OS), progression-free survival (PFS), and disease-specific survival (DSS) were plotted, and the Log-rank test was used to compare survival differences between groups; P < 0.05 was considered statistically significant.

[0037] 3. Experimental Results like Figure 1 As shown, in all 15 datasets included, the mRNA expression level of CNOT9 in hepatocellular carcinoma tissues was significantly higher than that in the corresponding adjacent normal tissues, cirrhotic tissues, and normal liver tissues (P<0.05).

[0038] like Figure 2 As shown, in the TCGA-LIHC and GSE76427 datasets, the OS, PFS, and DSS of patients in the CNOT9 high expression group were significantly shorter than those in the low expression group (Log-rank P<0.05).

[0039] 4. Conclusion CNOT9 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 CNOT9 may serve as a potential diagnostic biomarker and therapeutic target for liver cancer.

[0040] Example 2: Western blot analysis of CNOT9 expression differences between hepatocellular carcinoma (HCC) tissues and adjacent normal tissues, and evaluation of CNOT9 inhibition efficiency in HLF cell lines. 1. Experimental Objective To validate the expression of CNOT9 in clinical liver cancer samples at the protein level.

[0041] 2. Experimental 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.

[0042] 2.2 Preparation of protein extract 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.

[0043] 2.3 Methods for extracting cellular proteins Discard the culture medium and wash with PBS; add protein extraction buffer to lyse the 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] The PVDF membrane was cut and activated with methanol before being placed in transfer buffer. The gel and PVDF membrane were assembled in the order of sponge-filter paper-gel-membrane-filter paper-sponge and placed in a wet transfer apparatus. The membrane was transferred at 4℃ and 200mA for 60 min.

[0048] 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 (CNOT9, Proteintech, 22503-1-AP; 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.

[0049] The membrane was placed in an ECL chemiluminescent substrate for color development and then exposed in an imaging system to acquire a strip image.

[0050] 3. Experimental Results As shown in Figure 3, in the paired samples of 6 patients, the gray value of the CNOT9 protein band in cancerous 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).

[0051] 4. Conclusion At the protein level, CNOT9 was confirmed to be highly expressed in liver cancer tissues, consistent with the results at the mRNA level.

[0052] Example 3: Construction of CNOT9-targeting shRNA lentiviral vector and establishment of stable knockdown cell lines 1. Experimental Objective We constructed a lentiviral vector that could stably knock down the CNOT9 gene and established a human hepatocellular carcinoma (HLF) cell model with downregulated CNOT9 expression.

[0053] 2. Experimental Methods 2.1 shRNA sequence design and synthesis: A specific shRNA interference fragment was designed targeting the human CNOT9 (Gene ID: 9125) mRNA sequence. Its template sequence is as follows: Chain of Justice (SEQ ID NO.1): 5'- GAAGCATGTAGTGAGATGTTAttcaagagaTAACATCTCACTACATGCTTC-3' Antisense chain (SEQ ID NO.2): 3'-CTTCGTACATCACTCTACAATaagttctctATTGTAGAGTGATGTACGAAG-5'.

[0054] 2.2 Lentiviral vector construction: After annealing the synthesized double-stranded oligonucleotides to form double strands, they were cloned into the pLV3-U6-CopGFP-Puro lentiviral vector, which was double-digested with AgeI and EcoRI, to construct the recombinant plasmid. pLV3-U6-CNOT9(human)-shRNA1-CopGFP-Puro. The correctness of the inserted sequence was verified by colony PCR and sequencing.

[0055] 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 pLV3-U6-CNOT9(human)-shRNA1-CopGFP-Puro (or empty control plasmid pLV3-U6-CopGFP-Puro), 6 μg of packaging plasmid psPAX2, and 2 μg of envelope plasmid pMD2.G. After 6 h of transfection, the medium was replaced with complete medium (DMEM + 10% FBS). The virus-containing supernatant was collected after 48 h and 72 h of culture, filtered through a 0.45 μm filter, concentrated by ultracentrifugation, and stored at -80°C.

[0056] 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 stable CNOT9 knockdown HLF-shCNOT9 cell line and an empty vector control HLF-NC cell line were obtained.

[0057] 2.5 Knockout efficiency verification: Total protein was extracted from HLF-shCNOT9 and HLF-NC cells according to the method in Example 2, and Western blotting was performed to verify the knockdown effect of CNOT9 protein.

[0058] 3. Results like Figure 4As shown, compared with the HLF-NC group, the expression level of CNOT9 protein in HLF-shCNOT9 cells was significantly inhibited.

[0059] 4. Conclusion This invention successfully constructed a CNOT9-targeting shRNA lentivirus and established a stable CNOT9 knockdown liver cancer cell model, providing a reliable tool for subsequent functional experiments.

[0060] Example 4: CNOT9 expression inhibitors reduce the migration ability of liver cancer cells. 1. Experimental Objective To assess the effect of downregulating CNOT9 expression on the migration ability of hepatocellular carcinoma cells.

[0061] 2. Experimental Method (Scratch Healing Test) HLF-NC and HLF-shCNOT9 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 the 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 location was 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%.

[0062] 3. Results like Figure 8 As shown, after 48 h of culture, the scratch healing rate of the HLF-shCNOT9 group was significantly lower than that of the HLF-NC group (P<0.01).

[0063] 4. Conclusion The embodiments of the present invention demonstrate that knocking down CNOT9 can significantly inhibit the in vitro migration ability of liver cancer cells.

[0064] Example 5: CNOT9 expression inhibitors reduce the invasive ability of liver cancer cells. 1. Experimental Objective To assess the effect of downregulating CNOT9 expression on the invasive ability of hepatocellular carcinoma cells.

[0065] 2. Experimental Methods (Transwell Invasion Test) 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-shCNOT9 cells in serum-free medium and adjust the cell density to 5 × 10⁶ cells / mL. 5 / mL. Add 100 μL of cell suspension to the upper chamber and 600 μL of culture medium containing 20% ​​FBS to the lower chamber as a chemical inducer. 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.

[0066] 3. Experimental Results like Figure 7 As shown, the number of cells invading the lower chamber in the HLF-shCNOT9 group was significantly less than that in the HLF-NC group (P<0.05). 0.001).

[0067] 4. Conclusion The embodiments of the present invention demonstrate that knocking down CNOT9 can significantly inhibit the in vitro invasive ability of liver cancer cells.

[0068] Example 6: CNOT9 expression inhibitors weaken the ability of hepatocellular carcinoma cells to form colonies 1. Experimental Objective To assess the effect of downregulating CNOT9 expression on the monoclonal formation ability of hepatocellular carcinoma cells.

[0069] 2. Experimental Methods HLF-NC and HLF-shCNOT9 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 6As shown, the number of cell clones formed in the HLF-shCNOT9 group was significantly less than that in the HLF-NC group (P<0.05). 0.001).

[0071] 4. Conclusion The embodiments of the present invention demonstrate that knocking down CNOT9 can significantly weaken the long-term proliferation and self-renewal ability of liver cancer cells.

[0072] Example 7: CNOT9 expression inhibitors suppress the proliferation of liver cancer cells 1. Experimental Objective To assess the effect of downregulating CNOT9 expression on the proliferation ability of hepatocellular carcinoma cells.

[0073] 2. Experimental Methods 2.1 CCK-8 Experiment: HLF-NC and HLF-shCNOT9 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 (Biosharp) 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.

[0074] 2.2 EdU cell proliferation detection: Cells were seeded in 24-well plates and cultured to a density of approximately 50%. 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.

[0075] 3. Experimental 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-shCNOT9 group were significantly lower than those of the HLF-NC group (P<0.01), indicating that CNOT9 knockdown significantly inhibited cell proliferation.

[0076] like Figure 5As shown in the right figure, the EdU experiment results showed that the EdU-positive cell rate in the HLF-shCNOT9 group was significantly lower than that in the HLF-NC group (P<0.001), further confirming that CNOT9 knockdown can inhibit the DNA replication activity of liver cancer cells.

[0077] 4. Conclusion The embodiments of the present invention demonstrate that knocking down CNOT9 can significantly inhibit the in vitro proliferation 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 an LNP formulation encapsulating siRNA was prepared using microfluidic technology. This formulation can be used for intravenous injection to improve in vivo delivery efficiency.

[0082] The above examples fully demonstrate that targeting CNOT9 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. The application prospects of beds are broad.

[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. The application of the CNOT9 gene as a target gene in screening drugs for the prevention, alleviation, and / or treatment of liver cancer, characterized in that, The screening method includes screening for substances that can inhibit CNOT9 expression.

2. Application of CNOT9 expression inhibitors in the preparation of drugs for the prevention, relief and / or treatment of liver cancer.

3. The application according to claim 2, characterized in that, The CNOT9 expression inhibitor can suppress the transcriptional expression of the CNOT9 gene or inhibit the biological function of the CNOT9 protein.

4. The application according to claim 3, characterized in that, The CNOT9 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 CNOT9 protein.

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

6. The application according to claim 3, characterized in that, The CNOT9 expression inhibitor is a lentivirus capable of stably expressing the CNOT9 RNA interference fragment.

7. A drug for preventing, alleviating, and / or treating liver cancer, characterized in that, The drug contains a CNOT9 expression inhibitor.

8. The medicament according to claim 7, characterized in that, The drug also includes pharmaceutically acceptable excipients.

9. The medicament according to claim 8, characterized in that, The pharmaceutically acceptable excipient is selected from one of the following: fillers, disintegrants, binders, lubricants, sweeteners, or colorants.

10. The medicament according to claim 7, characterized in that, The dosage form of the drug includes one of the following: tablets, powders, granules, capsules, oral liquids, injections, or sustained-release formulations.

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