Application of ACOT2 upregulators in promoting liver regeneration and liver function recovery after partial hepatectomy

Abstract

The present invention relates to the field of biomedicine technology, and provides the use of an upregulator of ACOT2 in promoting liver regeneration and liver function recovery after partial hepatectomy. Specifically, it provides the use of an ACOT2 expression promoter in the preparation of a drug for promoting liver regeneration and liver function recovery after partial hepatectomy, as well as a pharmaceutical composition comprising the ACOT2 expression promoter. The present invention has developed new discoveries about the function of the ACOT2 gene. The increased expression of ACOT2 after hepatectomy has a certain protective effect on liver regeneration, or can serve as a potential marker for warning the degree of postoperative liver function damage and liver regeneration capacity, and a therapeutic target for promoting postoperative liver regeneration. By using the intervention method of the present invention, the decomposition and consumption of lipid droplets in the liver lipid metabolism process are targeted, thereby promoting the recovery of liver function and liver-to-body ratio.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and relates to the fields of gene function and liver regeneration therapy, and specifically to the discovery of new functions and regulatory methods of the ACOT2 gene, as well as the use of ACOT2 upregulators in promoting liver function recovery and liver regeneration after partial hepatectomy. Background Art

[0002] The liver has a powerful regenerative capacity, and can recover to near-original size after partial resection. In maintaining intrinsic homeostasis and regenerating after injury, mature hepatocytes demonstrate superior tissue repair and renewal capabilities. As the body's largest substantial metabolic organ, the liver regulates the synthesis, metabolism, storage, and redistribution of nutrients such as carbohydrates and lipids. Although cell proliferation and metabolic functions are incompatible in most organs, the liver maintains its essential glucose and lipid metabolism during liver regeneration. Decades of research have explored the regulatory mechanisms of key cytokines during liver regeneration, including IL-6, TGFα, and HGF. However, our understanding of the relationship between hepatocyte glucose and lipid metabolism and proliferation during liver regeneration remains limited, and insufficient attention has been given. There are no therapeutic targets targeting fatty acid oxidation to promote liver regeneration and repair after surgery.

[0003] In the early stages of liver regeneration, when glucose is depleted and no longer serves as the primary energy substrate, the liver relies on fatty acid oxidation to obtain ATP. During this period, decreased blood glucose and the reduced insulin-to-glucagon ratio stimulate adipose tissue lipolysis and the export of free fatty acids to the liver, leading to an increase in free fatty acids in the blood. Subsequently, increased fatty acid uptake by the liver leads to transient steatosis, a phenomenon critical for liver regeneration. Disruption of lipid droplet accumulation impairs subsequent regeneration. Lipids within lipid droplets are consumed through enhanced β-oxidation, accelerating hepatocyte hypertrophy and division. Fatty acid β-oxidation plays a crucial role in liver regeneration. Reduced levels of PPARα and CPT-1, key molecules involved in mitochondrial β-oxidation, lead to increased lipid accumulation and delayed DNA replication, potentially leading to oxidative stress and hepatocyte apoptosis. Protecting or activating CPT-1 activity can reverse lipotoxic damage. Mitochondrial β-oxidation produces a large amount of reducing equivalents for the respiratory chain reaction to produce ATP, and acetyl coenzyme A to participate in the subsequent tricarboxylic acid cycle. The NADPH produced is also used to maintain the glutathione-dependent antioxidant system.Therefore, how to ensure the integrity of mitochondrial function and the smooth progress of β-oxidation is particularly important during liver regeneration (Gazit V, Weymann A, Hartman E, Finck BN, Hruz PW, Tzekov A, Rudnick DA.Liver regeneration is impairedin lipodystrophic fatty liver dystrophy mice. Hepatology. 2010; 52: 2109-17; Ezaki H, Yoshida Y, Saji Y, Takemura T, Fukushima J, Matsumoto H, Kamada Y, Wada A, Igura T, Kihara S, Funahashi T, Shimomura I, Tamura S, et al. Delayed liver regeneration after partial hepatectomy in adiponectin knockout mice. Biochem Biophys Res Commun. 2009; 378: 68-72; Xiao W, Ren M, Zhang C, Li S, An W. Amelioration ofnonalcoholic fatty liver disease by hepatic stimulator substanceviapreservation ofcarnitine palmitoyl transferase-1activity. Am J Physiol CellPhysiol. 2015; 309:C215-27).

[0004] Acyl-CoA thioesterases (ACOTs) are a class of enzymes that hydrolyze acyl-CoA to free fatty acids (FFA) and coenzyme A (CoA-SH). They specifically inactivate fatty acids by reacting with CoA-containing lipids. Existing research evidence suggests that they control the oxidation rate of acyl-CoA in mitochondria and peroxisomes and also regulate fatty acid transport between organelles. The acyl-CoA thioesterase family is broadly divided into two classes based on their reactivity to peroxisome proliferators (PP): class I ACOTs are highly reactive to PP and have a molecular weight of approximately 40 kDa; class II ACOTs have varying reactivity to PP and have a molecular weight range of 110-150 kDa. ACOT2 belongs to the class I class of ACOTs and is primarily located in mitochondria. It is specific for long-chain acyl-CoAs. Currently, studies on its activity have been limited to skeletal muscle, myocardium, and adipose tissue, as well as under fasting conditions. Studies on ACOT2 knockdown or deletion in mice have not yet been conducted, and there are no reports on its role in liver regeneration after partial hepatectomy.

[0005] ACOT2 expression in vivo is regulated by the transcription factor PPAR. PPAR, short for peroxisome proliferator-activated receptor, belongs to the nuclear receptor subfamily. PPARα target genes are primarily involved in glucose and lipid metabolism, with some also participating in inflammatory pathways. Previous literature reports indicate that PPARα controls numerous lipid metabolism pathways and genes, including fatty acid oxidation in microsomes, peroxisomes, and mitochondria, fatty acid binding and activation, fatty acid elongation and desaturation, triglyceride synthesis and degradation, and bile acid metabolism.

[0006] Furthermore, RNA methylation to form N6-methyladenosine is the most abundant internal mRNA modification in eukaryotes, extensively regulating gene expression in a variety of physiological processes. m6A modification is added to specific sites in some transcripts in a highly specific manner by a multi-subunit methylase complex (writers). This complex, primarily composed of a heterodimer composed of METTL3 and METTL14, is the m6A methylase for the vast majority of mRNAs. m6A demethylases (erasers) include FTO and ALKBH5. m6A-modified mRNA is recognized by recruited m6A-binding proteins (readers), including YTHDF1 / 2 / 3, YTHDC1 / 2, and IGF2BP1 / 2 / 3, which affect mRNA alternative splicing, nuclear transport, translation, degradation, and stability (Geula S, Moshitch-Moshkovitz S, Dominissini D, Mansour AA, Kol N, Salmon-Divon M, Hershkovitz V, Peer E, Mor N, Manor YS, Ben-Haim MS, Eyal E, Yunger S, et al. Stem cells. m6A mRNA methylation facilitates resolution of naive pluripotency toward differentiation. Science. 2015; 347: 1002-6; Wang X, Feng J, Xue Y, Guan Z, Zhang D, Liu Z, Gong Z, Wang Q, Huang J, Tang C, Zou T, Yin). P.Structuralbasis ofN(6)-adenosine methylation by the METTL3-METTL14 complex.Nature.2016;534:575-8;Wang X,Lu Z,Gomez A,Hon GC,Yue Y,Han D,Fu Y,Parisien M,Dai Q,Jia G,Ren B,Pan T,He C.

[0007] N6-methyladenosine-dependent regulation of messenger RNA stability. Nature. 2014; 505:117-20). Methylation modification and related enzymes are closely related to lipid metabolism. Its effects on hepatic lipid metabolism are reported to be widespread in eukaryotes, but its effects on ACOT2 are rarely reported. Summary of the Invention

[0008] The present invention is based on the above research and, in response to the phenomena of liver function impairment and delayed liver-to-somatosal recovery that may occur after partial hepatectomy, provides the discovery of the promoting role of ACOT2 upregulation in postoperative liver regeneration and repair, as well as the dual regulatory pathway of this gene. It further provides the application of adeno-associated virus injection to influence ACOT2 expression and promote liver function recovery after partial hepatectomy.

[0009] After extensive and in-depth research, the inventors discovered that Acot2 may be involved in the lipid metabolism process of liver regeneration after hepatectomy and is closely related to the digestion of lipid droplets. Further studies have shown that overexpression of Acot2 significantly increases Acot2 protein levels in mouse livers, significantly reduces liver injury-related indicators such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and increases the liver-to-body ratio. Further research has revealed that the regulatory pathways of this gene are conditioned by the formation of a heterodimeric complex formed by PPARα:RXRα at the transcriptional level. Further studies have revealed that ACOT2 transcription levels can be upregulated through m6A modification, first by reducing the content of the m6A methylation modification enzyme Mettl14 and second by reducing the expression level of the m6A methylation reader protein YTHDF2.

[0010] The technical solutions adopted in the present invention are as follows:

[0011] In a first aspect, the present invention provides the use of an ACOT2 expression promoter in the preparation of a drug for promoting liver regeneration and liver function recovery after partial hepatectomy.

[0012] Preferably, the ACOT2 expression-promoting agent is an agent that directly or indirectly overexpresses ACOT2. The agent that directly overexpresses ACOT2 is selected from a recombinant expression vector that overexpresses ACOT2 or a substance that upregulates ACOT2 protein activity; the agent that indirectly overexpresses ACOT2 is selected from an agent that overexpresses the transcription factor PPARα or reduces the m6A modification level of ACOT2 mRNA.

[0013] Among them, the recombinant expression vector for overexpressing ACOT2 includes a plasmid, adeno-associated virus or CRISPR / Cas9 gene editing tool that overexpresses ACOT2 gene transcripts;

[0014] The reagent for overexpressing the transcription factor PPARα is a heterodimer complex formed by PPARα:RXRα; the reagent for reducing the m6A modification level of ACOT2 mRNA is a reagent for reducing the content of the m6A methylation modification enzyme Mettl14.

[0015] Specifically, the nucleotide sequence of the reagent (shRNA) for reducing the Mettl14 enzyme content is as described in any one of SEQ ID NOs. 5 to 10.

[0016] Conversely, the agent that reduces ACOT2 is a plasmid that overexpresses transcripts of the m6A reader protein YTHDF2 gene.

[0017] In the present invention, indirect overexpression of ACOT2 is a discovery of a dual regulatory pathway for the ACOT2 gene. The first regulatory pathway is to provide a pathway for upregulating ACOT2 expression at the transcriptional level, including overexpressing the transcription factor PPARα (NCBI Gene ID: 19013), such as by introducing a plasmid vector into cells, preferably a plasmid recombinant expression vector that forms a heterodimer complex of PPARα:RXRα. The second regulatory pathway is to upregulate ACOT2 transcription levels through m6A modification, which can be divided into two ways:

[0018] (1) Knockdown of m6A methylation modification enzyme METTL14 (NCBI Gene ID: 210529);

[0019] (2) Knockdown of the m6A methylation reader protein YTHDF2 (NCBI Gene ID: 213541).

[0020] Furthermore, the knockdown is achieved by using a lentivirus as a vector to infect cells.

[0021] Preferably, the partial liver resection is hemihepatectomy or staged liver resection combined with liver segmentation and portal vein ligation. Specifically, drugs that promote liver regeneration and liver function recovery include drugs that reduce intracellular lipid accumulation, lower the levels of alanine aminotransferase and aspartate aminotransferase, promote hepatocyte mitosis, and increase the liver-to-body ratio.

[0022] In a second aspect, the present invention provides a pharmaceutical composition for promoting liver regeneration and liver function recovery, comprising an active ingredient and a pharmaceutically acceptable carrier. The active ingredient comprises an agent that directly or indirectly overexpresses ACOT2, and the specific type is selected as described above.

[0023] The beneficial protection and effects of the present invention are as follows:

[0024] 1. This invention has pioneered new discoveries about the function of the ACOT2 gene. Increased expression of ACOT2 after hepatectomy has a certain protective effect on liver regeneration. ACOT2 may serve as a potential marker for early warning of the degree of postoperative liver damage and liver regeneration capacity, as well as a therapeutic target for promoting postoperative liver regeneration.

[0025] 2. By using the intervention method of the present invention, the decomposition and consumption of lipid droplets in the liver lipid metabolism process is accelerated in a targeted manner, thereby promoting the recovery of liver function and liver-to-body ratio.

[0026] 3. The present invention provides two ways to regulate ACOT2 content after hepatectomy, namely, transcriptional activation of PPARα and post-transcriptional regulation involving m6A-RNA methylation modification. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Detection of changes in ACOT2 expression during the early stage of liver regeneration after partial hepatectomy in Example 1: (A) Changes in the transcriptional levels of Acot family members before and 24 hours after hepatectomy; (B) Changes in the transcriptional levels of Acot2 at various time points during the early stage of liver regeneration. No significant difference; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

[0028] Figure 2 Figure 2 shows the expression of ACOT2 in mice after tail vein injection of AAV8 adeno-associated virus (AAV8) in Example 2: (A) Immunoblotting results of Acot2 in mouse livers after injection of AAV8-shAcot2 or AAV8-oeAcot2, or their control virus. shAcot2, Acot2 knockdown group; oeAcot2, Acot2 overexpression group.

[0029] Figure 3 Effects of ACOT2 knockdown or overexpression on mouse liver regeneration in Example 3: (A) Effects of Acot2 knockdown in mouse liver on liver-to-body ratio, hepatocyte proliferation, and liver injury markers; (B) Effects of Acot2 overexpression in mouse liver on liver-to-body ratio and liver injury markers. shAcot2, Acot2 knockdown group; oeAcot2, Acot2 overexpression group. PH, hepatectomy; ns, no significant difference; *P < 0.05.

[0030] Figure 4 Figure 4 shows the regulation of ACOT2 transcription by the transcription factor PPARα during liver regeneration after hepatectomy: (A) Relationship between cellular levels of PPARα and ACOT2; (B) Relationship between Pparα and Acot2 12 hours after hepatectomy and in mice under fasting conditions; (C) Changes in the expression of Pparα and Rxrα in relation to Acot2. No significant difference; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

[0031] Figure 5Figure 5 shows the correlation between changes in Acot2 m6A modification and its content in post-hepatectomy liver regeneration tissue: (A) Changes in m6A modification levels after hepatectomy in mice; (B) Changes in related methylation-modifying enzymes after hepatectomy in mice. Ctrl, control group; Sham, sham-operated group; PH12h, 12 hours after hepatectomy; IP, coimmunoprecipitation; Input, positive control. No significant difference; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

[0032] Figure 6 Figure 6 shows the regulation of m6A modification, transcription, and protein levels of Acot2 mRNA by the methyltransferase Mettl14 in Example 6: (A) Immunoblotting of Acot2 in the livers of Mettl14 conditional liver knockout mice; (B) qPCR of Acot2 in the livers of Mettl14 conditional liver knockout mice; (C) qPCR of PPARα in the livers of Mettl14 conditional liver knockout mice; (D) Immunoblotting of PPARα in the livers of Mettl14 conditional liver knockout mice; (E) Immunofluorescence and immunohistochemistry of Acot2 in Mettl14 conditional liver knockout mice. WT, wild-type mice; KO, Mettl14 conditional liver knockout mice. *P < 0.05; **P < 0.01.

[0033] Figure 7 Figure 7 shows the relationship between METTL14 and ACOT2 expression at the transcriptional and protein levels in the in vitro experiments. (A) METTL14 knockdown in AML12 and HepG2 cells; (B) Changes in ACOT2 expression in AML12 and HepG2 cells after METTL14 knockdown. shNC, control group; shM14, METTL14 knockdown group. Ctrl, control group; Ketogenic, ketogenic medium-treated group. No significant difference; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

[0034] Figure 8In Example 8, YTHDF2 is one of the readers of ACOT2 m6A modification: (A) PCR results of ACOT2 after anti-YTHDF2 RIP in AML12 and HepG2 cells; (B) Changes in ACOT2 after YTHDF2 overexpression; (C) Results of YTHDF2 RIP after Mettl14 knockdown. shNC, control group; shM14, METTL14 knockdown group. oeVec, empty vector control group; oeYTHDF2, YTHDF2 overexpression group. No significant difference; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. DETAILED DESCRIPTION

[0035] The following embodiments of the present invention are used to describe the implementation of the present invention in detail. The following embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operating procedures. However, the protection scope of the present invention is not limited to the following embodiments.

[0036] Example 1 Detection of ACOT2 expression changes in the early stage of liver regeneration after partial hepatectomy

[0037] 1. Experimental methods:

[0038] 1. Mouse 2 / 3PHx surgery:

[0039] Partial hepatectomy in mice was performed as previously reported. After anesthetizing mice with 1% sodium pentobarbital, a midline laparotomy was performed. Two-thirds of the liver (left lateral and middle lobes) were removed by ligating the corresponding branching vessels, while the right superior, right inferior, and caudate lobes were retained. The abdomen was closed for observation. Following these procedures, samples were collected at specific time points and cryopreserved or fixed.

[0040] 2. Fluorescence quantitative PCR

[0041] (1) Total RNA extraction from tissues:

[0042] This part uses an RNA rapid extraction kit. The usage of the kit is briefly described as follows: First, 30 mg of liquid nitrogen-frozen tissue was cut into 100 ul of normal saline, small steel balls were added, and the mixture was ground in a tissue grinder at -10°C for 2 minutes; after a brief centrifugation, the liquid was transferred to a new EP tube, 500 ul of RA2 solution was added, and the tube was immediately inverted to mix until the solution was clear, and then allowed to stand at room temperature for 1 minute; the RA2-treated sample was transferred to a purification column, centrifuged at 12,000 rpm at 4°C for 1 minute, and the waste liquid was discarded; 500 ul of Wash Buffer was added to the purification column, centrifuged at 12,000 rpm at 4°C for 1 minute, and the waste liquid was discarded; wash again as above; the empty purification column was centrifuged again for 1 minute; the purification column was transferred to a new 1.5 ml collection tube, 50 ul of Elute Buffer was added, the tube was allowed to stand for 1 minute, and centrifuged at 14,000 rpm for 1 minute; finally, the RNA concentration and quality were measured using Nanodrop and the sample was frozen at -80°C.

[0043] (2) Reverse transcription:

[0044] This study used a gDNA-removed reverse transcription reagent as follows: To remove genomic DNA, prepare the reaction mixture on ice: 1 μg total RNA, 4 μl of 4x genome wiper, and make up to 16 μl of DEPC-treated water. The reaction conditions used were: incubation at 42°C for 2 minutes, followed by cooling on ice. The reverse transcription reaction mixture consisted of adding 4 μl of 5xHiScript Enzyme Mix to the above mixture. The reaction conditions were: 37°C for 15 minutes, 85°C for 5 seconds, and finally cooling to 4°C.

[0045] (3) Real-time quantitative fluorescence PCR:

[0046] Prepare the reaction system on ice: 10 μl of SYBR Green Mix; 0.4 μl of Forward Primer (10 μM); 0.4 μl of Reverse Primer (10 μM); 2 μl of cDNA; and 7.2 μl of sterile ddH₂O, for a total of 20 μl. Reaction conditions were as follows: pre-denaturation phase (1 cycle) at 95°C for 5 min; cycling phase (40 cycles) at 95°C for 10 sec, annealing / extension at 60°C for 30 sec; and melting curve phase (1 cycle). Finally, gene expression was calculated using the ΔΔCt method.

[0047] 2. Experimental results:

[0048] We examined the changes in the transcript levels of acyl-CoA thioesterases located in the mouse Acot family, attempting to explore whether Acot family proteins participate in the mitochondrial β-oxidation process and play a role in the early regeneration period when lipids enter the liver in large quantities and the energy supply and demand balance fluctuates greatly. Among the six Acots reported to be located in the mitochondria, Acot2 was significantly increased, while Acot11 and 13 were significantly decreased ( Figure 1 A) This suggests that Acots on mitochondria may be associated with liver regeneration.

[0049] Based on the above findings, we conducted more intensive time-point detection on the three Acot genes with significant changes and found that the change trend of Acot2 among the three genes was very consistent with the dynamic changes of lipid droplets, that is, it began to increase 4 hours after surgery, gradually increased at 8 hours, reached the maximum at 12 hours, and then gradually decreased, and tended to recover at 48 hours ( Figure 1 B). Therefore, we believe that Acot2 may be involved in the lipid metabolism process of liver regeneration after hepatectomy and is closely related to the digestion of lipid droplets.

[0050] Example 2 Establishment of ACOT2 Liver-Specific Knockdown or Overexpression Mice

[0051] 1. Experimental methods:

[0052] 1. Tail vein injection of adeno-associated virus (AAV8-shAcot2 or AAV8-oeAcot2):

[0053] Table 1 Summary of Acot2 shRNA sequences

[0054]

[0055] The control group was pscAV-U6-CMV-GFP with a nonsense sequence inserted. The adeno-associated virus treatment group (AAV8-shAcot2) was a mixture of four sequences or a CMV promoter followed by the CDS region sequence of Acot2 (NCBI Gene ID: 171210) plus a FLAG tag (AAV8-oeAcot2). All AAV products were synthesized by Virgen. The virus titers of the treatment and control groups were diluted to 5×10 12 vg / ml, 200ul of each mouse was injected through the tail vein, and after successful injection, the mice were separated into cages and marked. After 14 days of feeding, partial liver resection was performed (as described above).

[0056] 2. Verification of knockdown or overexpression effects:

[0057] Tissue extraction and immunoblotting were performed to detect the knockdown or overexpression of ACOT2 protein in the liver. The tissue and cell protein extraction methods used in this study were all performed using the Yazyme PC201 kit, as briefly described below:

[0058] (1) Tissue protein extraction:

[0059] After removing the mouse liver from the living body, quickly rinse it with 1XPBS solution, wipe off excess water and place it in a labeled cryovial, immediately drop it into liquid nitrogen for quick freezing, and then store it at -80℃; before extracting protein, add PMSF and protease inhibitors at a ratio of 100:1 to the denaturing lysis buffer, quickly cut about 15-20mg of liver tissue on ice, place it in 200ul of lysis buffer, add 2 small steel balls, and grind it in a tissue grinder for 2min until no tissue pieces are visible to the naked eye; add the ground tissue suspension to the purification column, let it stand at room temperature for 2min, and then centrifuge it at 14000rpm in a 4℃ centrifuge for 2min; add an appropriate amount of 5xloading buffer to the purified tissue lysate, mix well, and boil it at 100℃ for 10min; finally, divide the sample into portions and freeze it at -80℃.

[0060] (2) Protein quantification:

[0061] Dilute 5 μl of the purified lysate with 20 μl of saline and add to a 96-well plate for protein quantification. Create a standard curve by diluting 2 mg / ml BSA standard with saline to 2, 1.5, 1, 0.75, and 0.5 mg / ml at 25 μl each and add to a 96-well plate. Mix colorimetric reagents A and B at a ratio of 50:1, add 200 μl to each well of the 96-well plate, incubate at 37°C for 30 minutes, and measure absorbance at 562 nm using a microplate reader. Calculate the protein concentration based on the standard curve.

[0062] (3) Immunoblotting:

[0063] Mount a 4-20% gradient precast gel in an electrophoresis tank. Load 30-50 μg of the aforementioned protein sample per well. Fill with MOPS running buffer and run at a constant voltage of 150 V for 1 hour. Remove the gel after electrophoresis, disassemble the plastic, and place the gel in pure ddH2O. Cut a PVDF membrane of the desired size, activate it in methanol, and then soak it in PVDF membrane equilibration buffer. Create a "sandwich" (from positive to negative): sponge-membrane-gel-sponge. Transfer the membrane using Transfer Buffer in the standard setting of a rapid wet transfer apparatus for 15 minutes. Remove the transferred PVDF membrane from the "sandwich" and quickly place it in 5% skim milk or ultrasensitive blocking buffer for 2 hours. After blocking, wash the membrane three times with PBST (5 minutes each). Washing is not required if ultrasensitive blocking buffer is used. Cut the membrane to identify the desired molecular weight band and place it in an incubator. Add the diluted primary antibody and incubate overnight at 4°C. Remove the strips incubated with primary antibodies from the 4°C refrigerator, recover the primary antibodies, wash the membrane three times with PBST, add the corresponding fluorescent secondary antibody, and incubate at room temperature in the dark for 1 hour. Finally, wash the strips incubated with secondary antibodies three times with PBST and image them on a multi-color fluorescent gel imaging analyzer. The reagents used in the detection process are shown in Table 2 below:

[0064] Table 2 Summary of reagents used in immunoblotting

[0065]

[0066] 2. Experimental results:

[0067] See also Figure 2 , the Acot2 protein level in the liver of mice in the AAV8-shAcot2 group was significantly reduced, while the Acot2 protein level in the AAV8-oeAcot2 group was significantly increased.

[0068] Example 3 Effects of ACOT2 knockdown or overexpression on liver regeneration in mice

[0069] 1. Experimental methods:

[0070] 1. Measurement of liver weight, body weight, and liver-to-body ratio

[0071] At specific time points during liver regeneration, mice were weighed and sacrificed. The livers were removed and rinsed with 1x PBS to remove excess blood. The livers were then dehydrated and weighed. Liver-to-body ratio = liver weight (g) / body weight (g).

[0072] 2. Blood biochemistry test

[0073] At the end of treatment, mice were bled using the eye enucleation method, with the mice anesthetized before blood collection. After clotting for 20 minutes at room temperature, the blood was centrifuged at 3000 rpm for 20 minutes at 4°C, and the supernatant was stored at -80°C. Alanine aminotransferase and aspartate aminotransferase were measured using an automated biochemical analyzer. Alanine aminotransferase was measured using the alanine substrate method, and aspartate aminotransferase was measured using the aspartate substrate method.

[0074] 2. Experimental results:

[0075] We synthesized an adeno-associated virus that knocked down Acot2 and injected it into the tail vein of mice. Hepatectomy was performed 14 days after the injection. We found that 24 hours (PH24h) and 36 hours (PH36h) after hepatectomy in wild-type mice, the number of Ki67-positive hepatocytes in AAV-shAcot2 mice was significantly less than that in the control group, indicating a delay in the peak of hepatocyte proliferation and a slightly smaller liver-to-body ratio than in the control group. However, liver injury-related indicators ALT and AST were significantly higher than those in the control group. In addition, more lipid droplets accumulated in hepatocytes than in the control group ( Figure 3 A). Injection of the virus overexpressing Acot2 via the tail vein can reduce liver damage-related indicators ALT and AST, and the liver volume at 24 hours after liver resection (PH24h) is slightly higher than that of the control group ( Figure 3 B).

[0076] Example 4 Overexpression of transcription factor PPARα upregulates ACOT2

[0077] 1. Experimental methods:

[0078] 1. Fasting of mice:

[0079] Mice were fasted for 24 h as previously described, with food removed but free access to water. Blood and liver samples were collected after fasting and fixed or cryopreserved.

[0080] 2. Mouse liver resection (same as above)

[0081] 3. Cell culture experiments

[0082] (1) HepG2 and 293T cell culture:

[0083] Use standard DMEM high-glucose medium supplemented with 10% FBS and 1% penicillin-streptomycin solution (P / S) and culture at 37°C in a 5% CO2 incubator. Passage cells at a ratio of 1:3-1:5 every 2-3 days. Cryopreserve cells using serum-free freezing medium at -80°C or in liquid nitrogen. Resuscitate cells in a 37°C water bath.

[0084] (2) AML12 cell culture:

[0085] Use DMEM / F12 basal medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution. Add ITS solution (10 μg / ml insulin, 5.5 μg / ml transferrin, 5 ng / ml selenium) and dexamethasone to a final concentration of 40 ng / ml for routine culture. The cell culture ratio is 1:3. All other procedures are the same as above.

[0086] (3) Preparation of ketogenic medium:

[0087] The ketogenic culture medium was prepared with reference to previous literature. The serum and insulin were removed from the basal culture medium of AML12, and 50 μM WY-14643 and 2 mM sodium octanoate were added and cultured for a specific time.

[0088] 4. Extraction of total RNA from cells:

[0089] This part uses the TRIzol method for RNA extraction. The method is briefly described as follows:

[0090] Discard the cell culture medium, wash once with 1x PBS buffer, immediately add 1 ml of TRIzol, and shake the culture dish to fully lyse the cells; pipette TRIzol into an RNase-free 1.5 ml eppendorf tube and let it stand at room temperature for 5 minutes; then add 200 μl of chloroform, tightly cap the tube and shake vigorously for 15 seconds, let it stand at room temperature for 5 minutes, and then centrifuge at 12,000 g and 4°C for 15 minutes; transfer the colorless supernatant layer of the layered homogenate to a new eppendorf tube, add 800 μl of isopropanol, mix by inversion, let it stand at room temperature for 10 minutes, and centrifuge at 12,000 g and 4°C for 10 minutes; discard the supernatant, wash the precipitate with 1 ml of 75% ethanol, and centrifuge at 7,500 g and 4°C for 5 minutes; discard the supernatant, dry the precipitate for 10 minutes, and dissolve it in 30-50 μl of DEPC-treated water; finally, measure the concentration and quality of RNA using Nanodrop and freeze it at -80°C.

[0091] 5. Cell protein extraction:

[0092] Digest the cells with trypsin and collect them by centrifugation. Resuspend them in ice-cold PBS and centrifuge them once. Use lysis buffer supplemented with PMSF and protease inhibitors before use, adjusting the amount of lysis buffer based on the cell population. Resuspend the cell pellet until there are no clumps. Subsequent steps are the same as for tissue protein extraction.

[0093] 6. Fluorescence quantitative PCR and immunoblotting (same as above). The reagents used are shown in Table 3:

[0094] Table 3 Summary of reagents used for quantitative PCR and immunoblotting

[0095]

[0096] 7. Plasmid construction

[0097] The vector used for mouse Ppara overexpression plasmid was pcDNA3.1 / V5-His A, and the selected restriction enzyme cutting sites were BamH I and Xho I. The vector used for mouse Rxra overexpression plasmid was pCMV-N-Flag, and the selected restriction enzyme cutting sites were EcoR I and BgI II.

[0098] (1) PCR gene fragment:

[0099] PCR primers were designed based on the CDS region sequence of the target gene found in NCBI and the vector used; then, the reaction system was prepared on ice: 2X PhantaMax Master Mix 25ul; upstream primer (10uM) 2ul; downstream primer (10uM) 2ul; cDNA 2ul; ddH2O filler 50ul; the reaction was carried out according to the following conditions: pre-denaturation (1 cycle) 95℃ 3min; cyclic (35 cycles) denaturation 95℃ 15sec → annealing 60℃ 15sec → extension 72℃ 1min / kb; complete extension (1 cycle) 72℃ 5min; after the reaction is completed, the PCR product was stored at 4℃ until use.

[0100] (2) Vector linearization:

[0101] Prepare the reaction system on ice: pcDNA3.1 plasmid 1ug, BamH I 1ul, Xho I 1ul, 10X Kbuffer 2ul, and make up to 20ul with sterile water; pCMV-N-Flag 1ug, QuickCut EcoR I 1ul, QuickCut BgI II 1ul, 10X QuickCut Buffer 2ul, and make up to 20ul with sterile water; select the buffer and enzyme digestion time according to the restriction site of the fragmented vector: EcoR I and BgI II are QuickCut restriction enzymes, and their activities in the universal buffer (10X QuickCut Buffer) are both 100%. Considering the reaction time of the two, choose 37°C water bath for 15min; BamH I and Xho I are common restriction enzymes, and Xho I has 100% activity in both H and Kbuffer, while BamH I has 80% activity in H buffer and 100% activity in Kbuffer. Based on the above two, choose Kbuffer. Incubate in a 37°C water bath for 1 h; upon completion of the reaction, place at 4°C for later use.

[0102] (3) Rubber recovery and purification:

[0103] Purify the PCR product and linearized vector by gel recovery. Run the PCR product and linearized vector on an agarose gel. After electrophoresis, excise the gel at the corresponding position, remove excess water, mince with a clean blade, and place in a new 1.5ml eppendorf tube. Tarre the empty tube and weigh the gel. Add the corresponding volume of Buffer GDP in grams. Heat in a 55°C water bath for 10 minutes until all gel pieces are fully dissolved, turning the tube upside down to check the degree of dissolution; place the adsorption column provided in the kit in a 2ml collection tube, transfer 700ul of gel solution at a time, centrifuge at 12000rpm for 1min, and discard the waste liquid until all the gel solution has passed through the adsorption column; add 300ul of Buffer GDP, let it stand for 1min, and centrifuge at 12000rpm for 1min; add 700ul of Buffer GW, press the tube cap tightly, turn it upside down 2-3 times, and centrifuge at 12000rpm for 1min; repeat once; discard the waste liquid and centrifuge the empty tube at 12000rpm for 2min; transfer the adsorption column to a new tube, add 30ul of elution buffer to the center of the membrane, let it stand at room temperature for 2min, and then centrifuge at 12000rpm for 1min; the concentration of the purified product was measured using Nanodrop.

[0104] (4) Recombination reaction:

[0105] Recombine the purified PCR product with the linearized vector. Calculate the amounts of PCR product and linearized vector required for the recombination reaction: PCR product (ng) = 0.02xbp; vector (ng) = 0.04xbp. Based on the concentrations measured in the gel recovery step, calculate the volumes X and Y, respectively. Then, prepare the reaction mixture on ice: insert (Xµl); linearized vector (Yµl); 5xCE II buffer (4µl); Exnase II (2µl), and ddH2O (20µl) to make up the volume. Mix thoroughly by pipetting. Finally, incubate at 37°C for 30 minutes and place on ice.

[0106] (5) Conversion coating:

[0107] Thaw DH5α on ice, add 5ul of the recombinant reaction product to 50ul of DH5α, gently pipette to mix, and let it stand on ice for 30min; heat shock in a 42℃ water bath for 45sec, and cool on ice for 2min; add 950ul of LB medium, shake at 37℃ and 220rpm for 1h, during which time preheat the solid plate containing resistance; centrifuge at 5000rpm for 5min, discard 900ul of the supernatant, resuspend in the remaining 100ul, and evenly spread on the plate; finally, place in a 37℃ incubator and incubate for 12h.

[0108] (6) Sequencing:

[0109] Select single clones from the plate, add corresponding antibiotics to LB, shake 5 ml microcentrifuge, and extract 1 ml of the bacterial solution for sequencing.

[0110] (7) Plasmid extraction:

[0111] Medium-volume and large-volume extractions were used, all based on the alkaline lysis method. The steps were based on the instructions of the kit used and are summarized as follows: 200 ml of bacterial solution with the corresponding antibiotics was added to the overnight culture, and the precipitate was collected after centrifugation; solution I was used to fully resuspend the bacterial solution to avoid agglomeration; alkaline SDS solution II was added to lyse the bacterial solution, and the solution was gently inverted, paying attention to the time; acid solution III was added to neutralize the alkaline solution to precipitate some proteins and most of the genomic DNA; the supernatant was obtained by filtration or centrifugation; the supernatant was ice-bathed with ETR solution or similar to prevent endotoxins from binding to the resin to remove endotoxins; the plasmid was purified using a purification column, and finally eluted and recovered in a sterile 1.5 ml ep tube; the concentration and quality of the plasmid were tested.

[0112] 2. Experimental results:

[0113] First, we examined the changes in PPARα levels in two previous animal models and found that PPARα transcripts increased 12 h after liver resection and 24 h after fasting ( Figure 4 B). In addition, we generated a gradient change in ACOT2 expression in human 293T and HepG2 cells by adding sodium octanoate in a gradient manner to the ketogenic medium. Under this gradient change, PPARA still showed a trend of co-upregulation with ACOT2 ( Figure 4 A). Therefore, we believe that there may be regulation between the two. Given the powerful transcriptional function of PPARα in fatty acid metabolism, we constructed a Ppara overexpression plasmid and transfected mouse hepatocytes to detect changes in Acot2 content. The content of AML12 cells increased significantly after Ppara transfection, but Acot2 only changed by 1.1 times and was not significant. On this basis, we overexpressed the PPARα heterodimer RXRα and detected the Acot2 content by PCR and WB. We found that compared with the transfection control plasmid, overexpression of the dual transcription factor increased Acot2 by nearly 4 times ( Figure 4 C). Therefore, we believe that the formation of a heterodimeric complex of PPARα:RXRα is the condition for the increase of Acot2.

[0114] Example 5 Changes in mA modification of ACOT2 mRNA during liver regeneration after hepatectomy

[0115] 1. Experimental methods:

[0116] 1. m6A-RIP

[0117] (1) RNA fragmentation:

[0118] First, extract total RNA from tissue (60-80mg of liver tissue) using the TRIzol method, requiring approximately 300µg of total RNA. After determining the concentration, adjust the RNA concentration to approximately 1µg / µL using DEPC-treated water. Aliquot the total RNA into 200µl PCR tubes, i.e., 18µl / tube, for a total of 17 tubes. Add 2µl of 10X Fragmentation Buffer to each tube, mix thoroughly by pipetting, briefly centrifuge, and place on ice. Preheat a thermal cycler to 94°C, processing 5 tubes at a time. Cover and heat for 5 minutes, then remove from the block. Immediately add 2µl of 0.5M EDTA, vortex, centrifuge, and place on ice. Repeat this operation until all RNA is fragmented; collect all fragmented RNA into a 1.5ml ep tube, add 30ul 3M sodium acetate (pH 5.2), 1.6ul 20mg / ml glycogen and 750ul anhydrous ethanol, mix by inversion, and place at -80℃ overnight to precipitate nucleic acids; after removal, centrifuge at 15000g 4℃ for 25min, carefully discard the supernatant, add 1ml 75% ethanol to wash the precipitate, and centrifuge at 15000g 4℃ for 15min; carefully discard the supernatant, air-dry the precipitate and dissolve it in 335ul DEPC-treated water; use Nanodrop to detect the concentration of fragmented RNA, and use 1.5% agarose gel to detect the RNA size distribution (mainly 100nt); after measurement, divide 30ul (10% Input) into a new tube, mark it as Input, and freeze it at -80℃; the remaining part will be used for IP later.

[0119] (2) Immunoprecipitation:

[0120] Dilute 5×IP buffer with DEPC-treated water and prepare 5 ml of 1x IP buffer for each sample; prepare new 1.5 ml EP tubes according to the number of samples and mark them. After fully resuspending the protein A / G magnetic beads, add 50ul to each tube, then resuspend them with 500ul IP buffer and gently pipette to mix several times; place the 1.5ml ep tube containing magnetic beads on a magnetic rack for 1 minute, aspirate the supernatant, and place it on ice; repeat the above two steps to wash again; resuspend the magnetic beads with 200ul 1x IP buffer, add 10ug anti-m6A antibody, and incubate with rotation at room temperature for 30min; after incubation, briefly centrifuge the ep tube, then place it on a magnetic rack for 1min, aspirate the supernatant; gently wash the magnetic beads three times with 500ul 1X IP buffer. After the last wash, only the magnetic beads remain, cover the tube tightly and place it on ice; prepare the IP reaction system: 300ug fragmented RNA, 10ul RNase inhibitor, 200ul 5X IP buffer, and make up to 1ml with DEPC-treated water, and add it to the magnetic beads coated with m6A antibody in the previous step to thoroughly resuspend the magnetic beads. Incubate with rotation at 4°C overnight; remove the magnetic beads after incubation, wash them three times with 500ul 1X IP buffer, and place on ice for immediate elution.

[0121] (3) Elution:

[0122] Before the experiment, dissolve 20mM m6A5' monophosphate sodium salt in 1.3mL DEPC-treated water, aliquot 150µl / tube, and store at -20°C. Thaw immediately before use. Prepare elution buffer (per sample): 45µl 5X IP buffer, 75µl 20mM m6A salt, 3.5µl RNase inhibitor, and 101.5µl DEPC-treated water, for a total of 225µl. Add 100µl of elution buffer to the magnetic beads washed in step 1 and gently resuspend the beads. Place in a 4°C constant-temperature shaking metal bath at 550rpm for 1 hour. After shaking, briefly centrifuge the tube and place on a magnetic stand for 1 minute. Transfer the eluted fragmented RNA to a new 1.5mL tube. Repeat this process to obtain a total of 200µl of elution product.

[0123] (4) RNA purification:

[0124] RNA was purified using the RNeasy (mini) kit as follows: 200 μl of the eluted product was transferred to a 15 ml centrifuge tube and 700 μl of Buffer RLT was added and mixed thoroughly; 1400 μl of 98% ethanol was added and pipetted to mix thoroughly; 700 μl of the sample was added to an RNeasy MinElute purification column (previously placed in a 2 ml collection tube), centrifuged at 10,000 rpm for 30 seconds, and the waste liquid was discarded. Repeat this step until all samples have passed through the purification column; transfer the RNeasy MinElute purification column to a new 2ml collection tube, add 500ul of Buffer RPE, centrifuge at 10,000rpm for 30s to wash the purification column membrane, and discard the waste liquid; add 500ul of 80% ethanol to the purification column, centrifuge at 10,000rpm for 2min, and discard the waste liquid; open the purification column and centrifuge at 12,000rpm for 5min, and discard the waste liquid; place the purification column in a new 1.5ml collection tube, add 14ul of DEPC-treated water to the center of the membrane, tightly cap the tube, and centrifuge at 15,000rpm for 1min to elute the RNA.

[0125] (5) PCR analysis:

[0126] This experiment uses One Step TB PrimeScript TM , that is, reverse transcription and PCR are in the same system, and the reverse transcription primers are the same as the PCR primers. The reaction system was prepared on ice: 2X One Step TB Green RT-PCR Buffer 4, 10ul; Prime Script 1Step Enzyme Mix 2, 0.8ul; Forward Primer (10uM), 0.8ul; Reverse Primer (10uM), 0.8ul; ROX Reference Dye (50X), 0.4ul; total RNA, 2ul; DEPC-treated water, 5.2ul; a total of 20ul; reaction conditions were as follows: stages one and two were reverse transcription reactions (1 cycle): 42°C for 5min; 95°C for 10sec; stage three was PCR reaction (40 cycles): 95°C for 5sec; 60°C for 30sec; stage four was melting curve stage; the obtained Ct value was calculated using the following formula: %Input = 2(-ΔCt[normalized IP]), -ΔCt[normalized IP] = CtIP-(CtInput-Log2[Input Dilution Factor]), where Input Dilution Factor=10.

[0127] 2. Tissue protein extraction and immunoblotting (same as above). The reagents used are shown in Table 4:

[0128] Table 4 Summary of reagents used for tissue protein extraction and immunoblotting

[0129]

[0130] 2. Experimental results:

[0131] Through the m6A-RIP-PCR experiment on the liver tissue of treated mice, we found that the abundance of m6A modification of Acot2 was significantly decreased (liver resection 12h group vs control group, P < 0.0001; fasting group vs control group, P < 0.0001) ( Figure 5 A). Combined with the reported m6A-modified methylases, we further detected the changes in the content of the major methylation modification enzymes Mettl3, Mettl14, Fto, and Ythdf2 by immunoblotting 12 hours after liver resection and found that Mettl14 was the methylation enzyme with the most significant change ( Figure 5 B).

[0132] Example 6 Knockout of Mettl14 reduces m6A modification of Acot2 mRNA and increases its transcription level and protein content

[0133] 1. Construction of Mettl14-flox; Alb-cre knockout mice

[0134] The knockout mice we constructed were the ones in which the second exon region of Mettl14 was excised by Cre enzyme mediated by albumin promoter.

[0135] 2. Genotype identification

[0136] (1) Toe number:

[0137] Three weeks after birth, mice were weaned and separated into cages. Their toes were clipped and numbered. The left forepaw was labeled with the digits, the right forepaw with the tens, and both hind paws were labeled with the hundreds from left to right. The clipped toes and tails were placed in a clean 1.5ml EP tube and frozen at -20°C pending DNA extraction and genotyping.

[0138] (2) Tissue DNA extraction. The reagents used are shown in Table 5:

[0139] Table 5 Summary of reagents used for tissue DNA extraction

[0140]

[0141] Prepare SNET buffer as shown in the table above and add proteinase K at a ratio of 50:1. Mix thoroughly and add 200 μl / tube to the pre-warmed tube containing the mouse tail and toe. Lyse the tube overnight in a 56°C incubator. Remove the tube and let it sit at room temperature for 5 minutes. Invert and mix twice. Centrifuge at 13,000 rpm for 10 minutes at room temperature. Transfer the supernatant to a new pre-warmed tube. Add 200 μl / tube of isopropanol and invert approximately 10 times to mix until a white flocculent mass appears. Centrifuge at 12,000 rpm for 10 minutes at room temperature and discard the supernatant. Wash the pellet with 500 μl of 75% ethanol per tube and centrifuge at 12,000 rpm for 5 minutes at room temperature. Aspirate the supernatant and air-dry for 10 minutes. Dissolve the DNA in 100 μl of ddH2O and heat in a 60°C metal bath for 2 hours to promote dissolution. Flick the pre-warmed tube to mix and check for dissolution. Assay the genomic DNA concentration and quality using a Nanodrop analyzer.

[0142] (3)PCR:

[0143] A 25ul reaction system was prepared according to the genotype identification product used: 100ng of mouse genomic DNA, 1uL of each upstream and downstream primers (10uM), 12.5uL of 2×Hieff PCR Master Mix, and the remainder was filled with ddH2O; the reaction conditions were: initial denaturation (1 cycle) at 94°C for 5min; denaturation at 94°C for 30sec, annealing at 60°C for 30sec, extension at 72°C for 30sec / kb, for 35 cycles; final extension (1 cycle) at 72°C for 10min.

[0144] (4) DNA electrophoresis:

[0145] Prepare 1%-3% agarose gel, heat and fully dissolve, cool to approximately 50°C, add 10,000x nucleic acid dye, mix thoroughly, and pour into a mold to cool. Add 10 μl of the above PCR product to each well, perform electrophoresis at 120 V constant voltage for 20 minutes, and image the gel using a UV gel imager. Sequentially check the flox and cre bands to confirm the final genotype of the mouse.

[0146] 3. Fluorescence quantitative PCR, immunoblotting, and m6A-RIP-PCR experimental procedures (same as described above)

[0147] 2. Experimental results:

[0148] We further verified the relationship between Mettl14 and Acot2 by taking tissues from mice with conditional knockout of Mettl14 in the liver. Both quantitative PCR and immunoblotting experiments showed that the transcript and protein levels of Acot2 were significantly increased in the liver tissues of knockout mice ( Figure 6A and B). Fluorescence quantitative PCR and immunoblotting were used to detect the changes in Pparα content in gene knockout mice, but it was found that the knockout of Mettl14 did not induce the upregulation of Pparα ( Figure 6 C and D). Therefore, we believe that the upregulation of Acot2 induced by Mettl14 may not be mediated by Pparα. We used the m6A-RIP method to detect the m6A modification of Acot2 in Mettl14 conditional knockout mice and found that the m6A level of this gene in the liver of Mettl14 knockout mice was significantly reduced ( Figure 6 B). We further confirmed the increase of Acot2 in knockout mice by immunohistochemistry and immunofluorescence, and found that the fluorescence signal of Acot2 mainly came from larger hepatocytes, and there was a regional distribution pattern with the highest content near the central vein ( Figure 6 E) This indicates that knocking out Mettl14 in hepatocytes alters Acot2 expression in parenchymal cells, not in non-parenchymal cells. Furthermore, Mettl14 affects Acot2 expression by affecting m6A modification.

[0149] Example 7 Knockdown of Mettl14 in vitro increases ACOT2 transcription level and protein content

[0150] 1. Experimental methods:

[0151] 1. Construction of stable lentiviral cell lines

[0152] Melt the lentivirus in an ice bath, take the amount needed for this experiment, and aliquot the remaining amount and freeze at -80°C; plate cells in a 6-well plate the day before infection and culture overnight with complete medium; on the day of infection, dilute HitransA with complete medium at 1:24, then add the virus according to the MOI and mix well; discard the old medium, add new culture medium containing HitransA and virus, and culture for 48 hours; replace the new medium and continue to culture for 48 hours, and check the infection effect by fluorescence under a microscope; add 2mg / ml of puromycin at 1:1000 to screen the infected cells for 2 weeks, and verify the effect by PCR and immunoblotting. The knockdown sequences targeted by the lentivirus are as follows in Table 6:

[0153] Table 6 Knockdown sequences targeted by lentivirus

[0154]

[0155]

[0156] Note: All virus products are synthesized by Genechem

[0157] 2. RNA extraction and qPCR (same as above)

[0158] 3. Protein extraction and immunoblotting (same as above)

[0159] 2. Experimental results:

[0160] We selected mouse liver cells AML12 and human hepatoblastoma HepG2 for in vitro culture and detected the expression of Acot2 in these two cells. We found that compared with AML12 cells, HepG2 cells had a higher ACOT2 content, and the Acot2 content of AML12 was consistent with the low content of normal wild-type mice. We designed three knockdown sequences for mouse Mettl14 and human METTL14 for lentiviral coating, and then infected AML12 and HepG2 cells respectively, and screened out successfully infected cells by puromycin. After screening and culturing for 2 weeks, we tested the knockdown effect and found that effective knockdown was achieved in both human and mouse cells (<40%), and as low as 10% in mouse cells ( Figure 7 A). After confirming the knockdown effect, we detected changes in Acot2 and found that in HepG2, the ACOT2 content of shMettl14 cells increased to 1.6 times that of the control group (P = 0.0098), while AML12 cells showed no significant changes under normal conditions. Considering that the background expression of Acot2 in AML12 cells is low, we adopted the ketogenic medium used in the first part, that is, adding sodium octanoate and WY14643 to AML12 cells for stimulation. We found that ketogenic medium can induce a significant increase in Acot2 in the shNC group, and the Acot2 expression level in the shMettl14 group is nearly 3 times that of the control group ( Figure 7 B) When the mRNA level of Acot2 in AML12 cells increased significantly, the effect of Mettl14 on Acot2 was more significant (P<0.0001).

[0161] Example 8 Overexpression of YTHDF2 reduces ACOT2 transcription levels

[0162] 1. Experimental methods:

[0163] 1. RNA-binding protein immunoprecipitation (RIP)

[0164] (1) Cell lysis: Prepare RIP lysis buffer: Add 0.5ul of protease inhibitor and 0.25ul of RNase inhibitor to every 100ul of RIP lysis. Prepare an equal volume of RIP lysis buffer according to the volume of the pellet after centrifugation. Wash the adherent cells twice with 3ml of pre-cooled 1xPBS. After adding 5ml of ice-cold PBS, scrape the adherent cells with a cell scraper and transfer them to a 15ml centrifuge tube. Wash the dish again with another 5ml of ice-cold PBS to collect the remaining cells and transfer them to a centrifuge tube. Centrifuge at 1500rpm at 4℃ for 5min to collect the cells and discard the supernatant. Add an equal volume of RIP lysis buffer to the cell pellet to fully resuspend the pellet and mix by pipetting until homogenous. Lyse on ice for 5min, then aliquot into approximately 200ul / tube and freeze at -80℃.

[0165] (2) Prepare magnetic beads: fully resuspend the magnetic beads, mark the tube for RIP reaction, and add 50ul of magnetic beads to each tube; wash the magnetic beads twice with 500ul of RIP Wash Buffer, and finally resuspend with 100ul; add 5ug of antibody (target antibody Anti-YTHDF2 or IgG) to the magnetic beads and incubate at room temperature for 30min; after incubation, centrifuge briefly, place the ep tube on the magnetic stand for 1min, and discard the supernatant; wash twice with 500ul of Wash Buffer, and finally resuspend the magnetic beads with 500ul and place on ice for subsequent operations.

[0166] Table 7 Antibodies used in the detection process

[0167]

[0168] (3) Immunoprecipitation: Prepare IP buffer: Each 900ul IP buffer consists of 860ul RIP Wash buffer with 35uL0.5M EDTA and 5uL RNase inhibitor. Each sample requires 900ul IP buffer. Place the resuspended magnetic beads on a magnetic stand for 1 minute, aspirate the supernatant, and add 900uL IP buffer. Quickly thaw the first part of the cell lysate, centrifuge at 14000rpm for 10 minutes at 4℃, take 100ul of each 200ul supernatant and add it to the antibody-magnetic bead complex or IgG-magnetic bead complex, so that the total IP system is 1ml. Take 10ul of the remaining lysate supernatant and add it to a new 1.5mlep tube, mark it as Input, and freeze it at -80℃. Place the IP reaction tube in a 4℃ rotating incubation overnight. After incubation, centrifuge briefly, place it on a magnetic stand for 1 minute, and aspirate the supernatant. Wash the magnetic bead complex with 500ul pre-cooled Wash buffer for a total of 6 times.

[0169] (4) RNA purification: Prepare proteinase K buffer: 150ul proteinase K buffer is prepared by adding 15ul 10% SDS and 18ul 10mg / ml proteinase K to 117ul RIP Wash buffer. Each sample requires 150ul proteinase K buffer; resuspend the magnetic beads in (3) with 150ul proteinase K buffer, and melt the input and add 107ul RIP Wash Buffer, 15ul 10% SDS and 18ul proteinase K; place all tubes with proteinase K in a 55℃ shaking metal bath for 30 minutes to digest the protein; after incubation, place the ep tube on a magnetic stand, transfer 150ul supernatant to a new tube, and add 250ul RIP wash buffer; add 400ul phenol-chloroform-isoamyl alcohol (125:24:1) mixture to each tube, shake for 15s, and centrifuge at 14000rpm at room temperature. 10 minutes for phase separation; transfer about 350ul of the upper aqueous phase to a new centrifuge tube, add 400ul of chloroform, and repeat the shaking and centrifugation operation; transfer about 300ul of the upper aqueous phase to a new tube, add 50ul of salt solution I, 15ul of salt solution II, 5ul of precipitation enhancer, and finally add 850ul of anhydrous ethanol, mix well, and place at -80℃ overnight to precipitate RNA; centrifuge at 14000rpm and 4℃ for 30min, and carefully discard the supernatant; wash the precipitate with 80% ethanol, centrifuge at 14000rpm and 4℃ for 15min, discard the supernatant, and air-dry the precipitate; dissolve the precipitate with 12ul of DEPC-treated water and place it on ice.

[0170] (5) Reverse transcription and PCR analysis:

[0171] Reverse transcription and PCR were performed as described above for quantitative fluorescence PCR. All IP products were reverse transcribed. Calculate %(IP / Input) = 2^(CtInput - CtIP) * DF * 100, where DF = amount of RNA used for input / amount of RNA used for IP. %(IgG / Input) was calculated similarly: Fold Enrichment = %(IP / Input) / %(IgG / Input).

[0172] 2. Experimental results:

[0173] We constructed a YTHDF2 overexpression plasmid and transfected it into HepG2 cells. We found that the Acot2 content in the overexpression group was reduced ( Figure 8B). We further used anti-YTHDF2 antibodies to perform RIP assays in both AML12 and HepG2 cells under normal culture conditions and found that compared with the IgG group, the YTHDF2 antibody could enrich more Acot2, and the enrichment multiple in mouse AML12 cells was more significant ( Figure 8 A). In addition, we used the Mettl14 knockdown strain to perform RIP experiments on YTHDF2. PCR confirmed that RIP of YTHDF2 enriched less Acot2 in the Mettl14 knockdown strain ( Figure 8 C).

[0174] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the described embodiments. Those skilled in the art may make various equivalent modifications or substitutions without departing from the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. Use of an ACOT2 expression promoter in the preparation of a drug for promoting liver regeneration and liver function recovery after partial hepatectomy, characterized in that: The ACOT2 expression promoting agent is selected from agents that overexpress the transcription factor PPARα or reduce the m6A modification level of ACOT2 mRNA. The reagent for overexpressing the transcription factor PPARα is a plasmid that overexpresses PPARα and RXRα; The reagent for reducing the m6A modification level of ACOT2 mRNA is RNAi that reduces the content of m6A methylation modification enzyme Mettl14, and the mRNA nucleotide sequence targeted by RNAi is as described in any one of SEQ ID NOs. 5 to 10.

2. The use according to claim 1, characterized in that: in, The partial liver resection is hemihepatectomy or staged liver resection combined with liver segmentation and portal vein ligation.

3. The use according to claim 1, characterized in that: in, The drug for promoting liver regeneration and liver function recovery is a drug that reduces intracellular lipid accumulation, lowers the levels of alanine aminotransferase and aspartate aminotransferase, promotes liver cell mitosis and increases the liver-to-body ratio.

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