Mutant gene for establishing primary liver cancer mouse model, and use thereof and method therefor

Abstract

Disclosed are a mutant gene for establishing a primary liver cancer mouse model, and the use thereof and a method therefor, wherein the mutant gene is injected via a caudal vein to induce spontaneous liver cancer in situ in mice. Compared with existing models, the method can better simulate the genesis and progression of liver cancer in a human body, is simple to operate and easily obtained, has a high and stable success rate, and has great significance in the exploration of the molecular mechanisms of the genesis and development of liver cancer, the search for new biomarkers, the development of new drugs and targeted drugs, etc.

Description

Mutant gene for establishing primary liver cancer mouse model and its application and method Technical Field

[0001] The present invention relates to the field of animal models, and in particular to a mutant gene for establishing a primary liver cancer mouse model, and further relates to using the mutant gene to establish a primary liver cancer mouse model, and further relates to a method for establishing a primary liver cancer mouse model. Background Art

[0002] Animal models are crucial tools for studying the pathogenesis of human diseases and developing therapeutic drugs. Mice are widely considered one of the most ideal animal models for tumor research due to their high similarity to the human genome, low breeding costs, and ease of genetic modification. Therefore, experimental animal models of liver cancer are essential for exploring the pathogenesis of liver cancer, identifying potential drug targets, and conducting preclinical research.

[0003] Currently, a variety of mouse liver cancer models are used in liver cancer research, including chemically induced models and xenograft models of human liver cancer tissue or cancer cells. However, existing modeling methods suffer from long development cycles, low success rates, and significant genetic modification of the model animals. Chemical induction methods, in particular, are known to be highly toxic, with a success rate of approximately 50%. In chemically induced models, DNA mutations are highly heterogeneous, and the cancer development cycle can last for at least several months or even a year. Commonly used chemical inducers of liver cancer include diethylnitrosamine (DEN), aflatoxin B1 (AFB1), and carbon tetrachloride (CCl4). These agents, administered through diet, oral gavage, gas inhalation, intraperitoneal injection, or subcutaneous injection, induce liver cancer in mice from an early age (day 14 after birth) over a prolonged period. However, chemically induced liver cancer models often exhibit insidious onset and high mortality, resulting in a low success rate. Furthermore, there is considerable heterogeneity in the timing, anatomical location, and number of liver cancer lesions in mice. Xenotransplantation uses irradiation or reduced immune defense, which leads to excessive modification of the animal's immune background and low ability to simulate and reproduce liver cancer diseases. Common transplantable liver cancer models are mainly divided into two types. One is to inoculate human liver cancer cell lines into immunodeficient mice, which is called the CDX (Cell Derived Xenograft) model. The other is to inoculate liver cancer tissue blocks from patients into immunodeficient mice, which is called the PDX (Patient-Derived Xenografts) model. The use of CDX and PDX in immunodeficient mice is based on the construction of tumors on the "immune damage basis". The application of these two model construction methods in the exploration of the pathogenesis of liver cancer and drug development is limited to immune-related. In addition, the CDX and PDX methods require in situ liver transplantation in mice, which has difficulties such as high operational difficulty and high technical requirements, and requires in vivo live labeling.

[0004] Genetically engineered mice are a method for producing model animals with a relatively high success rate, but the previous knock-in or knock-out process was relatively cumbersome, taking several years, and most of them changed the genetic background before the animal developed, making it difficult to simulate the timing and process of liver cancer in adults. For example, during the animal's pregnancy, the liver cancer gene c-MYC and other genes were specifically overexpressed, and tumors formed at 2 months after birth. This method has high modeling costs, is time-consuming, and has uneven tumor formation rates. When evaluating the role of related genes in the occurrence and development of liver cancer, it is necessary to co-cage with mice of related genes and transition from the F0 to the F2 generation before starting the evaluation. This process significantly increases the time and economic cost of the transgenic primary liver cancer mouse model.

[0005] Therefore, there is an urgent need for a method to prepare a mouse liver cancer model with a fast tumor formation time and simple operation, which is of great significance for exploring the molecular mechanism of liver cancer occurrence and development, searching for new biomarkers, and developing new drugs and targeted drugs.

[0006] Summary of the Invention

[0007] In view of this, one of the objects of the present invention is to provide a mutant gene for establishing a primary liver cancer mouse model; a second object of the present invention is to provide a mutant gene combination for establishing a primary liver cancer mouse model; a third object of the present invention is to provide the use of a mutant gene combination combined with an agent that interferes with mouse TP53 expression in establishing a primary liver cancer mouse model; a fourth object of the present invention is to provide a method for establishing a primary liver cancer mouse model.

[0008] In order to achieve the above object, the present invention provides the following technical solutions:

[0009] 1. Mutant genes used to establish a primary liver cancer mouse model, including mutant genes including mouse AKT1.

[0010] 2. A mutant gene combination for establishing a primary liver cancer mouse model, wherein the mutant gene combination includes a mutant gene of the mouse AKT1 gene and a mutant gene of the mouse NRAS gene.

[0011] Preferably, the mutant gene of the mouse AKT1 gene is mouse AKT1 gene c.49G>A; p.E17K; the mutant gene of the mouse NRAS gene is mouse NRAS gene c.35G>A; p.G12D, c.38G>A; p.G13D, c.182A>G; p.Q61R.

[0012] Preferably, in the present invention, the mutant gene sequence of the mouse AKT1 gene is shown as SEQ ID NO.1; the mutant gene sequence of the mouse NRAS gene is shown as SEQ ID NO.12.

[0013] 3. Application of the mutant gene combination combined with reagents that interfere with mouse TP53 expression in establishing a primary liver cancer mouse model.

[0014] Preferably, in the present invention, the reagent interfering with mouse TP53 expression is a pT2-shP53 vector.

[0015] 4. A method for establishing a primary liver cancer mouse model, comprising mixing the mutant gene described in claim 1 or the mutant gene combination described in claim 2 with an agent that interferes with mouse P53 expression and a vector that expresses a transposon enzyme to form a suspension, and injecting the suspension through the tail vein.

[0016] Preferably, the injection dose is injected at a volume-to-mass ratio of 20 μL / g to 100 μL / g.

[0017] Preferably, the vector for expressing the transposon enzyme is a pCMV(CAT)T7-SB100 vector.

[0018] The present invention has the beneficial effects of disclosing mutant genes for establishing a mouse model of primary liver cancer. By injecting the mutant genes into the tail vein, the mice develop spontaneous in situ liver cancer, which better simulates human liver cancer development than existing models for three reasons: First, the model is established after sexual maturity, avoiding the effects of gene transfection on mouse embryonic development. Compared with genetically engineered mice and chemically induced models, it closely mimics the age of human liver cancer development and avoids excessive interference with the model's underlying genetic background. Second, multiple oncogenes can be introduced simultaneously, better simulating the multi-gene mutation characteristic of human liver cancer development. Third, the model is constructed using immune-competent mice, rather than the immunodeficient mice used in transplanted tumor mouse models. This model essentially aligns with the immune progression of liver cancer development and progression in healthy humans. Therefore, this technique for inducing spontaneous in situ liver cancer by tail vein injection of mixed plasmids carrying mutant genes effectively simulates the development and progression of liver cancer, is simple to operate, readily available, and has a high and stable success rate. It is of great significance for exploring the molecular mechanisms of liver cancer development, identifying novel biomarkers, and developing new drugs and targeted therapies.

[0019] Furthermore, the present invention has the advantages of high construction success rate (nearly 100%), short cancer cycle (within 3 weeks), low accidental mortality, and wide applicability to strains (including but not limited to C57BL / 6J, FVB / N, and DBA / 2). BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to make the purpose, technical solutions and beneficial effects of the present invention more clear, the present invention provides the following drawings for illustration:

[0021] Figure 1 shows the abdominal and liver images of the control and model groups of different strains on the 10th and 20th days (yellow arrows represent liver cancer hyperplasia tissue);

[0022] Figure 2 shows the liver pathological sections of the control group and model group of different strains on the 10th and 20th day (yellow arrows represent pathological liver cancer lesions);

[0023] Figure 3 is a survival curve of mice in the control group (Ctrl) and the model group (Pos);

[0024] Figure 4 shows the abdominal circumference statistics of mice in the control group (Ctrl) and the model group (Pos) on the 9th and 21st days;

[0025] Figure 5 shows the abundance of liver cancer markers (tissue RNA sequencing) in mice in the model group (Pos) at different time points after injection;

[0026] Figure 6 shows the abdominal and liver photos of the control and model groups of different strains on the 10th and 20th days (yellow arrows represent liver cancer hyperplasia tissue). DETAILED DESCRIPTION

[0027] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0028] Example 1: Construction of pT3-mAKT1 mut -EGFP plasmid vector

[0029] The sequence shown in SEQ ID NO.1 was chemically synthesized. The sequence reference gene was Mouse AKT1 CCDS26194.1, and the mutations were c.49G>A; p.E17K. The specific sequence is shown in SEQ ID NO.1.

[0030] Using the pL-U6-sgRNA-SFFV-Puro-P2A-EGFP (Addgene: #175037) vector as a template, the P2A-EGFP fragment contained therein was amplified by PCR using the following primers:

[0031] F1: 5'-GCCACCAACTTCAGCCTGCTGAAGC-3' (Length: 25bp) (SEQ ID NO. 2);

[0032] R1:5'-TTACTTGTACAGCTCGTCCATGCCG-3' (Length: 25bp) (SEQ ID NO.3);

[0033] The amplified product was purified and set aside.

[0034] Overlapping PCR primers F2 and R2 were designed to express AKT1 mut The two amplified products were purified respectively, and then the two purified amplified products were amplified with F2 and R3 as templates. After purification, AKT1 was obtained. mut -P2A-EGFP fragment sequence.

[0035] The primer sequences are as follows:

[0036] F2: 5'-ATGAACGACGTAGCCATTGTGAAG-3' (Length: 24bp) (SEQ ID NO. 4);

[0037] R2: 5'-GCAGGCTGAAGTTGGTGGCGGCTGTGCCACTGGCTGAGTA-3' (Length: 41bp) (SEQ ID NO. 5);

[0038] F3: 5'-TACTCAGCCAGTGGCACAGCCGCCACCAACTTCAGCCTGC-3' (Length: 41bp) (SEQ ID NO. 6);

[0039] R3: 5'-CATGGACGAGCTGTACAAGTAA-3' (Length: 22bp) (SEQ ID NO.7);

[0040] To AKT1 mut -P2A-EGFG was inserted into the vector pT3-EF1αH (Addgene: #180149) and primers F4 and R4 were used to express AKT1 mut -P2A-EGFG was used as a template to amplify AKT1 with homology arms mut -P2A-EGFG fragment. Then primers F5 and R5 were used to amplify the pT3-EF1αH vector to obtain a linearized vector. After purification, AKT1 was cloned into the vector by seamless cloning (Biyuntian, CAT#D7010S). mut -P2A-EGFG was inserted into the vector pT3-EF1αH. The primers used were as follows:

[0041] F4: 5'-GTGTCGTGAGGAATTAGCTTGGTACATGAACGACGTAGCCATTGTGAAGG-3' (Length: 50bp) (SEQ ID NO. 8);

[0042] R4: 5'-TTACTTGTACAGCTCGTCCATGCCGTGCAGCGGCCGCGATATCCGTATTG-3' (Length: 50bp) (SEQ ID NO. 9);

[0043] F5: 5'-CCTTCACAATGGCTACGTCGTTCATGTACCAAGCTAATTCCTCACGACAC-3' (Length: 50bp) (SEQ ID NO. 10);

[0044] R5: 5'-CGGCATGGACGAGCTGTACAAGTAACAATACGGATATCGCGGCCGCTGCA-3' (Length: 50bp) (SEQ ID NO. 11);

[0045] Example 2: Construction of pT-mNRAS 3muts -mCherry vector

[0046] The following sequence was chemically synthesized, with reference to the Mouse NRAS gene (CCDS38570.1) (c.35G>A; G12D, c.38G>A; G13D, c.182A>G; Q61R), and the specific sequence is shown in SEQ ID NO.12.

[0047] Using the dCas9-VPR_P2A_mCherry (Addgene: #154193) vector as a template, the P2A-mCherry fragment contained therein was amplified by PCR using the following primers:

[0048] F6: 5'-GCCACCAACTTCAGCCTGCTGAAGC-3' (Length: 25bp) (SEQ ID NO. 13);

[0049] R6: 5'-TCACTTGTACAGCTCGTCCATGCCG-3' (Length: 25bp) (SEQ ID NO. 14);

[0050] The amplified product was purified and set aside.

[0051] Overlapping PCR primers F7 and R7 were designed to express mNRAS 3muts The two amplified products were purified respectively, and then the two purified amplified products were amplified with F7 and R8 as templates to obtain mNRAS after purification. 3muts-P2A-mCherry fragment sequence.

[0052] The primer sequences are as follows:

[0053] F7: 5'-ATGACTGAGTACAAACTGGTGGTGG-3' (Length: 25bp) (SEQ ID NO.15)

[0054] R7: 5'-AGCAGGCTGAAGTTGGTGGCCATCAGCACACAGGGCAGCC-3' (Length: 40bp) (SEQ ID NO.16)

[0055] F8: 5'-GGCTGCCCTGTGTGCTGATGGCCACCAACTTCAGCCTGCT-3' (Length: 40bp) (SEQ ID NO. 17)

[0056] R8: 5'-TCACTTGTACAGCTCGTCCATGCCG-3' (Length: 25bp) (SEQ ID NO.18)

[0057] To transform mNRAS 3muts -P2A-mCherry was inserted into the vector pT / CMV-SV40-LgT (Addgene: #20204) and primers F9 and R9 were used to express mNRAS 3muts -P2A-mCherry was used as a template to amplify mNRAS with homology arms 3muts -P2A-mCherry fragment. Then, primers F10 and R10 were used to linearize the vector using the pT / CMV-SV40-LgT vector as a template. After purification, the mNRAS was cloned into the vector by seamless cloning (Biyuntian, CAT#D7010S). 3muts -P2A-mCherry was inserted into the vector pT / CMV-SV40-LgT. The primers used were as follows:

[0058] F9: 5'-CTTTTGCAAAAAGCTTTGCAAAGATGACTGAGTACAAACTGGTGGTGG-3' (Length: 48bp) (SEQ ID NO. 19)

[0059] R9: 5'-GTTAACAACAACAATTGCATTCATTTCACTTGTACAGCTCGTCCATGCCG-3' (Length: 50bp) (SEQ ID NO. 20)

[0060] F10: 5'-CCACCACCAGTTTGTACTCAGTCATCTTTGCAAAGCTTTTGCAAAAG-3' (Length: 48bp) (SEQ ID NO. 21)

[0061] R10: 5'-CGGCATGGACGAGCTGTACAAGTGAAATGAATGCAATTGTTGTTGTTAAC-3' (Length: 50bp) (SEQ ID NO. 22)

[0062] Example 3: Construction of transgenic mice

[0063] The pT3-mAKT1 constructed above mut -EGFP vector, pT-mNRAS 3muts -mCherry vector, target sequence pT2-shP53 vector (Addgene: #124261) that interferes with mouse P53 expression, and pCMV (CAT) T7-SB100 vector (Addgene: #34879) that expresses transposon enzyme were suspended in a certain volume (volume to body weight ratio of 20 μL / g to 100 μL / g) of normal saline at a ratio of 0.7 μg: 0.7 μg: 1.8 μg: 0.05 μg to form a plasmid suspension. The control group was injected with wild-type gene expression plasmid pT3-mAKT1 without mutation WT -EGFP, pT-mNRAS WT -mCherry, pT2-shP53 vector and pCMV(CAT)T7-SB100 vector mixture, the vector construction method is the same as that of Example 1 and Example 2. The above plasmid suspension was injected into 6-8 week old mice through the tail vein.

[0064] Abdominal and liver photography was taken on days 10 and 20 in the control and model groups, and the results are shown in Figure 1. The results showed that the livers of the control mice on days 10 and 20 were normal in appearance, rosy in color, soft and brittle, with no visible lesions. The livers of the model group mice had very obvious lesions, as evidenced by a gradual yellowing of the liver from day 10 to day 20, an imbalance in the liver lobe proportions, and multiple, grayish-white nodular tumors of varying sizes visible on the surface on day 20.

[0065] Figure 2 shows liver pathological sections from the control and model groups of different strains on days 10 and 20. Pathological H&E staining revealed that the hepatocytes of the control mice were structurally normal on days 10 and 20, with hepatic cords arranged radially around the central vein, oval nuclei, and abundant hepatocyte cytoplasm with large, round nuclei. In the model group, vesicular degeneration was observed on day 10, with cytoplasmic changes consisting of multiple small vacuoles filling the cytoplasm and significant compression or displacement of the hepatocyte nuclei. Tumor cells were observed on day 20, presenting as solid nests with focal necrosis. The tumor cells showed significant atypia, abundant eosinophilic cytoplasm, round nuclei with distinct nucleoli, and frequent mitotic figures.

[0066] Survival curves for the control and model groups are shown in Figure 3. The results show that the mortality rate of mice in the control group (Ctrl) was 0% during the observation period, while the mortality rate of mice in the model group (Pos) reached or approached 100% during the 50-day observation period. Among these, the mortality rates were 100% for C57BL / 6 mice, 92.857% for DBA / 2 mice, and 87.5% for FVB / N mice. The median survival was 24 days for C57BL / 6 mice, 32.5 days for DBA / 2 mice, and 31.5 days for FVB / N mice. The earliest death occurred on day 20 for C57BL / 6 mice, day 28 for DBA / 2 mice, and day 10 for FVB / N mice.

[0067] Figure 4 shows the abdominal circumference statistics of the control (Ctrl) and model (Pos) mice on days 9 and 21. On day 9, there was no significant difference in abdominal circumference between the C57BL / 6, DBA / 2, and FVB / N mouse model groups compared to the control group. On day 21, the abdominal circumference of the three mouse model groups was significantly increased compared to the control group, indicating that the liver cancer model had further developed and undergone qualitative changes on day 21. In terms of the mechanism of ascites formation, factors such as impaired liver function, enlarged liver tumors, and portal hypertension may all contribute to the formation of ascites over time, leading to an increase in abdominal circumference. The increase in abdominal circumference observed on day 21 may be a critical time point in the development of the liver cancer model, and this finding may provide guidance for future research and treatment design.

[0068] The results of the sequencing abundance of liver cancer markers in the model group (Pos) mice at different time points after injection are shown in Figure 5. The RNA-seq results showed that the sequencing abundance of liver cancer markers in the model group mice continued to increase over time after injection, including Afp (α-fetoprotein), Krt18 (cytokeratin 18), Krt19 (cytokeratin 19) and Gpc3 (glioma protoplasm 3). These markers are usually highly expressed in liver cancer, so their increased abundance may be related to the growth and deterioration of liver cancer. These findings are part of the validation of the liver cancer model. Over time, the model group mice gradually developed liver cancer characteristics, including increased expression of the markers at the transcriptional level, which is consistent with the natural development process of liver cancer. In addition, the increased expression of these markers can be used to monitor the effect of drug treatment in the model.

[0069] Example 4: Construction of transgenic mice

[0070] The pT3-mAKT1 constructed above mut -EGFP vector, target sequence pT2-shP53 vector (Addgene: #124261) that interferes with mouse P53 expression, and pCMV (CAT) T7-SB100 vector (Addgene: #34879) that expresses transposon enzyme were suspended in a certain volume (volume to body weight ratio of 20 μL / g to 100 μL / g) of normal saline at a ratio of 0.7 μg: 1.8 μg: 0.05 μg to form a plasmid suspension. The control group was injected with wild-type gene expression plasmid pT3-mAKT1 without mutation WT -EGFP, pT2-shP53 vector and pCMV(CAT)T7-SB100 vector mixture. 6-8 week old mice were injected with the above plasmid suspension via the tail vein.

[0071] On day 75, open-abdomen and liver photography were taken from the control and model groups, and the results are shown in Figure 6. The results showed that the livers of the control mice on day 75 were normal in appearance, rosy in color, soft and brittle, with no visible lesions. However, the livers of the model group mice had very obvious lesions, manifested by yellowing of the liver, disproportionate liver lobes, and multiple gray-white nodular tumors of varying sizes on the surface.

[0072] The above embodiments are merely preferred embodiments for the purpose of fully illustrating the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are within the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

Claims

1. A mutant gene for establishing a primary liver cancer mouse model, characterized in that: The mutant genes include mutant genes including mouse AKT1.

2. A mutant gene combination for establishing a primary liver cancer mouse model, characterized in that: The mutant gene combination includes a mutant gene of the mouse AKT1 gene and a mutant gene of the mouse NRAS gene.

3. The mutant gene combination for establishing a primary liver cancer mouse model according to claim 2, characterized in that: The mutant gene of the mouse AKT1 gene is c.49G>A; p.E17K of the mouse AKT1 gene; the mutant gene of the mouse NRAS gene is c.35G>A; p.G12D, c.38G>A; p.G13D, c.182A>G; p.Q61R of the mouse NRAS gene.

4. The mutant gene combination for establishing a primary liver cancer mouse model according to claim 2, characterized in that: The mutant gene sequence of the mouse AKT1 gene is shown in SEQ ID NO.1; the mutant gene sequence of the mouse NRAS gene is shown in SEQ ID NO.

12.

5. Use of the mutant gene combination according to claim 2 in combination with an agent that interferes with mouse TP53 expression in establishing a primary liver cancer mouse model.

6. The use according to claim 5, characterized in that: The reagent interfering with mouse TP53 expression is a pT2-shP53 vector.

7. A method for establishing a primary liver cancer mouse model, characterized in that: The mutant gene according to claim 1 or the mutant gene combination according to claim 2 is mixed with an agent interfering with mouse P53 expression and a vector expressing a transposon enzyme to form a suspension, and the suspension is injected through the tail vein.

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