Construction method and application of miR-379 / 410 gene cluster knockout cell line

Abstract

The invention relates to a construction method and application of a miR-379 / 410 gene cluster knockout cell line, and the construction method of the miR-379 / 410 gene cluster knockout cell line comprises the following steps: respectively connecting sgRNA aiming at two ends of a miR-379 / 410 gene cluster with a carrier to obtain a recombinant plasmid, and transfecting a mouse liver cell line to obtain the miR-379 / 410 gene cluster knockout mouse liver cell line. The miR-379 / 410 gene cluster is knocked out, the early state of MAFLD is simulated, and an experimental model is provided for deeply researching the molecular mechanism of MAFLD and screening and evaluating novel therapeutic drugs.

Description

Technical Field

[0001] The present invention relates to the field of cell biology, and particularly to a method for constructing a miR-379 / 410 gene cluster knockout cell line and its application, specifically to a mouse hepatocyte cell line with miR-379 / 410 gene cluster knockout. Background Art

[0002] Metabolic associated fatty liver disease (MAFLD) is a chronic progressive disease caused by overnutrition and insulin resistance in genetically susceptible individuals, and is also one of the main risk factors leading to liver cancer. In addition to known susceptibility genes such as PNPLA3 and TM6SF2, more and more evidence shows that environmental exposure in early embryonic development (paternal caffeine exposure or maternal metabolic syndrome) may increase the risk of an individual developing MAFLD through epigenetic mechanisms.

[0003] As a special gene expression regulation method, imprinted genes do not follow Mendelian inheritance laws in their expression patterns, but are determined by parental origin. This unique expression pattern is determined by DNA methylation in the imprinting control region, and the methylation status of the imprinting control region is easily affected by the prenatal environment. Once established, the imprint will be maintained throughout an individual's life and have a long-term impact on the expression of imprinted genes. Therefore, modification of fetal imprinted genes by the prenatal environment may increase the risk of children developing metabolic diseases (such as MAFLD) in adulthood. Among them, the Dlk1-Dio3 gene cluster is an important imprinted gene cluster, encoding multiple non-coding RNAs, including the miR-379 / 410 gene cluster, and the miR-379 / 410 gene cluster plays an important role in embryonic development and metabolic regulation.

[0004] In summary, providing a MAFLD cell model related to epigenetic mechanisms has become one of the urgent problems to be solved in the current field. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides a method for constructing a miR-379 / 410 gene cluster knockout cell line and its application. By knocking out the miR-379 / 410 gene cluster in mouse hepatocytes, it simulates the early state of MAFLD, reveals the important role of the miR-379 / 410 gene cluster in the pathogenesis of MAFLD, and lays a foundation for clinical trials for evaluating the pharmacodynamics and safety of potential therapeutic drugs.

[0006] To achieve this purpose, the present invention adopts the following technical solutions:

[0007] In the first aspect, the present invention provides an sgRNA targeting the mouse miR-379 / 410 gene cluster, and the nucleic acid sequence of the sgRNA includes:

[0008] (1)Any one or a combination of at least two of the sequences shown in SEQ ID NO.1 to SEQ ID NO.6; and (2)Any one or a combination of at least two of the sequences shown in SEQ ID NO.7 to SEQ ID NO.12.

[0009] SEQ ID NO.1: CACCTCATAACGCCTACGTTCCATAGT.

[0010] SEQ ID NO.2: AAACACTATGGAACGTAGGCGTTATGA.

[0011] SEQ ID NO.3: CACCGATGGTAGACTATGGAACGTAGG.

[0012] SEQ ID NO.4: AAACCCTACGTTCCATAGTCTACCATC.

[0013] SEQ ID NO.5: CACCGGTAGACTATGGAACGTAGGCGT.

[0014] SEQ ID NO.6: AAACACGCCTACGTTCCATAGTCTACC.

[0015] SEQ ID NO.7: CACCCAGCGGATACGGACGGCTAGTGG.

[0016] SEQ ID NO.8: AAACCCACTAGCCGTCCGTATCCGCTG.

[0017] SEQ ID NO.9: CACCTGCAGCGGATACGGACGGCTAGT.

[0018] SEQ ID NO.10: AAACACTAGCCGTCCGTATCCGCTGCA.

[0019] SEQ ID NO.11: CACCATACGGACGGCTAGTGGACCAGG.

[0020] SEQ ID NO.12: AAACCCTGGTCCACTAGCCGTCCGTAT.

[0021] Based on the important role of the miR-379 / 410 gene cluster in embryonic development and metabolic regulation, the present invention proposes that the miR-379 / 410 gene cluster may increase the risk of an individual developing MAFLD after adulthood by affecting embryonic development, and is an important molecular bridge connecting embryonic development and MAFLD. Therefore, the present invention provides an sgRNA of the miR-379 / 410 gene cluster in mice, and by knocking out the miR-379 / 410 gene cluster in mouse hepatocytes, the changes in liver cells are observed from aspects such as liver function, liver pathology, and liver metabolism. The present invention for the first time reveals the important role of the miR-379 / 410 gene cluster in the pathogenesis of MAFLD, provides a new experimental platform for studying the early pathogenesis of MAFLD, and provides a new theoretical basis for the prevention and treatment of MAFLD.

[0022] Preferably, the nucleic acid sequence of the sgRNA includes the sequences shown in SEQ ID NO.1 to SEQ ID NO.12.

[0023] In a second aspect, the present invention provides a gene editing system targeting the miR-379 / 410 gene cluster in mice, and the gene editing system includes the sgRNA targeting the miR-379 / 410 gene cluster in mice described in the first aspect.

[0024] Preferably, the gene editing system further includes Cas9.

[0025] Preferably, the Cas9 includes Cas9 protein and / or mRNA of Cas9 protein.

[0026] In a third aspect, the present invention provides a method for constructing a miR-379 / 410 gene cluster knockout cell line, and the method for constructing the miR-379 / 410 gene cluster knockout cell line includes: respectively connecting the sgRNAs targeting both ends of the miR-379 / 410 gene cluster with a vector to obtain a recombinant plasmid, and transfecting cells to obtain the miR-379 / 410 gene cluster knockout cell line.

[0027] Preferably, the cells include hepatocytes.

[0028] Preferably, the cells are mouse hepatocytes, specifically the mouse hepatocyte line NCTC-1469.

[0029] Preferably, in the method for constructing the miR-379 / 410 gene cluster knockout cell line, the nucleic acid sequence of the sgRNA targeting the left end of the miR-379 / 410 gene cluster includes any one or a combination of at least two of the sequences shown in SEQ ID NO.1 to SEQ ID NO.6.

[0030] Preferably, in the method for constructing the miR-379 / 410 gene cluster knockout cell line, the nucleic acid sequence of the sgRNA targeting the right end of the miR-379 / 410 gene cluster includes any one or a combination of at least two of the sequences shown in SEQ ID NO.7 to SEQ ID NO.12.

[0031] Preferably, the vector includes pX459.

[0032] Preferably, after transfection of the cells, it further includes the steps of screening and identifying the miR-379 / 410 gene cluster knockout cell line using puromycin.

[0033] Fourthly, the present invention provides a cell line with the miR-379 / 410 gene cluster knocked out, and the cell line with the miR-379 / 410 gene cluster knocked out is prepared by the method for constructing the miR-379 / 410 gene cluster knockout cell line described in the third aspect.

[0034] Preferably, the cell line with the miR-379 / 410 gene cluster knocked out contains the sgRNA targeting the mouse miR-379 / 410 gene cluster described in the first aspect.

[0035] Preferably, the cell line with the miR-379 / 410 gene cluster knocked out contains the gene editing system targeting the mouse miR-379 / 410 gene cluster described in the second aspect.

[0036] Preferably, the cell line with the miR-379 / 410 gene cluster knocked out is a cell line in which the miR-379 / 410 gene cluster has been knocked out in the genome after being edited by the gene editing system targeting the mouse miR-379 / 410 gene cluster described in the second aspect.

[0037] Fifthly, the present invention provides the application of any one or a combination of at least two of the sgRNA targeting the mouse miR-379 / 410 gene cluster described in the first aspect, the gene editing system targeting the mouse miR-379 / 410 gene cluster described in the second aspect, the method for constructing the miR-379 / 410 gene cluster knockout cell line described in the third aspect, or the cell line with the miR-379 / 410 gene cluster knocked out described in the fourth aspect in the preparation of a cell model for metabolic associated fatty liver disease.

[0038] Sixthly, the present invention provides a cell model for metabolic associated fatty liver disease, and the cell model is prepared by the method for constructing the miR-379 / 410 gene cluster knockout cell line described in the third aspect, and the cell is a hepatocyte.

[0039] By knocking out the miR-379 / 410 gene cluster in mouse hepatocytes, the present invention simulates the early state of MAFLD, providing an experimental model for in-depth study of the molecular mechanism of MAFLD, screening and evaluating new therapeutic drugs.

[0040] Preferably, the cell model is the cell line with the miR-379 / 410 gene cluster knocked out as described in the fourth aspect.

[0041] In the seventh aspect, the present invention provides the application of any one or at least two combinations of the sgRNA targeting the miR-379 / 410 gene cluster in mice as described in the first aspect, the gene editing system targeting the miR-379 / 410 gene cluster in mice as described in the second aspect, the method for constructing the miR-379 / 410 gene cluster knockout cell line as described in the third aspect, the miR-379 / 410 gene cluster knockout cell line as described in the fourth aspect, or the cell model of metabolic associated fatty liver disease as described in the sixth aspect in studying the pathogenesis of metabolic associated fatty liver disease and screening therapeutic drugs for metabolic associated fatty liver disease.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] The present invention provides an sgRNA and a gene editing system targeting the miR-379 / 410 gene cluster in mice, constructs a miR-379 / 410 gene cluster knockout cell line, observes the changes of liver cells from aspects such as liver function, liver pathology, and liver metabolism, and shows the early onset phenomenon of MAFLD. The present invention successfully constructs a MAFLD cell model, provides a new experimental platform for studying the early pathogenesis of MAFLD, reveals the key role of the miR-379 / 410 cluster in connecting embryonic development and MAFLD, and can be used for in-depth study of the molecular mechanism of MAFLD, screening and evaluating new therapeutic drugs. Through this model, high-risk populations of MAFLD can be discovered earlier, so as to carry out early intervention and delay the progression of the disease. By detecting the miR-379 / 410 expression levels of different individuals, personalized treatment plans can be provided for patients.

[0044] Compared with the existing models, the cell model provided by the present invention has the following advantages: ① An earlier onset model: By gene manipulation at the embryonic development stage, it simulates the impact of pre-birth environmental factors on the metabolic health of offspring. ② More precise molecular mechanism research: It can more deeply study the specific mechanism of action of the miR-379 / 410 cluster in the pathogenesis of MAFLD. ③ A broader application prospect: It is not only applicable to the study of MAFLD, but also can be used to study other metabolic diseases and the impact of environmental factors on metabolic health. Brief Description of the Drawings

[0045] Figure 1 This is the technical roadmap for the construction of the miR-379 / 410KO cell line of the present invention.

[0046] Figure 2 This is the identification result diagram of the knockout efficiency of the miR-379 / 410KO cell line.

[0047] Figure 3 This is the result diagram of the ALT level in the culture supernatants of the WT cell line and the miR-379 / 410KO cell line.

[0048] Figure 4 This is the oil red O staining diagram of the WT cell line and the miR-379 / 410KO cell line.

[0049] Figure 5 This is the identification result diagram of the TC and TG contents of the WT cell line and the miR-379 / 410KO cell line.

[0050] Figure 6 This is the gene expression diagram of the cholesterol pathway of the WT cell line and the miR-379 / 410KO cell line.

[0051] Figure 7 This is the protein expression diagram of HMGCR, FABP2, and PMVK of the WT cell line and the miR-379 / 410KO cell line.

[0052] Figure 8 This is the gene expression diagram of the fatty acid synthesis and transport pathway of the WT cell line and the miR-379 / 410KO cell line.

[0053] Figure 9 This is the protein expression diagram of PPARγ, FAS, and APOA4 of the WT cell line and the miR-379 / 410KO cell line.

[0054] Figure 10 This is the protein expression diagram of CD36 and FGF21 of the WT cell line and the miR-379 / 410KO cell line. Detailed implementation manners

[0055] To further elaborate on the technical means and effects adopted by the present invention, the present invention will be further described below in conjunction with examples and drawings. It can be understood that the specific implementation manners described herein are only used to explain the present invention, rather than limiting the present invention.

[0056] For those not specifying specific techniques or conditions in the examples, the techniques or conditions described in the literature in this field or according to the product specifications are followed. For reagents or instruments not indicating the manufacturer, they are all conventional products that can be obtained through regular channels.

[0057] Example 1

[0058] This example provides an sgRNA targeting the miR-379 / 410 gene cluster in mice. According to the CDS sequences at both ends of the miR-379 / 410 gene cluster in the NCBI database, 3 pairs of gRNAs were designed through the website http: / / www.rgenome.net / cas-designer / , as shown in Table 1, and the single-stranded sequences were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0059] Table 1

[0060]

[0061]

[0062] Example 2

[0063] This example constructed a miR-379 / 410 gene cluster knockout (miR-379 / 410KO) cell line, and the construction technical roadmap is as Figure 1 shown. First, according to the principle of gene knockout by the CRISPR / Cas9 technology and the miR-379 / 410 gene cluster sequence, two sgRNAs were designed for both ends of the miR-379 / 410 gene cluster, named miR379-sgRNA and miR410-sgRNA respectively. Then, the recombinant plasmids pX459-miR379-sgRNA and pX459-miR410-sgRNA of sgRNA were constructed using the pX459 empty plasmid as the vector. After co-transfecting the above two recombinant plasmids into NCTC-1469 mouse hepatocytes, primers were designed for the recombinant plasmids and qPCR was performed to identify the transfection efficiency. Finally, puromycin was used to screen the NCTC-1469 mouse hepatocytes successfully transfected with the recombinant plasmids. The specific steps are as follows:

[0064] (1) The mouse normal hepatocyte cell line NCTE-1469 was placed in DMEM supplemented with 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 μg / mL streptomycin, and cultured in an incubator at 37 °C and 5% CO 2 2.

[0065] (2) The sgRNA single-strands provided in Example 1 were annealed. Referring to Table 2, the sgRNA annealing system was prepared, and the annealing program was: 37 °C for 30 min; 95 °C for 5 min; the temperature was lowered to 25 °C at a rate of 5 °C per minute to form a double-stranded sequence sgRNA with sticky ends.

[0066] Table 2

[0067] Component Dosage Forward primer (100 μm) 1 μL Reverse primer (100 μm) 1 μL PNK buffer 1 μL T4 PNK (Polynucleotide kinase) 1 μL Water Make up to 10 μL

[0068] (3) Digest the pX459 empty plasmid vector with the restriction endonuclease BbsI. The digestion system is shown in Table 3. Mix the digestion reaction system, centrifuge at low speed for 5 s, react overnight at 4 °C, and then take it out for electrophoresis analysis or ligation with sgRNA.

[0069] Table 3

[0070] Component Dosage pX459 2 μL BbsI enzyme 1 μL 10× buffer 2 μL Phosphatase 1 μL Water Make up to 20 μL

[0071] (4) Ligate the annealed product of sgRNA with the digested product of the pX459 vector. The reaction system is shown in Table 4. Incubate in a water bath at 16 °C overnight. Transform the recombinant plasmid obtained from overnight ligation into DH5α competent cells, and screen for positive bacteria for sequencing.

[0072] Table 4

[0073]

[0074]

[0075] (5) Co-transfect the recombinant plasmids pX459-miR379-sgRNA and pX459-miR410-sgRNA into NCTC-1469 cells using the cationic liposome-mediated transfection method, which uses a commercial lipo reagent. To optimize the transfection efficiency and determine the optimal transfection conditions, and to confirm the successful transfection of the recombinant plasmids pX459-miR379-sgRNA and pX459-miR410-sgRNA into cells, different mass ratios of different lipo reagents (lipo3000, lipo6000) to the recombinant plasmids were designed for co-transfection. Before the transfection experiment, when the cells were cultured to 60 - 70% confluence, the complete medium was replaced with DMEM without antibiotics to eliminate the possible interference of antibiotics on the transfection process.

[0076] (6) Screen the NCTC-1469 cells stably transfected with the recombinant plasmids px459-miR379-sgRNA and px459-miR410-sgRNA by Puro pressure

[0077] ① Establish the puro killing curve: The pX459 vector contains the PuroR gene, i.e., the pac gene. The puromycin N-acetyltransferase encoded by the pac gene can confer resistance to puromycin (puro). To obtain accurate cell viability, experiments were conducted using a 96-well plate in combination with the CCK-8 method. After the cells adhered and grew for 24 h, the medium was replaced with complete medium containing gradient concentrations of puro (0, 1, 2.5, 5, 7.5, 10 μg / mL), and 6 replicates were designed for each concentration. To reduce the error in observing cell viability and improve the accuracy of the data, the cells were grouped into the puro group, the control group, and the blank group. Puro group: Complete medium containing gradient concentrations of puro, cell suspension, and CCK-8 reagent accounting for 10% of the total volume; Control group: Complete medium without puro, cell suspension, and CCK-8 reagent accounting for 10% of the total volume, used to evaluate the natural survival state of cells without drug treatment; Blank group: Only complete medium and CCK-8 reagent, serving as the baseline of the experiment to exclude the influence of the medium and CCK8 reagent itself on the absorbance readings. After incubating the cell plate in a 37 °C constant temperature incubator for 90 min, the absorbance (OD value) at a wavelength of 450 nm was measured using a microplate reader. The experiment was repeated 3 times, and the average value of the experimental results was taken as the final experimental result.

[0078] Cell viability = (AY - AC / AK - AC) × 100%

[0079] Among them, AY is the OD value of the puro group; AC is the OD value of the control group; AK is the OD value of the blank group.

[0080] ② Screen pX459-miR379-sgRNA and pX459-miR410-sgRNA recombinant plasmid stably transfected cells with puro: After culturing the transfected cells in a 24-well plate for 48 h, the medium was changed to the culture medium containing gradient concentrations of puromycin according to the grouping. Continuously add puro for screening for 2 - 7 days until all the control group cells at the same puro concentration are observed to die, and the transfected group cells survive and resume growth. Then, it is considered that the cells in this transfected group are successfully transfected cells. After 2 - 15 days, pick cell clones, transfer the cells to a 48-well plate using a cloning ring for continued growth. When the confluence of the monolayer cells reaches 80%, passage the cells and transfer them to a 96-well plate. Measure the absorbance of NCTC-1469 at different concentrations of puromycin using CCK8, and plot the lethal concentration curves of puromycin at 24 h and 48 h. Use the minimum lethal concentration of puromycin to screen the stably knocked-out monoclonal cells after transfection, and obtain stably knocked-out monoclonal cells through the 96-well plate dilution method for sequencing and subsequent experiments.

[0081] In this example, the obtained miR-379 / 410 KO cell line was identified, and miR-300, miR-379, and miR-410 were detected at the RNA level by qPCR. The results are as Figure 2 shown. The expression levels in the knockout group (KO) were significantly lower than those in the wild type (WT). The expressions of miR-300 and miR-410 could not be detected in the KO cell line, indicating that the miR-379 / 410 cluster was successfully knocked out.

[0082] Example 3

[0083] In this example, functional tests were performed on the miR-379 / 410 KO mouse hepatocyte cell line, including the detection of the level of ALT in the cell supernatant, oil red staining of the cells, the detection of triglycerides and cholesteryl esters, the detection of cholesterol synthesis-related genes, the detection of fatty acid synthesis and transport-related genes, and the detection of cholesterol transport-related genes.

[0084] (1) Level of alanine aminotransferase (ALT) in the supernatant of the miR-379 / 410 KO mouse hepatocyte cell line

[0085] The supernatant of the miR-379 / 410 KO mouse hepatocyte cell line was detected for ALT. The results are as Figure 3 shown. Compared with the WT cell line, the level of ALT in the supernatant of the miR-379 / 410 KO mouse hepatocyte cell line was significantly increased. Therefore, the knockout of miR-379 / 410 in hepatocytes can cause hepatocyte damage.

[0086] (2) Oil red staining of the miR-379 / 410 KO cell line

[0087] The miR-379 / 410 KO cell line was stained with oil red. The operation steps refer to the existing technology. The results are as Figure 4 shown. Compared with the WT cell line, the miR-379 / 410 KO mouse hepatocyte cell line showed obvious fat accumulation. Therefore, the knockout of miR-379 / 410 in hepatocytes can cause the accumulation of lipid droplets.

[0088] (3) Detection of triglycerides and cholesteryl esters in the miR-379 / 410 KO mouse cell line

[0089] Oil red O can specifically stain neutral lipids such as triglycerides and cholesteryl esters in cells or tissues. The liver oil red staining of the miR-379 / 410 KO cell line was deeper, indicating an increase in neutral lipids. To clarify the components of the lipids, a cholesterol assay kit (including cholesterol and cholesteryl esters) and a triglyceride assay kit were used to detect the total cholesterol (TC) and triglyceride (TG) in the WT cell line and the miR-379 / 410 KO cell line, respectively. The results are asFigure 5 As shown, the TC content in miR-379 / 410KO cells is higher than that in WT cells, the TG content in miR-379 / 410KO cells is lower than that in WT cells, and the TG content in the culture supernatant of miR-379 / 410KO is higher than that in the WT cell line. It can be seen that the neutral lipids in the miR-379 / 410KO cell line are mainly cholesterol esters.

[0090] (4) Detection of genes related to cholesterol synthesis in the miR-379 / 410KO mouse cell line

[0091] To detect the cholesterol in the miR-379 / 410KO cell line, qPCR was performed on multiple genes (Fabp2, Pmvk, Hmgcr, Fdps, Lss, Dhcr7) involved in cholesterol synthesis and metabolism. The results are as Figure 6 shown. Except for no significant change in Hmgcr, the amounts of the other 5 cholesterol synthesis pathways are increased, indicating an increase in cholesterol synthesis in the miR-379 / 410KO cell line. In addition, WB was performed on multiple genes (HMGCR, FABP2, PMVK) involved in cholesterol synthesis and metabolism. The results are as Figure 7 shown. The expressions of HMGCR, FABP2, and PMVK proteins in the KO group are higher than those in the WT group.

[0092] (5) Detection of genes related to fatty acid synthesis and transport in the miR-379 / 410KO mouse cell line

[0093] Cholesterol esters are formed by the esterification of free cholesterol and fatty acids. Therefore, the synthesis of cholesterol esters requires not only free cholesterol but also free fatty acids. Free fatty acids have two sources: one is de novo synthesis within the cell, and the other is transported from outside the cell. To clarify the source of fatty acids, qPCR was performed on the key genes (PPARγ, FAS, FASN, ACACA) of the fatty acid synthesis pathway. Figure 8 The left figure shows that compared with WT cells, PPARγ, FAS, and FASN in the miR-379 / 410KO group are all decreased, indicating that de novo synthesis of fatty acids is not much.

[0094] The level of intracellular fatty acids depends on the ability of de novo synthesis of fatty acids and the ability of fatty acid decomposition. Therefore, qPCR was performed on the key transcription factor PPARα of intracellular fatty acid decomposition and the apolipoprotein APOA4. Figure 8 The right figure shows that compared with the WT cell group, the expressions of PPARα and APOA4 in the miR-379 / 410KO group are increased, indicating that after knocking out the miR-379 / 410 cluster, the decomposition of fatty acids in hepatocytes is increased. In addition, WB was performed on PPARγ, FAS, and APOA4. The results are asFigure 9 As shown, compared with the WT group, the expression of PPARγ in the KO group did not change significantly, FAS decreased, and the expression of APOA4 increased. This further indicates that after knocking out the miR-379 / 410 cluster, the fatty acid synthesis ability in mouse hepatocytes decreased, while the decomposition ability increased.

[0095] (6) Detection of cholesterol transport-related genes in the miR-379 / 410 KO mouse cell line

[0096] CD36 is a fatty acid transport protein. An increase in liver CD36 expression leads to more free fatty acids entering hepatocytes, exacerbating liver lipid accumulation. FGF21 is a hormone mainly secreted by the liver. FGF21 attempts to reduce hepatic steatosis by promoting fatty acid oxidation and improving insulin sensitivity. In patients with MAFLD, the expression of FGF21 is usually elevated, which is a compensatory response of the body to fatty liver. As Figure 10 shown, by detecting the expression of CD36 and FGF21 in WT cells and the miR-379 / 410 KO cell line by WB, it was found that compared with the WT cell line, CD36 was significantly increased in the miR-379 / 410 KO cell line, indicating that after knocking out the miR-379 / 410 cluster, the uptake of fatty acids by hepatocytes increased, while the change of FGF21 in both WT cells and the miR-379 / 410 KO cell line was not significant.

[0097] In summary, the neutral lipids in the miR-379 / 410 KO cell line mainly come from cholesterol esters; the increase in cholesterol esters has two aspects: one is the increase in cholesterol synthesis in the miR-379 / 410 KO cell line, and on the other hand, the ability of hepatocytes to uptake fatty acids increases. The decrease in triglycerides in the miR-379 / 410 KO cell line is because the fatty acid synthesis in hepatocytes decreases, and at the same time, the fatty acid decomposition increases, resulting in a decrease in triglyceride content.

[0098] Generally speaking, in the early stage of MAFLD, due to insulin resistance activating SREBP-1c, the expression of HMG-CoA reductase is up-regulated, leading to an increase in cholesterol synthesis. As the disease progresses, the expression of genes related to cholesterol synthesis tends to be down-regulated, while the expression of genes related to cholesterol esterification is up-regulated, resulting in a large accumulation of cholesterol esters in hepatocytes. In the miR-379 / 410 knockout cell line of the present invention, the increase in cholesterol synthesis is consistent with the early manifestation of MAFLD. The miR-379 / 410 knockout cell line mimics the early state of MAFLD, that is, the increase in cholesterol synthesis.

[0099] In summary, the present invention has successfully constructed a MAFLD cell model, deeply revealing the molecular characteristics in the early stage of MAFLD, and providing a theoretical basis for early diagnosis and intervention. Therefore, designing and synthesizing specific inhibitors targeting the miR-379 / 410 cluster or the PI3K / AKT pathway, validating them on the cell model, and performing pharmacodynamic and safety evaluations on potential therapeutic drugs lay the foundation for clinical trials.

[0100] The applicant declares that the above description is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by any person skilled in the art within the technical scope disclosed by the present invention fall within the protection scope and the disclosure scope of the present invention.

Claims

1. A sgRNA targeting the mouse miR-379 / 410 gene cluster, characterized in that: The nucleic acid sequence of the sgRNA includes: (1) any one or a combination of at least two of the sequences shown in SEQ ID NO.1 to SEQ ID NO.6; and (2) any one or a combination of at least two of the sequences shown in SEQ ID NO.7 to SEQ ID NO.

12.

2. The sgRNA targeting the mouse miR-379 / 410 gene cluster according to claim 1, characterized in that The nucleic acid sequence of the sgRNA includes the sequences shown in SEQ ID NO.1 to SEQ ID NO.

12.

3. A gene editing system targeting the mouse miR-379 / 410 gene cluster, characterized in that: The gene editing system includes the sgRNA targeting the mouse miR-379 / 410 gene cluster as described in claim 1 or 2.

4. The gene editing system according to claim 3, characterized in that The gene editing system also includes Cas9; Preferably, the Cas9 comprises Cas9 protein and / or mRNA of Cas9 protein.

5. A method for constructing a miR-379 / 410 gene cluster knockout cell line, characterized in that: The method for constructing the miR-379 / 410 gene cluster knockout cell line comprises: connecting sgRNAs targeting both ends of the miR-379 / 410 gene cluster to vectors respectively to obtain recombinant plasmids, transfecting cells, and obtaining the miR-379 / 410 gene cluster knockout cell line.

6. The method for constructing a miR-379 / 410 gene cluster knockout cell line according to claim 5, characterized in that: The cells include hepatocytes; Preferably, the nucleic acid sequence of the sgRNA targeting the left end of the miR-379 / 410 gene cluster in the method for constructing the miR-379 / 410 gene cluster knockout cell line includes any one or a combination of at least two of the sequences shown in SEQ ID NO.1 to SEQ ID NO.6; Preferably, the nucleic acid sequence of the sgRNA targeting the right end of the miR-379 / 410 gene cluster in the method for constructing the miR-379 / 410 gene cluster knockout cell line includes any one or a combination of at least two of the sequences shown in SEQ ID NO.7 to SEQ ID NO.12; Preferably, the vector comprises pX459; Preferably, after the cells are transfected, the method further comprises the step of using puromycin to screen and identify miR-379 / 410 gene cluster knockout cell lines.

7. A cell line with knockout of miR-379 / 410 gene cluster, characterized in that: The miR-379 / 410 gene cluster knockout cell line is prepared by the method for constructing the miR-379 / 410 gene cluster knockout cell line according to claim 5 or 6.

8. Use of any one or a combination of at least two of the sgRNA targeting the mouse miR-379 / 410 gene cluster as described in claim 1 or 2, the gene editing system targeting the mouse miR-379 / 410 gene cluster as described in claim 3 or 4, the method for constructing a miR-379 / 410 gene cluster knockout cell line as described in claim 5 or 6, or the miR-379 / 410 gene cluster knockout cell line as described in claim 7 in preparing a cell model of metabolic-related fatty liver disease.

9. A cell model of metabolic-related fatty liver disease, characterized in that: The cell model is prepared by the method for constructing the miR-379 / 410 gene cluster knockout cell line according to claim 5 or 6, and the cell is a hepatocyte.

10. Use of any one or a combination of at least two of the sgRNA targeting the mouse miR-379 / 410 gene cluster as described in claim 1 or 2, the gene editing system targeting the mouse miR-379 / 410 gene cluster as described in claim 3 or 4, the method for constructing a miR-379 / 410 gene cluster knockout cell line as described in claim 5 or 6, the miR-379 / 410 gene cluster knockout cell line as described in claim 7, or the cell model of metabolic-related fatty liver disease as described in claim 9 in studying the pathogenesis of metabolic-related fatty liver disease and screening therapeutic drugs for metabolic-related fatty liver disease.

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