Application of long noncoding RNA Gm17586 targeting SIRT1 to induce Smad3 acetylation and promote HSCs activation in liver fibrosis

Abstract

The application of long non-coding RNA Gm17586 in targeting SIRT1 to induce Smad3 acetylation and promote HSCs activation in liver fibrosis belongs to the field of research on the disease mechanism of liver fibrosis. The present invention establishes an in vitro HSCs cell model and determines that long non-coding RNA Gm17586 can target and regulate SIRT1 activity, inhibit Smad3 deacetylation modification, and then enhance Smad3 transcriptional activity, promote HSCs activation, and thus accelerate the occurrence and development of liver fibrosis through CCK8, RT-qPCR, WB, FISH, CO-IP, dual luciferase reporter gene experiments and other technologies.

Description

Technical Field

[0001] The present invention belongs to the field of research on the disease mechanism of liver fibrosis, and specifically relates to an application of a long non-coding RNA Gm17586 targeting SIRT1 to induce Smad3 acetylation and promote HSCs activation in liver fibrosis. Background Art

[0002] Liver fibrosis (LF) is a reversible healing response to acute or chronic liver injury and is the most common pathological process in various liver diseases. Fibrosis can lead to cirrhosis and even liver cancer. Activation of hepatic stellate cells (HSCs) is the primary driver of fibrosis, characterized by overexpression of α-smooth muscle actin (α-SMA) and collagen. Genetic disorders, alcoholism, medications, cholestasis, metabolic disorders, chronic viral hepatitis, and parasitic infections are the main causes of fibrosis. However, to date, effective and safe drugs for the treatment of LF are still lacking in clinical practice. Therefore, further understanding of the pathogenesis of LF is crucial for the development of effective drugs for its treatment.

[0003] Long non-coding RNA (lncRNA) can act as a molecular scaffold to recruit proteins or RNA to target genes, playing a vital regulatory function in pathophysiological processes, such as participating in angiogenesis and metastasis, chromatin modification, transcriptional activation and interference, and regulating cell tissue differentiation.

[0004] Silent information regulator 1 (SIRT1) is a nicotinamide adenine dinucleotide-dependent histone deacetylase and the most extensively studied sirtuin protein. It is commonly involved in physiological processes such as energy homeostasis and apoptosis, and exerts antioxidant and anti-inflammatory effects. The TGF-β1 / Smads pathway is a classic pro-fibrotic signaling pathway, in which Smad homolog 3 (SMAD family member 3, Smad3) is a key player in TGF-β1 / Smads signaling. Previous studies have reported that lysine 378 of Smad3 can be acetylated by p300 / CBP, enhancing Smad3's DNA binding ability and further promoting Smad3 transcriptional activity. Acetylation and deacetylation are dynamic processes that can transition between each other under the influence of various factors. Recent studies have confirmed that Smad3, a key regulator of TGF-β1 signaling, can also be deacetylated by SIRT1. Therefore, SIRT1-mediated deacetylation of Smad3 may be a key factor in the treatment of LF.

[0005] Based on the differentially expressed lncRNAs in LF obtained in the early screening, this paper uses primary HSCs of LF mice as an in vitro cell model. From the perspective of "Gm17586 targeted regulation of SIRT1 activity, inhibition of Smad3 deacetylation modification, and then enhancement of Smad3 transcriptional activity and promotion of HSCs activation", it clarifies the molecular mechanism of Gm17586 in the pathogenesis of liver fibrosis, in order to better understand the molecular mechanism of LF occurrence. Summary of the Invention

[0006] The present invention proposes an application of long non-coding RNA Gm17586 targeting SIRT1 to induce Smad3 acetylation and promote HSCs activation in liver fibrosis. Specifically, long non-coding RNA Gm17586 can target and regulate the activity of silent information regulator 1 (SIRT1), inhibit the deacetylation modification of Smad homolog 3 (Smad3), thereby enhancing Smad3 transcriptional activity, promoting the activation of hepatic stellate cells (HSCs), and thus accelerating the occurrence and development of liver fibrosis (LF).

[0007] The research team previously screened for 154 differentially expressed lncRNAs in the liver tissues of mice with liver fibrosis through RNA-Seq sequencing. Among them, Gm17586 was selected as a key differentially expressed lncRNA highly associated with the pathogenesis of liver fibrosis based on criteria such as differential fold change, functional enrichment, and species conservation. Gm17586 originates from chromosome 11, consists of three exons, and is a positive-sense non-coding RNA. The research team's preliminary sequencing and verification experiments showed that the expression level of Gm17586 is significantly reduced in liver fibrosis. To explore the mechanism by which Gm17586 mediates the pathogenesis of liver fibrosis, the research team previously used proteomic sequencing to discover that protein expression abnormalities also occur during the pathogenesis of liver fibrosis, and the expression level of SIRT1 is significantly reduced; bioinformatics analysis found that Gm17586 has potential binding sites with SIRT1.

[0008] To further study the relationship between Gm17586 and the occurrence of LF disease and its molecular mechanism, the present invention established an in vitro HSCs cell model. Through CCK8, RT-qPCR, WB, FISH, CO-IP, dual-luciferase reporter gene experiments and other technologies, it was determined that the long non-coding RNA Gm17586 can target and regulate SIRT1 activity, inhibit Smad3 deacetylation modification, thereby enhancing Smad3 transcriptional activity, promoting HSCs cell activation, and thus accelerating the occurrence and development of liver fibrosis.

[0009] The experimental results show that:

[0010] 1. Effects of Gm17586 overexpression / interference on the expression level of Gm17586 in primary HSCs of LF mice

[0011] Compared with the control group, the RNA expression of Gm17586 in HSCs of LF mice was significantly decreased; compared with the model group, the RNA expression level of Gm17586 was further decreased after Gm17586 interference; and the RNA expression level of Gm17586 was significantly increased after Gm17586 overexpression.

[0012] 2. Effects of Gm17586 overexpression / interference on HSCs cell viability, cell cycle, and HSCs activation markers

[0013] Overexpression of Gm17586 decreased HSC viability, increased the number of cells in the G1 phase, and decreased the mRNA expression of HSC activation markers α-SMA and Collagen I. Conversely, interference with Gm17586 increased HSC viability, decreased the number of cells in the G1 phase, and increased the mRNA expression of HSC activation markers α-SMA and Collagen I. Correlation analysis showed a significant negative correlation between Gm17586 and α-SMA and Collagen I.

[0014] 3. Effect of Gm17586 overexpression / interference on SIRT1 expression level

[0015] SIRT1 mRNA and protein expression were significantly decreased in primary HSCs from LF mice. Gm17586 knockdown further reduced SIRT1 mRNA and protein expression, as well as enzyme activity. Conversely, Gm17586 overexpression significantly increased SIRT1 mRNA and protein expression, as well as enzyme activity.

[0016] 4. Co-localization analysis of Gm17586 and SIRT1 cells

[0017] Gm17586 binds to SIRT1 with a binding free energy of -24.35 kcal / mol. Both Gm17586 and SIRT1 are primarily expressed in the nucleus. Therefore, from a cellular localization perspective, direct binding is possible.

[0018] 5. Effects of SIRT1 overexpression / interference on Smad3 mRNA and protein expression levels

[0019] Smad3 is highly expressed at both mRNA and protein levels in HSCs. After SIRT1 overexpression, Smad3 mRNA and protein expressions were significantly decreased, while after SIRT1 interference, Smad3 mRNA and protein expressions were significantly increased.

[0020] 6. Effect of SIRT1 overexpression / interference on Smad3 acetylation level

[0021] CO-IP results showed that the expression levels of Smad3 and its acetylation were significantly decreased in the SIRT1 overexpression group, while the expression levels of Smad3 and its acetylation were significantly increased in the SIRT1 interference group.

[0022] 7. Effects of SIRT1 overexpression / interference on Smad3 transcriptional activity

[0023] The results of the dual luciferase reporter experiment showed that the activity of Smad3 luciferase was significantly decreased in the SIRT1 overexpression group, while the activity of Smad3 luciferase was significantly increased in the SIRT1 interference group.

[0024] Therefore, the present invention experimentally confirms that Gm17586 can reduce SIRT1 activity, inhibit Smad3 deacetylation modification, thereby enhancing Smad3 transcriptional activity, promoting HSCs activation, and participating in the pathogenesis of liver fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1The expression of Gm17586 in primary HSCs of LF mice.

[0026] Note: RT-qPCR was used to detect the expression of Gm17586 in primary HSCs of LF mice. Compared with the control group, ## P<0.01, indicating that the difference is statistically significant.

[0027] Figure 2 Screening of Gm17586 overexpression plasmids and small interfering RNA.

[0028] Note: A: RT-qPCR verifies the effect of the recommended transfection concentration and time of Gm17586 overexpression plasmid. B: RT-qPCR screening of Gm17586 small interfering RNA. Compared with the siRNA1 group, ** P<0.01 indicates that the difference is statistically significant. ## P<0.01, indicating that the difference is statistically significant.

[0029] Figure 3 This is the effect of Gm17586 overexpression and interference on its expression level.

[0030] Note: Compared with the control group, ## P<0.01; compared with the NC group, ** P<0.01, indicating that the difference is statistically significant.

[0031] Figure 4 This is the effect of Gm17586 overexpression and interference on HSCs cell proliferation and cell cycle.

[0032] Note: A: CCK8 assay for HSCs cell viability. B: Cell cycle quantification statistics. C: Flow cytometry assay for cell cycle. a: Control group; b: Model group; c: Gm17586 overexpression group; d: Overexpression NC group; e: Gm17586 interference group; f: Interference NC group. Compared with the control group, ## P<0.01; compared with the NC group, ** P<0.01, indicating that the difference is statistically significant.

[0033] Figure 5 This is the effect of Gm17586 overexpression and interference on α-SMA and Collagen Ⅰ.

[0034] Note: A: RT-qPCR detection of α-SMA mRNA expression level. B: RT-qPCR detection of Collagen I mRNA expression level. Compared with the control group, ## P<0.01; compared with the NC group, * P<0.05,** P<0.01 indicates that the difference is statistically significant.

[0035] Figure 6 This is the correlation analysis between Gm17586, α-SMA and CollagenⅠ.

[0036] Note: A: Correlation analysis between the expression levels of Gm17586 and α-SMA in HSCs. B: Correlation analysis between the expression levels of Gm17586 and Collagen Ⅰ in HSCs.

[0037] Figure 7 Figure 2 shows the expression of SIRT1 in primary HSCs of LF mice.

[0038] Note: A: RT-qPCR detection of SIRT1 mRNA expression in LF mouse HSCs. B: Western blot detection of SIRT1 protein expression in LF mouse HSCs. C: Semi-quantitative analysis of protein expression. Compared with the control group, ## P<0.01, indicating that the difference is statistically significant.

[0039] Figure 8 This is the effect of Gm17586 overexpression and interference on SIRT1.

[0040] Note: A: RT-qPCR detection of SIRT1 mRNA expression level. B: Western blot detection of SIRT1 protein expression level. C: Semi-quantitative analysis of protein expression. Compared with the control group, ## P<0.01; compared with the NC group, ** P<0.01, indicating that the difference is statistically significant.

[0041] Figure 9 This is the effect of Gm17586 overexpression and interference on SIRT1 activity.

[0042] Note: SIRT1 activity was detected by SIRT1 activity fluorescence detection kit. Compared with the control group, ## P<0.01; compared with the NC group, ** P<0.01, indicating that the difference is statistically significant.

[0043] Figure 10 Prediction of Gm17586 binding sites to SIRT1 and their localization in HSCs.

[0044] Note: A: Schematic diagram of the binding sites of Gm17586 and SIRT1. B: FISH staining of the localization of Gm17586 and SIRT1 in HSC cells (×400).

[0045] Figure 11 The expression of Smad3 in primary HSCs of LF mice.

[0046] Note: A: RT-qPCR detection of Smad3 mRNA expression level. B: Western blot detection of Smad3 protein expression level. C: Semi-quantitative analysis of protein expression. Compared with the control group, ## P<0.01, indicating that the difference is statistically significant.

[0047] Figure 12 Screening of SIRT1 overexpression plasmids and small interfering RNA.

[0048] Note: A: RT-qPCR verifies the effect of the recommended concentration of SIRT1 overexpression plasmid. B: RT-qPCR screens the concentration of SIRT1 overexpression plasmid. Compared with the siRNA1 group, ** P<0.01, indicating that the difference was statistically significant; compared with the NC group, ## P<0.01.

[0049] Figure 13 This is the effect of SIRT1 overexpression and interference on Smad3 expression level.

[0050] Note: A: RT-qPCR detection of Smad3 mRNA expression level. B: Western blot detection of Smad3 protein expression level. C: Semi-quantitative analysis of protein expression. Compared with the control group, ## P<0.01; compared with the NC group, ** P<0.01, indicating that the difference is statistically significant.

[0051] Figure 14 The effects of SIRT1 overexpression and interference on Smad3 protein and acetylation levels.

[0052] Note: A: Immunoprecipitation detection of Smad3 and its acetylated protein expression. B: Semi-quantitative analysis of Smad3 protein expression. C: Semi-quantitative analysis of Smad3 acetylated protein expression. Compared with the control group, ## P<0.01; compared with the NC group, ** P<0.01, indicating that the difference is statistically significant.

[0053] Figure 15 This is the effect of SIRT1 overexpression / interference on Smad3 transcriptional activity.

[0054] Note: Compared with the control group, ## P<0.01; compared with the NC group, **P<0.01, indicating that the difference is statistically significant. DETAILED DESCRIPTION

[0055] Example: Study on the mechanism by which LncRNA Gm17586 targets SIRT1 to induce Smad3 acetylation and promote HSCs cell activation

[0056] 1 Experimental Materials

[0057] 1.1 Experimental Animals

[0058] Male C57BL / 6 mice, weighing 20 ± 2 g, SPF-grade, and 6–8 weeks old, were purchased from Hangzhou Ziyuan Laboratory Animal Technology Co., Ltd. All mice were weighed and recorded twice weekly. They were housed in an environment with a 12-hour day / night cycle and a room temperature of 18–22°C. They were provided with ample food and water, with free access to both food and water. Laboratory Animal License Number: SCXK (Zhejiang) 2019-00004.

[0059] 1.2 Experimental Ethics Approval

[0060] All animal experiments involved in this invention were reviewed and approved by the Ethics Committee of Anhui University of Chinese Medicine, with the ethics approval number: AHUCM-mouse-2023116.

[0061] 2 Experimental methods

[0062] 2.1 Establishment of liver fibrosis model

[0063] SPF male C57BL / 6 mice were adaptively fed for 1 week, and then a mixture of carbon tetrachloride (CCl4) and olive oil (ratio of 1:4) was injected subcutaneously on the back at a rate of 0.1 mL / 10 g, twice a week, for a total of 12 weeks.

[0064] 2.2 Isolation of HSCs

[0065] Mice were anesthetized with an intraperitoneal injection of sodium pentobarbital. The limbs were immobilized, the abdomen disinfected, and sodium heparin injection injected for anticoagulation. The abdominal cavity was opened, and the liver and portal vein exposed. 50 mg of collagenase IV was dissolved in 25 mL of DMEM medium and diluted 4-fold to prepare a 0.05% collagenase perfusion solution. This solution was perfused into the inferior vena cava, using gentle pressure to ensure adequate perfusion. Once the blood drained and the liver turned grayish white, the perfusion solution was replaced with a digestive enzyme solution to digest the liver tissue and thoroughly dissect the liver. 1% BSA was removed and used to resuspend the cells, which were then filtered through a 200-mesh filter. The cell suspension was collected and centrifuged at 40g for 4 minutes. The supernatant was collected and centrifuged again for 10 minutes. The pellet was resuspended in serum-free RPMI-1640 medium and centrifuged at 400g for 10 minutes. The pellet was then added to 2 mL of the cell suspension, 4 mL of 35% Percoll, and 4 mL of 50% Percoll, followed by centrifugation at 900g for 30 minutes. The cells at the interface of 35% percoll and PBS were collected, diluted and centrifuged, and the supernatant was discarded. The cells were cultured with RPMI-1640 medium containing 10% FBS and seeded into culture dishes to obtain HSCs.

[0066] 2.3 Synthesis of overexpression plasmids and small interfering RNA and cell transfection

[0067] 2.3.1 Synthesis of overexpression plasmid

[0068] The Gm17586 and SIRT1 overexpression plasmid vectors and the control plasmid empty vector were commissioned to Shanghai Jima Pharmaceutical Technology Co., Ltd. for construction.

[0069] 2.3.2 Synthesis of small interfering RNA

[0070] Primer sequences for Gm17586 and SIRT1 were obtained from the NCBI database (https: / / www.ncbi.nlm.nih.gov / ). Small interfering RNAs (siRNAs) and negative controls were designed based on these sequences. The siRNAs for Gm17586 and SIRT1 were designed and synthesized by Shanghai Gene Pharmaceutical Technology Co., Ltd. Sequence information for the siRNAs for Gm17586 and SIRT1 is shown in Table 1.

[0071] Table 1 Gm17586 and SIRT1 small interfering RNA sequences

[0072]

[0073] 2.3.3 Cell transfection

[0074] Cells were seeded into 6-well plates and incubated overnight in a 37°C, 5% CO2 incubator with 2 mL of DMEM supplemented with 10% FBS. Subsequently, 75 μL of serum-free DMEM was added and divided into two portions. One portion was mixed with the corresponding overexpression plasmid / small interfering RNA, while the other portion was added with the transfection reagent Lip 2000. After incubation for 20 minutes, the two solutions were mixed and allowed to stand at room temperature for 20 minutes. The solution was then added to a 96-well plate washed with PBS and incubated in a 37°C incubator for 5 hours to complete transfection.

[0075] Overexpression plasmid and small interfering RNA name:

[0076] Gm17586 and SIRT1 overexpression plasmids, namely pc-Gm17586 and pc-SIRT1; their control empty plasmid, namely pc-NC. Gm17586 and SIRT1 small interfering RNA, namely siRNA; their negative control, namely siRNA-NC.

[0077] 2.4CCK8 detection of cell viability

[0078] The cells were resuspended in complete culture medium under the action of trypsin and the cell density was adjusted to 1×10 5 / mL. Cells were seeded into a 96-well plate to prepare a cell suspension. 100 μL was added to each well. The edge wells were filled with sterile PBS. Blank wells containing only culture medium were also set up. After incubation in a 5% CO2 incubator for 24, 48, and 72 hours, 10 μL of CCK8 was added to each well and incubated for an additional hour. Finally, the absorbance of each well was measured at 450 nm using an enzyme-linked immunosorbent assay (ELISA).

[0079] 2.5 Flow cytometry detection of cell cycle

[0080] Prepare cells by separating cell clumps into single cells. Gently vortex the cells and slowly add 2.7 mL of pre-chilled anhydrous ethanol dropwise to a final concentration of 90%. Fix at -20°C for 1 hour. Wash the fixed cells with pre-chilled PBS and centrifuge at 2000 rpm for 5 minutes. Aspirate the PBS and place them in an EO tube. Add 500 μL of PBS and gently tap the bottom of the tube to prevent cell clumping. Resuspend the cells in 20 μL of RNase A and incubate in a 37°C water bath for 30 minutes. Then, resuspend in 400 μL of PI staining solution and stain at 4°C in the dark for 30 minutes. Cell cycle analysis was performed by flow cytometry.

[0081] 2.6 RT-qPCR detection of gene expression

[0082] 2.6.1 RNA extraction

[0083] Collect the cell pellet and lyse it by adding 1 mL of TRIzol. Place the pellet in a centrifuge tube and add 0.2 mL of chloroform. Vortex the tube for 10 seconds. After standing at room temperature for 5 minutes, centrifuge at 12,000 rpm at 4°C for 10 minutes. Remove the supernatant and add 0.5 mL of isopropanol. Mix by inversion and centrifuge again. Discard the supernatant. Add 1 mL of pre-chilled 75% ethanol and centrifuge. Discard the supernatant and repeat this step. Set aside the pellet, add an appropriate amount of DEPC water, and store at -80°C until needed.

[0084] 2.6.2 RT reaction

[0085] After removing genomic DNA, RNA, 5× gDNA Eraser Buffer, and gDNA Eraser were added to a PCR tube, and the volume was made up to 10 μL with DEPC water. After centrifugation, the tube was placed in a PCR instrument and incubated at 42°C for 2 minutes. RT Primer Mix, PrimeScript RT Enzyme Mix I, and RNase-free dH2O were then added on ice. Reverse transcription was performed in a PCR instrument at 37°C for 15 minutes and then at 85°C for 5 seconds. The resulting cDNA was stored at -20°C until further use.

[0086] 2.6.3 Fluorescence quantitative PCR reaction

[0087] Using the above cDNA as a fluorescent quantitative template, the reaction system in Table 2 was established, and the fluorescent quantitative PCR reaction was performed under the reaction conditions in Table 3 to detect the expression level of each indicator. The primer information of each detection indicator is shown in Table 4. The results include amplification curves, melting curves and relative expression levels. The analysis method is the relative quantitative method, and the calculation method is 2 -△△CT .

[0088] Table 2 Fluorescence quantitative PCR reaction system

[0089]

[0090] Table 3 Fluorescence quantitative PCR reaction conditions

[0091]

[0092] Table 4 Primer sequences

[0093]

[0094] 2.7 Western blot detection of protein expression

[0095] Cell pellets were collected and 100 μL of RIPA lysis buffer (containing 1 mM PMSF) was added to each 6-well plate. The cells were lysed on ice for 30 minutes and centrifuged at 12,000 rpm for 10 minutes. The supernatant was collected to obtain total cellular protein. 5× SDS-PAGE protein loading buffer (1:4) was added to the protein sample and heated in a boiling water bath for 10 minutes to fully denature the protein. An SDS-PAGE gel was prepared and 30 μg of protein sample was loaded onto the SDS-PAGE gel at 80 V for 30 minutes, followed by 120 V. A PVDF membrane was activated by soaking in methanol, cut, and soaked in transfer buffer for 5 minutes. A piece of filter paper was placed on each anode and cathode plate and transferred to an electroporator at 300 mA. After blocking with 5% nonfat dry milk at room temperature for 2 hours, the PVDF membrane was incubated in the corresponding primary antibody working solution at 4°C on a shaker overnight. The primary antibodies used and their dilution ratios are detailed in Table 5. The PVDF membrane was then washed with PBST buffer and transferred to the corresponding secondary antibody diluted 1:10,000. The membrane was incubated on a shaker for 2 hours. Equal volumes of ECL solution and ECL solution B were mixed to prepare fresh luminescent solution. The PVDF membrane was placed in the exposure plate of an automatic light emitting device and 100 μL of ECL solution was added to capture the image. Finally, grayscale analysis of the image was performed using Image J software.

[0096] Table 5 Antibody information

[0097]

[0098] 2.8 Immunofluorescence experiments

[0099] Culture cells on a culture plate, remove excess medium, and gently rinse with PBS for 3 minutes, repeat twice. Fix with 4% paraformaldehyde for 15 minutes, then rinse with PBS for 3 minutes, repeat three times. Permeabilize in 0.1% Buffer A for 15 minutes, then rinse with PBS. Immediately, add 200 μL of 1x blocking solution and block for 30 minutes. After blocking, add 200 μL of primary antibody diluted 1:200 in PBS and incubate at 37°C for 1.5 hours. Add 200 μL of the corresponding secondary antibody (diluted 1:200) and incubate at room temperature for 30 minutes. Wash with PBS for 5 minutes three times. Add 200 μL of diluted DAPI working solution to the cell wells and wash with PBS for 5 minutes twice in the dark. Finally, add anti-quencher to the slides and observe fluorescence distribution and intensity changes under a fluorescence microscope.

[0100] 2.9 Fluorescence in situ hybridization

[0101] After paraffin sections were thawed, they were deparaffinized in xylene I and II, followed by dehydration in graded ethanol. Proteinase K was added dropwise and incubated at 37°C for 20 minutes. The proteinase K working solution was aspirated, and 100 μL of 1x blocking buffer was added dropwise. The sections were incubated at 37°C for 30 minutes. The blocking buffer was aspirated and discarded. 100 μL of 2x Buffer C was added dropwise, and the sections were washed three times at room temperature for 1 minute. Subsequently, 100 μL of preheated denaturing buffer was added dropwise to each section, and the sections were incubated at 78°C for 8 minutes. Dehydration was performed using graded ethanol and dried. Buffer E was pre-incubated in a 73°C waterbath for 30 minutes until clear. The probe was diluted with DEPC water to a concentration of approximately 100 μM. The probe stock solution was diluted to 1 μM and denatured in a 75°C waterbath for 10 minutes. The probe was then added to PBS in the appropriate proportions with SA-Cy3 and incubated at 37°C for 30 minutes. Then, 100 μL of the denatured probe mixture was added dropwise, the sections were sealed with parafilm, and hybridization was carried out overnight at 37°C for 16 hours. The next day, add 100 μL of 2× Buffer C and wash three times at 60°C for 10 minutes each time. Aspirate the Buffer C, repeat the wash cycle, and air-dry at room temperature. Add 100 μL of the diluted DAPI working solution to each section and incubate at room temperature in the dark for 10 to 20 minutes. Aspirate the DAPI working solution, wash twice with PBS for 5 minutes, and mount the sections with an antifade reagent. Observe under a fluorescence microscope.

[0102] 2.10 Dual-luciferase reporter assay

[0103] Following the cell transfection steps in 2.3.3, transfect the Smad3 promoter fragment in triplicate wells. Luciferase activity was measured using the Beyotime Dual Luciferase Assay Kit, using firefly luciferase and Renilla luciferase as standard.

[0104] 2.11 SIRT1 enzyme activity detection

[0105] Total cell protein was extracted, and SIRT1 enzyme activity was measured using a SIRT1 enzyme activity detection kit according to the manufacturer's instructions.

[0106] 2.12 Co-immunoprecipitation experiments

[0107] For each 6-well plate, add lysis buffer to an EP tube and lyse thoroughly on ice. Centrifuge at 14,000 rpm for 5 minutes. Collect the supernatant. For the normal group, divide the supernatant into three tubes, each labeled IP, IgG, and Input. For the other groups, divide the supernatant into two tubes, each labeled IP and Input. Magnetic separation was performed on the two tubes of Protein A / G magnetic beads. Aspirate the supernatant and add 500 μL of Smad3 antibody working solution and 500 μL of Normal Rabbit IgG working solution, respectively. Incubate on a rocking platform for 1 hour. Add 500 μL of TBS and gently pipette to resuspend the Protein A / G magnetic beads. Incubate the sample with Protein A / G magnetic beads conjugated with the target antibody and Normal Rabbit IgG. Add the protein samples labeled IP and IgG to the Protein A / G magnetic beads conjugated with the target antibody and Normal Rabbit IgG, respectively. Incubate overnight at 4°C on a side-by-side shaker or rotary mixer. Magnetic separation was performed. After incubation, separate the beads on a magnetic rack for 10 seconds and remove the supernatant. Add 0.5 mL of lysis buffer containing inhibitors and gently pipette to resuspend the beads. Separate the beads on a magnetic rack for 10 seconds and remove the supernatant. Repeat washing three times and detect the relevant proteins as in step 2.7.

[0108] 2.13 Statistical Analysis

[0109] The experiments involved in this invention were repeated 3 times independently, and SPSS software was used for data analysis and drawing. The results are expressed as mean ± standard deviation. . Differences between groups were analyzed using the t-test; comparisons between multiple means were performed using one-way analysis of variance; and correlations were analyzed using the Pearson correlation method. Results were considered statistically significant when the p-value was < 0.05. Figures were assembled using Adobe Illustrator software.

[0110] 3 Experimental results

[0111] 3.1 Expression of Gm17586 in primary HSCs of LF mice

[0112] In order to study the expression of Gm17586 in liver fibrosis, RT-qPCR was used to detect the changes in the expression level of Gm17586 in HSCs. Figure 1 As shown in Figure 3, the expression of Gm17586 in HSCs of the model group was significantly decreased compared with that of the control group.

[0113] 3.2 Transfection efficiency of Gm17586 in primary HSCs of LF mice

[0114] Gm17586 small interfering RNA and overexpression plasmid were transfected into HSCs, and the expression level of Gm17586 was detected by RT-qPCR to screen the optimal transfection conditions. Figure 2 As shown in A, compared with the NC group, the expression of Gm17586 overexpression group was significantly increased. Figure 2 As shown in Figure 2, compared with the siRNA-NC group, the expression of Gm17586 in the siRNA group was significantly reduced, and the inhibitory effect of siRNA3 on Gm17586 expression was more significant than that of siRNA1 and siRNA2.

[0115] 3.3 Effects of Gm17586 overexpression / interference on Gm17586 expression in primary HSCs of LF mice

[0116] In order to investigate the effects of Gm17586 overexpression / interference on Gm17586 expression in HSCs, RT-qPCR was used to detect the changes in Gm17586 expression levels in HSCs. Figure 3 As shown, compared with the NC group, the expression level of Gm17586 in the Gm17586 overexpression group was significantly increased, while the expression level of Gm17586 in the Gm17586 small interference group was significantly decreased.

[0117] 3.4 Effects of Gm17586 overexpression / interference on HSCs viability, cell cycle, and HSCs activation indicators

[0118] 3.4.1 Effects of Gm17586 Overexpression / Interference on HSC Viability and Cell Cycle

[0119] CCK8 and flow cytometry were used to detect the effects of Gm17586 overexpression / interference on HSCs proliferation and cell cycle. Figure 4 As shown in A, compared with the control group, the HSCs cell viability in the LF model group was significantly increased; compared with the NC group, Gm17586 overexpression inhibited the HSCs cell viability, while Gm17586 interference significantly increased the HSCs cell viability. Figure 4 As shown in B and 4C, compared with the control group, the number of G1 HSCs in the LF model group was significantly reduced; compared with the NC group, Gm17586 overexpression significantly increased the number of G1 HSCs, while Gm17586 interference significantly decreased the number of G1 HSCs.

[0120] 3.4.2 Effects of Gm17586 Overexpression / Interference on the Expression of HSCs Activation Markers α-SMA and Collagen I

[0121] RT-qPCR was used to detect the effects of Gm17586 overexpression / interference on HSCs activation markers α-SMA and Collagen I. Figure 5 As shown in A and 5B, compared with the control group, the expression levels of α-SMA and CollagenⅠ mRNA in the LF model group were significantly increased; compared with the NC group, the expression levels of α-SMA and CollagenⅠ mRNA in the Gm17586 overexpression group were significantly decreased, while in contrast, the expression levels of α-SMA and CollagenⅠ mRNA in the Gm17586 interference group were further increased.

[0122] 3.4.3 Correlation Analysis between Gm17586 and Liver Fibrosis

[0123] In order to further explore the relationship between Gm17586 and LF disease, the present invention conducted a correlation analysis and used Pearson correlation to analyze the correlation between Gm17586 and α-SMA and Collagen I at the cellular level. Figure 6 As shown in A and 6B, Gm17586 was significantly negatively correlated with α-SMA and Collagen Ⅰ in HSCs, further confirming that the expression levels of α-SMA and Collagen Ⅰ decreased with the increase of Gm17586.

[0124] 3.5 SIRT1 expression in primary HSCs of LF mice

[0125] To investigate the expression of SIRT1 in liver fibrosis, RT-qPCR and WB were used to detect the changes in SIRT1 mRNA and protein expression levels in HSCs. Figure 7 As shown in AC, compared with the control group, the mRNA and protein expression levels of SIRT1 in HSCs of the model group were significantly decreased.

[0126] Effects of Gm17586 overexpression / interference on SIRT1 expression in primary HSCs of LF mice

[0127] RT-qPCR and WB were used to detect the expression of SIRT1. RT-qPCR results showed that compared with the control group, the expression level of SIRT1 mRNA in the LF model group was significantly decreased; compared with the NC group, the expression level of SIRT1 mRNA in the Gm17586 overexpression group was significantly increased, while in contrast, the expression level of SIRT1 mRNA in the Gm17586 interference group was further decreased ( Figure 8A). WB results showed that compared with the control group, the expression level of SIRT1 protein in the LF model group was significantly decreased, while compared with the NC group, the expression level of SIRT1 protein in the Gm17586 overexpression group was significantly increased. On the contrary, the expression level of SIRT1 protein in the Gm17586 interference group was further decreased ( Figure 8 B and 8C).

[0128] Effects of Gm17586 overexpression / interference on SIRT1 activity in primary HSCs of LF mice

[0129] The SIRT1 activity fluorescence detection kit was used to detect the effect of Gm17586 overexpression / interference on SIRT1 enzyme activity. The results showed that compared with the control group, SIRT1 activity in the LF model group was significantly reduced; compared with the NC group, SIRT1 activity in the Gm17586 overexpression group was significantly increased, while SIRT1 activity in the Gm17586 interference group was further reduced ( Figure 9 ).

[0130] 3.8 Prediction of Gm17586-SIRT1 Binding Sites and Localization in HSCs

[0131] In order to further clarify the localization of Gm17586 expression, the present invention used IF and fluorescence in situ hybridization (FISH) experiments to detect the expression location of Gm17586 in HSCs. The target prediction results showed that Gm17586 had a binding site with SIRT1 ( Figure 10 A), the binding free energy is -24.35kcal / mol. The results of IF and FISH experiments showed that ( Figure 10 B) Gm17586 and SIRT1 are primarily expressed in the nucleus, thus enabling direct binding from a cellular localization perspective.

[0132] 3.9 Expression of Smad3 in primary HSCs of LF mice

[0133] To investigate the expression of Smad3 in liver fibrosis, RT-qPCR and WB were used to detect the changes in Smad3 mRNA and protein expression levels in HSCs. Figure 11 As shown in Figures AC, compared with the control group, the expression levels of Smad3 mRNA and protein in HSCs of the model group were significantly increased.

[0134] 3.10 SIRT1 transfection efficiency in primary HSCs of LF mice

[0135] SIRT1 small interfering RNA and overexpression plasmid were transfected into HSCs, and the expression of SIRT1 was detected by RT-qPCR to screen the optimal transfection conditions. Figure 12 As shown in A, the expression of SIRT1 overexpression group was significantly increased compared with the NC group. Figure 12 As shown in Figure B, compared with the siRNA-NC group, the expression of SIRT1 in the siRNA group was significantly reduced, and the inhibitory effect of siRNA3 on SIRT1 expression was most significant compared with siRNA1 and siRNA2.

[0136] 3.11 Effects of SIRT1 overexpression / interference on Smad3 expression in primary HSCs of LF mice

[0137] To investigate the effects of SIRT1 overexpression / interference on Smad3 expression in HSCs, RT-qPCR and WB were used to detect changes in Smad3 expression levels in HSCs. RT-qPCR results showed that compared with the control group, the Smad3 mRNA expression level in the LF model group was significantly increased; compared with the NC group, the Smad3 mRNA expression level in the SIRT1 overexpression group was significantly decreased, while the Smad3 mRNA expression level in the SIRT1 small interference group was significantly increased ( Figure 13 A). WB results showed that compared with the control group, the expression of Smad3 protein was increased in the LF model group; compared with the NC group, the expression level of Smad3 protein was significantly decreased in the SIRT1 overexpression group, while the expression level of Smad3 protein was further increased in the SIRT1 interference group ( Figure 13 B and 13C).

[0138] 3.12 Effects of SIRT1 overexpression / interference on Smad3 acetylation levels in primary HSCs of LF mice

[0139] In order to study the effect of SIRT1 on the acetylation level of Smad3 in HSCs, CO-IP was used to detect the changes in Smad3 and its acetylation level. Figure 14 As shown in Figures AC, compared with the NC group, the expression levels of Smad3 and its acetylation were significantly decreased in the SIRT1 overexpression group, while the expression levels of Smad3 and its acetylation were significantly increased in the SIRT1 small interference group. These results indicate that SIRT1 can inhibit the deacetylation level of Smad3.

[0140] 3.13 Effects of SIRT1 overexpression / interference on Smad3 transcriptional activity in primary HSCs of LF mice

[0141] A dual-luciferase reporter assay was used to examine the effects of SIRT1 overexpression / interference on Smad3 transcriptional activity in HSCs. The results showed that compared with the control group, the Smad3 luciferase activity in the model group was significantly increased; compared with the NC group, the Smad3 luciferase activity in the SIRT1 overexpression group was significantly decreased, while the Smad3 luciferase activity in the SIRT1 small interference group was significantly increased ( Figure 15 The results showed that SIRT1 could significantly enhance the transcriptional activity of Smad3.

[0142] 4 Summary

[0143] In this study, primary HSCs from LF mice were used as an in vitro cell model. Transfection with Gm17586 and SIRT1 overexpression plasmids and small interfering RNA (siRNA) was performed. We determined that Gm17586 expression was reduced in HSCs, affecting HSC activation and promoting HSC proliferation. mRNA expression levels of HSC activation markers α-SMA and Collagen I were significantly increased. Correlation analysis revealed a significant negative correlation between Gm17586 and α-SMA and Collagen I mRNA. Similarly, SIRT1 expression was significantly reduced in HSCs, and Gm17586 could specifically enhance SIRT1 mRNA and protein expression and enzyme activity. FISH experiments and bioinformatics predictions demonstrated that Gm17586 and SIRT1 share common subcellular localization and multiple binding sites. In contrast, Smad3 expression was elevated in HSCs, and SIRT1 inhibited Smad3 expression. CO-IP experiments showed that SIRT1 inhibited the deacetylation modification of Smad3, and dual luciferase experiments showed that SIRT1 enhanced the transcriptional activity of Smad3.

[0144] Through the above experimental results, the present invention determined that Gm17586 can target SIRT1, inhibit Smad3 deacetylation modification, thereby enhancing Smad3 transcriptional activity, promoting HSCs activation, and participating in the pathogenesis of liver fibrosis.

[0145] The above content is merely an example and explanation of the concept of the present invention. Those skilled in the art may make various modifications or additions to the described specific embodiments or replace them in a similar manner. As long as they do not deviate from the concept of the invention or exceed the scope defined by the claims, they should all fall within the scope of protection of the present invention.

Claims

1. Application of a long non-coding RNA Gm17586 in the preparation of targeted therapeutic preparations for liver fibrosis.

2. The use according to claim 1, characterized in that This long non-coding RNA Gm17586 can target and regulate SIRT1 activity, promote Smad3 deacetylation modification, reduce Smad3 transcriptional activity, inhibit HSCs activation, and thus exert a therapeutic effect on liver fibrosis.

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