Application of long-chain non-coding RNA Gm17586 targeting SIRT1 to induce Smad3 acetylation to promote HSCs activation in hepatic fibrosis

Abstract

The invention discloses application of long-chain non-coding RNA Gm17586 targeting SIRT1 to induce Smad3 acetylation to promote HSCs activation in hepatic fibrosis, and belongs to the field of hepatic fibrosis disease mechanism research. An HSCs in-vitro cell model is established, and through CCK8, RT-qPCR, WB, FISH, CO-IP and dual luciferase reporter gene experiments and other technologies, it is determined that the long-chain non-coding RNA Gm17586 can regulate the activity of SIRT1 in a targeted mode and inhibit Smad3 deacetylation modification, so that the transcriptional activity of Smad3 is enhanced, HSCs activation is promoted, and occurrence and development of hepatic fibrosis are accelerated.

Description

Technical Field

[0001] The present invention belongs to the field of research on the mechanism of liver fibrosis, and specifically relates to an application of long-chain 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. Liver fibrosis can lead to cirrhosis and even liver cancer. Activation of hepatic stellate cells (HSCs) is the main driving factor for the formation of liver fibrosis, which is characterized by overexpression of α-smooth muscle actin (α-SMA) and collagen. Genetic diseases, alcoholism, drugs, cholestasis, metabolic disorders, chronic viral hepatitis, parasitic infections, etc. are the main causes of liver fibrosis. However, to date, there is still a lack of effective and safe drugs for the treatment of liver fibrosis in clinical practice. Therefore, further understanding of the pathogenesis of LF is of great significance for finding effective drugs for the treatment of LF.

[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 pathological and physiological 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 widely studied Sirtuin protein. It is usually involved in physiological processes such as energy homeostasis and cell apoptosis in organisms, and plays an antioxidant and anti-inflammatory role. The TGF-β1 / Smads pathway is a classic pro-fibrotic signaling pathway, among which Smad homolog 3 (SMAD family member 3, Smad3) is a key factor in the TGF-β1 / Smads signaling process. It has been reported in the literature that lysine 378 of Smad3 can be acetylated by p300 / CBP, which enhances the ability of Smad3 to bind to DNA and further promotes the transcriptional activity of Smad3. Acetylation and deacetylation are dynamic processes that can be transformed into each other under the influence of multiple factors. Recent studies have confirmed that Smad3, as an important regulator in 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 LF differentially expressed lncRNA screened in the early stage, the primary HSCs of LF mice were used 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, promoting HSCs activation", the molecular mechanism of Gm17586 involved in the pathogenesis of liver fibrosis was elucidated 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 the transcriptional activity of Smad3, promoting the activation of hepatic stellate cells (HSCs), and thus accelerating the occurrence and development of liver fibrosis (LF).

[0007] In the early stage, the research group screened out 154 differentially expressed lncRNAs in the liver tissue of mice with liver fibrosis through RNA-Seq sequencing. Among them, Gm17586 is a key differential lncRNA that is highly correlated with the onset of liver fibrosis, which was screened out based on the criteria of differential multiples, functional enrichment, species conservation, etc. Gm17586 originates from chromosome 11, consists of 3 exons, and is a positive non-coding RNA. The early sequencing and verification experiments of the research group showed that the expression level of Gm17586 was significantly reduced in liver fibrosis. In order to find the mechanism by which Gm17586 mediates the onset of liver fibrosis, the research group used proteomic sequencing to find that there is also abnormal protein expression in the process of liver fibrosis, and the expression level of SIRT1 is significantly reduced; bioinformatics analysis found that Gm17586 has potential binding sites with SIRT1.

[0008] In order to further study the relationship between Gm17586 and the occurrence of LF disease and its molecular mechanism, the present invention established an HSCs in vitro cell model, and determined that the long non-coding RNA Gm17586 could target and regulate SIRT1 activity, inhibit Smad3 deacetylation modification, and then enhance Smad3 transcriptional activity, promote HSCs cell 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.

[0009] The experimental results show that:

[0010] 1. Effect 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, after Gm17586 interference, the RNA expression level of Gm17586 was further decreased; after Gm17586 overexpression, the RNA expression level of Gm17586 was significantly increased.

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

[0013] After Gm17586 overexpression, HSCs cell viability decreased, the number of cells in G1 phase increased, and the mRNA expression of HSCs activation indicators α-SMA and Collagen Ⅰ decreased; conversely, after Gm17586 interference, HSCs cell viability increased, the number of cells in G1 phase decreased, and the mRNA expression of HSCs activation indicators α-SMA and Collagen Ⅰ increased. The results of correlation analysis showed that Gm17586 was significantly negatively correlated with 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 of LF mice. After Gm17586 interference, SIRT1 mRNA and protein expression and enzyme activity levels were further reduced. On the contrary, after Gm17586 overexpression, SIRT1 mRNA and protein expression and enzyme activity levels were significantly increased.

[0016] 4. Analysis of co-localization of Gm17586 and SIRT1 in cells

[0017] Gm17586 and SIRT1 have binding sites with a binding free energy of -24.35 kcal / mol. Both Gm17586 and SIRT1 are mainly expressed in the cell nucleus. Therefore, from the perspective of cell localization, they have direct binding conditions.

[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 reduced, 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. Effect 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, and significantly increased in the SIRT1 interference group.

[0024] Therefore, the present invention experimentally confirms that Gm17586 can reduce SIRT1 activity, inhibit Smad3 deacetylation modification, and then enhance Smad3 transcription activity, promote HSCs activation, and participate 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 detected the expression of Gm17586 in primary HSCs of LF mice. Compared with the control group, ## P<0.01, indicating that the difference was statistically significant.

[0027] Figure 2 Screening of Gm17586 overexpression plasmid 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 screens Gm17586 small interfering RNA. Compared with the siRNA1 group, ** P<0.01, indicating that the difference was statistically significant. ## P<0.01, indicating that the difference was 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 was 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 detection of HSCs cell viability. B: Cell cycle quantification statistics. C: Flow cytometry detection of 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 was 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 to detect the expression level of α-SMA mRNA. B: RT-qPCR to detect the expression level of Collagen I mRNA. 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 of Gm17586 and α-SMA in HSCs. B: Correlation analysis between the expression of Gm17586 and CollagenⅠ in HSCs.

[0037] Figure 7 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 was statistically significant.

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

[0040] Note: A: RT-qPCR to detect SIRT1 mRNA expression level. B: Western blot to detect 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 was statistically significant.

[0041] Fig. 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 was statistically significant.

[0043] Fig.10 Prediction of Gm17586 and SIRT1 binding sites 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] Fig.11 The expression of Smad3 in primary HSCs of LF mice.

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

[0047] Fig.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] Fig.13 This is the effect of SIRT1 overexpression and interference on Smad3 expression level.

[0050] Note: A: RT-qPCR to detect Smad3 mRNA expression level. B: Western blot to detect 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 was statistically significant.

[0051] Fig.14 This is the effect of SIRT1 overexpression and interference on Smad3 protein and acetylation levels.

[0052] Note: A: Immunoprecipitation to detect the expression of Smad3 and its acetylated protein. 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 was statistically significant.

[0053] Fig.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 was statistically significant. DETAILED DESCRIPTION

[0055] Example Study on the mechanism of LncRNA Gm17586 targeting SIRT1 to induce Smad3 acetylation and promote HSCs cell activation

[0056] 1 Experimental Materials

[0057] 1.1 Experimental animals

[0058] Male C57BL / 6 mice were purchased from Hangzhou Ziyuan Experimental Animal Technology Co., Ltd., weighing 20±2g, SPF grade, 6-8 weeks old. The weight of all mice was measured and recorded twice a week. They were raised in an environment with a 12-hour day and night alternation and an indoor temperature of 18-22°C, with sufficient food and water, and free drinking and food intake. Experimental Animal License Number: SCXK (Zhejiang) 2019-00004.

[0059] 1.2 Experimental Ethics Approval

[0060] The animal experiments involved in this invention were reviewed and approved by the Ethics Committee of Anhui University of Chinese Medicine, and the ethics approval number is: 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 injected subcutaneously with a mixture of carbon tetrachloride (CCl4) and olive oil (ratio of 1:4) at 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 by intraperitoneal injection of sodium pentobarbital, the limbs of mice were fixed, the abdomen was disinfected and sodium heparin injection was injected for anticoagulation. The abdominal cavity was opened to expose the liver and portal vein. 50 mg collagenase IV was dissolved in 25 mL DMEM medium, diluted 4 times, and prepared into 0.05% collagenase perfusion solution and perfused into the inferior vena cava. Gently press with fingers to ensure full perfusion. When the blood was discharged until the liver turned gray, the perfusion solution was replaced with digestive enzyme solution to digest the liver tissue and fully tear the liver. 1% BSA was taken out to resuspend the cells and filtered with a 200 mesh filter. The cell suspension was collected and centrifuged at 40g×4min, the supernatant was collected, and centrifuged again at high speed for 10min. The precipitate was added to serum-free RPMI-1640 medium to resuspend the cells. After centrifugation at 400g×10min, the precipitate was added to 2mL cell suspension, 35% percoll 4mL and 50% percoll 4mL in sequence, and centrifuged at 900g×30min. The cells at the interface of 35% percoll and PBS were collected, the supernatant was discarded after dilution and centrifugation, the cells were cultured with RPMI-1640 medium containing 10% FBS, and seeded in a culture dish 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. to construct.

[0069] 2.3.2 Synthesis of small interfering RNA

[0070] The primer sequences of Gm17586 and SIRT1 were obtained through the NCBI database (https: / / www.ncbi.nlm.nih.gov / ), and small interfering RNA and negative control were designed based on the sequence information of Gm17586 and SIRT1. Gm17586 and SIRT1 small interfering RNA were commissioned to Shanghai Jima Pharmaceutical Technology Co., Ltd. for design and synthesis. The sequence information of Gm17586 and SIRT1 small interfering RNA is shown in Table 1.

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

[0072]

[0073] 2.3.3 Cell transfection

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

[0075] Overexpression plasmid and small interfering RNA name:

[0076] Gm17586, SIRT1 overexpression plasmids, namely pc-Gm17586, pc-SIRT1; their control plasmid empty vector, namely pc-NC. Gm17586, 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 medium under the action of trypsin and the cell density was adjusted to 1×10 5 / mL. Inoculate in a 96-well plate to make a cell suspension, add 100 μL to each well, fill the edge wells with sterile PBS, and set up blank wells containing only culture medium. After culturing in a 5% CO2 incubator for 24h, 48h and 72h, add 10 μL CCK8 to each well and continue incubation for 1h. Finally, the absorbance of each well was measured at OD 450nm by an enzyme-linked immunosorbent assay.

[0079] 2.5 Flow cytometry detection of cell cycle

[0080] Prepare cells to separate single cells from cell clumps, gently vortex the cells and slowly add 2.7mL of pre-cooled anhydrous ethanol to adjust the final concentration to 90%, and fix at -20℃ for 1h. The fixed cells were washed with pre-cooled PBS, centrifuged at 2000rpm for 5min, and placed in an EO tube after absorbing PBS. Then add 500μL PBS and flick the bottom of the tube to avoid cell clumping. Add 20μL RNase to resuspend the cells, bathe at 37℃ for 30min, then add 400μL PI dye solution to resuspend, stain at 4℃ in the dark for 30min, and detect the cell cycle by flow cytometry.

[0081] 2.6 RT-qPCR detection of gene expression

[0082] 2.6.1 RNA extraction

[0083] Collect the cell pellet and add 1mL TRIzol to lyse it. Put it in a centrifuge tube, add 0.2mL chloroform, oscillate the centrifuge tube for 10s, place it at room temperature for 5min, centrifuge it at 4℃12000rpm for 10min, take the supernatant and add 0.5mL isopropanol, mix it upside down and centrifuge it, discard the supernatant. Add 1mL pre-cooled 75℅ ethanol and centrifuge it, discard the supernatant, and repeat this step. Place the pellet, add an appropriate amount of DEPC water, and store it at -80℃ for later use.

[0084] 2.6.2 RT reaction

[0085] Remove genomic DNA, add RNA, 5×gDNA Eraser Buffer and gDNAEraser to the PCR tube, and finally add DEPC water to 10μL. Place it on the PCR instrument after centrifugation. After reacting at 42℃ for 2min, add RTPrimer Mix, PrimeScript RT Enzyme Mix I and RNase Free dH2O on ice. Perform reverse transcription on the PCR instrument, set the temperature to 37℃ for 15min and 85℃ for 5s. The cDNA obtained was stored at -20℃ for later use.

[0086] 2.6.3 Fluorescence quantitative PCR reaction

[0087] The above cDNA was used as a fluorescent quantitative template to establish the reaction system in Table 2, and the fluorescent quantitative PCR reaction was performed under the reaction conditions in Table 3 to detect the expression level of each index. The primer information of each detection index 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] Collect the cell pellet, add 100μL RIPA cell lysis buffer (containing 1mM PMSF) to each 6-well plate cell, place on ice for lysis for 30min, centrifuge at 12000rpm for 10min, and take the supernatant to obtain the total cell protein. Add 5×SDS-PAGE protein loading buffer (1:4) to the protein sample, heat in a boiling water bath for 10min to fully denature the protein. Prepare SDS-PAGE gel, load the protein sample into SDS-PAGE according to 30μg per well, adjust the voltage to 80V, electrophoresis for 30min, and then adjust the voltage to 120V for electrophoresis. Soak the PVDF membrane in methanol for activation, cut it and soak it in transfer buffer for 5min. Put a piece of filter paper on each anode and cathode plate and put it into the electrotransfer instrument for transfer at 300mA. After blocking with 5% skim milk powder at room temperature for 2h, put the PVDF membrane in the corresponding primary antibody working solution and incubate it overnight on a shaker at 4℃. The primary antibodies used and the dilution ratios are detailed in Table 5. Then wash with PBST buffer, transfer the PVDF membrane to the diluted corresponding secondary antibody, the dilution ratio is 1:10000, and incubate on a shaker for 2h. Mix equal volumes of ECLA solution and ECL B solution to prepare fresh luminescent solution, place the PVDF membrane in the exposure plate of the automatic exposure instrument, add 100μL ECL luminescent solution and take pictures. Finally, use Image J software to analyze the gray value of the picture.

[0096] Table 5 Antibody information

[0097]

[0098] 2.8 Immunofluorescence experiments

[0099] Culture cells on the culture plate, remove excess culture medium, wash gently with PBS for 3 minutes, repeat twice. Then fix with 4% paraformaldehyde for 15 minutes, wash gently with PBS for 3 minutes, repeat three times. Permeate in 0.1% Buffer A permeabilization solution for 15 minutes and then wash with PBS. Then add 200μL 1x blocking solution for 30 minutes. After blocking, add 200μL of primary antibody diluted with PBS at a ratio of 1:200 and incubate at 37℃ for 1.5h. Add 200μL of corresponding secondary antibody (dilution ratio 1:200) and incubate at room temperature for 30min. Wash with PBS for 5min×3 times. Add 200μL of diluted DAPI working solution to the cell well plate and wash with PBS for 5min×2 times in the dark. Finally, add anti-fluorescence quencher to seal the slide and observe the fluorescence distribution and intensity changes under a fluorescence microscope.

[0100] 2.9 Fluorescence in situ hybridization

[0101] After the paraffin sections are melted, they are immersed in xylene I and II for dewaxing, and then dehydrated in gradient alcohol. Add proteinase K at 37°C and incubate for 20 minutes. Remove the proteinase K working solution, add 100μL 1x blocking solution, incubate at 37°C for 30 minutes, discard the blocking solution, add 100μL 2×Buffer C, and wash the slides 3 times at room temperature for 1 minute. Then add 100μL of preheated denaturing solution to each slide, incubate at 78°C for 8 minutes, dehydrate with gradient ethanol and dry. Buffer E is incubated in a 73°C water bath for 30 minutes in advance until it is clear and transparent, and the probe is diluted with DEPC water to a concentration of about 100μM. Dilute the probe storage solution to 1μM, put it in a 75°C water bath for denaturation for 10 minutes, and then add it to PBS in proportion with SA-Cy3, and incubate at 37°C for 30 minutes. Then add 100μL of the denatured probe mixture, seal the slide with a sealing film, and hybridize overnight at 37°C for 16 hours. The next day, add 100 μL of 2× Buffer C, wash 3 times at 60°C for 10 min each time, discard Buffer C, repeat the wash, and dry naturally at room temperature. Add 100 μL of diluted DAPI working solution to each slice, incubate at room temperature in the dark for 10 to 20 min. Discard the DAPI working solution, wash 2 times with PBS for 5 min, add antifade agent to seal the slice, and observe the results under a fluorescence microscope.

[0102] 2.10 Dual luciferase reporter assay

[0103] According to the cell transfection steps in 2.3.3, the Smad3 promoter fragment was transfected and 3 replicate wells were set up. The luciferase activity was detected using the dual luciferase detection kit of Beyotime, and firefly luciferase and Renilla luciferase were used as standardization.

[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 instructions.

[0106] 2.12 Co-immunoprecipitation experiments

[0107] According to the cells in each 6-well plate, add lysis buffer to the EP tube and fully lyse on ice, put it into the centrifuge, 14000rpm×5min, take the supernatant, the normal group divides the supernatant into 3 tubes with 0.5mL and marks IP, IgG and Input respectively, and the other groups divide the supernatant into 2 tubes with 0.5mL and 0.2mL and marks IP and Input respectively. The prepared 2 tubes of protein A / G magnetic beads are magnetically separated, the supernatant is removed, and 500μL Smad3 antibody working solution and Normal Rabbit IgG working solution are added respectively, and incubated on a shaker for 1h. Add 500μL TBS and gently blow and resuspend the protein A / G magnetic beads with a pipette. The sample is incubated with protein A / G magnetic beads combined with the target antibody and Normal Rabbit IgG. The protein samples marked as IP and IgG are added to the protein A / G magnetic beads containing the target antibody and Normal Rabbit IgG respectively, placed on a side-swinging shaker or a rotating mixer, and incubated overnight at 4℃. Magnetic separation. After incubation, place on a magnetic stand for 10 seconds and remove the supernatant. Add 0.5 mL of lysis buffer containing inhibitors and gently pipette to resuspend the magnetic beads. Place on a magnetic stand for 10 seconds and remove the supernatant. Repeat washing three times and detect related proteins according to experimental step 2.7.

[0108] 2.13 Statistical analysis

[0109] The experiments involved in this invention included 3 independent repeated experiments, and SPSS software was used for data analysis and processing and drawing pictures. The results are presented as mean ± standard deviation The difference between the groups was analyzed by t-test; the comparison between multiple means was performed by one-way analysis of variance; the correlation analysis was performed by Pearson correlation analysis. When the p value was <0.05, the result was considered statistically significant. The result images were assembled by 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, 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 B, compared with the siRNA-NC group, the expression of Gm17586 in the siRNA group was significantly reduced, and the inhibitory effect of siRNA3 on the expression of Gm17586 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 study the effect of Gm17586 overexpression / interference on the expression of Gm17586 in HSCs, RT-qPCR was used to detect the changes in the expression level of Gm17586 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 cell viability, cell cycle and HSCs activation indicators

[0118] 3.4.1 Effects of Gm17586 overexpression / interference on HSCs cell 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 HSCs in the G1 phase in the LF model group was significantly reduced; compared with the NC group, the number of HSCs in the G1 phase was significantly increased after Gm17586 overexpression, while the number of HSCs in the G1 phase was significantly reduced after Gm17586 interference.

[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, on the contrary, the expression levels of α-SMA and CollagenⅠ mRNA in the Gm17586 interference group were further increased.

[0122] 3.4.3 Analysis of the correlation 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] In order to study 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] 3.6 Effect 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. The results of RT-qPCR 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 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, while in contrast, the expression level of SIRT1 protein in the Gm17586 interference group was further decreased ( Figure 8 B and 8C).

[0128] 3.7 Effect of Gm17586 overexpression / interference on SIRT1 enzyme 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 ( Fig. 9 ).

[0130] 3.8 Prediction of Gm17586-SIRT1 binding sites and localization in HSCs

[0131] In order to further clarify the location of Gm17586 expression, the present invention uses IF and fluorescence in situ hybridization (FISH) experiments to detect the expression location of Gm17586 in HSCs. The targeted prediction results show that Gm17586 has a binding site with SIRT1 ( Fig.10 A), the binding free energy is -24.35 kcal / mol. The results of IF and FISH experiments show that ( Fig.10 B), Gm17586 and SIRT1 are mainly expressed in the cell nucleus. Therefore, from the perspective of cellular localization, they have direct binding conditions.

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

[0133] In order to study the expression of Smad3 in liver fibrosis, RT-qPCR and WB were used to detect the changes in the expression levels of Smad3 mRNA and protein in HSCs. Fig.11 As shown in 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 Transfection efficiency of SIRT1 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. Fig.12 As shown in A, the expression of SIRT1 overexpression group was significantly increased compared with the NC group. Fig.12 As shown in 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] In order to study the effect of SIRT1 overexpression / interference on Smad3 expression in HSCs, RT-qPCR and WB were used to detect the changes in Smad3 expression levels in HSCs. RT-qPCR results showed that compared with the control group, the expression level of Smad3 mRNA in the LF model group was significantly increased; compared with the NC group, the expression level of Smad3 mRNA in the SIRT1 overexpression group was significantly decreased, while the expression level of Smad3 mRNA in the SIRT1 small interference group was significantly increased ( Fig.13 A). WB results showed that compared with the control group, the expression of Smad3 protein in the LF model group was increased; compared with the NC group, the expression level of Smad3 protein in the SIRT1 overexpression group was significantly decreased, while in contrast, the expression level of Smad3 protein in the SIRT1 interference group was further increased ( Fig.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. Fig.14 As shown in AC, compared with the NC group, the expression levels of Smad3 and its acetylation in the SIRT1 overexpression group were significantly decreased, while the expression levels of Smad3 and its acetylation in the SIRT1 small interference group were significantly increased. The results showed that SIRT1 can inhibit the deacetylation level of Smad3.

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

[0141] The dual luciferase reporter assay was used to detect the effect 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 ( Fig.15 ). The results showed that SIRT1 could significantly enhance the transcriptional activity of Smad3.

[0142] 4 Summary

[0143] In this study, LF mouse primary HSCs were used as an in vitro cell model, and Gm17586 and SIRT1 overexpression plasmids and small interfering RNA were transfected as intervention methods. It was determined that Gm17586 expression was reduced in HSCs, affecting HSCs activation and promoting HSCs proliferation. The expression levels of HSCs activation markers α-SMA and CollagenⅠ mRNA were significantly increased; and the results of correlation analysis showed that Gm17586 was significantly negatively correlated with α-SMA and CollagenⅠ mRNA. Similarly, SIRT1 expression was significantly reduced in HSCs, and Gm17586 could target and enhance SIRT1 mRNA, protein expression levels and enzyme activity. FISH experiments and bioinformatics predictions proved that Gm17586 and SIRT1 had common subcellular localization and there were multiple binding sites between the two. In contrast, Smad3 expression was increased in HSCs, and SIRT1 could inhibit the expression of Smad3. 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, and then enhance Smad3 transcription activity, promote HSCs activation, and participate in the mechanism of liver fibrosis.

[0145] The above contents are merely examples and explanations of the concept of the present invention. The technicians in this technical field may make various modifications or additions to the specific embodiments described 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 protection scope of the present invention.

Claims

1. Application of long non-coding RNA Gm17586 targeting SIRT1 to induce Smad3 acetylation and promote HSCs activation in liver fibrosis, characterized in that: Long noncoding 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 the transcriptional activity of Smad3, promoting the activation of hepatic stellate cells (HSCs), and thus accelerating the occurrence and development of liver fibrosis (LF).

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