Double-stranded siRNA (small interfering Ribonucleic Acid) targeting liver Sam68 and application of double-stranded siRNA in metabolic syndrome related diseases

Abstract

The invention discloses double-stranded siRNA (small interfering Ribonucleic Acid) targeting liver Sam68 and application of the double-stranded siRNA in metabolic syndrome related diseases, and relates to the field of biological medicines. The double-stranded siRNA comprises a positive-sense strand and an antisense strand complementary to the positive-sense strand, the positive-sense strand is a modified or unmodified sequence of which the nucleotide sequence is shown as SEQ ID NO.41; the antisense strand is a modified or unmodified sequence of which the nucleotide sequence is as shown in SEQ ID NO. 42. The double-chain siRNA designed and synthesized on the basis of Sam68 can realize potent, dose-dependent and liver-limited Sam68 knock-down in vivo, inhibits liver gluconeogenesis by down-regulating glucagon signals, can reduce fasting blood-glucose, improve insulin sensitivity and protect diabetic liver from being damaged, and can be used for treating diabetic liver diseases such as diabetes mellitus, diabetes mellitus, diabetes mellitus, diabetes mellitus, diabetes mellitus and diabetes mellitus. A novel long-acting nucleic acid medicine is provided for diabetes, obesity, hypercholesterolemia, atherosclerosis and other metabolic syndrome related diseases.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to double-stranded siRNA targeting liver Sam68 and its use in metabolic syndrome-related diseases. Background Technology

[0002] Sam68 (Src-Associated in Mitosis of 68 kDa) is a member of the Signal Transduction and RNA Activation (STAR) family. US Patent US20220119511A1 discloses a method for treating metabolic syndrome-related diseases such as diabetes, obesity, hypercholesterolemia, and atherosclerosis by inhibiting Sam68. However, existing drugs targeting Sam68 are mostly small molecule chemicals and antibody drugs, with limited specificity, stability, and long-term efficacy.

[0003] Oligonucleotide drugs are a highly anticipated type of drug in new drug development, considered the third generation of therapeutic agents after small molecule drugs and antibody drugs. Because they act directly on the gene expression regulation level, small nucleic acid drugs offer numerous advantages over protein-targeting therapies, including shorter development cycles, higher success rates, lower in vivo toxicity, and longer duration of action. siRNA is a double-stranded RNA oligonucleotide containing 19-21 base pairs. In organisms, siRNA-mediated RNA interference (RNAi)-induced gene silencing is an important mechanism for gene expression regulation, specifically inducing the degradation of target mRNA and inhibiting gene expression. Especially in terms of long-term efficacy, some currently marketed small nucleic acid drugs achieve effectiveness for 6-9 months, a feat unmatched by small molecule drugs and antibody drugs. Taking Leqvio as an example, compared to the once-daily dosing frequency of statins and the subcutaneous injection frequency of monoclonal antibodies every two weeks, Leqvio only requires a subcutaneous injection every six months to achieve a good effect in lowering low-density lipoprotein cholesterol (LDL-C), significantly reducing the frequency of medication for patients and thus improving patient compliance. However, there are also technical barriers in the development of small nucleic acid drugs, including the optimization of target sequence design, chemical modification, synthesis of small nucleic acid drugs (raw materials and equipment), and delivery vectors.

[0004] Therefore, existing technologies still need improvement and development. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of this invention is to provide a double-stranded siRNA targeting liver Sam68 and its use in metabolic syndrome-related diseases, aiming to solve the problem of the lack of efficient double-stranded siRNAs targeting liver Sam68.

[0006] The technical solution of the present invention is as follows: In the first aspect, a double-stranded siRNA targeting liver Sam68 is provided, the double-stranded siRNA comprising: a sense strand and an antisense strand complementary to the sense strand; The positive strand is a modified or unmodified sequence containing the nucleotide sequence shown in SEQ ID NO.41; The antisense strand is a modified or unmodified sequence containing the nucleotide sequence shown in SEQ ID NO.42.

[0007] Optionally, the modification is selected from one or more of nucleotide chemical modification, overhang modification, and liver-targeting ligand modification.

[0008] Preferably, the nucleotide chemical modification includes: chemical modification of the 2' position of the nucleotide ribose, and / or chemical modification of the 3',5'-phosphodiester bond between nucleotides.

[0009] More preferably, the chemical modification at the 2' position of the ribose in the nucleotide is selected from one or more of 2'-methoxy, 2'-O-methoxyethyl, 2'-fluoro, 2'-benzyloxy, 2'-methylcarbonylamino, and 2'-pyridinemethoxy. The 2' position of the ribose is a hydroxyl group; modifying it can prevent side reactions and improve the stability of the double-stranded siRNA.

[0010] More preferably, the chemical modification of the 3',5'-phosphodiester bonds between the nucleotides is a thiomodification, replacing oxygen atoms with sulfur atoms. That is, the phosphodiester bonds between the nucleotides are replaced with thiophosphate bonds. Sulfur atoms have lower electronegativity than oxygen; thiomodification can reduce the electron cloud density of the phosphodiester bonds, increase bond length, enhance the resistance of double-stranded siRNA to nucleases, prolong its half-life in plasma, and give it suitable pharmacokinetic properties.

[0011] Optionally, the protruding end modification involves adding 1-3 protruding nucleotides to the 3' end of the antisense strand.

[0012] Preferably, the protruding end modification involves adding two protruding nucleotides to the 3' end of the antisense strand.

[0013] More preferably, the protruding end modification involves adding a protruding UU to the 3' end of the antisense chain. Adding a protruding UU to the 3' end of the antisense chain can mimic the natural structure, enhance functional activity, and improve stability.

[0014] Optionally, the liver-targeting ligand is modified by covalently conjugating a liver-targeting ligand to the 3' end of the positive chain.

[0015] Preferably, the liver-targeting ligand is a GalNAc ligand. For example, the trivalent GalNAc ligand: L96 (CAS No.: 1159408-62-4). L96 is chemically synthesized and covalently conjugated to the 3' end of the positive strand of siRNA. GalNAc ligand conjugation can improve the liver tissue delivery efficiency of double-stranded siRNA.

[0016] Optionally, the justice chain is 5'-Cm*Am*GmAmCmAmAfGmUfAfAfUmUmGmUmCmUmAmAmGmUm-3'-L96; The antisense chain is 3'-Um*Um*GmUmCmUmGmUfUmCfAmUmUmAmAmCmAmGfAmUmUm*Cf*Am-5'.

[0017] In this context, the suffix m indicates that the ribose of the nucleotide has a 2'-methoxy modification, the suffix f indicates that the ribose of the nucleotide has a 2'-fluoride modification, and the asterisk between the nucleotides indicates that the two nucleotides are linked by a thiophosphate bond.

[0018] Secondly, the use of double-stranded siRNA as described in the first aspect in the preparation of medicaments for the prevention and / or treatment of metabolic syndrome-related diseases is provided.

[0019] In a preferred embodiment, the metabolic syndrome-related disease is selected from one or more of diabetes, obesity, hypercholesterolemia, and atherosclerosis.

[0020] Beneficial Effects: This invention provides a double-stranded siRNA targeting liver Sam68 and its use in metabolic syndrome-related diseases. The double-stranded siRNA comprises a sense strand and an antisense strand complementary to the sense strand; the sense strand is a modified or unmodified nucleotide sequence as shown in SEQ ID NO. 41; the antisense strand is a modified or unmodified nucleotide sequence as shown in SEQ ID NO. 42. The double-stranded siRNA designed and synthesized based on Sam68 in this invention can achieve potent, dose-dependent, and liver-restricted Sam68 knockdown in vivo. By downregulating glucagon signaling and inhibiting hepatic gluconeogenesis, it can reduce fasting blood glucose, improve insulin sensitivity, and protect diabetic liver from damage. This provides a novel long-acting nucleic acid drug for metabolic syndrome-related diseases such as diabetes, obesity, hypercholesterolemia, and atherosclerosis. Attached Figure Description

[0021] Figure 1This is a figure showing the results of integrated analysis of two public datasets: APOE*3-Leiden mouse GSE229188 (A) and wild-type hamster GSE227411 (B). It shows that both a 32-week Western diet and a 12-week high-fat diet significantly upregulated hepatic Sam68 (KHDRBS1) mRNA expression (*p<0.05, **p<0.01, ****p<0.0001), suggesting that high-fat load itself can drive increased intrahepatic Sam68 transcription. Among them, Chow represents a normal diet, WTD represents a Western diet, WTD_Atorv represents a Western diet + atorvastatin, ND represents a normal diet, HFD represents a high-fat diet, and HFD_Totum_070 represents a high-fat diet + Totum-070.

[0022] Figure 2 This figure shows the results of Sam68 protein expression detection in the liver of diabetic patients, HFD-induced diabetic mice, and db / db 2 diabetic mice. A indicates that the expression of key gluconeogenesis proteins Sam68, CRTC2, PGC-1α, PEPCK, and G6Pase is elevated in the liver of diabetic patients. B indicates that the levels of Sam68 and CRTC2 proteins in the nucleus and cytoplasm of hepatocytes from HFD-induced diabetic mice and db / db 2 diabetic mice are also significantly increased. **p<0.01, ***p<0.001.

[0023] Figure 3 This image shows the results of gluconeogenesis, blood glucose, and insulin sensitivity assays in a diabetic mouse model with liver-specific Sam68 knockout or knockdown. A indicates a significant decrease in liver Sam68 protein after tail vein injection of AAV8-TBG-Sam68 shRNA (db / db;sh-Sam68) in db / db mice. BE indicates that in congenital liver Sam68 knockout mice fed a high-fat diet, Sam68 protein levels decreased significantly. LKO HFD (Comparison with Sam68) f / fIn db / db genetic model mice with HFD and acquired liver-specific Sam68 knockdown (db / db;sh-Sam68 mice, control db / db;sh-Scr mice), compared with their respective controls, fasting blood glucose was reduced, pyruvate tolerance (PTT), glucagon tolerance (GcTT), and glucose tolerance (GTT) were significantly improved. F and G showed that the mRNA and protein levels of hepatic gluconeogenesis genes PGC-1α, G6Pase, and PEPCK were synchronously downregulated. ChIP-PCR of H confirmed that Sam68 deletion reduced the occupancy of CRTC2 in the promoters of the above genes. The hyperinsulin-eukaryotic clamp assay of IL further showed that liver Sam68 knockout mice had reduced fasting blood glucose, pyruvate tolerance (PTT), glucagon tolerance (GcTT), and glucose tolerance (GTT) in the last 40 minutes of liver-specific Sam68 knockdown. Increased min glucose infusion rate (GIR), increased glucose processing rate, decreased hepatic glycogen production rate, and increased glycogen synthesis rate; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; indicating that liver-specific deletion / knockdown of Sam68 can inhibit gluconeogenesis, reduce blood glucose, and significantly enhance systemic insulin sensitivity in both diabetes models.

[0024] Figure 4 This image shows the results of cholesterol synthesis, blood lipid levels, and the degree of atherosclerosis in a mouse model of hypercholesterolemia and atherosclerosis after knocking out Sam68 in the liver; where A and B represent Sam68. f / f ApoE - / - Mice were injected via tail vein into AAV8-TBG-iCre (Sam68) f / f -iCre;ApoE - / - After [the following], the levels of Sam68 mRNA (A) and protein (B) in the liver were significantly reduced; C indicates that under the Western diet, the serum of the mice was grossly turbid (left), and the serum total cholesterol (TC, middle) and triglycerides (TG, right) were significantly increased; DF indicates that the expression of mRNA (DE) and protein (F) of genes related to cholesterol synthesis and uptake in the liver was upregulated; GI indicates that Oil Red O staining of longitudinal section and transverse section of the aorta showed an increased area of ​​atherosclerotic plaques; J indicates that MOMA-2 / Hoechst-33342 immunofluorescence of the aortic root showed a large number of mononuclear / macrophage infiltrations; *p<0.05, **p<0.01, ***p<0.001.

[0025] Figure 5This figure shows the design and screening results of Sam68-siRNA sequences. A and B represent the expression levels of Sam68 protein in HepG2 cells 72 h after transfection with the specified Sam68-siRNA candidate sequences (A is a representative band from Western blot, B is quantified by optical density). Results were corrected using β-actin as an internal control. CE represents the detection results of Sam68 mRNA (C) and protein levels in AML-12 cells 72 h after transfection (D is a representative band from Western blot, E is quantified by optical density). Data are expressed as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001 vs. scramble group (two-way ANOVA). #13 and #21 showed good knockdown efficiency in both AML-12 and HepG2 cell lines.

[0026] Figure 6 This diagram shows the design and detection results of GalNAc–siSam68. A is a schematic diagram of the chemical modification of the GalNAc–siSam68#21 sequence and GalNAc conjugation. BD shows the results of detecting liver Sam68 protein (B: representative bands from Western blot, C: optical density quantification) and mRNA (D) 72 hours after a single subcutaneous injection of 8 µg / g GalNAc-siSam68#13, #15, or #21 into mice. E and F verify the dose-effect: liver Sam68 protein was analyzed 72 hours after subcutaneous administration of 0, 4, 8, and 16 µg / g GalNAc-siSam68#21 (E: representative bands from Western blot, F: optical density quantification). G and H verify tissue specificity: liver, heart, testis, epididymal white fat (epiWAT), and gastrocnemius muscle Sam68 protein were analyzed 24 days after a single subcutaneous injection of 16 µg / g into db / db mice (G: Western blot). Representative blot bands, H for optical density quantification); IK validation of persistence: liver Sam68 protein in db / db mice after 24 days with the same dose (I for representative Western blot bands, J for optical density quantification) and time course (K) from 0 to 51 days; L and M show the changes in liver Sam68 protein in HFD / STZ T2D rats after a single subcutaneous dose of 16 µg / g for 24 days (L for representative Western blot bands, M for optical density quantification); Data: mean ± sem *p<0.05, **p<0.01, ***p<0.001; B, C, and H were analyzed using two-way ANOVA, and J and M were analyzed using unpaired t-tests.

[0027] Figure 7This is a graph showing the results of GalNAc–siSam68 in reducing fasting blood glucose, improving insulin sensitivity, and protecting diabetic liver from damage and fatty liver disease. AC represents the results of a single subcutaneous injection of GalNAc-siSam68 #21 or GalNAc-siScramble (16µg / g) in db / db mice: A is the 16-hour fasting blood glucose on day 8 after injection (n=12 / group); B and C are the blood glucose curves (B) and area under the curve (AUC) (C) of the insulin tolerance test (ITT) on day 16 (n=12 / group); DG is the liver sample taken 16 hours after fasting on day 24: D and E are the serum AST (D) and ALT (E) levels (GalNAc-siSam68 n=9; GalNAc-siScramble n=11); F and G are representative H&E images of liver tissue (F) and quantitative lipid droplet area (G) (30 low power fields, n=6-7, ≥4 fields per mouse); HK is the HFD / STZ. T2D rats were treated with the same dose: H was the fasting blood glucose on day 3 (n=12 / group); I was the serum insulin measured by ELISA (n=12 / group); J and K were the ITT blood glucose curve (J) and AUC (K) (n=12 / group); Data: mean±sem *p<0.05, **p<0.01, ***p<0.001, ns=no significant difference (two-way ANOVA).

[0028] Figure 8 This is a graph showing the results of GalNAc-siSam68 inhibiting hepatic gluconeogenesis by downregulating glucagon signaling. AC represents the mRNA levels of gluconeogenesis genes G6Pase (A), PEPCK (B), and PGC-1α (C) by qRT-PCR 72 h after AML-12 hepatocytes were transfected with GalNAc-siSam68 #12, #13, #15, or #21 (n=9 / group). DG represents a single subcutaneous injection of GalNAc-siSam68 into male db / db mice. #21 or GalNAc-siScramble (16µg / g), liver was harvested on day 24; qRT-PCR was used to detect the expression of G6Pase (D, E), PEPCK (D, F) and PGC-1α (D, G) mRNA in the liver (n=8-9 / group); Data: mean±sem*p<0.05, **p<0.01, ***p<0.001, ns=no significant difference (two-way ANOVA). Detailed Implementation

[0029] This invention provides a double-stranded siRNA targeting liver Sam68 and its use in metabolic syndrome-related diseases. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below.

[0030] Current hypoglycemic drugs require daily administration, leading to low adherence and minimal lipid-regulating effects. However, 67% of patients with type 2 diabetes (T2DM) also have dyslipidemia, while only 4.4% achieve target levels for blood glucose, blood pressure, and blood lipids. Switching to statins or other lipid-lowering drugs with increased dosages can cause significant side effects, and the risk of atherosclerotic cardiovascular disease (ASCVD) remains high. Therefore, developing new "dual-drug" drugs that provide long-lasting effects with a single dose and simultaneously lower both blood glucose and blood lipids is an urgent clinical need to improve target achievement rates, reduce patient burden, and prevent macrovascular complications.

[0031] Sam68 (Src-Associated in Mitosis of 68 kDa) is a member of the signal transduction and RNA activation (STAR) family. In recent years, the inventors' research group has conducted extensive research on the pathological role of Sam68. First, their group discovered that systemic Sam68 knockout mice can resist obesity, hyperglycemia, and insulin resistance induced by a high-fat diet. Importantly, high expression of Sam68 and gluconeogenesis signaling was also confirmed in liver samples from type 2 diabetic patients. Based on this, their group subsequently constructed liver-specific knockout Sam68 mice (Sam68... f / f :Alb-Cre + / – Sam68 LKO ), found to be similar to the Sam68 whole-body knockout mouse, Sam68 LKO Mice can also resist hyperglycemia and insulin resistance induced by a high-fat diet. In db / db (type II diabetes model) mice, knocking down liver Sam68 expression with a specific interfering RNA (shRNA) targeting liver Sam68 can also significantly reduce blood glucose, maintain hepatic and peripheral insulin sensitivity, and is accompanied by a decrease in hepatic gluconeogenesis (the protein abundance of CRTC2, a key transcriptional coactivator of gluconeogenesis, and the transcription of the rate-limiting enzyme of gluconeogenesis are both significantly downregulated).

[0032] In Sam68 LKO In a transcriptomic study of mouse liver tissue, our research group found that in addition to the gluconeogenesis pathway, the cholesterol synthesis pathway was also significantly downregulated, as evidenced by the significant downregulation of transcription factor-sterol regulatory element-binding protein 2 (SREBP2) and approximately 80% of cholesterol synthesis-related genes. Furthermore, in Sam68... f / f ApoE – / – In mice, specific knockout of liver Sam68 expression (i.e., Sam68) was achieved by tail vein injection of the AAV8-TGB-Cre viral vector. f / f -iCre;ApoE – / – After 16 weeks of feeding a Western diet, compared with control Sam68...f / f -iGFP;ApoE – / – Compared to mice, Sam68 f / f -iCre;ApoE – / – Serum total cholesterol (TC) and triglyceride (TG) levels in mice decreased significantly by approximately 36% and 30%, respectively. Furthermore, serum ApoB levels, as well as total cholesterol and triglyceride levels, also decreased significantly. (Sam68) f / f -iCre;ApoE – / – In mice, the levels of SREBP-2, HMGCR, and PCSK9 in the liver were significantly decreased, while the level of LDLR protein was significantly increased. This was accompanied by a decrease in blood lipid levels. (Sam68) f / f -iCre;ApoE – / – The atherosclerotic plaques and inflammatory cell infiltration in the mouse aorta were significantly reduced. Both in vivo and in vitro results indicate that specific knockout of Sam68 in the liver significantly reduces hepatic gluconeogenesis and cholesterol synthesis.

[0033] The inventors filed a U.S. patent application (Publication No.: US20220119511A1, Application No.: US17503113) on October 15, 2021, to protect "compositions and methods for treating diabetes, obesity, hypercholesterolemia, and atherosclerosis by inhibiting sam68". Based on this patent, this invention develops a double-stranded siRNA targeting liver Sam68 and performs chemical and GalNAc modifications, providing a novel long-acting nucleic acid drug for the treatment of type II diabetes and hypercholesterolemia.

[0034] The specific research content of this invention is as follows: 1. In hyperlipidemic rodents, Sam68 mRNA expression was significantly elevated: This invention, through data retrieval and analysis in the database, found that in animal models of hyperlipidemia, APOE*3-Leiden mice fed a Western diet (WTD) for 32 weeks and wild-type (WT) hamsters fed a high-fat diet (HFD) for 12 weeks showed significantly increased levels of Sam68 (KHDRBS1) mRNA expression in their livers. Figure 1 ).

[0035] 2. In patients with type 2 diabetes (T2D), high-fat fed mice, and db / db hereditary diabetic mice, the protein expression of Sam68 in the liver was significantly increased: This invention collected liver samples from 10 patients with type 2 diabetes (T2D) and 10 control individuals at UAB (University of Alabama at Birmingham) Medical Center; Western blot and quantitative analysis showed that the protein level of Sam68 in the livers of T2D patients was significantly elevated. Figure 2The study also showed significant upregulation of key gluconeogenesis factors, including transcriptional coactivator 2 (CRTC2), transcription factor 1α (PGC-1α), and rate-limiting enzyme 6Pase (PEPCK). The inventors also analyzed data from normal diet (ND) WT mice and high-fat diet (HFD) WT mice fed for 3 months to obtain gluconeogenesis enzymes. Figure 2 (Middle B, left) and in 2-3 month old db / db (diabetic) and control db / m mice ( Figure 2 The expression levels of Sam68 and CRTC2 proteins in the hepatocyte nucleus and cytoplasm of HFD-fed WT mice or db / db mice were found to be significantly higher than those in the corresponding control group.

[0036] 3. In a diabetic mouse model, liver-specific knockout or knockdown of Sam68 significantly reduced gluconeogenesis, lowered blood glucose, and enhanced insulin sensitivity. Compared with mice without Sam68 knockout, mice fed a high-fat diet and mice with liver-specific Sam68 elimination, as well as mice with liver Sam68 knockdown specifically induced by AAV8-TBG-Sam68, showed significantly lower blood glucose levels under both fed and fasted conditions, and in PTT (Partial Thromboplastin Time), GTT (Glucose Tolerance Test), and ITT (Insulin Tolerance Test). The expression levels of mRNA and protein of key gluconeogenesis genes PGC-1α, PEPCK, and G6Pase were significantly reduced, accompanied by a significant decrease in CRTC2 protein levels. Furthermore, the inventors found that Sam68... / A sharp decrease in CRTC2 occupancy at the promoters of PGC-1α, G6Pase, and PEPCK was observed in hepatocytes. Furthermore, the hyperinsulinic-euglycemic clamp (the gold standard for assessing insulin sensitivity) demonstrated that Sam68... LKO Mice showed significantly higher insulin sensitivity than their Sam68 counterparts. f / f Control mice. These observations suggest that silencing and downregulating Sam68 expression in the liver can alleviate the hyperglycemic phenotype in HFD-fed and db / db mice, respectively, thereby reducing the transcription of key gluconeogenesis factors and rate-limiting enzymes. Therefore, Sam68 may be a molecular target for the treatment of T2D. Figure 3 ).

[0037] 4. In mouse models of hypercholesterolemia and atherosclerosis, knocking out liver Sam68 significantly reduced cholesterol synthesis, downregulated blood lipid levels, and alleviated atherosclerosis. In a study of liver transcriptomics in mice after fasting and refeeding, the inventors discovered Sam68 LKO The cholesterol synthesis pathway in mice (including the SREBP2 gene and approximately 80% of cholesterol synthesis-related genes) was significantly downregulated. This was observed in the Sam68 liver knockout atherosclerosis mouse model (Sam68). f / f -iCre;ApoE – / – In this study, serum total cholesterol (TC) and triglycerides (TG), serum cholesterol and triglyceride levels in very low-density lipoprotein fractions, and AopB levels were significantly decreased in mice. The mRNA levels of SREBP-2 and many SREBP-2-regulated cholesterol synthesis genes and PCSK9 in the liver of these mice were also significantly decreased. At the protein level, the precursor (P-SREBP-2) and mature (M-SREBP-2) forms of SREBP-2, HMGCR, HMGCS1, and PCSK9 in the liver of these mice were all significantly decreased, while LDLR protein levels were significantly increased. Furthermore, Oil Red O staining of the aorta indicated a reduction in atherosclerotic lesions in these mice. Figure 4 ).

[0038] 5. GalNAc-siSam68 #21 provides a long-lasting reduction of Sam68 expression in the liver with good tissue specificity: The expression level of Sam68 protein in the liver of mice was measured after subcutaneous injection of 16 μg / g GalNAc-siSam68 #21. Sam68 expression levels decreased by approximately 70% three days after injection, and remained at an 85% knockdown rate on day 21. No significant Sam68 knockdown was observed in the heart, kidney, spleen, testis, adipose tissue, or muscle tissue of these mice, indicating the good tissue specificity of GalNAc-siSam68 #21. Figure 5 and Figure 6 ).

[0039] 6. GalNAc-siSam68 #21 significantly reduced blood glucose in drug-induced and hereditary type 2 diabetes models, improved insulin resistance, and downregulated hepatic gluconeogenesis-related pathways. In db / db mice and HFD+streptozotocin (STZ)-induced type 2 diabetes rat models, subcutaneous injection of GalNAc-siSam68 #21 significantly reduced fasting blood glucose levels at 16 hours and in the ITT test. In the HFD+STZ-induced type 2 diabetes rat model, GalNAc-siSam68 #21 significantly reduced fasting insulin levels in rats. In the db / db mouse model, liver G6Pase, Pepck, and PGC-1α protein levels were all significantly reduced. Figure 7 and Figure 8 ).

[0040] 7. GalNAc-siSam68 #21 significantly reduced drug-induced fatty liver and metabolic liver injury in a hereditary type 2 diabetes model: In db / db mice, subcutaneous injection of GalNAc-siSam68 #21 significantly reduced serum ALT and AST. HE staining indicated a reduction in fatty liver. Figure 7 ).

[0041] The present invention will be further described below through specific embodiments.

[0042] Experimental animals and their rearing: 8-10 week old, weighing 22-25g, male C57BL / 6 wild-type mice (WT) and BKS-Lepr were selected. em2Cd479 / Gpt mice and HFD+STZ-induced type 2 diabetic rats were used as experimental subjects. All experimental mice were housed at the SPF-grade Laboratory Animal Center of Southern University of Science and Technology or the SPF-grade Laboratory Animal Center of Shenzhen Lingfu Top Technology Co., Ltd. Housing conditions: room temperature between 22-24℃, humidity between 40-70%, alternating light and dark lighting for 12 hours, and free access to water and food.

[0043] Example 1: siRNA design and synthesis: The target sequence of human Sam68 mRNA was obtained using the NCBI (National Center for Biotechnology Information) online database (code: NM_001271878.2). The ideal siRNA sequence was searched starting 50-100 nucleotides downstream of the target gene's start codon AUG. Exon-exon junctions and single nucleotide polymorphism (SNP) regions were avoided; regulatory protein binding sites were also avoided, as regulatory proteins compete with RISC (RNA-induced silencing complex) for binding to the siRNA sequence. The three classic structural features of siRNA are: a length of 21-23 nt; two protruding bases at the 3' end of each siRNA double strand; and a phosphate group at the 5' end of the siRNA double strand. The preferred siRNA sequence is AA(Nn)UU (where N represents any base; n is the number of bases, between 19-29 nt), or NA(Nn)UU and NA(Nn)NN sequences are also acceptable. In the siRNA sequence, the choice of G / C content is more important than determining the AA-starting sequence. Sequences with a balanced base ratio (G / C ratio of 30%-70%) can be used as candidate siRNA sequences; while siRNA sequences with a lower G / C ratio (30%-52%) have better gene silencing effects. Replacing the two 3′ protrusions with dTdT enhances the stability of the siRNA double-stranded complex. Multiple software programs, including siDIRET, DSIR, siDESIGN, Oligowalk, and Genescript, were used to screen siRNA sequences, obtaining design results with identical sequences. Combining the high-score screening results from different software programs, multiple siRNA molecules were initially obtained according to the siRNA design guidelines. To ensure that the candidate siRNA sequences silence only a single target gene, the candidate siRNA sequences should be BLAST homology compared with the EST or Unigene database of NCBI in the United States. The comparison showed that the screened siRNAs did not have homologous sequences with any housekeeping genes; therefore, housekeeping genes such as β-actin and GADPH can be used as internal controls. The siRNA sequences designed in this invention are shown in Table 1. These sequences can be used for subsequent synthesis and screening.

[0044] Table 1. siRNAs designed and synthesized in this invention

[0045] Example 2: Preparation method of siRNA: Unmodified siRNA molecules were synthesized in high throughput using a 32-channel RNA solid-phase synthesizer in the laboratory. The synthesized oligonucleotides were desalted and purified by HPLC. Equal proportions of the chemically synthesized siRNA sense and antisense oligonucleotides were added to 0.1×TE buffer, and the mixture was incubated at 90°C. Hold at -80°C for 1 minute, then allow to cool naturally to room temperature to obtain the prepared double-stranded siRNA. Store frozen at C.

[0046] Example 3: siRNA transfection conditions: The procedure was performed according to the Lipofectamine RNAiMAX (Invitrogen #13778075) product instructions. The recommended reverse transfection method, best suited for HepG2 cells, was used to transfect the siRNA (this method ensures sufficient contact between the cells and the transfection complex, thereby improving transfection efficiency). The specific steps are as follows.

[0047] Preparation of the transfection system: First, dilute the synthesized double-stranded siRNA to a 20 μM stock solution with enzyme-free water. Add 100 μL of Opti-MEM™ I serum-depleted medium to a 24-well plate, add 18 pmol of siRNA, mix well by pipetting, then add 1.5 μL of RNAiMAX reagent, and mix again by pipetting. Incubate at room temperature for 10-20 minutes. After passage the newly revived HepG2 cells twice, digest the HepG2 cells in logarithmic growth phase with 0.25% trypsin + 0.02% EDTA, gently pipetting to disperse the cells evenly. After staining the HepG2 single-cell suspension with 0.4% trypan blue (BD, #1450003), adjust the cell density to 5 × 10⁶ cells / well with antibiotic-free DMEM medium (containing 10% fetal bovine serum). 5 Cells / mL, viable cell percentage >95%. Add 500µL of diluted cells to each well containing the RNAi duplex-Lipofectamine™ RNAiMAX complex. The final volume per well is 600µL, and the final siRNA concentration is 30nM. Each siRNA is processed in triplicate. Shake the culture plate to ensure adequate contact between the cells and the transfection complex. Samples can be taken for subsequent analysis 72 hours after transfection. The HepG2 human liver cancer cells used in this invention were obtained from the Chinese Academy of Sciences Type Culture Collection Committee Cell Bank (SCSP-510) and cryopreserved in our laboratory.

[0048] Example 4: Detection of the inhibitory efficiency of siRNA on target proteins in HepG2 cells by Western blotting: HepG2 cells were transfected with 30 nM Sam68-siRNA, GADPH-siRNA, or scramble-siRNA, respectively, and then cultured at 37°C. Incubate at 5% CO2 for 72 h. Add 1 mL of pre-chilled PBS to each well and wash twice. Then add 100 μL of cell lysis buffer (RIPA-Strong, Beyotime P0013B) and 1 μL of protease inhibitor (MedChemExpress, HY-K0010). Scrape cells from the wells with a cell scraper, vortex to mix, and incubate on ice for 30 min. Incubate at 13000 rpm for 4 hours. Centrifuge at C for 20 minutes. Collect the supernatant into a new centrifuge tube. Determine protein concentration using the Brandford method (Novizan, E112-02), and adjust the final protein concentration to 2 mg / mL after adding 5× loading buffer. 95 Heating at 1°C for 10 min ensures complete protein denaturation. Prepare a 10% SDS-PAGE gel, loading 10 μL (20 µg protein) per well. Electrophoresis at 120 V for 1.5 h, followed by transfer. 0.45 μm PVDF was pre-activated with methanol and transferred at 30 V for 1.5-2 h. The PVDF membrane was then immersed in PBST blocking buffer containing 5% skim milk powder and blocked at room temperature for one hour. The PVDF membrane was washed three times with PBST for 5 min to remove unbound protein. Rabbit anti-Sam68 monoclonal antibody (abcam, EPR3231) diluted 1:2000 with primary antibody dilution buffer (Beyotime, P0023A) or mouse anti-β-actin monoclonal antibody (Beyotime, AF5001) diluted 1:1000 was added. Incubate overnight with shaking. After washing three times with PBST, dilute horseradish peroxidase-labeled goat anti-rabbit or mouse antibody with PBST containing 5% skim milk powder as a secondary antibody, dilute 1:5000, and incubate at room temperature for 2 hours. Wash three more times with PBST. Due to the large number of siRNAs screened and the use of duplicate wells, to avoid differences in band signal intensity caused by inconsistent exposure times, all membranes were exposed together in this embodiment. Add ECL chemiluminescence detection solution (Absin, abs920) according to the instructions, covering the entire membrane, and let stand for 1 minute. Acquire images using a Tanon-6100C chemiluminescence imaging system, and perform WB band quantification analysis using ImageJ software. The results are shown in Table 1: Compared with the control group Neg-siRNA (NC), the best Sam68-siRNAs #12, #13, #16, and #10 showed protein inhibition efficiencies exceeding 80%, namely 81.71%, 80.82%, 81.73%, and 80.78%, respectively. In addition, the inhibition efficiencies of #8, #9, #11, #15, #21 and #18 all exceeded 70%, namely 72.70%, 76.17%, 73.49%, 76.13%, 74.36% and 72.67%, respectively.

[0049] Example 5: Quantitative-PCR detection of the inhibitory efficiency of siRNA on target mRNA: HepG2 cells were transfected with 30 nM Sam68-siRNA, GADPH-siRNA, or scramble-siRNA, respectively, and then cultured at 37°C. Incubate at 5% CO2 for 72 h. Wash twice with 1 mL of pre-chilled PBS in each well, then add 1 mL of Trizol to each well and repeatedly pipette until all cells are expelled. Incubate on ice for 5 min, add 200 μL of chloroform, shake vigorously for 15 s, incubate on ice for 2-3 min, centrifuge at 12000g for 15 min at 4°C. Carefully transfer the upper aqueous phase to a new centrifuge tube, add an equal volume of pre-chilled isopropanol (pre-chilled at -20°C), shake 4-5 times, incubate for 10 min to precipitate RNA, centrifuge at 12000g for 15 min at 4°C. Discard the supernatant, wash the RNA precipitate with 1 mL of 75% ethanol, shake vigorously for a few seconds, centrifuge at 7500g for 10 min at 4°C. After centrifugation, carefully discard the supernatant, incubate to allow the ethanol to evaporate, then add 60 μL of DEPC water preheated in a 60°C oven to each tube to resuspend the RNA precipitate. RNA concentration was detected using a Nano-300 (Ausun), and sample quality was ensured (A260 / A280 values ​​between 1.8 and 2.1). RNA was reverse-transcribed into cDNA according to the reverse transcription kit instructions (TaKaRa, RR037A). The silencing efficiency of 30 nM Sam68-siRNA was detected using qPCR (Novizan, Q711). The reaction mixture contained 10 μL of 2×ChamQ Universal SYBR qPCR Master Mix, 0.4 μL of forward primer (10 μM), 0.4 μL of reverse primer (10 μM), and an equal volume of cDNA template, with ddH2O added to a final volume of 20 μL. Primers used are listed in Table 2. Reactions were performed according to instructions. Each treatment was run in triplicate. GraphPad Prism software was used for data analysis, and the results are shown below. Figure 5 As shown in Figure C. Based on the results in Example 4, #15, #13, #21, #16, and #12, with different inhibition efficiencies, were selected. Their Sam68 transcription levels were only 23.80%, 24.47%, 27.75%, 24.26%, and 26.20% of the control group (Neg-siRNA, NC), respectively. This result also corroborates that the selected siRNA can effectively inhibit Sam68 expression in HepG2 cells at the transcriptional level.

[0050] Table 2. qPCR primers used in this invention

[0051] Example 6: Detection of the inhibitory efficiency of siRNA on target proteins in the AML-12 cell line by Western blotting: After passaged twice, newly revived AML-12 cells in logarithmic growth phase were adjusted to a cell density of 1×10⁶ cells / years in antibiotic-free DMEM medium (containing 10% fetal bovine serum and 1×ITS).5 Cells / mL, viable cell percentage >95%. Seed 500µL of cells into a 24-well plate and incubate overnight. Follow the Lipofectamine RNAiMAX product instructions. Transfect siRNA using the recommended forward transfection method best suited for AML-12 cells, as detailed below.

[0052] The transfection complex was prepared as in Example 3. After standing for 10-15 minutes, 100 µL of the RNAiduplex-Lipofectamine™ RNAiMAX complex was added to each well. The final volume of each well was 600 µL, and the final siRNA concentration was 30 nM. Each siRNA was processed in triplicate. Samples could be taken for subsequent detection 72 hours after transfection. To further validate the role of the screened siRNAs in disease models in animals, this embodiment performed homology sequence alignment between the screened siRNA sequences targeting human (Homosapiens) Sam68 and those from rodents (Mus musculus, GenBank: NM_011317.4), rats (Rattus norvegicus, GenBank: NM_130405.2), and Chinese hamsters (Cricetulus griseus, GenBank: XM_027399403.2). The results are shown in Table 3: #12, #9, #10, and #16 specifically target the human Sam68 sequence, while #13, #15, and #21 can simultaneously target the Sam68 sequences of humans, mice, rats, and hamsters. Based on this, this embodiment used Western blotting to detect the inhibitory efficiency of the siRNAs on the target protein in mice. The results showed that... Figure 5 As shown in the DE, #13, #15, and #21 of Sam68-siRNA can effectively reduce the expression of Sam68 in AML-12 cells, with silencing efficiencies of 64.06%, 41.72%, and 45.52%, respectively, while #16, which specifically targets human Sam68, has no significant inhibitory effect.

[0053] Table 3 shows the sequence conservation of the screened siRNAs between humans and rodents.

[0054] Example 7: qPCR detection of gluconeogenesis pathway gene expression in HepG2 cells transfected with Sam68-siRNA: HepG2 cells were transfected with 30 nM Sam68-siRNA, GADPH-siRNA, or scramble-siRNA, respectively, and then cultured at 37°C. Cells were cultured at 5% CO2 for 48 hours. Cells were collected for qPCR, following the same experimental procedures as in Example 5, and the primers used are listed in Table 2. Gluconeogenesis is the process of synthesizing glucose from non-carbohydrate precursors (such as lactate, glycerol, and amino acids). This process primarily occurs in the liver (and to a lesser extent, the kidneys), maintaining blood glucose levels during starvation or low carbohydrate intake. In diabetes, particularly in cases of insulin resistance or deficiency, hepatic gluconeogenesis is often abnormally activated, leading to abnormally high blood glucose levels. In diabetes management, controlling gluconeogenesis is crucial for lowering fasting blood glucose levels, typically achieved through the use of medications (such as metformin, which can reduce hepatic gluconeogenesis) and lifestyle interventions (such as diet and exercise). Hepatic gluconeogenesis is regulated by a series of transcription factors, among which PGC-1α is a key transcription factor initiating the transcription of the G6Pase and PEPCK genes. Glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxylase (PEPCK) are rate-limiting enzymes in gluconeogenesis; the amount of transcription of these two enzymes determines the rate of gluconeogenesis. qPCR results showed that, among the screened siRNAs, #15 could significantly inhibit the transcription of PGC-1α, G6Pase and PEPCK simultaneously.

[0055] Example 8: siRNA modification: This experiment selected three advantageous siRNA sequences based on Examples 4-7 and performed extensive and patterned chemical modifications using the ESC mode to maximize stability and specificity while minimizing off-target effects. Delivery system: A high-affinity GalNAc (L96) ligand was used to achieve efficient and specific hepatocyte targeting via receptor-mediated endocytosis. Specific modifications are shown in Table 4; the modified Sam68-siRNA is abbreviated as GalNAc-siSam68.

[0056] Table 4. Sequences and modifications of GalNAc-siSam68 and GalNAc-siScramble

[0057] Example 9: Western blotting to detect the inhibitory efficiency of GalNAc-siSam68 on the target protein: WT mice were subcutaneously injected with 4 μg / g GalNAc-siSam68 and GalNAc-siScramble, respectively. After 72 hours, the mice were euthanized with an excess of CO2. Immediately afterwards, the thorax was opened, the right atrial appendage was cut open, and the left ventricle was perfused using a syringe containing 5-10 mL of cold PBS. The liver was then removed using ophthalmic scissors, quickly placed on sterile gauze, and the surface fluid was absorbed using sterile absorbent paper. The liver was then placed in corresponding cryovials and rapidly stored in liquid nitrogen at -80°C for molecular biological analysis. Proteins extracted from the collected liver were subjected to SDS-PAGE-Western blot assays. Antibodies specifically recognizing Sam68 protein and an internal control antibody for β-actin were used to detect Sam68 expression, with β-actin serving as the internal control. The results are as follows: Figure 6 As shown in BC, compared with the control group GalNAc-siScramble, the inhibition efficiencies of #13, #15 and #21 in GalNAc-siSam68 at the protein level were -21.64%, 23.63% and 67.72%, respectively.

[0058] Example 10: Determination of the effective dose of GalNAc-siSam68 #21 in vivo: WT mice were subcutaneously injected with 4 μg / g, 8 μg / g, and 16 μg / g of GalNAc-siSam68 #21 or GalNAc-siScramble, respectively. They were sacrificed 72 hours later, and their livers were collected, following the same procedure as in Example 9. Proteins were extracted from the collected livers and subjected to SDS-PAGE-Western blot analysis. The expression of Sam68 was measured using antibodies specifically recognizing Sam68 protein and an internal control antibody for β-actin. β-actin was used as the internal control. The results are as follows: Figure 6 As shown in the middle EF, compared with the control group GalNAc-siScramble, the protein inhibition efficiencies of GalNAc-siSam68 #21 at 4 μg / g, 8 μg / g and 16 μg / g were 37.37%, 30.09% and 77.68%, respectively.

[0059] Example 11: GalNAc-siSam68 #21 significantly improves hyperglycemia and insulin resistance. (1) Animal model: Male BKS-DB mice aged 8-10 weeks and HFD+STZ rats aged 10-12 weeks were used as diabetes models. Among them, BKS-DB mice (db / db mice) were spontaneous diabetes models. The HFD+STZ rat model was established as follows: 4-6 week old rats were selected, fed a high-fat, high-sugar diet, and 5% glucose drinking water for 45 days. Then, 35 mg / kg STZ was injected intraperitoneally. Random blood glucose was measured for three consecutive days after injection, and the high-fat, high-sugar diet was maintained. Rats with three random blood glucose levels greater than 16.7 mmol / L were considered to have successfully established the model and were allowed to resume normal diet and water intake.

[0060] (2) Administration: GalNAc-siSam68 #21 and GalNAc-siScramble solutions of appropriate concentrations were prepared using PBS. For db / db rats and HFD+STZ rats, the dosage was 16 μg / g, administered subcutaneously to the neck and back after cleaning with alcohol.

[0061] (3) Measurement of fasting blood glucose: For db / db rats and HFD+STZ rats, tail blood was collected after fasting for 16 hours overnight, and blood glucose values ​​were measured and read using a blood glucose meter. Blood glucose values ​​were remeasured for each rat every 5 minutes, and the average of the two blood glucose values ​​was taken as the final reading.

[0062] (4) Measurement of body weight: For db / db rats and HFD+STZ rats, body weight was measured after fasting overnight for 16 hours.

[0063] (5) Determination of ITT (insulin tolerance): db / db mice were fasted for 6 hours in the morning, and HFD+STZ rats were fasted for 5 hours in the morning. Then, mice and rats were intraperitoneally injected with 2 μ / kg and 0.75 U / kg insulin, respectively, and blood glucose values ​​were measured at 0 min, 30 min, 60 min, 90 min and 120 min.

[0064] (6) Serum insulin level measurement: Blood was collected from the orbital cavity of rats after fasting for 16 hours overnight. After centrifugation, the supernatant was collected and the serum insulin concentration was measured by ELISA.

[0065] (7) Tissue insulin tolerance assay: db / db mice were fasted for 16 hours and then divided into two groups. One group was injected intraperitoneally with 2 U / kg insulin, and the other group was injected with an equal volume of PBS. Fifteen minutes later, the mice were euthanized with an excess of CO2. Immediately afterwards, the thorax was opened, the right atrial appendage was cut open, and a syringe containing 5-10 mL of cold PBS was inserted into the left ventricle for cardiac perfusion. Then, the peritoneum of the mice was cut open, and the liver, epididymal fat, and gastrocnemius muscle were removed with ophthalmic scissors. After drying, the samples were stored in corresponding centrifuge tubes and then flash-frozen in liquid nitrogen at -80°C for molecular biological detection.

[0066] Example 12: Measurement of blood AST and ALT after GalNAc-siSam68 #21 treatment: After fasting for 16 hours, db / db mice were anesthetized with isoflurane. The epidermis of the left chest was removed, and blood was collected from the heart by inserting a needle at the pulsation point. The collected blood was placed in the corresponding centrifuge tube, mixed by inverting, and allowed to stand at room temperature for 45 minutes. The tubes were then centrifuged at 1500g, and the supernatant was collected. ALT and AST levels were then measured using a rapid recording method.

[0067] Example 13: Histopathological examination of liver tissue after GalNAc-siSam68 #21 treatment: (1) Sampling: For liver HE staining, prepare centrifuge tubes containing 20 mL of formalin and label them. Sacrifice the mice, then quickly perfuse the heart with PBS, and immediately inject 100 μL of 10% KCl into the left ventricle. Remove the liver, blot dry with absorbent paper, and transfer it to the corresponding formalin centrifuge tube for fixation for 24 hours for pathological examination. After blotting dry a portion of the liver tissue, embed it directly using OCT. Use a -25℃ freezer for embedding, and then transfer it to -80℃ for storage.

[0068] (2) Paraffin embedding and sectioning: After trimming the liver, the tissue is dehydrated and cleared by gradient and then embedded in paraffin. The tissue is then sectioned and spread. After the sections are dried and baked, they are ready for use.

[0069] (3) H&E staining: After baking, the sections were dewaxed with xylene and rehydrated with alcohol in a gradient decreasing range. Then, hematoxylin staining was performed for 5 min. After washing with water for 1 min, the sections were separated by 1% hydrochloric acid alcohol for 1-3 s. After washing with water for 1 min, eosin staining was performed for 3-5 min. After washing away the floating color with water, the sections were dehydrated with alcohol in a gradient increasing range. After soaking in xylene, the sections were immediately mounted with neutral resin. After drying in a fume hood, the sections were photographed under a microscope.

[0070] The results above indicate that in rat or mouse models of type 2 diabetes, Sam68 gene knockout significantly reduced blood glucose, inhibited hepatic gluconeogenesis, and downregulated cholesterol synthesis-related pathways. Therefore, knocking out / inhibiting the Sam68 gene has a blood glucose and cholesterol-lowering effect. Meanwhile, the nucleic acid drug GalNAc-siSam68 #21, which inhibits hepatic Sam68, also has the effect of lowering blood glucose and improving insulin resistance.

[0071] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A double-stranded siRNA targeting liver Sam68, characterized in that, The double-stranded siRNA comprises: a sense strand and an antisense strand complementary to the sense strand; The positive strand is a modified or unmodified sequence containing the nucleotide sequence shown in SEQ ID NO.41; The antisense strand is a modified or unmodified sequence containing the nucleotide sequence shown in SEQ ID NO.

42.

2. The double-stranded siRNA according to claim 1, characterized in that, The modification is selected from one or more of nucleotide chemical modification, overhang modification, and liver-targeting ligand modification.

3. The double-stranded siRNA according to claim 2, characterized in that, The nucleotide chemical modifications include: chemical modifications at the 2' position of the ribose of the nucleotide, and / or chemical modifications of the 3',5'-phosphodiester bonds between nucleotides; The protruding end modification involves adding 1-3 protruding nucleotides to the 3' end of the antisense strand; The liver-targeting ligand modification involves covalently attaching a liver-targeting ligand to the 3' end of the positive chain.

4. The double-stranded siRNA according to claim 3, characterized in that, The chemical modification at the 2' position of the nucleotide ribose is selected from one or more of 2'-methoxy, 2'-O-methoxyethyl, 2'-fluoro, 2'-benzyloxy, 2'-methylcarbonylamino, and 2'-pyridinemethoxy.

5. The double-stranded siRNA according to claim 3, characterized in that, The chemical modification of the 3',5'-phosphodiester bond between the nucleotides is a thiomodification that replaces oxygen atoms with sulfur atoms.

6. The double-stranded siRNA according to claim 3, characterized in that, The protruding end modification is to add a protruding UU to the 3' end of the antisense chain.

7. The double-stranded siRNA according to claim 3, characterized in that, The liver-targeting ligand is GalNAc ligand.

8. The double-stranded siRNA according to claim 1, characterized in that, The justice chain is 5'-Cm*Am*GmAmCmAmAfGmUfAfAfUmUmGmUmCmUmAmAmGmUm-3'-L96; The antisense chain is 3'-Um*Um*GmUmCmUmGmUfUmCfAmUmUmAmAmCmAmGfAmUmUm*Cf*Am-5'.

9. The use of the double-stranded siRNA as described in any one of claims 1-8 in the preparation of medicaments for the prevention and / or treatment of metabolic syndrome-related diseases.

10. The application according to claim 9, characterized in that, The metabolic syndrome-related diseases are selected from one or more of diabetes, obesity, hypercholesterolemia, and atherosclerosis.

Images