CN114606235A - Circular RNA SIRT5 and its application in the diagnosis and treatment of nonalcoholic fatty liver disease - Google Patents

Abstract

The invention relates to a circular RNA SIRT5 and its application in the diagnosis and treatment of non-alcoholic fatty liver disease. The invention discloses the role and mechanism of ci rcRNA S I RT5 in the occurrence and development of NAFLD, provides a theoretical basis for enriching the pathogenesis and clinical treatment of NAFLD, and is helpful for 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 longevity, and the change of living habits, the incidence of non-alcoholic fatty liver disease (NAFLD) shows a rapid upward trend. 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 diagnostic level and the in-depth study of the mechanism, people have a certain understanding of the occurrence and development of fatty liver. However, for the treatment of NAFLD, there is currently no effective targeted drug or means for the treatment of NAFLD at home and abroad. Therefore, clarifying the pathogenesis 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 in the world, including Chinese scholars, have reached a consensus and suggested that NAFLD be renamed as metabolic disorder-related 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 fat accumulation in liver cells, to nonalcoholic steatohepatitis (NASH), characterized by inflammation, hepatocyte damage, and liver fibrosis, NASH can further develop into cirrhosis and hepatocellular carcinoma. Although it is now clear that the excessive accumulation of fatty acids in the liver caused by obesity is the main cause of fatty liver, not all obese patients are complicated by NAFLD, and not all patients with NAFLD are obese. This phenomenon occurs in Asian populations. More significant; in addition, only about 20-30% of patients with simple fatty liver will further develop NASH and liver necrosis when fatty acids accumulate in the liver. 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 only a symptom, so what causes the abnormal deposition of lipids in the liver? How does the excessive accumulation of lipids in the liver cause the occurrence and development of NAFLD? These important questions remain to be further elucidated.

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

Circular RNAs (circRNAs) are a newly discovered endogenous non-coding RNA with a closed-loop structure. . CircRNAs are not affected by RNA exonuclease, their expression is more stable and not easily degraded, 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 to RNA-binding proteins ( RNA-binding protein, RBP) and translation of short peptides, etc.

In the field of NAFLD research, due to factors such as limited access to clinical liver tissue specimens, most of the past studies were limited to using palmitic acid or oleic acid to intervene 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 was then investigated in vitro by means of sequencing, silencing and overexpression. There are few research reports on the expression profile of circRNAs in liver tissue of NAFLD patients and their potential molecular mechanisms and molecular targets. Recently, we have noticed that the research published on CELL by the team of Professor Su Shicheng of Sun Yat-sen University in 2020 suggested 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 liver cirrhosis, and the It was named as Steatohepatitis-associated circRNA ATP5B regulator (SCAR). Further phenotypic and mechanistic studies found that circRNA SCAR can directly bind to ATP5B of the mPTP complex ATP synthase, and block the interaction of CypD and mPTP in the resting state. This study is the first to focus on the expression of circRNAs in hepatic stellate cells in patients with NASH and liver cirrhosis, and further reveal the important molecular mechanism of the development of human NASH to liver fibrosis and cirrhosis. The development of NASH to liver cirrhosis belongs to the relatively late stage of NAFLD disease progression, and there are few relevant literature reports at home and abroad on the role and mechanism of circRNAs in patients with early NAFLD, and corresponding molecular markers and drugs are also lacking.

SUMMARY 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, and the nucleotide sequence of the circular RNA SIRT5 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 containing 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 preparing a product for the diagnosis, treatment or condition monitoring of non-alcoholic fatty liver disease.

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

The present invention also provides a medicine for treating non-alcoholic fatty liver disease, comprising the circular RNASIRT5, DNA fragment, recombinant expression vector or host cell, and pharmaceutically acceptable excipients.

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

The present invention firstly 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 in the mouse NAFLD model, observed that circRNASIRT5 regulates the imbalance of mitochondrial metabolic homeostasis in hepatocytes To explore whether SIRT5 can be used as an epigenetic regulatory target to intervene in NAFLD process, and whether rational regulation of circRNA SIRT5 expression in hepatocytes can help maintain the balance of mitochondrial metabolic homeostasis, thereby improving the prognosis of NAFLD patients. . The invention discloses 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 is helpful for the development of molecular markers of NAFLD disease and the development of clinical drugs.

Description of drawings

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

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

Figure 3 shows the results of reducing fat deposition, ROS and inflammatory factor release in liver tissue of NAFLD mice by circRNA SIRT5 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 levels of inflammatory factors in plasma; C: Body shape of normal mice and NAFLD mice; D: General view of mouse liver tissue and oil red staining to observe steatosis.

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

Figure 5 shows the correlation detection and target prediction results of the potential mechanism of action of circRNA SIRT5 according to an embodiment of the present invention; wherein, A: the expression level of has-miR-150-5P in human liver tissue; B: 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 : expression level of SIRT5 in hepG2 cells; G: predicted binding sites 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; wherein, A: expression of SIRT5, ECH1, VLCAD and SOD1 in mouse liver tissue; B: SIRT5, ECH1, VLCAD and SOD1 in hepG2 cells Expression of ECH1, VLCAD and SOD1;.

Detailed ways

In order to show the technical solutions, objects and advantages of the present invention more concisely and clearly, the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It can be understood that those skilled in the art can refer to the content of this document to appropriately improve the process parameters for implementation. It should be particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are deemed to be included in the present invention. The method and application of the present invention have been described through the preferred embodiments, and it is obvious that relevant persons can make changes or appropriate changes and combinations of the methods and applications described herein without departing from the content, spirit and scope of the present invention to achieve and Apply the technology of the present invention.

The present invention selects human normal liver tissue, mild NAFLD (bullous steatosis 5%-10%), moderate NAFLD (bullous steatosis 30-60%) and severe NAFLD (bullous steatosis>60%) %) 5 cases of liver tissue were subjected to next-generation sequencing of circRNAs. The results showed that, compared with normal liver, with the severity of the disease, there were 10 circRNAs that were expressed in correlation with the severity of the disease, indicating that these circRNAs may be It plays an important role in the occurrence and development of NAFLD. Among them, the expression levels of 3 circRNAs were decreased, and the expression levels of 7 circRNAs were increased. Then, the related data were subjected to differential expression analysis, Venn analysis, gene function analysis, signaling pathway analysis, and expression verification experiments. We found that novel_circ_0029917 had the largest expression change among these 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 SIRT5 gene. For the convenience of understanding, we named novel_circ_0029917 as 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 localization and function. SIRT1, 6, and 7 were mainly located in the nucleus, while SIRT3, 4, and 5 were distributed in the mitochondria. Sirtuins are known to regulate a variety of biological processes: DNA repair, gene expression, cell survival, metabolism, aging, and more. Among mitochondrial sirtuins, SIRT5 exhibits a unique affinity for negatively charged acyl-lysine modifications and undergoes protein desuccinylation, demalonylation, and deglutarylation reactions. SIRT5 is widely distributed in the body, with the highest content in the brain, heart, liver, kidney, muscle and testis. SIRT5 maintains 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 resulted in impaired β-oxidation and accumulation of medium- and long-chain acylcarnitines in the liver and muscle of mice. SIRT5 directly binds and activates copper/zinc superoxide dismutase via desuccinylation, thereby enhancing SOD1-mediated ROS detoxification. SIRT5 knockdown or knockout cells showed reduced levels of NADPH and GSH, resulting in an impaired ability to scavenge ROS and increased sensitivity to oxidative stress. For a long time, the Sirtuins family has been a hot spot in the field of metabolic disease research. In the field of NAFLD, studies have shown that SIRT5 alleviates hepatic steatosis by regulating the deacetylation of metabolism-related proteins in ob/ob mice. In addition, SIRT5 knockout impaired mitochondrial medium-chain fatty acid oxidation in the liver of high-fat-fed mice, aggravating 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 changes of SIRT5 protein expression in human liver tissue were closely related to the accumulation of fat in hepatocytes and the development of NAFLD. These studies suggest that simple obesity and insulin resistance are not determinants of hepatocyte fat accumulation and NAFLD, and that SIRT5-mediated mitochondrial metabolic homeostasis may play a more important role in the occurrence and development of NAFLD. So what are the initiating factors and intrinsic motivations for abnormal expression of SIRT5 protein? Does the circRNA SIRT5 formed by back-splicing of SIRT5 play an important role in this process?

To this end, we preliminarily explored the effect of circRNA SIRT5 on SIRT5-mediated imbalance in mitochondrial metabolic homeostasis. The lentivirus was overexpressed by synthetic circRNA SIRT5 and transfected into HepG2 cells. In the establishment of 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 cellular lipotoxicity. Therefore, we speculate that circRNA SIRT5 may regulate the occurrence and development of NAFLD by regulating mitochondrial metabolic homeostasis in hepatocytes, but the specific mechanism of its involvement in regulating NAFLD is still unclear. We further found through bioinformatics analysis that SIRT5 was targeted and regulated by hsa-miR-150-5p, and there was a binding site for hsa-miR-150-5p on circRNA SIRT5. We also found that although the circRNA SIRT5 has poor homology between humans and mice, SIRT5 and hsa-miR-150-5p are highly conserved in sequences between humans and mice. The precedent of source non-coding RNA interfering with the disease process in mice 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 in vitro lipotoxicity model. We further speculate that circRNA SIRT5 may upregulate the expression of SIRT5 by adsorbing hsa-miR-150-5p and participate in the regulation of the occurrence and development of NAFLD, as shown in Figure 1.

To sum up, the present invention firstly confirmed whether there is an imbalance of mitochondrial metabolic homeostasis and the correlation between circRNA SIRT5 and the development of NAFLD in normal liver and NAFLD liver tissue samples, and then observed the regulation of circRNA SIRT5 in hepatocytes in a mouse NAFLD model. The dynamic process of the imbalance of internal mitochondrial metabolic homeostasis, to explore whether SIRT5 can be used as an epigenetic regulatory target to interfere with NAFLD process, and whether rational regulation of the expression of circRNA SIRT5 in hepatocytes can help maintain the balance of mitochondrial metabolic homeostasis. And then improve the prognosis of NAFLD patients. The invention discloses 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 is helpful for the development of molecular markers of NAFLD disease and the development of clinical drugs.

circRNA SIRT5 sequence:

UAAAUGGAAAUGUUUUCUAACAUAUAAAAACCUACAGAAGAAGAAAAUAAUUUUCUGGAUCAAAUUAGAAGUCUGUAUUAUAUUGAUGUCUCCAGAUUCAAAUAUAUUAGAAAGCAGCCGUGGAGACAACCAUCUUCAUUUUGGGAGAAAUAACUAAAGUAGCUUAUUUAAAACUCGAUGUACCUCUUGUGGAGUUGUGGCUGAGAAUUACAAGAGUCCAAUUUGUCCAGCUUUAUCAGGAAAAGGGCUCCAGAACCUGGAACUCAAGAUGCCAGCAUCCCAGUUGAGAAACUUCCCCG

circRNA SIRT5 cDNA gene sequence:

TAAATGGAAATGTTTTCTAACATATAAAAACCTACAGAAGAAGAAAATAATTTTCTGGATCAAATTAGAAGTCTGTATTATATTGATGTCTCCAGATTCAAATATATTAGAAAGCAGCCGTGGAGACAACCATCTTCATTTTGGGAGAAATAACTAAAGTAGCTTATTTAAAACTCGATGTACCTCTTGTGGAGTTGTGGCTGAGAATTACAAGAGTCCAATTTGTCCAGCTTTATCAGGAAAAGGGCTCCAGAACCTGGAAGACTCAGTCAGTCAGT

The specific research methods are as follows:

1. To analyze 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 aggregation and morphological changes; qRT-PCR, Western Blotting or immunohistochemical methods were used to detect inflammatory mediators (TNF-α, IL- 1. IL-6) mRNA and protein levels;

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

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

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

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

b. Biochemical detection: Before the experiment, 1, 8 weeks and 16 weeks after the start of the experiment, the blood of the animals was drawn. By detecting serum cytokines (IL-1β, TNFα, IL-6, etc.), the inflammatory activation state of experimental animals was reflected; liver function indexes (ALT, AST) were detected;

c. Morphology of lipid accumulation: the degree of hepatic steatosis was detected on frozen sections with Oil Red O staining;

d. Detection of mitochondrial metabolic homeostasis-related indicators: 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 mitochondrial metabolic homeostasis-related indexes, and the correlations among the three were analyzed by combining the above indexes sex;

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

a. NAFLD in vitro model construction: Palmitic acid-induced lipotoxicity model of HepG2 liver cell line, 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: the degree of cellular fatty degeneration was detected by Oil Red O staining;

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

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

Key technical description:

Seahorse assay: To observe the link between changes in mitochondrial function and NAFLD, we will perform Seahorse assay on liver tissue samples from NAFLD patients and normal people. The Seahorse XFp Analyzer is an ideal tool for routine testing of metabolic phenotypes in vitro and other limited samples, and is now widely used in basic experimental research. We first performed thread purification using the sucrose method, and these compounds (oligomycin, FCCP, and a mixture of rotenone and antimycin a) were injected sequentially to measure ATP production, maximal respiration, and non-mitochondrial respiration, respectively. These parameters and basal respiration are then used to calculate proton leak and basal respiratory capacity.

Experimental results:

1. 5 cases of liver tissue samples from healthy people, NAFLD (bullous fatty liver 5-10%), NAFLD (bullous fatty liver 30-60%) and NAFLD (bullous fatty liver> 60%) CircRNAs were sequenced and hundreds of potential pathogenic circRNAs were obtained. NAFLD in each group was compared with healthy people for difference analysis, 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. Subsequently, gene function analysis, signaling pathway analysis and expression verification experiments were performed on these 10 circRNAs. We found that novel_circ_0029917 had the largest change in these 10 differential genes, and the expression change had the strongest correlation with the severity of the disease. We further identified the cyclization properties of circRNA SIRT5 using RNase R digestion experiments. The results indicated that circRNA SIRT5 was circular and was resistant to RNase R digestion, as shown in Figure 2.

2. Construct the NAFLD model of wild-type C57BL/6J mice fed a high-fat diet, and transfected with circRNA SIRT5 overexpressing lentivirus. The results suggest that circRNA SIRT5 can significantly alleviate fatty lesions in mouse liver tissue and reduce the levels of mROS and cROS. At the same time, it can inhibit the release of inflammatory factors in hepatocytes 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 overexpressing lentivirus and transfected HepG2 cells. In the establishment of 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 cellular lipotoxicity, see Figure 4 .

4. In order to explore the potential mechanism of circRNA SIRT5's role, we analyzed whether it has protein-coding potential by using three common protein potential prediction softwares, CPC, CNCI and PFAM. Has protein translation potential. Further, we used prediction software such as miRDB, TargetScan and RNA Hydrad 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 and bind SIRT5 mRNA. We then 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 in the HepG2 cytolipotoxicity model. In the lipotoxicity model of HepG2 cells overexpressed by circRNA SIRT5, circRNA SIRT5 could significantly inhibit the expression of hsa-miR-150-5P and relieve the inhibitory effect of hsa-miR-150-5P on SIRT5 mRNA, as shown in Figure 5.

5. Subsequently, we observed the effect of circRNA SIRT5 on SIRT5 protein and mitochondrial metabolic homeostasis-related indicators in the circRNA SIRT5-overexpressed mouse NAFLD model and the HepG2 cytolipotoxicity model. 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 represent several embodiments of the present invention, and the descriptions thereof are specific and detailed, but should not be construed as a limitation on the scope of the invention patent. It should be pointed out that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can also be made, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention should be subject to 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, wherein the nucleotide sequence of the circular RNA SIRT5 is shown as SEQ ID NO. 1.

2. A DNA fragment encoding the circular RNA SIRT5 according to claim 1.

3. A recombinant expression vector comprising the DNA fragment of claim 2.

4. The recombinant expression vector of claim 3, wherein the recombinant expression vector is a lentiviral vector.

5. A host cell comprising the recombinant expression vector of claim 3 or 4.

6. The host cell of claim 4, wherein the host cell is a HepG2 cell.

7. Use of the circular RNA SIRT5 of claim 1, the DNA fragment of claim 2, the recombinant expression vector of any one of claims 3 to 4, or the host cell of any one of claims 5 to 6 in the preparation of a product for the diagnosis, treatment, or monitoring of non-alcoholic fatty liver disease.

8. Use according to claim 7, wherein the product is a reagent, kit, medicament or device.

9. A medicament for treating non-alcoholic fatty liver disease, comprising the cyclic RNA SIRT5 of claim 1, the DNA fragment of claim 2, the recombinant expression vector of any one of claims 3 to 4 or the host cell of any one of claims 5 to 6, and a pharmaceutically acceptable excipient.

10. Use of the circular RNA SIRT5 of claim 1, the DNA fragment of claim 2, the recombinant expression vector of any one of claims 3 to 4, or the host cell of any one of claims 5 to 6 in the preparation of a product for inhibiting expression of hsa-miR-150-5P or increasing expression of a SIRT5 protein.

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