Application of lncRNAHOXA-AS3 as treatment target of metabolism-related fatty liver disease

Abstract

The invention relates to the technical field of medicine and biological medicine, and discloses application of lncRNA HOXA-AS3 as a treatment target of metabolism-related fatty liver diseases. According to the present invention, it is found that the lncRNA HOXA-AS3 in the human liver cancer cell HepG2 is subjected to targeted knock-down through siRNA transfection, such that the expression of the lncRNA HOXA-AS3 is reduced, and the content of total cholesterol and the content of triglyceride are significantly reduced; the lncRNA HOXA-AS3 in the mouse hepatocyte AML12 is knocked down by transfecting siRNA (small interfering ribonucleic acid), so that the expression of the lncRNA HOXA-AS3 is reduced; two mouse MAFLD models are established and analyzed to find that the expression of lncRNA HOXA-AS3 in the mouse liver of the MAFLD group is obviously increased compared with that of a control group, and the lncRNA HOXA-AS3 is prompted to be a drug development target related to MAFLD. A treatment action mechanism of the lncRNA HOXA-AS3 on the MAFLD by inhibiting the generation of fat and cholesterol is definite.

Description

Technical Field

[0001] The present invention belongs to the field of medicine and biomedicine technology, and specifically relates to the application of lncRNA HOXA-AS3 as a therapeutic target for metabolism-related fatty liver disease. Background Art

[0002] Nowadays, gene technology is developing rapidly, and scientists have shown great interest in using genetic engineering to develop drugs. Long non-coding RNA (lncRNA) is roughly defined as non-coding transcripts of more than 200 nucleotides (200nt). lncRNA was originally considered to be transcriptional "noise", but with the in-depth study, it was found that it plays an important intermediary role in the information transmission process caused by higher-order chromosome interactions and chromatin structure changes. They may coordinate the organization of chromatin domains and promote the long-range activation of specific genes. For example, cell cycle, chromatin remodeling, activation of transcription factors, and post-transcriptional processes of gene expression. In addition, one point repeatedly observed in various disease models is that lncRNA forms a complex network, coordinates with numerous chromatin regulatory factors, and precisely guides their enzymatic activity to the location within the genome. In a broader sense, lncRNA represents a class of multifunctional genes that participate in various biological functions. In different cellular processes, they are expressed differently according to cell types, thus localizing to specific organelles and participating in the development of human diseases. From the perspective of RNA regulation, it provides a theoretical basis and new gene therapy targets for people to reveal the mechanism of disease occurrence.

[0003] Disadvantages of existing technologies: The diagnostic criteria for metabolic associated fatty liver disease (MAFLD) are based on histological (liver biopsy), imaging and blood biomarker evidence of liver fat accumulation (hepatocyte steatosis), combined with one of the following three conditions: overweight / obesity, type 2 diabetes, and metabolic dysfunction. Metabolic dysfunction is defined as the presence of at least two metabolic abnormality risk factors, which was formerly known as nonalcoholic fatty liver disease (NAFLD). The global prevalence is as high as 25%, which seriously endangers human health and imposes a huge economic burden on society. On March 14, 2024, the U.S. FDA approved Rezdiffra (resmetirom) as the first drug to treat adult patients with non-cirrhotic nonalcoholic steatohepatitis (NASH), marking an important milestone in this field. Currently, adverse reactions in some patients treated with Rezdiffra include diarrhea, nausea, itching, abdominal pain, vomiting, constipation and dizziness. Therefore, it is necessary to find and develop new therapeutic drugs that are effective and have no serious adverse reactions. At present, there is no relevant research to verify that lncRNA HOXA-AS3 can be used to prepare drugs for the treatment of MAFLD, nor is there any relevant research to verify that lncRNA HOXA-AS3 can be used as a target for the development of MAFLD drugs, and there is no report on the treatment of MAFLD with lncRNA HOXA-AS3. Summary of the invention

[0004] In view of the above, it is necessary to study new target sites for MAFLD, find out new targets suitable for the preparation and treatment of MAFLD, and verify the model of the target sites.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is:

[0006] Application of lncRNA HOXA-AS3 as a therapeutic target for MAFLD or hepatic lipid metabolism disorders.

[0007] Use of an inhibitor that inhibits the functional expression of lncRNA HOXA-AS3 in the preparation of a drug for treating MAFLD.

[0008] Furthermore, the inhibitor is siRNA, the sense strand of siRNA is shown in SEQ ID NO.1, and the antisense strand is shown in SEQ ID NO.2. Wherein, the sequence of SEQ ID NO.1 is: 5'-ACGGAUAAAGGCACAUAUA-3', and the sequence of SEQ ID NO.2 is: 5'-UAUAUGUGCCUUUAUCCGU-3'.

[0009] Furthermore, the inhibitor is siRNA, the sense strand of siRNA is shown in SEQ ID NO. 3, and the antisense strand is shown in SEQ ID NO. 4. Wherein, the sequence of SEQ ID NO. 3 is: 5'-CAGUAAAUGUGCAAAUCUC-3', and the sequence of SEQ ID NO. 4 is: 5'-GAGAUUUGCACAUUUACUG-3'.

[0010] The present invention has the following beneficial effects

[0011] The lncRNAHOXA-AS3 of the present invention provides a new target for the preparation and treatment of MAFLD. Through the analysis of mouse HFMCD and MCD models, it was found that lncRNAHOXA-AS3 was expressed in liver tissues of mouse HFMCD and MCD models. The results showed that lncRNAHOXA-AS3 binds to miR-29a-3p, mediates miR-29a-3p to negatively regulate HMGCR, causing increased expression of HMGCR, thereby leading to an increase in cholesterol content. Cholesterol, as a lipotoxic molecule, can induce macrophage activation, promote a significant increase in the number of macrophages, and form a liver microenvironment with a pro-inflammatory state. Therefore, we speculate that lncRNAHOXA-AS3 may affect the cholesterol metabolism signaling pathway regulated by miR-29a-3p, ultimately promoting the development of liver inflammation and accelerating the progression of MAFLD. In addition, siRNA that downregulates lncRNAHOXA-AS3 acts as an inhibitor of HMGCR in MAFLD, indicating that downregulation of lncRNAHOXA-AS3 can reduce the production of cholesterol and has a protective effect on the liver. It can be used to prepare drugs for the treatment of MAFLD, which is beneficial to reduce the occurrence of cirrhosis, liver fibrosis, liver damage and liver cancer caused by MAFLD. It also has a good market prospect for clinical application. lncRNAHOXA-AS3 plays an important role in the production of cholesterol by regulating the miR-29a-3p / HMGCR axis and is a new therapeutic target for MAFLD. Silencing the lncRNAHOXA-AS3 target with siRNA can effectively reduce the levels of total cholesterol (TC) and triglyceride (TG) in cells to play a therapeutic role in MAFLD. lncRNAHOXA-AS3 inhibitors can be used to prepare oral drugs and injectable drugs for the treatment of MAFLD, as well as other measures for the treatment of MAFLD. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1:lncRNAHOXA-AS3 may regulate the level of miR-29a-3pA. Sequence-complementary binding sites; B. Levels of lncRNA HOXA-AS3; C. Results of dual-luciferase reporter gene experiment.

[0013] Figure 2 :Levels of related genes in the cell modelA. Total cholesterol (TC) level; B. Triglyceride (TG) level; C. miR-29a-3p level; D. lncRNA HOXA-AS3 level; E. HMGCR level.

[0014] Figure 3 : The levels of each gene in HepG2 cells and AML12 cells after knocking down lncRNA HOXA-AS3A. The level of lncRNA HOXA-AS3 in HepG2 cells; B. The level of HMGCR in HepG2 cells; C. The level of miR-29a-3p in HepG2 cells; D. The level of lncRNA HOXA-AS3 in AML12 cells; E. The level of HMGCR in AML12 cells; F. The level of miR-29a-3p in AML12 cells; G. The level of total cholesterol (TC); H. The level of triglyceride (TG).

[0015] Figure 4 :The level of lncRNA HOXA-AS3 in MAFLD animal modelA. HE staining and Oil red staining results of liver tissue in HFMCD model; B. The level of lncRNA HOXA-AS3 in HFMCD model; C. HE staining and Oil red staining results of liver tissue in MCD model; D. The level of lncRNA HOXA-AS3 in MCD model. DETAILED DESCRIPTION

[0016] Example 1

[0017] 1. Experimental Methods

[0018] 1.1 Levels of miR-29a-3p overexpression in HepG2 cells

[0019] Cell transfection: HepG2 cells with good growth status were transfected at 2×10 5 The cells were seeded at a density of 100 μg / mL in 6-well plates and grown to a confluence of 50-60% on the second day. Then, miR-29a-3p was overexpressed in the cells by transfection with miR-29a-3p mimics. The control group was transfected with the same dose of negative control (NC), and cell samples were collected after 72 h.

[0020] 1.2 Establishment of MAFLD cell model

[0021] Cell culture: HepG2 cells were cultured in a high glucose medium (DMEM) containing 10% fetal bovine serum (FBS) at 37°C and 5% CO2, and passaged every 2-3 days; Cell modeling: HepG2 cells with good growth status were passaged at 2×10 5 The cells were seeded at a density of 100 μg / mL in a 6-well plate. When the confluence reached 50-60% on the second day, the cells in the control group continued to be cultured with DMEM containing 10% FBS, and the cells in the modeling group were cultured with DMEM containing 50% FBS. Cell samples were collected after 48 h.

[0022] 1.3 Knockdown of lncRNA HOXA-AS3 in HepG2 cells and AML12 cells

[0023] Cell transfection: HepG2 cells and AML12 cells with good growth status were transfected at 2×10 5 The cells were inoculated at a density of 100 μg / cm2 in a 6-well plate. After the cells grew to a confluency of 50-60% on the second day, lncRNA HOXA-AS3 was knocked down in the cells by transfecting siRNAs. The SEQ ID NO.1 sequence of the human siRNA transfected in HepG2 cells was 5'-ACGGAUAAAGGCACAUAUA-3', and the SEQ ID NO.2 sequence was 5'-UAUAUGUGCCUUUAUCCGU-3'. The SEQ ID NO.3 sequence of the mouse siRNA transfected in AML12 cells was 5'-CAGUAAAUGUGCAAAU CUC-3', and the SEQ ID NO.4 sequence was 5'-GAGAUUUGCACAUUUACUG-3'. The control group was transfected with the same dose of negative control (NC), and the cell samples were collected 72 hours later.

[0024] 1.4 Extraction of total cell RNA, reverse transcription and cDNA synthesis, and real-time fluorescence quantitative PCR

[0025] Total RNA was isolated from cells or liver tissue using trizol reagent. RNA quality and concentration were determined using a NanoDrop2000 spectrophotometer (Thermo Fisher Scientific). After RNA extraction, cDNA was synthesized using MightyScript Plus first-strand cDNA, and miRNA was prepared using a miRNA first-strand cDNA synthesis kit. RT-qPCR used SGExcel Fast SYBR Mixture (Sangon Biotech, Shanghai, China). qRT-RCR was performed in a fluorescent quantitative gene amplification instrument qTOWER3G (Jena Analytical Instruments (Shanghai) Co., Ltd.) to detect mRNA and miRNA levels. The primers used for RT-qPCR are shown in Table 1. The internal reference gene for detecting mRNA is GAPDH, and the internal reference gene for detecting miRNA is U6. The sequences of PCR primers and related primers (all synthesized by Shanghai Biotech) are as follows:

[0026] Table 1

[0027]

[0028] Note: miR-29a-3p-R and U6-R both use the universal primer R provided in the kit, and U6-F is also provided in the kit

[0029] 1.5 Detection of total cholesterol and triglyceride levels

[0030] The operation was performed according to the instructions of the TC and TG kits. The 6-well plate used for culturing model cells and normal cells was removed from the incubator, and 1.5 mL of PBS was added to each well to wash the cells, and the cells were washed twice in total. 200 μL of lysis solution was added to each well, and the cells were scraped off with a pipette tip. The liquid was transferred to a new 1.5 mL EP tube and mixed. A portion of the homogenate was centrifuged at 2000 g for 5 min, and the supernatant was placed in a new EP tube for later use. (1) Cholesterol detection: 20 μL of purified water (blank group), 10 μL of standard and 10 μL of distilled water (standard group), 20 μL of treated normal cell supernatant (control group) and 20 μL of treated model cell supernatant (experimental group) were added to the 96-well plate. In addition, 190 μL of working solution was added to each well, and then the 96-well plate was placed in a 37°C incubator for 30 min. After 30 minutes, the 96-well plate was taken out and placed on the microplate reader. On this basis, the wavelength was selected as 490 nm, and then the detection was started after shaking for 10 seconds. (2) Triglyceride detection: 15 μL of distilled water (blank group), 5 μL of standard and 10 μL of pure water (standard group), 15 μL of treated normal cell supernatant (control group) and 15 μL of treated model cell supernatant (experimental group) were added to the 96-well plate. In addition, 190 μL of working solution was added to each well, and then the 96-well plate was placed in a 37°C incubator for 30 minutes. After 30 minutes, the 96-well plate was taken out and placed on the microplate reader. On this basis, the wavelength was selected as 490 nm, and then the detection was started after shaking for 10 seconds. (3) Protein detection: The working solution was prepared according to the ratio of BCA:Cu of 50:1, and the remaining homogenate was the sample required for protein detection. Add 1 μL of distilled water, standard and sample to be tested to the 96-well plate, add 19 μL of distilled water to each well, then add 190 μL of working solution to each well, and place the 96-well plate in a 37°C incubator for 30 minutes. After 30 minutes, take out the 96-well plate and place it on the microplate reader, select the wavelength of 562nm, and then start the detection after shaking for 10 seconds.

[0031] 1.6 Establishment of MAFLD mouse model

[0032] Male C57BL / 6 wild-type mice weighing 20-22g and aged 6 weeks were randomly divided into four groups, with 6 mice in each group. After at least three days of feeding adaptation, the model feed was given. The mice in the MCD experimental group were fed with a methionine / choline-deficient diet (MCD), and the mice in the MCS control group were fed with a methionine / choline diet (MCS) for three weeks. The mice in the HFMCD experimental group were fed with a methionine / choline-deficient diet (HFMCD), and the mice in the LFMCS control group were fed with a methionine / choline diet (LFMCS) for 6 weeks. The mice had free access to food and water, and the mouse diet was changed every day. After the modeling was completed, the mice were killed by dislocation of the neck and blood was collected, and the mice were fasted but not watered the night before the killing. The mouse liver was collected on the day of sampling, the middle lobe of the liver was trimmed as required, the position of the liver tissue was adjusted after placing it in the embedding box, the frozen section OCT embedding agent was dripped into the embedding box, and the embedded tissue was quickly frozen and stored at -80℃. The left lateral lobe of the mouse liver was fixed with a universal tissue fixative, and the remaining liver was placed in a 2 mL EP tube and stored at -80°C for later use.

[0033] 1.7 HE staining and oil red staining

[0034] HE staining: The mouse liver tissue was fixed with a universal tissue fluid, dehydrated by gradient treatment, and then embedded in paraffin, and finally prepared into 4μm thick tissue sections, dried, dewaxed, and hydrated for HE staining, and finally placed under a microscope to observe the morphological changes of the mouse liver tissue. Oil red staining: The pre-prepared 10mm thick frozen sections were taken out of the -20℃ refrigerator, fixed with tissue fixative, stained with Oil Red O staining solution, soaked and dried in 60% isopropanol solution, distilled water, hematoxylin counterstaining solution, and blueing solution, and then sealed with glycerol gelatin and placed under a microscope for observation.

[0035] 2. Experimental results and analysis

[0036] 2.1 lncRNA HOXA-AS3 may regulate the level of miR-29a-3p in HepG2 cells

[0037] According to the results of bioinformatics prediction, the seed region sequence of miR-29a-3p may be complementary to the sequence of lncRNA HOXA-AS3 ( Figure 1 A). After overexpression of miR-29a-3p in HepG2 cells, qRT-PCR was used to detect the level of lncRNA HOXA-AS3, and it was found that the level of lncRNA HOXA-AS3 was significantly decreased compared with the control group ( Figure 1 B). The results of the dual luciferase reporter gene experiment showed that the relative luciferase activity of cells in the lncRNA HOXA-AS3+miR-29a-3p group was lower than that in the lncRNA HOXA-AS3+NC group ( Figure 1 C), suggesting that lncRNA HOXA-AS3 may regulate miR-29a-3p by pairing with its sequence complementarity.

[0038] 2.2 Changes in lncRNA HOXA-AS3 levels in MAFLD cell models

[0039] HepG2 cells were cultured in DMEM medium containing 50% FBS to induce cell fat accumulation, thereby establishing a MAFLD cell model. Figure 2 A) shows that compared with the blank control group, the total cholesterol level in the MAFLD model group established in the experiment was significantly increased; ( Figure 2 B) shows that the triglyceride level of the model group cells is also significantly higher than that of the blank group cells, indicating that the MAFLD cell model was successfully established in this experiment. Compared with the blank control group, the level of miR-29a-3p in the MAFLD model cells increased ( Figure 2 C), while the relative expression of lncRNAHOXA-AS3 was significantly decreased compared with the blank control group ( Figure 2 D), and the mRNA expression of HMGCR, the downstream target gene of miR-29a-3p, was also significantly decreased compared with the blank control group ( Figure 2 E). These results suggest that the level of lncRNA HOXA-AS3 may be related to lipid metabolism.

[0040] 2.3 Changes in the levels of various genes after knockdown of lncRNA HOXA-AS3 in HepG2 and AML12

[0041] The expression of lncRNA HOXA-AS3 in HepG2 and AML12 cells was knocked down by transfection of siRNA. Figure 3 A and Figure 3 D) It can be seen that the expression of lncRNAHOXA-AS3 is reduced, indicating that lncRNA HOXA-AS3 is successfully knocked down. Then the expression of miR-29a-3p and HMGCR mRNA levels in the corresponding cells were detected. The results are shown in ( Figure 3 As shown in (B, 3C, 3E, 3F), in both cells, lncRNA HOXA-AS3 expression was reduced, miR-29a-3p levels were increased, and HMGCR mRNA expression was reduced. Figure 3 G, 3H), the TC and TG contents in HepG2 were also significantly reduced. Based on the above results, it can be concluded that lncRNA HOXA-AS3 is negatively correlated with miR-29a-3p levels and positively correlated with HMGCR levels. Reducing lncRNA HOXA-AS3 can significantly reduce the levels of TC and TG in cells.

[0042] 2.4 Changes in lncRNA HOXA-AS3 levels in the MAFLD mouse model

[0043] The MAFLD mouse model was established, and the results of HE staining and Oil Red O staining of liver sections ( Figure 4 A, 4C) showed that compared with the control group, the HFMCD and MCD groups had obvious lipid accumulation in the liver, and some inflammatory infiltration, indicating that the in vivo model of this experiment was successfully established. qRT-PCR detected the level of lncRNA HOXA-AS3, and found that compared with the control group, the expression of lncRNA HOXA-AS3 in the liver of mice in the HFMCD and MCD groups was significantly upregulated ( Figure 4 B, 4D), suggesting that lncRNA HOXA-AS3 may be involved in the lipid metabolism process in the liver in MAFLD.

[0044] In summary, downregulating lncRNA HOXA-AS3 in the present invention is beneficial to avoiding lipid accumulation and has a protective effect on the liver. It can be promoted and applied in the preparation of drugs for treating MAFLD, has good application prospects, and also provides a new direction and technical basis for finding and developing new therapeutic drugs for MAFLD.

Claims

1. Application of lncRNA HOXA-AS3 as a therapeutic target for metabolic-related fatty liver disease or hepatic lipid metabolism disorders.

2. Application of inhibitors that inhibit the functional expression of lncRNA HOXA-AS3 in the preparation of drugs for the treatment of metabolic-related fatty liver disease.

3. The use according to claim 1 or 2, characterized in that: The inhibitor is siRNA, the sense strand of the siRNA is shown in SEQ ID NO.1, and the antisense strand is shown in SEQ ID NO.

2.

4. The use according to claim 1 or 2, characterized in that: The inhibitor is siRNA, the sense strand of the siRNA is shown in SEQ ID NO.3, and the antisense strand is shown in SEQ ID NO.4.

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