Application of lncRNA TUG1 as target spot in preparation of medicine for treating metabolism-related fatty liver disease

Abstract

The invention belongs to the technical field of molecular biology and medicine, and particularly relates to application of lncRNA TUG1 serving as a target spot to preparation of a medicine for treating metabolism-related fatty liver diseases. According to the application disclosed by the invention, it is found that lncRNA TUG1 can be knocked down in a targeted manner by transfecting siRNA in human hepatoma carcinoma cells HepG2, so that the expression of the lncRNA TUG1 is reduced, and the contents of total cholesterol, free cholesterol and triglyceride are remarkably reduced; meanwhile, the mouse hepatoma carcinoma cell Hepa1-6 is transfected with siRNA to knock down lncRNA TUG1, so that the expression of the mouse hepatoma carcinoma cell Hepa1-6 is reduced. Besides, an MAFLD animal model is established, and it is found that compared with a control group, the expression of lncRNA TUG1 in the mouse liver of the model group is remarkably improved. The results indicate that the lncRNA TUG1 possibly becomes a new target for developing MAFLD related drugs, and it is clear that the lncRNA TUG1 affects generation of fat and cholesterol to achieve treatment intervention on MAFLD.

Description

Technical Field

[0001] The present invention belongs to the technical fields of molecular biology and medicine, and particularly relates to the application of lncRNA TUG1 as a target in the preparation of drugs for treating metabolic associated fatty liver disease. Background Art

[0002] Metabolic Associated Fatty Liver Disease (MAFLD) is a chronic liver disease closely related to metabolic dysfunction. In recent years, its global prevalence has shown a significant upward trend and it has become one of the most common liver diseases worldwide. MAFLD not only poses a serious threat to the health of patients, but also brings a huge economic burden to the social medical system. Given that the pathogenesis of MAFLD involves a complex pathological process of multiple factors and multiple links, the current drug treatment strategies mainly intervene in its pathogenic factors, key pathological links and related metabolic disorders. It is worth noting that during the occurrence and development of MAFLD, the epigenetic regulation mechanism plays an important role, especially the regulatory role of long non-coding RNA (lncRNA) has been increasingly concerned. Based on the key regulatory role of lncRNA in the pathological process of MAFLD, as a potential new diagnostic marker and therapeutic target, it provides a new research direction and treatment strategy for the precise diagnosis and treatment of MAFLD.

[0003] LncRNA is a class of RNA molecules with a length exceeding 200 nucleotides that do not encode proteins. However, it plays an important role in gene expression regulation. LncRNA participates in intracellular biological processes through various mechanisms, including transcriptional regulation, translational regulation, and protein stability regulation, etc. In recent years, studies have shown that lncRNA plays a key role in the occurrence and development of MAFLD, and the abnormal expression of lncRNA is closely related to disease progression. For example, the dysregulation of H19 expression may lead to lipid accumulation and insulin resistance, thus promoting the progression of MAFLD; MALAT1 promotes liver lipid synthesis and accumulation by regulating the expression of SREBP-1c, and it also exacerbates the liver inflammatory response by regulating the expression of inflammatory factors (such as TNF-α and IL-6); MEG3 inhibits liver lipid accumulation and fibrosis by regulating the p53 signaling pathway, and the deletion of MEG3 will lead to lipid metabolism disorders and hepatocyte damage, thus accelerating the progression of MAFLD. Due to the extensive functions and disease correlations of lncRNA, it has become a hot field in biomedical research and is expected to provide new biomarkers and targets for the diagnosis and treatment of diseases.

[0004] Rezdiffra (resmetirom) is the world's first drug for the treatment of adult patients with non-cirrhotic non-alcoholic steatohepatitis, and it is also accompanied by side effects such as diarrhea and nausea. Therefore, it is necessary to develop new therapeutic drugs with definite efficacy, high safety, and the ability to avoid serious adverse reactions to provide a more effective means for clinical treatment. Long non-coding RNA taurine upregulated gene 1 (lncRNA TUG1) is one of the earliest discovered lncRNAs related to human diseases and is also a regulator of various physiological and pathological processes, participating in the regulation of multiple biological behaviors and molecular mechanisms. Currently, there is no study confirming that lncRNA TUG1 can be used as a potential drug target for the treatment of MAFLD, nor is there any relevant verification study on its use as a drug development target for MAFLD. Based on the above situation, this application proposes the application of lncRNA TUG1 as a therapeutic target for metabolic associated fatty liver disease. Summary of the Invention

[0005] The object of the present invention is to provide the application of lncRNA TUG1 as a target in the preparation of drugs for the treatment of metabolic associated fatty liver disease and to verify the target through a model.

[0006] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] The application of lncRNA TUG1 as a target in the preparation of drugs for the treatment of metabolic associated fatty liver disease, wherein the ID number of lncRNA TUG1 in the NCBI database (https: / / www.ncbi.nlm.nih.gov / ) is 55000 (human source) and 544752 (mouse source).

[0008] Further, in the above application, the drug composition contains an inhibitor that targets and inhibits the functional expression of lncRNA TUG1.

[0009] Furthermore, the inhibitor is a human-derived siRNA, and the sequence of the sense strand of the human-derived siRNA SEQ ID NO.1 is: 5'-GACCGACUUAUUCUUAGAUTT-3', and the sequence of the antisense strand SEQ ID NO.2 is 5'-AUCUAAGAAUAAGUCGGUCTT-3'.

[0010] Furthermore, the inhibitor is a mouse-derived siRNA, and the sequence of the sense strand of the mouse-derived siRNA SEQ ID NO.3 is: 5'-GGCCUAUGCUUUUGCGAUCTT-3', and the sequence of the antisense strand SEQ ID NO.4 is: 5'-GAUCGCAAAAGCAUAGGCCTT-3'.

[0011] The present invention has the following beneficial effects:

[0012] In the present invention, lncRNA TUG1 regulates the expression of key enzymes in the lipid metabolism pathway by binding to miR-29a-3p. After silencing the lncRNA TUG1 target with siRNA, the levels of TC, FC, and TG in cells can be significantly reduced, and lipid accumulation can be alleviated, thereby exerting a therapeutic effect on MAFLD. In addition, in the MAFLD mouse model, the expression of lncRNA TUG1 is significantly increased. In summary, the present invention provides the application of lncRNA TUG1 as a target in the preparation of drugs for MAFLD, and lncRNA TUG1 inhibitors can be applied to the preparation of oral or injectable drugs for the treatment of MAFLD. Description of the Drawings

[0013] Figure 1 Changes in lipid metabolism indexes and lncRNA TUG1 levels in the MAFLD cell model, (a) TC level; (b) TG level; (c) lncRNA TUG1 level.

[0014] Figure 2 Regulation of miR-29a-3p expression in HepG2 cells by lncRNA TUG1, (a) Binding site of lncRNA TUG1 and miR-29a-3p; (b) lncRNA TUG1 level; (c) miR-29a-3p level; (d) Results of dual-luciferase reporter gene assay.

[0015] Figure 3 Regulation of HMGCR expression in HepG2 cells by lncRNA TUG1 through miR-29a-3p, (a) HMGCR mRNA level; (b) HMGCR protein level.

[0016] Figure 4 Regulation of SREBF-2 expression in HepG2 cells by lncRNA TUG1 through miR-29a-3p, (a) Binding site of miR-29a-3p and SREBF-2; (b) Results of dual-luciferase reporter gene assay; (c) SREBF-2 mRNA level; (d) SREBP-2 protein level.

[0017] Figure 5Changes in related genes and indicators after knocking down lncRNA TUG1 in HepG2 and Hepa1-6. (a) lncRNA TUG1 level in HepG2 cells; (b) TC level in HepG2 cells; (c) FC level in HepG2 cells; (d) TG level in HepG2 cells; (e) lncRNA TUG1 level in Hepa1-6 cells; (f) miR-29a-3p level in Hepa1-6 cells; (g) HMGCR mRNA level in Hepa1-6 cells; (h) SREBF-2 mRNA level in Hepa1-6 cells.

[0018] Figure 6 Changes in related genes and levels in the MAFLD mouse model. (a) Serum AST level; (b) Serum ALT level; (c) H&E staining; (d) Oil Red O staining; (e) lncRNA TUG1 level; (f) HMGCR mRNA level; (g) SREBF-2 mRNA level; (h) Liver TC level; (i) Liver FC level; (j) Liver TG level.

[0019] Figure 7 Correlation between lncRNA TUG1 and various indicator levels in the liver of HFMCD model mice. (a) AST level; (b) ALT level; (c) TC level; (d) FC level; (e) TG level. Specific implementation mode

[0020] Example 1

[0021] 1. Experimental method

[0022] 1.1 HepG2 cell culture and construction of cell fatty degeneration model

[0023] Cell culture: The cell culture medium used is a mixture of 10% fetal bovine serum + 90% DMEM basal medium (high glucose). HepG2 cells are placed in a 37°C incubator and cultured at a CO2 concentration of 5%. Passage can be carried out when the cell density reaches 70%-80%. Model construction: Use a cell fatty degeneration model induced by fetal bovine serum. Increase the fetal bovine serum concentration from 10% to 50%, add 50% DMEM basal medium (high glucose), and culture the cells in a 37°C incubator with a CO2 concentration of 5% for 48 hours.

[0024] 1.2 Overexpression of miR-29a-3p in HepG2 cells

[0025] HepG2 cells in good growth state were seeded at a density of 1×10 5Inoculate at a density of

[0026] 1.3 Dual - luciferase reporter gene assay

[0027] Seed HepG2 cells at a density of 2.5×10 4 cells / mL in a 24 - well plate until the cell confluence reaches 40%. Mix the reporter gene vector and the internal reference vector with the transfection reagent in a certain proportion to form a transfection complex. Then add the transfection complex to the cell culture plate and co - incubate with the cells to allow the vector to enter the cells. After 72 h, remove the cells and wash them with PBS. Subsequently, add 200 μL of cell lysis buffer to each well and incubate on ice for 5 min to fully lyse the cells. Centrifuge at 13,000 rpm for 1 min, take the supernatant, and transfer it to an EP tube. Finally, use the dual - luciferase reporter gene detection system for detection and perform statistical analysis on the detected luciferase activity data.

[0028] 1.4 Western Blot assay

[0029] Extract proteins using RIPA lysis buffer, determine the protein concentration by the BCA method, and complete the sample preparation after heat denaturation. After SDS - PAGE electrophoresis and membrane transfer, place the PVDF membrane containing the target band in 5% non - fat milk powder and incubate on a shaker at room temperature for 2 h for blocking. After blocking, incubate the different bands with the corresponding primary antibodies overnight at 4°C; the next day, add TBST and place it on the shaker to wash three times, then incubate the bands with the corresponding secondary antibodies at room temperature for 1 h. After incubation, wash three times with TBST on the shaker; take out the washed PVDF membrane, slightly blot the liquid on the membrane, immerse it in the ECL luminescent solution, and use a chemiluminescent imaging system to detect the protein bands.

[0030] 1.5 Knockdown of lncRNA TUG1 in HepG2 and Hepa1 - 6 cells

[0031] Seed well - growing HepG2 and Hepa1 - 6 cells at a density of 1×10 5Inoculate at a density of

[0032] 1.6 cells / mL in a 6-well plate. After the cell density reaches 50%-60% the next day, transfect siRNA into the cells to knockdown lncRNA TUG1. Among them, the SEQ ID NO.1 sequence of the human-derived siRNA transfected into HepG2 cells is: 5'-GACCGACUUAUUCUUAGAUTT-3', and the SEQ ID NO.2 sequence is: 5'-AUCUAAGAAUAAGUCGGUCTT-3'; the SEQ ID NO.3 sequence of the mouse-derived siRNA transfected into Hepa1-6 cells is: 5'-GGCCUAUGCUUUUGCGAUCTT-3', and the SEQ ID NO.4 sequence is: 5'-GAUCGCAAAAGCAUAGGCCTT-3'. The control group was transfected with the same dose of negative control (NC), and cell samples were collected after 72 h.

[0033] Use Trizol reagent to lyse cells or liver tissues to extract total RNA. Determine the RNA quality and concentration using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). Reverse transcribe mRNA and miRNA using the HiScript III RT SuperMix for qPCR (+gDNA wiper) kit (Vazyme, Nanjing, China) and the miRNA First Strand cDNA Synthesis kit (Sangon Biotech, Shanghai, China), respectively. qRT-PCR uses the 2×Universal SYBR Green Fast qPCR Mix (Abclonal, Wuhan, China) and the miRNA Fluorescent Quantitative PCR kit (Sangon Biotech, Shanghai, China). 3 Perform qRT-RCR to detect the expression of mRNA and miRNA in a fluorescence quantitative gene amplifier qTOWER

[0034] Table 1 Primer sequences (human)

[0035]

[0036]

[0037] Note: Both miR-29a-3p-R and U6-R use the universal primer R provided in the kit, and U6-F is also provided in the kit.

[0038] Table 2 Primer sequences (mouse)

[0039]

[0040] Note: Both miR-29a-3p-R and U6-R use the universal primer R provided in the kit, and U6-F is also provided in the kit.

[0041] 1.7 Detection of total cholesterol (TC), free cholesterol (FC), and triglyceride (TG)

[0042] The operation was performed according to the instructions of the TC, FC, and TG kits.

[0043] (1) First, prepare the samples to be tested: ① Cell samples: Take out the 6-well plate used for culturing cells from the incubator and wash the cells with PBS. Add 200 μL of lysis buffer to each well, scrape the cells, transfer the liquid to a new EP tube and mix well, centrifuge the homogenate at 2000g for 5 minutes, take the supernatant and place it in a new EP tube for later use; ② Liver tissue: Take liver tissue, add an appropriate volume of lysis buffer, use a tissue grinder to homogenize, take an appropriate amount of homogenate and centrifuge it at 2000g for 5 minutes, take the supernatant and place it in a new EP tube for later use.

[0044] (2) Total cholesterol detection: Add samples to a 96-well plate in sequence and divide them into blank group, standard group, control group and experimental group, then add an appropriate amount of working solution to each well. Place the 96-well plate in a 37°C incubator and incubate for 30 minutes. After the incubation, take out the 96-well plate, put it into the microplate reader, set the wavelength to 490nm, shake it for 10 seconds, and start the detection.

[0045] (3) Free cholesterol detection: Add samples to the 96-well plate in sequence and divide them into blank group, standard group, control group and experimental group, then add an appropriate amount of working solution to each well (prepared in the ratio of R1:R2=4:1). Place the 96-well plate in a 37°C incubator and incubate for 30 minutes. After the incubation, take out the 96-well plate, put it into the microplate reader, set the wavelength to 562nm, shake for 10 seconds, and start the detection.

[0046] (4) Triglyceride detection: Add samples to a 96-well plate in sequence and divide them into blank group, standard group, control group and experimental group, then add an appropriate amount of working solution to each well. Place the 96-well plate in a 37°C incubator and incubate for 30 minutes. After incubation, take out the 96-well plate and place it in an ELISA reader. Set the detection wavelength to 490nm, shake for 10 seconds, and start the detection.

[0047] (5) Protein detection: Prepare the working solution according to the ratio of BCA to Cu of 50:1. Use the remaining homogenate as the sample for protein measurement. Add distilled water, standards, and test samples to the 96-well plate respectively, and then supplement each well with distilled water to make the total volume reach 20 μL. Add an appropriate amount of the prepared working solution to each well. Place the 96-well plate in an incubator at 37 °C and incubate statically for 30 min. After incubation, take out the 96-well plate and place it on the microplate reader. Set the wavelength to 562 nm, shake for 10 s, and then start the detection.

[0048] 1.8 Establishment of the MAFLD mouse model

[0049] Male C57BL / 6 wild-type mice at 6 weeks of age with a body weight in the range of 20 - 22 g were randomly divided into two groups, with 6 mice in each group. After at least three days of adaptation, they were given the model diet. The experimental group of mice was fed a high-fat methionine / choline-deficient diet (HFMCD), and the control group of mice was given a low-fat methionine / choline-supplemented diet (LFMCS) for 6 weeks. The mice had free access to food and water, and the mouse food was changed daily.

[0050] Collection and preservation of animal samples: The mice were sacrificed and sampled after the modeling was completed. The mice were fasted overnight before sacrifice but had free access to water.

[0051] After blood collection, the mice were sacrificed by cervical dislocation, and the liver was quickly separated. According to the experimental requirements, the middle lobe of the liver was trimmed, placed in an embedding cassette and adjusted to the appropriate position, and OCT embedding agent was added dropwise. Then the embedded tissue was placed in a -80 °C freezer for quick freezing and preservation. The left lateral lobe of the liver was fixed with a universal tissue fixative, and the remaining part was placed in an EP tube and stored at -80 °C for later use.

[0052] 1.9 Blood biochemical detection of mice

[0053] Take out the mouse blood samples previously stored at -80 °C, melt them at room temperature, and use a Chemray 800 fully automatic biochemical analyzer to detect blood biochemical indexes, including TC, TG, aspartate transaminase (AST), and alanine aminotransferase (ALT).

[0054] 1.10 H&E staining and Oil Red O staining

[0055] H&E staining: Mouse liver tissues were dehydrated through a gradient dehydration process and then embedded in paraffin. Finally, tissue sections with a thickness of 4 μm were prepared. After dewaxing and rehydration, the sections were immersed in hematoxylin staining solution for 5 minutes. After differentiation and blueing, the sections were placed in eosin solution for 2 minutes. After dehydration and clearing, the sections were sealed and observed under a microscope by taking pictures. Oil red staining: The pre-prepared frozen sections with a thickness of 10 mm were taken out from the -20°C refrigerator, fixed with tissue fixative, stained with oil red O staining solution, immersed in 60% isopropanol solution, washed with distilled water, counterstained with hematoxylin for 3 minutes, then washed with distilled water again, blueed, and gently immersed in tap water. After sealing, the sections were observed under a microscope by taking pictures.

[0056] 2. Experimental results and analysis

[0057] 2.1 Changes in the level of lncRNA TUG1 in the MAFLD cell model

[0058] HepG2 cells were induced to accumulate fat with a medium containing 50% FBS to establish a MAFLD cell model. As shown in ( Figure 1 a, 1b), compared with the control group, the total cholesterol level and triglyceride level in the model group were significantly higher than those in the control group, indicating that the MAFLD cell model was successfully established. In addition, the expression of lncRNA TUG1 in the model group was significantly lower than that in the control group ( Figure 1 c). The above results suggest that the level of lncRNATUG1 may be related to lipid metabolism.

[0059] 2.2 lncRNATUG1 regulates the expression of miR-29a-3p in HepG2 cells

[0060] According to the results of bioinformatics prediction, the seed region sequence of miR-29a-3p may be complementary to the sequence of lncRNA TUG1 ( Figure 2 a). Compared with the control group, after overexpressing miR-29a-3p in HepG2 cells, the level of lncRNA TUG1 decreased significantly ( Figure 2 b); while after knocking down the expression of lncRNA TUG1, the expression of miR-29a-3p increased significantly ( Figure 2 c). In addition, the results of the dual-luciferase reporter gene assay showed that the luciferase activity transfected with miR-29a-3p mimics was significantly lower than that of the other two groups ( Figure 2 d), suggesting that lncRNA TUG1 may regulate miR-29a-3p by complementary base pairing with its sequence.

[0061] 2.3 lncRNATUG1 regulates the expression of HMGCR in HepG2 cells through miR-29a-3p

[0062] The expression level of HMGCR mRNA was detected by overexpressing miR-29a-3p and knocking down the expression of lncRNA TUG1. In addition, the expression level of HMGCR protein was analyzed by Western Blot experiment. The results are as follows ( Figure 3 ). After transfection with miR-29a-3p mimics, the levels of HMGCR mRNA and protein were significantly downregulated; after transfection with si-TUG1, the levels of HMGCR mRNA and protein were also significantly downregulated. The above results showed that with the decrease of lncRNA TUG1 expression or the increase of miR-29a-3p expression, the expression of HMGCR was significantly reduced, suggesting that lncRNA TUG1 affects the expression of HMGCR by regulating miR-29a-3p.

[0063] 2.4 lncRNA TUG1 regulates the expression of SREBF-2 in HepG2 cells through miR-29a-3p

[0064] First, prediction was performed through a database and it was found that there was a binding site between miR-29a-3p and SREBF-2, as shown in ( Figure 4 a). And it was verified by the dual-luciferase reporter gene experiment that the luciferase activity transfected with miR-29a-3p mimics was significantly lower than the other two groups, as shown in ( Figure 4 b). Subsequently, the expression of SREBF-2 mRNA and protein levels was detected, as shown in ( Figure 4 c, 4d). With the decrease of lncRNA TUG1 expression or the increase of miR-29a-3p expression, the expressions of SREBF-2 mRNA and protein levels were significantly reduced, suggesting that lncRNA TUG1 affects the change of SREBP-2 by regulating miR-29a-3p.

[0065] 2.5 Changes in related genes and indexes after knocking down lncRNA TUG1 in HepG2 and Hepa1-6

[0066] The expression of lncRNA TUG1 in HepG2 and Hepa1-6 was knocked down by transfecting siRNA. As shown in ( Figure 5 a, 5e), the expression of lncRNA TUG1 was decreased, indicating that lncRNA TUG1 was successfully knocked down. Subsequently, the expressions of miR-29a-3p, HMGCR and SREBF-2 mRNA levels were detected, and the results are shown in ( Figure 2 c, 3a, 4c, 5f, 5g, 5h). With the decrease of lncRNA TUG1 expression, the contents of TC, FC and TG in HepG2 cells also decreased significantly ( Figure 5b, 5c, 5d). According to the above results, the expression of lncRNA TUG1 was negatively correlated with miR-29a-3p, and positively correlated with the contents of HMGCR, SREBF-2, TC, FC, and TG.

[0067] 2.6 Changes in related genes and levels in the MAFLD mouse model

[0068] For the contents of AST and ALT in mouse serum, as shown in ( Figure 6 a, 6b), the contents of AST and ALT in the serum of the model group were significantly higher than those of the control group. At the same time, through H&E staining of liver tissues ( Figure 6 c) and Oil Red O staining ( Figure 6 d), it was found that compared with the control group, there were lymphocyte infiltrations distributed in dots, ballooning degeneration and swelling of hepatocytes, and obvious lipid accumulation in the liver of mice in the model group. The above results indicated that the animal model was successfully established. Detection showed that compared with the control group, the expressions of lncRNA TUG1, HMGCR, and SREBF-2 in the liver of mice in the model group were significantly increased ( Figure 6 e, 6f, 6g), and the levels of TC, FC, and TG were also significantly up-regulated ( Figure 6 h, 6i, 6j). Combining the experimental results, it was found that the expression of lncRNA TUG1 was positively correlated with the levels of AST, ALT in mouse serum and TC, FC, TG in mouse liver ( Figure 7 ), suggesting that lncRNA TUG1 might be involved in the process of liver lipid metabolism in MAFLD.

[0069] In summary, this study showed that down-regulating the expression of lncRNA TUG1 could effectively inhibit liver lipid accumulation, thereby playing a liver-protective role. This finding not only provided a new potential target for the development of therapeutic drugs for MAFLD, but also opened up a new research direction for the preparation and application of therapeutic drugs for MAFLD, and provided reliable technical support for the research and development of related drugs.

Claims

1. Application of lncRNA TUG1 as a target in the preparation of drugs for treating metabolic associated fatty liver disease.

2. The application according to claim 1, characterized in that, The drug composition contains an inhibitor that targets and inhibits the functional expression of lncRNA TUG1.

3. The application according to claim 2, characterized in that The inhibitor is a human-derived siRNA. The sense strand of the human-derived siRNA is shown in SEQ ID NO.1, and the antisense strand is shown in SEQ ID NO.

2.

4. The application according to claim 2, characterized in that, The inhibitor is a mouse-derived siRNA. The sense strand of the mouse-derived siRNA is shown in SEQ ID NO.3, and the antisense strand is shown in SEQ ID NO.4.

Images