CN114606235B - Cyclic RNA SIRT5 and application thereof in diagnosis and treatment of non-alcoholic fatty liver disease - Google Patents

Abstract

The invention relates to a circular RNA SIRT5 and its application in the diagnosis and treatment of nonalcoholic fatty liver disease. The present invention reveals the function and mechanism of c i rcRNA SIRT5 in the occurrence and development of NAFLD, provides a theoretical basis for enriching the pathogenesis and clinical treatment of NAFLD, and contributes to the development of molecular markers of NAFLD disease and the research and development of clinical drugs.

Description

Circular RNA SIRT5 and its application in the diagnosis and treatment of nonalcoholic fatty liver disease

technical field

The invention relates to the technical field of medicine, in particular to a circular RNA SIRT5 and its application in the diagnosis and treatment of non-alcoholic fatty liver disease.

Background technique

With the improvement of the economy, the improvement of people's living standards and life expectancy, and the change of living habits, the incidence of nonalcoholic fatty liver disease (NAFLD) is on the rise rapidly. NAFLD is expected to replace chronic viral hepatitis in the future. It has become the most important chronic liver disease and seriously threatens the life and health of the people. At present, with the improvement of the diagnosis level and the deepening of the mechanism research, people have a certain understanding of the occurrence and development of fatty liver. Clinically, therefore, clarifying the mechanism of fatty liver and finding effective prevention and treatment measures are still important issues that we face for a long time.

NAFLD is usually a component of metabolic syndrome and is the manifestation of metabolic syndrome in the liver. Scholars from 22 different countries, including Chinese scholars, have reached a consensus and suggested changing the name of NAFLD to metabolic disorder-associated fatty liver disease (Metabolic-dysfunction-associated fatty liver disease, MAFLD). NAFLD involves a wide range of clinical symptoms, ranging from steatosis, a benign liver disease characterized by intracellular fat accumulation, to nonalcoholic steatohepatitis (NASH), characterized by inflammation, hepatocellular injury, and liver fibrosis, NASH can further develop into cirrhosis and hepatocellular carcinoma. Although it is clear that the excessive accumulation of fatty acids in the liver caused by obesity is the main cause of fatty liver, clinically, not all obese patients are concurrent with NAFLD, and not all patients with NAFLD are obese. This phenomenon is more common in Asian populations. It is more significant; in addition, when fatty acids accumulate in the liver, only about 20-30% of patients with simple fatty liver will further develop into NASH and liver necrosis. Furthermore, the relationship between the development of steatosis, inflammation and fibrosis, and key clinical outcomes appears to vary widely between individuals. This shows that the excessive accumulation of lipids in the liver is just an appearance, so what factors lead to the abnormal deposition of lipids in the liver? How does the excessive accumulation of lipid in the liver cause the occurrence and development of NAFLD? These important issues still await further elucidation.

Mitochondria are key organelles in cells. They not only provide energy for cells, but are also an important place for the generation of free radicals in cells, and even participate in the regulation of cell apoptosis. Under physiological conditions, mitochondria in cells are undergoing dynamic changes, including changes in the shape and structure of mitochondria, division, regeneration and fusion of mitochondria, and mitophagy. Through their own homeostasis, mitochondria maintain metabolic homeostasis in the body. During the pathogenesis of NAFLD, mitochondria first respond to excessive lipid overload in hepatocytes by balancing the redox state of NAD+/NADH and increasing mitochondrial elongation. As the disease progresses, the adaptability and flexibility of mitochondria decrease, resulting in increased production of reactive oxygen species (Reactive oxygen species, ROS), which in turn leads to oxidative damage to mitochondrial DNA (Mitochondrial DNA, mtDNA), abnormal mitochondrial structure (shown as giant mitochondria , mitochondrial cristae loss and mitochondrial particle turbidity), lipid peroxidation and other mitochondrial metabolic homeostasis imbalances, which worsen the disease process. It can be seen that liver mitochondrial damage is not only the initial event in the early stage of NAFLD, but also continues to aggravate with the progress of NAFLD, and it runs through the entire course of NAFLD. In view of the fact that fatty acid β-oxidation and oxidative stress that affect the occurrence and progression of NAFLD mainly occur Regarding mitochondria, some scholars even proposed that NAFLD is a mitochondrial disease, so mitochondrial damage has always been the focus of NAFLD pathogenesis research. If the dynamic balance of mitochondria is disrupted, it will affect the function of mitochondria and even affect the survival of cells. Mitochondrial energy metabolism disorder is the main cause of cellular oxidative stress, manifested as the formation of ROS. Therefore, the imbalance of mitochondrial metabolic homeostasis may be the key factor affecting the abnormal accumulation of liver lipids and the occurrence and development of NAFLD. So what factors lead to the imbalance of mitochondrial metabolic homeostasis?

Circular RNAs (circular RNAs, circRNAs) are a newly discovered endogenous non-coding RNA with a closed-loop structure, which has the characteristics of stability, generality, conservation and tissue specificity, and plays an important role in the regulation of gene expression. . circRNAs are not affected by RNA exonucleases, their expression is more stable and not easy to degrade, and they are widely expressed in the eukaryotic cell transcriptome. Studies have found that circRNAs are abnormally expressed in many human diseases, including cancer, neurodegenerative changes, and cardiovascular diseases. Although a large number of circRNAs have been identified, the functions of only a few circRNAs have been revealed so far, and the functions involved are mainly concentrated in miRNA sponges, cis-regulation of parental genes, and competitive binding of RNA-binding proteins ( RNA-binding protein, RBP) and translated short peptides, etc.

In the field of NAFLD research, due to factors such as limited access to clinical liver tissue samples, most past studies were limited to using palmitic acid or oleic acid to interfere with human liver cancer cell lines or normal liver cell lines to establish in vitro models of NAFLD, or using high-fat Diet-induced mouse NAFLD model, followed by in vitro studies by means of sequencing, silencing and overexpression, rarely involves the expression profile of circRNAs in the liver tissue of NAFLD patients and further explores the potential molecular mechanisms and molecular targets. Recently, we have noticed that in 2020, the team of Professor Su Shicheng from Sun Yat-Sen University published a study on CELL, suggesting that by extracting primary fibroblasts from human liver tissue for gene chip detection, it was found that hsa_circ_0089762 was lowly expressed in the liver tissue of NASH cirrhosis, and It was named steatohepatitis-associated circRNA ATP5B regulator (SCAR). Further phenotype and mechanism studies found that circRNA SCAR can directly bind to ATP5B of mPTP complex ATP synthase, and block the interaction between CypD and mPTP in a resting state. This study is the first to focus on the expression of circRNAs in hepatic stellate cells in patients with NASH and cirrhosis, and further reveals the important molecular mechanism of the development of human NASH to liver fibrosis and cirrhosis. The development of NASH to cirrhosis belongs to the relatively late stage of NAFLD disease progression. However, there are few relevant literature reports at home and abroad on the role and mechanism of circRNAs in early NAFLD patients, and there is also a lack of corresponding molecular markers and drugs.

Contents of the invention

The purpose of the present invention is to provide a circular RNA that can be used for the diagnosis, treatment or condition monitoring of non-alcoholic fatty liver disease.

The present invention provides a circular RNA SIRT5, the nucleotide sequence of which is shown in SEQ ID NO.1.

The present invention also provides a DNA fragment encoding the circular RNA SIRT5, as shown in SEQ ID NO.2.

The present invention also provides a recombinant expression vector, which contains the DNA fragment.

In some embodiments, the recombinant expression vector is a lentiviral vector.

The present invention also provides a host cell containing the recombinant expression vector.

In some embodiments, the host cell is a HepG2 cell.

The present invention also provides the application of the circular RNA SIRT5, DNA fragment, recombinant expression vector or host cell in the preparation of products for the diagnosis, treatment or condition monitoring of non-alcoholic fatty liver disease.

In some embodiments, the product is a reagent, kit, medicament or device.

The present invention also provides a medicine for treating non-alcoholic fatty liver disease, including the circular RNA SIRT5, DNA fragment, recombinant expression vector or host cell, and pharmaceutically acceptable auxiliary materials.

The present invention also provides the application of the circular RNA SIRT5, DNA fragment, recombinant expression vector or host cell in the preparation of products for inhibiting the expression of hsa-miR-150-5P or increasing the expression of SIRT5 protein.

The present invention first confirmed the imbalance of mitochondrial metabolic homeostasis and the correlation between circRNA SIRT5 and the occurrence and development of NAFLD in normal liver and NAFLD liver tissue samples, and then observed that circRNA SIRT5 regulates the imbalance of mitochondrial metabolic homeostasis in liver cells in a mouse NAFLD model To explore whether SIRT5 can be used as an epigenetic regulatory target to intervene in the process of NAFLD, and whether rationally regulating the expression of circRNA SIRT5 in hepatocytes can help maintain the balance of mitochondrial metabolic homeostasis, thereby improving the prognosis of NAFLD patients . The present invention reveals the role and mechanism of circRNA SIRT5 in the occurrence and development of NAFLD, provides a theoretical basis for enriching the pathogenesis and clinical treatment of NAFLD, and contributes to the development of molecular markers of NAFLD disease and the research and development of clinical drugs.

Description of drawings

Fig. 1 is a schematic diagram of the regulation of the occurrence and development of circRNA SIRT5 involved in NAFLD according to an embodiment of the present invention;

Figure 2 shows the results of NAFLD liver tissue circRNA next-generation sequencing and analysis verification in an embodiment of the present invention; wherein, A: human liver tissue HE staining; B: human liver tissue circRNA next-generation sequencing heat map; C: Venn analysis D: Expression verification of differentially expressed circRNAs; E: Human liver tissue mitochondrial ECAR; F: Human liver tissue mitochondrial OCR; G: Circular RNA circle formation verification.

Figure 3 shows the results of circRNA SIRT5 reducing fat deposition, ROS and inflammatory factor release in the liver tissue of NAFLD mice according to an embodiment of the present invention; wherein A: detection of ROS expression in mouse liver tissue cells and mitochondria; B: mouse The expression of inflammatory factors in plasma; C: the body shape of normal mice and NAFLD mice; D: the general view of mouse liver tissue and oil red staining to observe the fatty degeneration.

Figure 4 is the result of circRNA SIRT5 inhibiting fat deposition and inflammatory factor release in HepG2 cell lipotoxicity model of an embodiment of the present invention; wherein, A: circRNA expression after palmitic acid treatment; B: circRNA expression after palmitic acid treatment; C : the level of liver enzymes in cells after palmitic acid treatment; D: the effect of circRNA SIRT5 on fatty degeneration of hepG2 cells treated with PA under light microscope; E: the detection of ROS expression in hepG2 cells and mitochondria; F: the expression level of SIRT5 mRNA in cells .

Fig. 5 is the correlation detection and target prediction results of the potential mechanism of action of circRNA SIRT5 according to an embodiment of the present invention; among them, A: the expression level of has-miR-150-5P in human liver tissue; B: the expression level of has-miR-150-5P in mouse liver tissue -expression level of miR-150-5P; C: expression level of has-miR-150-5P in hepG2 cells; D: expression level of SIRT5 in human liver tissue; E: expression level of SIRT5 in mouse liver tissue; F : the expression level of SIRT5 in hepG2 cells; G: the predicted binding site of circRNA SIRT5 and hsa-miR-150-5P.

Figure 6 shows the results that circRNA SIRT5 can regulate the expression of SIRT5 and its downstream signaling pathway proteins according to an embodiment of the present invention; among them, A: the expression of SIRT5, ECH1, VLCAD and SOD1 in mouse liver tissue; B: SIRT5, Expression of ECH1, VLCAD and SOD1;.

Detailed ways

In order to demonstrate the technical solutions, objectives and advantages of the present invention more concisely and clearly, the present invention will be further described in detail below in conjunction with specific embodiments and accompanying drawings. It can be understood that those skilled in the art can refer to the contents herein and appropriately improve the process parameters to realize. In particular, it should be pointed out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and the relevant personnel can obviously make changes or appropriate changes and combinations to the method and application described herein without departing from the content, spirit and scope of the present invention to realize and Apply the technology of the present invention.

The present invention selects human normal liver tissue, mild NAFLD (big steatosis 5%-10%), moderate NAFLD (big steatosis 30-60%) and severe NAFLD (big steatosis>60%) %) liver tissues of 5 cases each were subjected to next-generation sequencing of circRNAs, and the results indicated that, compared with normal liver, with the aggravation of lesion severity, there were 10 circRNAs that were correlated with the severity of the disease, indicating that these circRNAs may It plays an important role in the occurrence and development of NAFLD. Among them, the expression levels of 3 circRNAs decreased, and the expression levels of 7 increased. Subsequently, differential expression analysis, Venn analysis, gene function analysis, signaling pathway analysis and expression verification experiments were performed on the relevant data. We found that novel_circ_0029917 had the largest expression change among the 10 differential genes, and had the strongest correlation with the severity of the disease. We further analyzed its maternal gene and found that novel_circ_0029917 was back-spliced from the exon of the SIRT5 gene. For ease of understanding, we named novel_circ_0029917 circRNA SIRT5.

Sirtuins are a class of nicotinamide adenine dinucleotide (NAD+)-dependent protein deacylases and/or ADP ribosyltransferases, including seven members, namely SIRT1-SIRT7. Different sirtuin members have different subcellular localizations and functions. SIRT1, 6, and 7 are mainly located in the nucleus, and SIRT3, 4, and 5 are distributed in the mitochondria. It is known that sirtuins can regulate a variety of biological processes: DNA repair, gene expression, cell survival, metabolism, aging, etc. Among mitochondrial sirtuins, SIRT5 displays a unique affinity for negatively charged acyllysine modifications and performs protein desuccinylation, demalonylation, and deglutarylation reactions. SIRT5 is widely distributed in the body, with the highest content in brain, heart, liver, kidney, muscle and testis. SIRT5 can maintain mitochondrial metabolism and cellular homeostasis by regulating biological processes such as glucose oxidation, ketone body formation, fatty acid oxidation, ammonia and ROS detoxification. SIRT5 knockout results in impaired β-oxidation and accumulation of medium- and long-chain acylcarnitines in liver and muscle of mice. SIRT5 can directly bind and activate copper/zinc superoxide dismutase through desuccinylation, thereby enhancing the ROS detoxification function mediated by SOD1. SIRT5 knockdown or knockout cells showed reduced levels of NADPH and GSH, resulting in impaired ability to scavenge ROS and increased sensitivity to oxidative stress. Sirtuins family has always been a hotspot in the field of metabolic disease research. In the field of NAFLD, studies have shown that SIRT5 can alleviate hepatic steatosis by regulating the deacetylation of metabolism-related proteins in ob/ob mice. In addition, knockout of SIRT5 can impair the oxidation capacity of medium-chain fatty acids in liver mitochondria of high-fat-fed mice and aggravate fatty liver. Therefore, we believe that SIRT5, as a metabolic sensor protein, plays an important role in maintaining mitochondrial metabolic homeostasis and participating in the occurrence and development of NAFLD. In addition, we also found that the expression of SIRT5 protein in human liver tissue is closely related to the accumulation of fat in liver cells and the development of NAFLD. These studies show that obesity and insulin resistance alone are not the decisive factors for hepatic fat accumulation and NAFLD, while the imbalance of mitochondrial metabolic homeostasis mediated by SIRT5 may play a more important role in the occurrence and development of NAFLD. So what are the initiating factors and internal dynamics of the abnormal expression of SIRT5 protein? Does the circRNA SIRT5 formed by SIRT5 backsplicing play an important role in this process?

To this end, we initially explored the effect of circRNA SIRT5 on the imbalance of mitochondrial metabolic homeostasis mediated by SIRT5. The lentivirus was overexpressed by synthesizing circRNA SIRT5 and transfected into HepG2 cells. In an in vitro lipotoxicity model established using palmitic acid, we found that overexpression of circRNA SIRT5 can significantly promote the β-oxidation of long-chain fatty acids and inhibit the generation of mitochondrial ROS, thereby reducing the release of inflammatory factors and alleviating cell lipotoxicity. Therefore, we speculate that circRNA SIRT5 may regulate the occurrence and development of NAFLD by regulating the mitochondrial metabolic homeostasis of liver cells, but the specific mechanism of its participation in regulating NAFLD is still unclear. We further found through bioinformatics analysis that SIRT5 is targeted and regulated by hsa-miR-150-5p, and there is a binding site of hsa-miR-150-5p on circRNA SIRT5. We also found that although the homology of circRNA SIRT5 between humans and mice is poor, the sequences of SIRT5 and hsa-miR-150-5p are highly conserved between humans and mice. The precedent of source non-coding RNA interfering with the disease process in mice has laid a solid foundation for our subsequent in vivo model to verify the phenotype and function of circRNA SIRT5. At the same time, we also found that the expression of circRNA SIRT5 was negatively correlated with hsa-miR-150-5p and positively correlated with the expression of SIRT5 in human NAFLD liver tissue and HepG2 lipotoxicity model in vitro. We further speculated that circRNA SIRT5 may be involved in the regulation of the occurrence and development of NAFLD by adsorbing hsa-miR-150-5p, thereby up-regulating the expression of SIRT5, as shown in Figure 1.

In summary, the present invention first confirms whether there is an imbalance of mitochondrial metabolic homeostasis and the correlation between circRNA SIRT5 and the occurrence and development of NAFLD in normal liver and NAFLD liver tissue samples, and then observes that circRNA SIRT5 regulates liver cells in a mouse NAFLD model. The dynamic process of mitochondrial metabolic homeostasis imbalance, to explore whether SIRT5 can be used as an epigenetic regulatory target to intervene in the process of NAFLD, and whether rational regulation of circRNA SIRT5 expression in liver cells can help maintain the balance of mitochondrial metabolic homeostasis, Thus improving the prognosis of NAFLD patients. The present invention reveals the role and mechanism of circRNA SIRT5 in the occurrence and development of NAFLD, provides a theoretical basis for enriching the pathogenesis and clinical treatment of NAFLD, and contributes to the development of molecular markers of NAFLD disease and the research and development of clinical drugs.

circRNA SIRT5 sequence:

UAAAUGGAAAUGUUUUCUAACAUAUAAAAACCUACAGAAGAAGAAAAUAAUUUUCUGGAUCAAAUUAGAAGUCUGUAUUAUAUAUUGAUGUUCCCAGAUUCAAAUAUAUAUAGAAAGCAGCCGUGGAGACAACCAUCUUCAUUUUGGGAGAAAUAACUAAAGUAGCUUAUUUAAAACUCGAUGUACCUCUUGUGGAG UUGUGGCUGAGAAUUACAAGAGUCCAAUUUGUCCAGCUUUAUCAGGAAAAGGGCUCCAGAACCUGGAACUCAAGAUGCCAGCAUCCCAGUUGAGAAACUUCCCCG

circRNA SIRT5 cDNA gene sequence:

TAAATGGAAATGTTTTTCTAACATATAAAAAACCTACAGAAGAAGAAAATAATTTTCTGGATCAAATTAGAAGTCTGTATTATATTGATGTCTCCAAGATTCAAATATATTAGAAAGCAGCCGTGGAGACAACCATCTTCATTTTGGGAGAAATAACTAAAGTAGCTTATTTAAAACTCGATGTACCTCTTGTGGAGTTGTGGCTGAGAATTACAAGAGTCCAATTTGTCC AGCTTTATCAGGAAAAGGGCTCCAGAACCTGGAACTCAAGATGCCAGCATCCCAGTTGAGAAACTTCCCCG

The specific research methods are as follows:

1. Analysis of the correlation of circRNA SIRT5, hsa-miR-150-5p and mitochondrial metabolic homeostasis-related proteins in liver tissues of NAFLD patients and healthy people

a. Determination of biochemical indicators: cytokines (IL-1β, TNF-α, IL-6, etc.) in peripheral blood or liver tissue reflect the activation state of inflammation; detect liver function indicators (ALT, AST);

b. Detection of NAFLD-related indicators: oil red O and HE staining were used to observe liver lipid accumulation and morphological changes; qRT-PCR, Western Blotting or immunohistochemical methods were used to detect liver tissue inflammatory mediators (TNF-α, IL- 1. IL-6) mRNA and protein levels;

c. Detection of indicators related to mitochondrial metabolic homeostasis: Seahorse detects the metabolic phenotype of liver mitochondria; Western Blotting detects SOD1, ECH1, VLCAD, SIRT5; DCFH-DA method and mitoSOX method detect ROS in cytoplasm and mitochondria respectively; Seahorse detects liver cell mitochondria changes in energy metabolism;

d. Expression and localization of circRNA SIRT5 in the liver tissue of NAFLD patients: qRT-PCR and other methods were used to detect the expression of circRNA SIRT5; RNaseR digestion tolerance test was used to verify the circularization of circRNA SIRT5; combined with the above indicators, circRNA SIRT5 and hsa-miR were analyzed The correlation between -150-5p and related indicators of mitochondrial metabolic homeostasis;

2. Verify the role of circRNA SIRT5 in NAFLD mouse model

a. Animal model construction: High-fat diet (High-fat diet, HFD) was used to feed wild-type C57BL/6J mice to establish the NAFLD mouse model, and CD-fed wild-type C57BL/6J mice were used as the control group. AAV8 liver-specific viral vector transfection established a mouse model of circRNA SIRT5 overexpression;

b. Biochemical detection: Animal blood was drawn before the experiment and at 1, 8 and 16 weeks after the start of the experiment. Reflect the inflammatory activation state of experimental animals by detecting serum cytokines (IL-1β, TNFα, IL-6, etc.); detect liver function indicators (ALT, AST);

c. Morphology of lipid accumulation: oil red O staining was used to detect the degree of hepatic steatosis on frozen sections;

d. Detection of indicators related to mitochondrial metabolic homeostasis: Western Blotting to detect SOD1, ECH1, VLCAD, SIRT5; DCFH-DA method and mitoSOX method to detect ROS in cytoplasm and mitochondria respectively;

e. Expression and localization of circRNA SIRT5 in liver tissue of NAFLD mice: qRT-PCR was used to detect the expression levels of circRNA SIRT5, hsa-miR-150-5p and indicators related to mitochondrial metabolic homeostasis, and the correlation between the three was analyzed in combination with the above indicators sex;

3. Validation of the role of circRNA SIRT5 in the NAFLD liver cell in vitro model at the cellular level

a. Construction of NAFLD in vitro model: HepG2 liver cell line lipotoxicity model induced by palmitic acid, circRNASIRT5 overexpressed lentivirus transfected cells, in order to clarify the role of circRNA SIRT5 in NAFLD in vitro model;

b. Determination of biochemical indicators: liver function indicators and cytokines (IL-1β, TNFα, IL-6, etc.) in the supernatant and cell homogenate;

d. Morphology of lipid accumulation: detect the degree of cell steatosis with Oil Red O staining;

e. Detection of indicators related to mitochondrial metabolic homeostasis: Western Blotting detection of SOD1, ECH1, VLCAD, SIRT5;

f. Expression and identification of circRNA SIRT5 in NAFLD in vitro model: qRT-PCR detection of circRNA SIRT5 expression level, combined with the above indicators to analyze the correlation among circRNA SIRT5, hsa-miR-150-5p and mitochondrial metabolic homeostasis related indicators ;

Key technical description:

Seahorse test: In order to observe the relationship between mitochondrial function changes and NAFLD, we will perform Seahorse test on liver tissue samples of NAFLD patients and normal people. The Seahorse XFp analyzer is an ideal tool for routine testing of metabolic phenotypes in vitro and in other limited samples, and has been widely used in basic experimental research. We first performed mitochondrial purification using the sucrose method, and these compounds (oligomycin, FCCP, and a mixture of rotenone and antimycin a) were separately and serially injected to measure ATP production, maximal respiration, and non-mitochondrial respiration. These parameters and basal respiration are then used to calculate proton leak and basal respiration volume.

Experimental results:

1. 5 cases of liver tissue samples from healthy people, NAFLD (large vesicular fatty liver 5-10%), NAFLD (large vesicular fatty liver 30-60%) and NAFLD (large vesicular fatty liver > 60%) Perform circRNAs sequencing to obtain hundreds of potential pathogenic circRNAs. NAFLD in each group was analyzed for differences with healthy people, and then Venn analysis was performed to find potential common pathogenic circRNAs. The results showed that 10 common differential circRNAs were found, of which 3 were low-expressed and 7 were highly expressed. Then, gene function analysis, signal pathway analysis and expression verification experiments were carried out on these 10 circRNAs. We found that novel_circ_0029917 had the largest variation among the 10 differential genes, and the expression change had the strongest correlation with the severity of the disease. We further used RNase R digestion experiments to identify the circularization properties of circRNA SIRT5, and the results suggested that circRNA SIRT5 was circular and resistant to RNase R enzyme digestion, as shown in Figure 2.

2. Construct a NAFLD model of wild-type C57BL/6J mice fed a high-fat diet, and use circRNA SIRT5 overexpression lentivirus to transfect, the results suggest that circRNA SIRT5 can significantly alleviate fatty lesions in mouse liver tissue and reduce the expression of mROS and cROS At the same time, it can also inhibit the release of hepatic inflammatory factors caused by NAFLD, suggesting that circRNASIRT5 can correct the imbalance of mitochondrial metabolic homeostasis caused by NAFLD, as shown in Figure 3.

3. We synthesized circRNA SIRT5 overexpression lentivirus and transfected HepG2 cells. In establishing an in vitro lipotoxicity model using palmitic acid, we found that overexpression of circRNA SIRT5 can significantly promote the β-oxidation of long-chain fatty acids and inhibit the generation of mitochondrial ROS, thereby reducing the release of inflammatory factors and alleviating cell lipotoxicity, as shown in Figure 4 .

4. In order to explore the potential mechanism of circRNA SIRT5 function, we analyzed whether it has protein coding potential by using three common protein potential prediction software, CPC, CNCI and PFAM. Possess protein translation potential. Further, we used prediction software such as miRDB, TargetScan, and RNA Hydrid to analyze the potential sites of mutual binding between circRNA/miR/mRNA and found that circRNA SIRT5 has three sites that can bind to hsa-miR-150-5p , and hsa-miR-150-5p can target SIRT5 mRNA. Subsequently, we found that the expression of hsa-miR-150-5P was increased, while the mRNA expression of SIRT5 was decreased in human and mouse NAFLD liver tissues and HepG2 cell lipotoxicity model. In the lipotoxicity model of HepG2 cells overexpressed by circRNA SIRT5, circRNA SIRT5 can significantly inhibit the expression of hsa-miR-150-5P, and at the same time, it can relieve the inhibitory effect of hsa-miR-150-5P on SIRT5 mRNA, as shown in Figure 5.

5. Subsequently, we observed the effects of circRNA SIRT5 on SIRT5 protein and mitochondrial metabolic homeostasis-related indicators in the mouse NAFLD model and HepG2 cell lipotoxicity model overexpressed in circRNA SIRT5. The results suggest that circRNASIRT5 can up-regulate the expression of SIRT5 protein in mouse liver and HepG2 cells, and then up-regulate the expression of mitochondrial metabolic homeostasis-related proteins (ECH1, VLCAD and SOD1) downstream of the signaling pathway, as shown in Figure 6.

The above-mentioned embodiments only express several implementation modes of the present invention, and the descriptions thereof are relatively specific and detailed, but should not be construed as limiting the patent scope of the invention. It should be pointed out that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent for the present invention should be based on the appended claims.

sequence listing

<110> West China Hospital of Sichuan University

<120> Circular RNA SIRT5 and its application in the diagnosis and treatment of nonalcoholic fatty liver disease

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Claims (10)

1. a circular RNA SIRT5, characterized in that the nucleotide sequence of the circular RNA SIRT5 is as shown in SEQ ID NO.1. 2. A DNA fragment, characterized in that it encodes the circular RNA SIRT5 according to claim 1. 3. A recombinant expression vector, characterized in that the recombinant expression vector contains the DNA fragment according to claim 2. 4. The recombinant expression vector according to claim 3, characterized in that, the recombinant expression vector is a lentiviral vector. 5. A host cell, characterized in that the host cell contains the recombinant expression vector according to claim 3 or 4. 6. The host cell according to claim 4, characterized in that, the host cell is a HepG2 cell. 7. The circular RNA SIRT5 according to claim 1, the DNA fragment according to claim 2, the recombinant expression vector according to any one of claims 3 to 4 or the host cell according to any one of claims 5 to 6 Application in preparing products for diagnosis, treatment or condition monitoring of non-alcoholic fatty liver disease. 8. The application according to claim 7, wherein the product is a reagent, kit, medicine or device. 9. A medicine for the treatment of non-alcoholic fatty liver disease, characterized in that it comprises the circular RNA SIRT5 according to claim 1, the DNA fragment according to claim 2, and any one of claims 3 to 4. The recombinant expression vector described above or the host cell described in any one of claims 5-6, and pharmaceutically acceptable adjuvants. 10. The circular RNA SIRT5 according to claim 1, the DNA fragment according to claim 2, the recombinant expression vector according to any one of claims 3 to 4 or the host cell according to any one of claims 5 to 6 Application in the preparation of products for inhibiting the expression of hsa-miR-150-5P or increasing the expression of SIRT5 protein.

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