Use of RNF125 inhibitor in preparation of drug for preventing or treating obesity and related complications

Abstract

The present invention belongs to the technical field of biomedicine, and particularly relates to the use of an RNF125 inhibitor in the preparation of a drug for preventing or treating obesity and related complications. The present invention constructs an obesity model by means of knocking out RNF125 in mice, and feeding the mice with a high-fat feed. The result shows that RNF125 deletion can improve diseases or symptoms such as obesity phenotypes, hyperglycemia, hyperlipidemia, fat accumulation and non-alcoholic fatty liver induced by a high-fat diet. The injection of an siRNA specifically targeting RNF125 in the obesity mouse model can effectively improve weight gain, reduced glucose tolerance and insulin sensitivity, as well as diseases such as hyperglycemia, hyperlipidemia, fat accumulation and hepatic steatosis caused by a high-fat diet. It is determined therefrom that the RNF125 gene or protein is a new target for preventing or treating obesity and related complications. On the basis of the target, a series of human siRNAs for targeted inhibition of RNF125 are developed in the present invention, and the stability of the siRNAs in vivo and the silencing efficiency of the siRNAs on target genes are further improved by means of modification, which is beneficial to drug preparation.

Description

Application of RNF125 inhibitors in the preparation of drugs for preventing or treating obesity and related complications Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to the use of RNF125 inhibitors in the preparation of drugs for preventing or treating obesity and related complications. Background Art

[0002] Obesity refers to the abnormal distribution or excessive accumulation of body fat, which ultimately affects health. The main characteristic of obesity is excessive fat accumulation. The World Health Organization (WHO) defines overweight adults as having a body mass index (BMI) between 25.0-29.9 kg / m 2 Obesity is defined as BMI ≥ 30.0 kg / m 2 .

[0003] Obesity, a health risk, affects the entire metabolic system, leading to various complications such as type 2 diabetes mellitus (T2DM), fatty liver disease, cardiovascular disease, stroke, lipid metabolism disorders, hypertension, gallbladder problems, arthritis, sleep apnea and other respiratory problems, and cancers (such as breast cancer, liver cancer, ovarian cancer, prostate cancer, gallbladder cancer, and colon cancer). These conditions all increase the risk of death. The most studied complications directly related to obesity are type 2 diabetes, non-alcoholic fatty liver disease, and cardiovascular disease. Obesity is the primary risk factor for type 2 diabetes. The main cause of T2DM is obesity-induced insulin resistance in the liver, white adipose tissue (WAT), and skeletal muscle, coupled with insufficient insulin secretion by pancreatic beta cells, which makes it difficult to overcome this resistance. Furthermore, obesity promotes cellular senescence in various organs, including adipose tissue, liver, brain, and pancreas, shortening lifespan and increasing the risk of various age-related diseases and complications, including diabetes, hypertension, cancer, cognitive impairment, atherosclerosis, and vascular dysfunction. Therefore, the need to treat / prevent obesity and improve quality of life is urgent. However, the development of obesity treatments is fraught with difficulties. For example, most weight loss therapies currently approved by regulatory agencies have significant side effects and cannot be used long-term. Surgical treatments are expensive and not a permanent solution, requiring long-term dietary control. Long-term dietary control can easily lead to malnutrition or recurrent obesity. Identifying a target for obesity treatment and developing safe and effective new drugs would be a boon for those suffering from obesity.

[0004] The RNF125 (Ring Finger Protein 125) gene encodes a novel E3 ubiquitin ligase containing a ring finger domain at its N-terminus and three zinc-binding motifs and a ubiquitin interaction motif at its C-terminus. This encoded protein may play a positive regulatory role in T cell receptor signaling. RNF125 is ubiquitously expressed in adipose tissue, bone marrow, and 25 other tissues. However, studies have yet to confirm the association between RNF125 and metabolic status, obesity, insulin resistance, or fatty liver disease.

[0005] Summary of the Invention

[0006] (1) Technical issues to be solved

[0007] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides the use of RNF125 inhibitors in the preparation of drugs for preventing or treating obesity and related complications, providing a reliable solution for the development of safe and effective obesity treatment drugs.

[0008] (2) Technical solution

[0009] The present invention provides use of an RNF125 inhibitor in preparing a medicament for preventing or treating obesity and related complications.

[0010] Animal experiments have shown that knocking out RNF125 or injecting siRNA targeting RNF125 into a mouse obesity model constructed with a high-fat diet can effectively improve symptoms such as weight gain, fat accumulation, weakened glucose tolerance, insulin resistance, hyperglycemia, hyperlipidemia, and liver fatty degeneration caused by a high-fat diet.

[0011] The RNF125 inhibitor can produce any of the following changes in metabolic levels: (1) inhibiting obesity caused by a high-fat diet in mice; (2) inhibiting fat accumulation caused by a high-fat diet; (3) inhibiting glucose tolerance and insulin resistance caused by a high-fat diet and thus preventing hyperglycemia and hyperlipidemia; (4) inhibiting liver fatty degeneration caused by a high-fat diet.

[0012] Among them, RNF125 inhibitors can inhibit the activity or expression level of RNF125 protein, thereby achieving the effect of preventing or treating obesity and related complications.

[0013] According to a preferred embodiment of the present invention, the RNF125 inhibitor is any protein molecule, nucleic acid molecule (siRNA or shRNA or miRNA, etc.), small compound molecule, viral particle or any combination of two or more of the foregoing that can reduce the activity or expression level of the RNF125 protein; or, the RNF125 inhibitor is any protein molecule, nucleic acid molecule, small compound molecule, viral particle, gene editing kit or any combination of two or more of the foregoing that can knock out or knock down or silence the RNF125 gene or protein.

[0014] According to a preferred embodiment of the present invention, the RNF125 inhibitor is any one of the following siRNAs:

[0015] siRNA formed by annealing two single strands shown in SEQ ID No. 9-10;

[0016] siRNA formed by annealing two single strands shown in SEQ ID NOs. 41-42;

[0017] siRNA formed by annealing two single strands shown in SEQ ID NOs. 43-44;

[0018] siRNA formed by annealing two single strands represented by SEQ ID NOs. 111-112;

[0019] siRNA formed by annealing two single strands shown in SEQ ID NO. 1-2;

[0020] siRNA formed by annealing two single strands shown in SEQ ID NO.37-38;

[0021] siRNA formed by annealing two single strands shown in SEQ ID NOs. 85-86;

[0022] siRNA formed by annealing two single strands shown in SEQ ID NOs. 205-206;

[0023] siRNA formed by annealing two single strands shown in SEQ ID NO. 3-4;

[0024] siRNA formed by annealing two single strands shown in SEQ ID NOs. 87-88;

[0025] siRNA formed by annealing two single strands represented by SEQ ID NO. 45-46;

[0026] siRNA formed by annealing two single strands shown in SEQ ID NO. 5-6;

[0027] siRNA formed by annealing two single strands shown in SEQ ID NOs. 55-56;

[0028] siRNA formed by annealing two single strands shown in SEQ ID NOs. 159-160;

[0029] siRNA formed by annealing two single strands shown in SEQ ID NO. 23-24;

[0030] siRNA formed by annealing two single strands shown in SEQ ID NO. 29-30;

[0031] siRNA formed by annealing two single strands represented by SEQ ID NOs. 175-176;

[0032] siRNA formed by annealing two single strands shown in SEQ ID NO.11-12;

[0033] siRNA formed by annealing two single strands shown in SEQ ID NO. 33-34;

[0034] siRNA formed by annealing two single strands shown in SEQ ID NO. 17-18;

[0035] siRNA formed by annealing two single strands shown in SEQ ID NOs. 93-94;

[0036] siRNA formed by annealing two single strands shown in SEQ ID NOs. 49-50;

[0037] siRNA formed by annealing two single strands shown in SEQ ID NO.35-36;

[0038] siRNA formed by annealing two single strands shown in SEQ ID NOs. 75-76;

[0039] siRNA formed by annealing two single strands shown in SEQ ID NOs. 79-80;

[0040] siRNA formed by annealing two single strands represented by SEQ ID NOs. 109-110;

[0041] siRNA formed by annealing two single strands shown in SEQ ID NOs. 153-154;

[0042] siRNA formed by annealing two single strands shown in SEQ ID NO. 15-16;

[0043] siRNA formed by annealing two single strands represented by SEQ ID NOs. 131-132;

[0044] siRNA formed by annealing two single strands shown in SEQ ID NOs. 59-60;

[0045] siRNA formed by annealing two single strands shown in SEQ ID NOs. 51-52;

[0046] siRNA formed by annealing two single strands represented by SEQ ID NOs. 167-168;

[0047] siRNA formed by annealing two single strands represented by SEQ ID NOs. 133-134;

[0048] siRNA formed by annealing two single strands shown in SEQ ID NOs. 77-78;

[0049] siRNA formed by annealing two single strands shown in SEQ ID NOs. 219-220;

[0050] siRNA formed by annealing two single strands shown in SEQ ID NO. 19-20;

[0051] siRNA formed by annealing two single strands shown in SEQ ID NO. 7-8;

[0052] siRNA formed by annealing two single strands shown in SEQ ID NO. 13-14;

[0053] siRNA formed by annealing two single strands shown in SEQ ID NO. 27-28;

[0054] siRNA formed by annealing two single strands shown in SEQ ID NOs. 165-166;

[0055] siRNA formed by annealing two single strands shown in SEQ ID NOs. 201-202;

[0056] siRNA formed by annealing two single strands represented by SEQ ID NOs. 145-146;

[0057] siRNA formed by annealing two single strands shown in SEQ ID NOs. 123-124;

[0058] siRNA formed by annealing two single strands shown in SEQ ID NOs. 195-196;

[0059] siRNA formed by annealing two single strands shown in SEQ ID NOs. 61-62;

[0060] siRNA formed by annealing two single strands shown in SEQ ID NOs. 207-208;

[0061] siRNA formed by annealing two single strands shown in SEQ ID NOs. 39-40;

[0062] siRNA formed by annealing two single strands shown in SEQ ID NOs. 83-84;

[0063] siRNA formed by annealing two single strands shown in SEQ ID NOs. 69-70;

[0064] siRNA formed by annealing two single strands represented by SEQ ID NOs. 107-108;

[0065] siRNA formed by annealing two single strands shown in SEQ ID NOs. 47-48;

[0066] siRNA formed by annealing two single strands represented by SEQ ID NOs. 125-126;

[0067] siRNA formed by annealing two single strands shown in SEQ ID NO. 25-26;

[0068] siRNA formed by annealing two single strands shown in SEQ ID NOs. 199-200;

[0069] siRNA formed by annealing two single strands shown in SEQ ID NO.31-32;

[0070] siRNA formed by annealing two single strands represented by SEQ ID NOs. 171-172;

[0071] siRNA formed by annealing two single strands shown in SEQ ID NOs. 135-136;

[0072] siRNA formed by annealing two single strands shown in SEQ ID NOs. 143-144;

[0073] siRNA formed by annealing two single strands shown in SEQ ID NOs. 89-90;

[0074] siRNA formed by annealing two single strands shown in SEQ ID NO. 21-22;

[0075] siRNA formed by annealing two single strands shown in SEQ ID NOs. 97-98;

[0076] siRNA formed by annealing two single strands represented by SEQ ID NOs. 151-152;

[0077] siRNA formed by annealing two single strands represented by SEQ ID NOs. 221-222;

[0078] siRNA formed by annealing two single strands represented by SEQ ID NOs. 105-106;

[0079] siRNA formed by annealing two single strands shown in SEQ ID NOs. 57-58;

[0080] siRNA formed by annealing two single strands represented by SEQ ID NOs. 129-130;

[0081] siRNA formed by annealing two single strands represented by SEQ ID NOs. 229-230;

[0082] siRNA formed by annealing two single strands represented by SEQ ID NOs. 141-142;

[0083] siRNA formed by annealing two single strands shown in SEQ ID NO. 155-156;

[0084] siRNA formed by annealing two single strands represented by SEQ ID NOs. 147-148;

[0085] siRNA formed by annealing two single strands shown in SEQ ID NOs. 65-66;

[0086] siRNA formed by annealing two single strands shown in SEQ ID NOs. 67-68;

[0087] siRNA formed by annealing two single strands shown in SEQ ID NOs. 189-190;

[0088] siRNA formed by annealing two single strands shown in SEQ ID NOs. 193-194;

[0089] siRNA formed by annealing two single strands shown in SEQ ID NOs. 53-54;

[0090] siRNA formed by annealing two single strands shown in SEQ ID NOs. 73-74;

[0091] siRNA formed by annealing two single strands shown in SEQ ID NOs. 213-214;

[0092] siRNA formed by annealing two single strands represented by SEQ ID NOs. 181-182;

[0093] siRNA formed by annealing two single strands shown in SEQ ID NOs. 101-102;

[0094] siRNA formed by annealing two single strands shown in SEQ ID NOs. 139-140;

[0095] siRNA formed by annealing two single strands shown in SEQ ID NOs. 91-92;

[0096] siRNA formed by annealing two single strands shown in SEQ ID NOs. 197-198;

[0097] siRNA formed by annealing two single strands shown in SEQ ID NOs. 99-100;

[0098] siRNA formed by annealing two single strands represented by SEQ ID NOs. 137-138;

[0099] siRNA formed by annealing two single strands shown in SEQ ID NOs. 71-72;

[0100] siRNA formed by annealing two single strands represented by SEQ ID NOs. 127-128;

[0101] siRNA formed by annealing two single strands represented by SEQ ID NOs. 121-122;

[0102] siRNA formed by annealing two single strands represented by SEQ ID NOs. 115-116;

[0103] siRNA formed by annealing two single strands shown in SEQ ID NOs. 215-216;

[0104] siRNA formed by annealing two single strands represented by SEQ ID NOs. 173-174;

[0105] siRNA formed by annealing two single strands shown in SEQ ID NOs. 103-104;

[0106] siRNA formed by annealing two single strands represented by SEQ ID NOs. 177-178;

[0107] siRNA formed by annealing two single strands shown in SEQ ID NOs. 95-96;

[0108] siRNA formed by annealing two single strands represented by SEQ ID NOs. 231-232;

[0109] siRNA formed by annealing two single strands shown in SEQ ID NOs. 209-210;

[0110] siRNA formed by annealing two single strands represented by SEQ ID NOs. 119-120;

[0111] siRNA formed by annealing two single strands shown in SEQ ID NOs. 149-150;

[0112] siRNA formed by annealing two single strands shown in SEQ ID NOs. 81-82;

[0113] siRNA formed by annealing two single strands shown in SEQ ID NOs. 203-204;

[0114] siRNA formed by annealing two single strands represented by SEQ ID NOs. 157-158;

[0115] siRNA formed by annealing two single strands represented by SEQ ID NOs. 163-164;

[0116] siRNA formed by annealing two single strands represented by SEQ ID NOs. 217-218;

[0117] siRNA formed by annealing two single strands shown in SEQ ID NOs. 191-192;

[0118] siRNA formed by annealing two single strands represented by SEQ ID NOs. 187-188;

[0119] siRNA formed by annealing two single strands represented by SEQ ID NOs. 169-170;

[0120] siRNA formed by annealing two single strands represented by SEQ ID NOs. 113-114;

[0121] siRNA formed by annealing two single strands represented by SEQ ID NOs. 117-118;

[0122] siRNA formed by annealing two single strands shown in SEQ ID NOs. 63-64;

[0123] siRNA formed by annealing two single strands represented by SEQ ID NOs. 161-162;

[0124] siRNA formed by annealing two single strands shown in SEQ ID NOs. 223-224;

[0125] siRNA formed by annealing two single strands represented by SEQ ID NOs. 185-186;

[0126] siRNA formed by annealing two single strands represented by SEQ ID NOs. 211-212;

[0127] siRNA formed by annealing two single strands shown in SEQ ID NOs. 215-216;

[0128] siRNA formed by annealing two single strands represented by SEQ ID NOs. 227-228;

[0129] siRNA formed by annealing two single strands shown in SEQ ID NOs. 183-184;

[0130] The siRNA is formed by annealing two single strands shown in SEQ ID NO. 179-180.

[0131] According to a preferred embodiment of the present invention, the RNF125 inhibitor is a modified product obtained by modifying the antisense strand and sense strand of any of the above-mentioned siRNAs; the nucleotide modification includes one or a combination of methoxy modification, fluoro modification, GalNAc coupling modification, thio modification, and ethylene glycol, which is used to improve the stability of the siRNA in vivo, reduce the degradation rate, and facilitate the silencing efficiency of the target gene or form drug delivery. GalNAc is covalently coupled to the 3' end of the nucleic acid in a trivalent state. Thio modification is a phosphorothioate group connection between two adjacent nucleotides.

[0132] According to a preferred embodiment of the present invention, the modification method is any of the following:

[0133] Method 1: Among the 21 nucleotides of the sense strand, there are two thio-modified nucleotides at the 5' end, fluorinated nucleotides at positions 9, 10, and 11, and methoxy-modified nucleotides at the remaining positions, and the 3' end is coupled with GalNAc; among the 23 nucleotides of the antisense strand, there are two thio-modified nucleotides at the 5' end, fluorinated nucleotides at positions 2, 5, 14, and 16, and two thio-modified nucleotides at the 3' end, and methoxy-modified nucleotides at the remaining positions;

[0134] Method 2: Among the 21 nucleotides of the sense strand, two 5'-end thiolation modifications are performed, and positions 9, 10, and 11 are fluorinated. The remaining nucleotides are modified with methoxy and fluorine in sequence, and two 3'-end thiolation modifications are performed, and the 3'-end is coupled with GalNAc. The 23 nucleotides of the antisense strand are modified with methoxy and fluorine in sequence, and two 5'-end thiolation modifications are performed, and two 3'-end thiolation modifications are performed.

[0135] Method 3: Among the 21 nucleotides of the sense strand, there are two thio-modified nucleotides at the 5' end, fluorinated nucleotides at positions 7, 9, 10, and 11, and methoxy-modified nucleotides at the remaining positions. There are two thio-modified nucleotides at the 3' end, and the 3' end is coupled with GalNAc. Among the 23 nucleotides of the antisense strand, there are fluorinated nucleotides at positions 2, 6, 8, 9, 15, and 17, and methoxy-modified nucleotides at the remaining positions. There are two thio-modified nucleotides at the 5' end and two thio-modified nucleotides at the 3' end.

[0136] Method 4: Among the 21 nucleotides of the sense chain, there are 2 thio-modified nucleotides at the 5' end, fluorinated modifications at positions 7, 9, 10, and 11, and methoxy modifications at the remaining positions. There are 2 thio-modified nucleotides at the 3' end, and the 3' end is coupled with GalNAc; among the 23 nucleotides of the antisense chain, positions 2, 14, and 16 are fluorinated, position 7 is ethylene glycol nucleic acid, and the remaining positions are methoxy modified. There are 2 thio-modified nucleotides at the 5' end and 2 thio-modified nucleotides at the 3' end.

[0137] For the purposes of this application, obesity refers to a chronic metabolic disease characterized by excessive accumulation or abnormal distribution of body fat, leading to weight gain, and is caused by the interaction of multiple factors, including genetic and environmental factors. Obesity-related complications include type 2 diabetes, hypertension, hyperlipidemia, atherosclerosis, vascular dysfunction, non-alcoholic fatty liver disease, stroke, lipid metabolism disorders, and sleep apnea.

[0138] In a fourth aspect, the present invention provides a drug for preventing or treating obesity and related complications, wherein the active ingredient of the drug is an RNF125 inhibitor; the drug comprises the active ingredient RNF125 inhibitor and a pharmaceutically acceptable excipient. The percentage of the RNF125 inhibitor in the drug is 1-99%.

[0139] In a fifth aspect, the present invention provides the use of RNF125 gene or protein as a target in constructing a model for treating obesity.

[0140] Preferably, the construct is achieved by increasing the expression level of the RNF125 gene or protein in the target animal.

[0141] RNF125 is a known gene. RNF125, also known as TRAC-1, is an E3 ubiquitin ligase composed of an N-terminal RING domain, three zinc finger structures (C2HC, C2H2, C2H2) in the middle region, and a C-terminal ubiquitin interaction motif (UIM). RNF125 was originally discovered as a regulator of T cell activation and is highly expressed in lymphoid tissues within the bone marrow, spleen, and thymus. Subsequent studies have also found that RNF125 is associated with various diseases, including inflammatory responses, the development and progression of cancer, and innate antiviral immune responses. However, no reports have yet revealed its connection with obesity.

[0142] This invention discloses for the first time that inhibiting the expression of the RNF125 gene or protein can effectively improve symptoms such as weight gain, fat accumulation, impaired glucose tolerance, insulin resistance, and hepatic steatosis caused by a high-fat diet. Animal experiments have shown that using CRISPER-Cas9 technology to knock out RNF125 in mice and then feeding them a high-fat diet to create an obesity model, the experimental results suggest that RNF125 deficiency can improve the obesity phenotype, hyperglycemia, hyperlipidemia, fat accumulation, and non-alcoholic fatty liver disease induced by a high-fat diet. Injecting siRNA targeting RNF125 into the obese mouse model created by a high-fat diet for treatment showed that the siRNA can effectively improve symptoms such as weight gain, impaired glucose tolerance and insulin sensitivity, hyperglycemia, hyperlipidemia, fat accumulation, and hepatic steatosis caused by a high-fat diet. This confirms that the RNF125 gene or protein is a new target for the treatment or treatment of obesity and related complications.

[0143] It is not difficult to understand that based on the above research conclusions, the innovation of the present invention mainly lies in providing a new target RNF125 for the treatment or prevention of obesity and its complications, and the existing known RNF125 inhibitors (including knockout or knockdown agents) can be used as active ingredients of drugs for the prevention or treatment of obesity and related complications.

[0144] For the sake of convenience and more concise description, the meaning of "RNF125 inhibition" in this application includes targeted knockout, knockdown or silencing of RNF125 and other functions, and the meaning of "RNF125 inhibitor" includes any protein molecule, nucleic acid molecule, small compound molecule, gene editing kit, viral particle or any combination of two or more of the foregoing that can knock out, knockdown or silence RNF125, such as but not limited to neutralizing antibodies, siRNA, shRNA, miRNA, knockout reagents, gene editing crispr_cas9 kits or lentivirus, etc.

[0145] (3) Beneficial effects

[0146] 1. The present invention provides a new target related to obesity, RNF125. RNF125 has been shown to have at least one of the following activities: (1) promoting obesity caused by a high-fat diet; (2) promoting fat accumulation caused by a high-fat diet; (3) promoting glucose tolerance and insulin resistance caused by a high-fat diet, leading to hyperglycemia and hyperlipidemia; and (4) promoting hepatic steatosis caused by a high-fat diet. Animal experiments have shown that knocking out RNF125 or injecting siRNA targeting RNF125 into an obesity mouse model established by a high-fat diet can effectively improve symptoms such as weight gain, fat accumulation, impaired glucose tolerance, insulin resistance, hyperglycemia, hyperlipidemia, and hepatic steatosis caused by a high-fat diet. Based on the above experimental results, it is inferred that obesity and related complications can be prevented or treated by inhibiting RNF125 gene or protein expression.

[0147] In summary, the present invention provides the application of inhibiting RNF125 as a therapeutic target in preventing or treating obesity and related complications. This target provides new direction and theoretical guidance for the development of safe and effective treatment plans and drugs for obesity and its complications.

[0148] 2. The present invention provides a series of siRNA sequences targeting RNF125 for knocking down or silencing the expression of the gene. It has been confirmed at the cellular level that the siRNA sequences provided by the present invention can effectively silence the expression of the target gene RNF125 in the cells when transfected into experimental cells at an extremely low concentration (1 nM).

[0149] 3. By further modifying the siRNA sequence targeting RNF125 provided by the present invention, coupling it with a GalNAc delivery system, and injecting it into a transgenic mouse model, the researchers collected blood and separated the serum to test the silencing effect of the modified siRNA on the target gene mRNA. The experiments showed that the modified siRNA could improve the silencing efficiency of the target gene RNF125 in the animal model, and different nucleotide modification methods could effectively silence the target gene. Nucleotide modification is mainly used to reduce the degradation of nucleic acid molecules in animals and improve drugability. BRIEF DESCRIPTION OF THE DRAWINGS

[0150] FIG1 shows the experimental statistical results of the effect of RNF125 deficiency on the food intake and weight gain rate of mice in Example 1.

[0151] FIG2 is the experimental statistical results of the effect of RNF125 deficiency on high-fat diet-induced hyperglycemia and hyperlipidemia in Example 2.

[0152] FIG3 shows the experimental statistical results of the effect of RNF125 deficiency on fat accumulation induced by a high-fat diet in Example 3.

[0153] FIG4 shows the experimental statistical results of the effect of RNF125 deficiency on non-alcoholic fatty liver disease induced by a high-fat diet in Example 4.

[0154] FIG5 shows the therapeutic effect of the siRNA sequence of RNF125 in Example 5 on obesity induced by a high-fat diet.

[0155] FIG6 shows the changes in glucose tolerance, insulin sensitivity, and blood lipids in mice fed a high-fat diet after treatment with RNF125-specific siRNA in Example 6.

[0156] FIG7 shows the changes in adipose tissue of mice fed a high-fat diet after treatment with RNF125-specific siRNA in Example 7.

[0157] FIG8 shows the therapeutic effect of the specific siRNA sequence of RNF125 in Example 8 on hepatic fatty lesions induced by a high-fat diet. DETAILED DESCRIPTION

[0158] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0159] The present invention first uses CRISPER-Cas9 technology to construct RNF125 knockout mice, which are fed with normal chow diet (NCD) and high fat diet (HFD), respectively, to analyze the role of RNF125 deficiency in obesity, hyperglycemia, hyperlipidemia, fat accumulation and non-alcoholic fatty liver disease induced by a high-fat diet. Afterwards, a mouse obesity model is established using a high-fat diet, and the mice are divided into two groups. While continuing to be fed a high-fat diet, one group is injected with a specific siRNA sequence targeting RNF125 through the tail vein, which is the treatment group (siRNF125); the other group is injected with a control sequence that has no homology with the transcriptomes of humans, rats, mice, etc., which is the control group (siCtrl). At the same time, mice of the same age fed a normal diet are used as controls to study the therapeutic effect of the RNF125 siRNA sequence on obesity and related diseases. The ingredients of normal feed and high-fat feed are as follows:

[0160] The experimental methods and experimental conclusions of various embodiments of the present invention are described in detail below.

[0161] 1. Screening and synthesis of RNF125-specific siRNA sequences

[0162] First, Jiangsu Jima Company synthesized multiple RNAi oligos targeting different sites of RNF125 in mice and transfected them into H22 mouse liver cancer cells. RT-qPCR was used to analyze RNF125 knockdown and select RNAi sequences with better knockdown efficiency. The selected RNF125 RNAi oligos (RNF125siRNA) sequences were synthesized and their sense strands were modified by cholesterol modification at the 3' end, two thiolate backbone modifications at the 5' end, four thiolate backbone modifications at the 3' end, and methoxyl modification throughout the entire strand. Finally, three RNF125-specific mouse siRNAs were obtained as shown in Table 1:

[0163] Table 1:

[0164] 2. Construction of obese mouse model and treatment plan

[0165] Six- to eight-week-old male C57BL / 6 mice were purchased from Spayfor Biotechnology Co., Ltd. and housed in an SPF-grade animal room at a temperature of 22±2°C. After one week of adaptation, the mice were divided into two groups: one group was fed with a normal diet, and the other was fed with a high-fat diet. During the feeding process, the body weight, random blood glucose, fasting blood glucose, and blood TG and TC levels of the mice were monitored. When the body weight, blood glucose, TG, and TC levels of the high-fat diet mice were significantly higher than those of the normal diet group, the high-fat diet mice were randomly divided into two groups. siRNA specifically targeting RNF125 and a control sequence (siCtrl) were designed and synthesized. They were injected into the mice via tail vein at a dose of 0.5 OD (1.25 nM) per mouse. The injections were performed every four days, and the weight changes of the mice were monitored. Columbus Instruments was used to monitor the changes in the diet of the mice.

[0166] 3. During the experiment, the blood glucose, TG and TC levels in the blood, TG and TC levels in the liver, glucose tolerance test (GTT) and tissue H&E staining of the mice can be measured by conventional methods. You can also refer to the following methods:

[0167] ① Blood glucose measurement of mice: under normal diet conditions, random blood glucose of mice was measured using a Roche blood glucose meter; under conditions of overnight starvation for 12 hours, fasting blood glucose of mice was measured using a Roche blood glucose meter.

[0168] ② Detection of TG and TC in mouse blood: After the mice were starved overnight for 12 hours, 100 μL of blood was collected from the tail of the mouse. After being placed at room temperature for 2 hours, the blood was centrifuged at 3000 rpm for 10 minutes. The serum was collected and the levels of TG, TC, LDL-C and FFA in the serum were detected using a biochemical analyzer.

[0169] ③ TG Assay in Mouse Liver: 50 mg of liver tissue was homogenized with n-propane / isopropanol at a ratio of 1:5-10 tissue mass (g): n-propane / isopropanol volume (mL). The mixture was then centrifuged at 8000 g for 10 min at 4°C. The supernatant was collected and the TG content in the liver tissue was determined using the Solebol Triglyceride (TG) Assay Kit (Cat. No. BC0625). TC Assay in the liver was determined as follows: isopropanol was added at a ratio of 1:5-10 tissue mass (g): isopropanol volume (mL). The mixture was homogenized on ice. The mixture was centrifuged at 10000 g for 10 min at 4°C. The supernatant was collected and the TC content in the liver tissue was determined using the Solebol Total Cholesterol (TC) Assay Kit (Cat. No. BC1985).

[0170] ④ The method of glucose tolerance test (GTT) is as follows: after fasting for 6 hours, mice were intraperitoneally injected with glucose at a dose of 2 g / kg. The blood glucose of mice was measured at 0, 15, 30, 45, 60, 90, and 120 minutes after injection using a Roche blood glucose meter. The glucose tolerance curve of mice was drawn, and the area under the blood glucose curve was calculated using the AOC method. The method of insulin tolerance test (ITT) is as follows: after fasting for 6 hours, mice were intraperitoneally injected with insulin (Novo Nordisk, insulin aspart injection) at a dose of 0.75 U / kg. The blood glucose of mice tail vein was measured at 0, 15, 30, 45, 60, 90, and 120 minutes after injection using a Roche blood glucose meter (20% glucose was prepared at the same time so that 100 μL of 20% glucose could be injected intraperitoneally to rescue mice from hypoglycemia). The insulin tolerance curve of mice was drawn, and the area under the blood glucose curve was calculated using the AOC method.

[0171] ⑤ The method of tissue HE staining is as follows: take tissue samples and fix them in 4% paraformaldehyde (Sevier, G1101). After 24 hours, dehydrate, clear, dip in wax, embed, section, and dewax are performed. After dewaxing, the sections are first stained with hematoxylin and then with eosin. After dehydration, the sections are sealed and observed.

[0172] Example 1

[0173] The experiment in this example demonstrates that RNF125 deficiency can improve the obesity phenotype induced by a high-fat diet. The experiment includes experimental methods and experimental results:

[0174] Experimental Methods: RNF125 knockout mice were constructed using CRISPR-Cas9 technology. Six- to eight-week-old wild-type (WT) and RNF125 knockout (KO) mice were divided into two groups and fed either a standard diet or a high-fat diet. Body size and weight were monitored during the feeding process, and dietary changes were monitored using Columbus Instruments.

[0175] Experimental results: As shown in Figure 1. Figure A shows that under normal diet conditions, RNF125 knockout mice consume more feed daily; however, after 6 months of feeding, the weight gain is significantly lower than that of wild-type mice, and the body size is also significantly smaller than that of wild-type mice (B). Under high-fat diet conditions, RNF125 knockout mice also consume significantly more feed daily than wild-type mice (C); however, after 6 months of feeding, the weight gain is significantly lower than that of wild-type mice, and the body size is also significantly smaller than that of wild-type mice (D).

[0176] As shown, RNF125 knockout mice, when fed a normal diet, consumed significantly more food per day than wild-type mice (A). However, after six months of feeding, RNF125 knockout mice were slightly smaller than wild-type mice and gained significantly less weight than wild-type mice (B). When fed a high-fat diet, RNF125 knockout mice still consumed significantly more food per day than wild-type mice (C). After six months of high-fat diet, wild-type mice more than doubled their weight, but knockout mice only gained approximately 75%, a significantly lower weight gain than wild-type mice, and were significantly smaller than wild-type mice (D). This suggests that RNF125 deficiency significantly ameliorates the obesity phenotype in mice, allowing them to consume more food while still gaining significantly less weight than wild-type mice. In the figure: *, P < 0.05; **, P < 0.01; ***, P < 0.001; NCD, normal diet; HFD, high-fat diet; WT, wild-type mice; KO, RNF125-deficient mice.

[0177] In summary, RNF125 knockout can significantly improve the obesity phenotype induced by a high-fat diet. At the same time, RNF125 affects the appetite of mice. The lack of RNF125 causes mice to consume more food, but their weight gain is less.

[0178] Example 2

[0179] The experiment in this example demonstrates that RNF125 deficiency can effectively improve high blood sugar and high blood lipids induced by a high-fat diet. The experiment includes experimental methods and experimental results:

[0180] Experimental method: The blood glucose of the mice in Example 1 during the high-fat diet was detected using a Roche blood glucose meter, and the changes in blood lipids of the mice were analyzed using a biochemical analyzer. The glucose tolerance of the mice was analyzed by a glucose tolerance test (GTT).

[0181] Experimental results: As shown in Figure 2. Figure A shows that the random blood glucose of RNF125 knockout mice was significantly lower than that of wild-type mice; Figure B shows that after the mice fasted overnight, the fasting blood glucose of RNF125 knockout mice was significantly lower than that of wild-type mice; Figure C shows that when fed a normal diet, the GTT curve of RNF125 knockout mice was significantly lower than that of wild-type mice; Figure D shows that when fed a high-fat diet, the GTT curve of RNF125 knockout mice was significantly lower than that of wild-type mice; Figure E shows that the area under the curve of Figures C and D shows that the glucose tolerance of RNF125 knockout mice was significantly higher than that of wild-type mice; Figure F shows that when fed a normal diet and Under high-fat diet conditions, the triglyceride (TG) level in the blood of RNF125 knockout mice was significantly lower than that of wild-type mice; Figure G shows that under normal diet and high-fat diet conditions, the cholesterol (TC) level in the blood of RNF125 knockout mice was significantly lower than that of wild-type mice; Figure H shows that under normal diet and high-fat diet conditions, the LDL-C level in the blood of RNF125 knockout mice was significantly lower than that of wild-type mice; Figure I shows that under normal diet and high-fat diet conditions, the free fatty acid (FFA) level in the blood of RNF125 knockout mice was significantly lower than that of wild-type mice.

[0182] As can be seen from this, the random blood glucose (A) and fasting blood glucose (B) of mice after RNF125 knockout are significantly reduced. For wild-type mice, a high-fat diet can cause the random blood glucose of wild-type mice to increase significantly, but RNF125 knockout can restore blood glucose to the level of a normal diet or slightly below the normal level, indicating that RNF125 deficiency is conducive to blood glucose control. To further verify the regulatory ability of RNF125 on blood glucose, the present embodiment analyzed the blood glucose changes of mice before and after RNF125 knockout by a glucose tolerance test (GTT), and the experimental results are shown in Figures C to E of Figure 2. Whether it is a normal diet (C) or a high-fat diet (D), the blood glucose levels of mice after RNF125 knockout are significantly lower than those of wild-type mice, and the area under the curve (AOC) of mice in the KO group is also significantly reduced (E), indicating that the glucose tolerance of mice after RNF125 knockout is significantly enhanced. Continuing to analyze changes in blood lipids, the results are shown in Figures 2F to 2I. RNF125 knockout mice showed significant reductions in serum triglyceride (F), cholesterol (G), LDL-C (H), and serum free fatty acid (I) levels, regardless of whether they were fed a normal or high-fat diet. These results indicate that RNF125 knockout can effectively alleviate high blood sugar, high blood lipids, and impaired glucose tolerance induced by a high-fat diet in animals.

[0183] These results suggest that RNF125 deficiency can effectively alleviate high-fat diet-induced hyperglycemia, hyperlipidemia, and impaired glucose tolerance. In the figure: *, P < 0.05; **, P < 0.01; ***, P < 0.001. NCD, normal diet; HFD, high-fat diet.

[0184] Example 3

[0185] The experiment in this example demonstrates that RNF125 deficiency can effectively improve fat accumulation induced by a high-fat diet. The experiment includes experimental methods and experimental results:

[0186] Experimental method: The mice fed a high-fat diet for 6 months in Example 1 were dissected to observe the fat accumulation in the abdominal cavity of the mice. The inguinal subcutaneous white adipose tissue (iWAT) and epididymal white adipose tissue (eWAT) of the mice were weighed, fixed with 4% paraformaldehyde, and then stained with H&E.

[0187] Experimental results: As shown in Figure 3. Panel A shows that after 6 months of normal or high-fat diet, RNF125 knockout mice had significantly less abdominal fat than wild-type mice; Panel B shows that after 6 months of normal or high-fat diet, the body weight ratio of subcutaneous fat in RNF125 knockout mice was significantly lower than that in wild-type mice; Panel C shows that after 6 months of normal or high-fat diet, the body weight ratio of epididymal visceral fat in RNF125 knockout mice was significantly lower than that in wild-type mice; H&E staining results in Panel D show that after 6 months of normal or high-fat diet, the adipocytes in subcutaneous fat of RNF125 knockout mice were significantly smaller than those in wild-type mice; H&E staining results in Panel E show that after 6 months of normal or high-fat diet, the adipocytes in epididymal visceral fat of RNF125 knockout mice were significantly smaller than those in wild-type mice.

[0188] Thus, after 6 months of a normal diet, there was fat accumulation around the organs of wild-type mice on a normal diet, but in contrast, the fat around the organs of RNF125-deficient mice was almost invisible (Figure A, top); similarly, after 6 months of a high-fat diet, it was found that long-term high-fat diet caused the abdomen of wild-type mice to be filled with a large amount of fat, and almost all organs were wrapped in thick fat, while the fat in the abdomen of RNF125-deficient mice was significantly less (Figure A, bottom). It was determined that after 6 months of a normal diet, the subcutaneous fat of RNF125 knockout mice was significantly reduced; long-term (6 months) high-fat diet caused the subcutaneous fat of wild-type mice to increase exponentially, and RNF125 deficiency significantly reduced the content of subcutaneous fat (Figure B); in addition, after 6 months of a normal diet or a high-fat diet, RNF125 deficiency also significantly reduced the epididymal visceral fat of mice (Figure C). H&E staining results showed that after 6 months of either a normal or high-fat diet, the adipocytes in the subcutaneous fat of RNF125-knockout mice were significantly smaller than those in wild-type mice (D). Similarly, after 6 months of either a normal or high-fat diet, the adipocytes in the epididymal visceral fat of RNF125-knockout mice were significantly smaller than those in wild-type mice (E). Overall, RNF125 deficiency significantly reduced adipose tissue accumulation and the size of lipid droplets in fat.

[0189] These results indicate that RNF125 deficiency can effectively ameliorate high-fat diet-induced fat accumulation. In the figure: *, P < 0.05; ***, P < 0.001. NCD, normal diet; HFD, high-fat diet.

[0190] Example 4

[0191] The experiment in this example demonstrates that RNF125 deficiency can effectively improve non-alcoholic fatty liver disease induced by a high-fat diet. The experiment includes the following experimental methods and experimental results:

[0192] Experimental method: The mice fed a high-fat diet for 6 months in Example 1 were dissected, and the livers were removed, weighed, and fixed with 4% paraformaldehyde and stained with H&E or Oil Red O to analyze the effects of RNF125 deficiency on the mouse livers.

[0193] Experimental results: As shown in Figure 4. Figure A shows the effect of RNF125 deficiency on the morphology, size, and color of the mouse liver after 6 months of normal diet or high-fat diet; Figure B shows that after 6 months of normal diet or high-fat diet, RNF125 deficiency significantly reduced the liver weight ratio of mice; The H&E staining results in Figure C show that after 6 months of normal diet or high-fat diet, RNF125 deficiency significantly alleviated the liver vacuolization caused by high-fat diet in mice; Figure D shows that after 6 months of normal diet or high-fat diet, RNF125 deficiency significantly reduced the triglyceride (TG) level in the mouse liver. Figure 1 shows that RNF125 deficiency significantly reduced liver lipid droplet levels after 6 months of either a normal or high-fat diet. Figure 2 shows that RNF125 deficiency significantly reduced liver cholesterol (TC) levels after 6 months of either a normal or high-fat diet. Figure 3 shows that RNF125 deficiency significantly reduced blood ALT levels after 6 months of either a normal or high-fat diet. Figure 4 shows that RNF125 deficiency significantly reduced blood AST levels after 6 months of either a normal or high-fat diet. These results indicate that RNF125 deficiency effectively ameliorates high-fat diet-induced nonalcoholic fatty liver disease. In the figures: *, P < 0.05; **, P < 0.01; ***, P < 0.001. NCD, normal diet; HFD, high-fat diet.

[0194] As shown in the results, compared with a normal diet, a long-term high-fat diet caused liver enlargement in mice, with the liver color changing from dark red to beige, becoming greasy and exhibiting severe steatosis. However, liver enlargement and steatosis were significantly alleviated in mice deficient in RNF125 (Panel A). Regardless of the diet, the liver-to-body weight ratio of RNF125 knockout mice was significantly lower than that of wild-type mice (Panel B). Hematoxylin and eosin staining of the livers revealed that a long-term high-fat diet caused liver blebbing and fatty lesions in mice, and that blebbing was significantly reduced after RNF125 knockout (Panel C). Analysis of triglycerides (Panel D) and cholesterol (Panel E) in the livers of mice, as well as Oil Red O staining (Panel F), showed that RNF125 knockout significantly reduced hepatic lipid accumulation caused by a high-fat diet. Furthermore, blood tests for ALT (Panel G) and AST (Panel H) showed that ALT (alanine aminotransferase) and AST (aspartate aminotransferase) levels were significantly reduced in RNF125 knockout mice. If the liver is damaged or destroyed, transaminases in liver cells enter the blood, causing elevated ALT and AST levels in the blood, signaling liver disease. The aforementioned experimental results indicate that RNF125 deficiency alleviates liver damage caused by a high-fat diet.

[0195] In summary, RNF125 knockout can effectively alleviate obesity-induced non-alcoholic fatty liver disease, and RNF125 may be an important target for the treatment of non-alcoholic fatty liver disease.

[0196] Example 5

[0197] The experiment in this example demonstrates that RNF125-specific siRNA can treat obesity induced by a high-fat diet. The experiment includes the following experimental methods and experimental results:

[0198] Experimental methods: To investigate the effects of RNF125 inhibition on the obese phenotype during a high-fat diet, we first designed and synthesized mouse siRNA sequences specifically targeting RNF125: siRNF125-117 (siRNA formed by annealing two single strands shown in SEQ ID No. 234 and SEQ ID No. 233), siRNF125-118 (siRNA formed by annealing two single strands shown in SEQ ID No. 236 and SEQ ID No. 235), and siRNF125-119 (siRNA formed by annealing two single strands shown in SEQ ID No. 238 and SEQ ID No. 237), and analyzed the knockdown efficiency of these sequences. Subsequently, C57BL / 6 mice were fed a high-fat diet. When their blood glucose, body weight, and TC levels were significantly higher than those in the normal diet group, the mice were randomly divided into two groups. The siRNF125 sequence and the control siCtrl sequence (siRNA formed by annealing two single strands shown in SEQ ID No. 240 and SEQ ID No. 239) were injected into the mice via the tail vein at a dose of 0.5 OD (1.25 nM) per mouse. The injections were performed once every 4 days, and the weight changes of the mice were monitored. The diet changes of the mice were also monitored using Columbus Instruments.

[0199] Experimental results: As shown in Figure 5. Figure A shows the knockdown efficiency of three RNA sequences (siRNF125-117, siRNF125-118, and siRNF125-119) that specifically target RNF125 in H22 cells analyzed by RT-qPCR. As can be seen from the figure, all three siRNAs can reduce the expression level of RNF125 to below 50%. Figure B shows the general process of the experiment, that is, the mice were first divided into a normal diet group and a high-fat diet group. When the blood glucose, body weight, TG, and TC levels of the high-fat diet group were significantly higher than those of the normal diet group, the high-fat diet mice were randomly divided into two groups and injected with the siRNF125 sequence and the control sequence siCtrl respectively. Figure C shows that the control group consumed 1.80±0.22g of high-fat diet per mouse per day. After treatment with the three RNF125-targeting siRNA sequences, each mouse consumed 2.5±0.16g, 2.8±0.39g, and 3.1±0.39g of high-fat diet per day, respectively. The daily food intake of mice in the treatment groups was significantly higher than that of the control group. Figure D shows that after two months of treatment with the three RNF125-targeting siRNA sequences, the body weight of mice was significantly lower than that of the control group, closer to that of mice on a normal diet. The body weight of mice on a normal diet was 31.7±1.0g, while that of the high-fat diet control group was 47.3±6.1g. The body weights of the high-fat diet-treated groups were 38.9±1.0g, 36.2±1.0g, and 35.6±1.5g, respectively. The body weight of mice treated with RNF125-specific siRNA was closer to that of mice on a normal diet. Figure E shows that two months after treatment with three RNF125-targeting RNAs, mice were significantly smaller than controls and closer to the size of mice on a normal diet. Specifically, two months after treatment with RNF125-specific siRNA sequences, mice became thinner. All siRNA-injected groups in the figure were on a high-fat diet; NCD, normal diet. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

[0200] These results demonstrate that targeting RNF125 with specific siRNA sequences significantly improved the obese phenotype in mice, allowing them to consume more high-fat food while maintaining body weight comparable to mice on a normal diet. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

[0201] Example 6

[0202] The experiments in this example demonstrate that three RNF125-specific siRNAs (siRNF125-117, siRNF125-118, and siRNF125-119) can improve glucose tolerance, insulin resistance, and hyperlipidemia induced by a high-fat diet. The experiments include the following experimental methods and results:

[0203] Experimental methods:

[0204] (1) Glucose tolerance test (GTT): After fasting for 6 hours, mice were intraperitoneally injected with glucose at a dose of 2 g / kg. Blood glucose levels in the tail vein of the mice were measured using a Roche blood glucose meter at 0, 15, 30, 45, 60, 90, and 120 min after injection. The glucose tolerance curve of the mice was drawn, and the area under the blood glucose curve was calculated using the AOC method.

[0205] (2) Insulin tolerance test (ITT): After fasting for 6 hours, mice were intraperitoneally injected with insulin at a dose of 0.75 U / kg. Blood glucose levels were measured using a Roche blood glucose meter at 0, 15, 30, 45, 60, 90, and 120 min after injection (20% glucose was prepared at the same time, and 100 μL of 20% glucose was injected intraperitoneally to rescue hypoglycemia in mice). The insulin tolerance curve of the mice was drawn, and the area under the blood glucose curve was calculated using the AOC method.

[0206] (3) After the mice were fasted overnight, serum was collected and blood lipids were analyzed using a biochemical analyzer.

[0207] Experimental results: As shown in Figure 6. The experiment conducted a glucose tolerance test on mice on a normal diet, control mice on a high-fat diet, and mice treated with the three RNF125-specific siRNAs. Figures A and B are the results of the glucose tolerance test (GTT); the experimental results showed that after RNF125 was inhibited by specific siRNA, the blood glucose level of mice was significantly lower than that of the control group when responding to high sugar stimulation. The AOC area of ​​the control group was 2410.2±500.3, and the AOC areas of the mice treated with the three siRNAs were 1168.4±531.5, 729.7±206.3, and 462.8±160.3, respectively. The AOC area of ​​the normal diet group was 580.9±120.4. This shows that RNF125 knockdown enhances the glucose tolerance of mice during a high-fat diet and can alleviate the effect of high sugar on the blood glucose of mice. Figures C and D show the results of a glucose tolerance test (ITT). The results show that the three RNF125-specific siRNA-treated groups were more sensitive to insulin. The area of ​​interest (AOC) in the control group was 293.1±172.9, while the AOC areas in the three siRNA-treated groups were 345.0±24.4, 382.0±66.8, and 479.1±64.7, respectively. The AOC area in the normal diet group was 388.1±44.9. This figure demonstrates that the siRNA-treated groups were more sensitive to insulin and effectively improved insulin resistance caused by a high-fat diet. Figure E shows that a high-fat diet increases blood triglyceride levels. After treatment with the three RNF125-specific siRNAs, blood triglyceride levels were significantly reduced, approaching those in the normal diet, demonstrating the therapeutic effect of the three RNF125-specific siRNAs on elevated triglycerides caused by a high-fat diet. Panel F shows that a high-fat diet elevates blood TC levels. Treatment with three RNF125-specific siRNAs significantly reduces blood TC levels, approaching those in a normal diet. This demonstrates the therapeutic efficacy of these three RNF125-specific siRNAs against high cholesterol caused by a high-fat diet. Figure: siRNF125-117, siRNF125-118, and siRNF125-119 compared with the control group, siCtrl. All groups were treated with a high-fat diet. Figure: *, P < 0.05; **, P < 0.01; ***, P < 0.001. NCD, normal chow diet.

[0208] In summary, specific inhibition of RNF125 can improve problems such as weakened glucose tolerance and hyperlipidemia caused by a high-fat diet.

[0209] Example 7

[0210] The experiments in this example demonstrate that three RNF125-specific siRNAs (siRNF125-117, siRNF125-118, and siRNF125-119) can reduce fat accumulation caused by a high-fat diet. The experiments include experimental methods and experimental results:

[0211] Experimental method: After the experiment in Example 6, mice were dissected, and their epididymal visceral fat (eWAT) and subcutaneous fat (iWAT) were taken and weighed using a 1 / 1000 analytical balance to calculate the body weight ratio; after weighing, a portion of the adipose tissue was fixed in 4% paraformaldehyde, and the changes in the adipose tissue were observed by H&E staining.

[0212] Experimental results: As shown in Figure 7 . As shown in Figure 7A , after treatment with three RNF125-specific siRNAs, the body weight ratio of visceral adipose tissue (eWAT) decreased from 9.0±1.2% in the high-fat diet control group to 6.8±0.8%, 4.4±1.2%, and 4.1±1.0% in the high-fat diet treatment group (Figure A); the body weight ratio of subcutaneous adipose tissue (iWAT) decreased from 7.9±1.6% in the high-fat diet control group to 4.1±0.9%, 1.6±0.3%, and 1.9±0.7% in the high-fat diet treatment group (Figure B). H&E staining results showed that after treatment with the three siRNAs, the cell volume of visceral and subcutaneous adipose tissue decreased, and inflammatory cell infiltration was alleviated (Figure C). In the figure, siRNF125-117, siRNF125-118, and siRNF125-119 were compared with the control group (siCtrl) (sequence-disrupted RNAi oligos), all in the high-fat diet group. **, P < 0.01; ***, P < 0.001. NCD, normal chow diet.

[0213] These experimental results show that using specific siRNA to inhibit RNF125 during a high-fat diet can significantly reduce visceral fat and subcutaneous fat accumulation, as well as inflammatory infiltration in mice.

[0214] Example 8

[0215] The experiments in this example demonstrate that three RNF125-specific siRNAs (siRNF125-117, siRNF125-118, and siRNF125-119) can improve hepatic fatty lesions induced by a high-fat diet. The experiments include the following experimental methods and results:

[0216] Experimental method: After the experiment in Example 6, mice were dissected, the mouse livers were removed, photographed, and weighed. Part of the liver samples were fixed in 4% paraformaldehyde, and the changes in liver tissue were observed by H&E staining and Oil Red O staining; 50 mg of liver tissue was taken, ground and crushed with a tissue homogenizer, and the TG content in the liver was analyzed using the Solebo triglyceride (TG) content detection kit; 50 mg of liver tissue was taken, ground and crushed with a tissue homogenizer, and the TC content in the liver was analyzed using the Solebo cholesterol (TC) content detection kit.

[0217] Experimental results: As shown in Figure 8. As shown in Figure 8A, a long-term high-fat diet causes fatty liver lesions, with enlarged livers, soft texture, light yellow or khaki color, and a greasy feel (siCtrl control group). The livers of the groups treated with the three RNF125-specific siRNA injections were similar in size to the livers of mice on a normal diet and were reddish-brown. As shown in Figure B, the liver weights of the different treatment groups are shown. The high-fat diet causes an increase in the liver weight of mice (siCtrl control group). After treatment with the three RNF125-specific siRNAs, the liver weights were significantly reduced, approaching the level of a normal diet. As shown in Figure C, the results of H&E staining and Oil Red O staining of the liver are shown. A long-term high-fat diet causes a large number of large lipid droplets to accumulate in the liver, and the liver becomes vacuolated (siCtrl control group). After treatment with the three RNF125-specific siRNAs (siRNF125-117, siRNF125-118, and siRNF125-119), the liver tissue morphology of the mice basically returned to normal, with only a small number of small lipid droplets present. As shown in Figure D, the analysis results of triglyceride (TG) content in the liver are as follows: the TG content in the liver of mice fed a normal diet was 30.6±6.0 mg / g tissue (NCD), and the TG content in the liver of mice fed a high-fat diet was 70.1±16.5 mg / g tissue (siCtrl control group). After treatment with three RNF125 siRNAs, the TG content in the liver of mice fed a high-fat diet was 37.4±6.3 mg / g, 29.8±5.5 mg / g, and 19.2±3.8 mg / g tissue, respectively. After treatment, the TG level in the liver of mice was significantly reduced and significantly lower than that in the control group siCtrl. Figure E shows the analysis of liver cholesterol (TC) levels. The TC content in the livers of mice fed a normal diet was 34.2±4.4μmol / g tissue (NCD), while the TC content in the livers of mice fed a high-fat diet was 56.0±15.3μmol / g tissue (siCtrl control group). After treatment with RNF125-specific siRNA, the TC content in the livers of mice fed a high-fat diet was 38.9±3.7μmol / g, 33.4±5.0μmol / g, and 32.3±4.3μmol / g tissue, respectively. This indicates that TC levels in the livers of treated mice were significantly reduced, similar to those in mice fed a normal diet. In summary, a high-fat diet significantly elevated TG and TC levels in the livers of mice, and RNF125-specific siRNA significantly reduced these levels. Therefore, inhibiting RNF125 may help improve hepatic fatty lesions caused by obesity. Figure: siRNF125-117, siRNF125-118, and siRNF125-119 were compared with the control group siCtrl, all in the high-fat diet group. *, P < 0.05; **, P < 0.01; ***, P < 0.001. NCD, normal chow diet.

[0218] In summary, RNF125 deficiency or targeted inhibition can effectively address obesity, hyperglycemia, hyperlipidemia, fat accumulation, and non-alcoholic fatty liver disease caused by a high-fat diet. RNF125 is an important target for the treatment of metabolic diseases such as obesity, diabetes, and non-alcoholic fatty liver disease.

[0219] Example 9

[0220] In order to develop human siRNA targeting RNF125, this example designs siRNA sequences targeting any position of the gene RNF125 (gene bank ID: 54941) to knock down or silence the expression of the gene. Each siRNA sequence includes at least one sense strand (21 bases) and one antisense strand (23 bases), and the 3' end of the antisense strand of each siRNA has 2 more bases than its sense strand. The siRNA sequences designed by the present invention are shown in Table 2:

[0221] Table 2:

[0222] Next, we conducted cell experiments to verify the silencing effect of the above-listed siRNA on the target gene RNF125. The experimental steps are as follows:

[0223] (1) Since the background expression level of RNF125 in Huh7 cells is low, it is suitable for dual reporter gene screening of siRNA. Therefore, Huh7 cells were selected for this experiment.

[0224] (2) Construction of a dual-reporter fluorescent reporter system plasmid: encoding the mRNA sequence of the human RNF125 gene, inserted into the psiCHECK TM -2 vector backbone, constructing human RNF125-psiCHECK TM -2 overexpression plasmid. psiCHECK TMThe -2 vector is a dual-luciferase reporter gene system capable of monitoring expression changes of target genes fused to the reporter gene. The Renilla luciferase gene (hRluc) expression cassette promoter is a T7 RNA polymerase promoter and serves as the primary reporter gene. The target gene is cloned into the multiple cloning region downstream of the hRluc translation stop codon and expressed in fusion with hRluc. Fluorescence intensity is used to monitor changes in mRNA expression after siRNA induction. The firefly luciferase gene (hluc+) expression cassette promoter is an HSV-TK promoter, independent of hRluc expression and unrelated to target gene expression. This allows monitoring of cell transfection efficiency and normalization of the fluorescence signal expressed by hRluc.

[0225] (3) RNF125-psiCHECK TM Huh7 cells were co-transfected with the -2 plasmid and different concentrations of siRNA (1nM, 10nM). After 24 hours, the cells were washed with PBS, and the fluorescence signal intensity values ​​of hRluc and hluc+ were detected on a multifunctional microplate reader. The hRluc detection wavelength was 480nm, and the hluc+ detection wavelength was 560nm.

[0226] The formula for calculating the relative expression fold is as follows:

[0227] Normalized value = hRluc fluorescence value (main reporter gene) / hluc + (reference gene)

[0228] Relative expression fold = normalized value of experimental group / normalized value of control group.

[0229] The relative expression levels of target mRNAs were calculated according to the above calculation method, and the target mRNA expression levels after 1 nM siRNA transfection were ranked from low to high as shown in Table 3. The lower the relative expression level of target mRNA, the higher the silencing efficiency of siRNA on the target gene.

[0230] Table 3:

[0231] Among them, the siRNAs ranked 1-48 in Table 3 can reduce the mRNA expression in Huh7 cells to ≤40%, indicating that they have a silencing efficiency of more than 60% for the target gene RNF125. The siRNAs ranked 49-84 can reduce the mRNA expression in Huh7 cells to between 41-60%, indicating that they have a silencing efficiency of 40-59% for the target gene RNF125. The siRNAs ranked 85-105 can reduce the mRNA expression in Huh7 cells to between 62-80%, indicating that they have a silencing efficiency of 20-38% for the target gene RNF125. The siRNAs ranked 106-116 have a silencing efficiency of only 7-19% for the target gene RNF125 in Huh7 cells. Although the siRNAs numbered 106-116 have low silencing efficiency on the target gene RNF125, they may be able to improve their knockdown effect on the target gene in animal models after nucleotide modification or delivery system coupling modification, and they also have the potential to become drugs.

[0232] Example 10

[0233] Fifteen siRNAs were selected from the siRNAs ranked 1-48 in Table 3 of Example 9 and transfected into human colon cancer cells HCT116 at final concentrations of 50 nM, 10 nM, 2 nM, 0.4 nM, 0.08 nM, and 0.016 nM. qPCR was used to detect the relative expression of target gene mRNA, and the IC50 values ​​of each siRNA were tested. The experimental results are shown in Table 4. The IC50 values ​​of the selected siRNAs for the target genes were all less than 10 nM, which is within the clinical range.

[0234] Table 4

[0235] Example 11

[0236] In this example, some human siRNA targeting RNF125 provided by the invention was modified and coupled with a GalNAc structure to obtain a deliverable complex. The complex was then injected into a human transgenic mouse model to perform a target gene silencing experiment. The experiment included the following parts:

[0237] 1. Construction of a human RNF125 overexpression mouse model

[0238] C57BL / 6J male mice (Beijing Biotechnology Co., Ltd.) aged 6-8 weeks and weighing approximately 25 g were acclimated to the environment for 1 week and then randomly assigned according to body weight for human transgenic mouse modeling.

[0239] A human RNF125 mRNA overexpression plasmid was constructed using the PiggyBac mammalian expression vector. The PiggyBac vector system consists of two vectors: a helper plasmid encoding the transposase; and a transposon plasmid containing two terminal repeats (TRs) and a transposed region between them, where the target gene expression cassette is cloned. The PiggyBac transposon vector and helper plasmid are transfected into mammalian cells. Due to the action of the transposase, the DNA sequence on the transposon is stably integrated into the host cell chromosome. The mRNA sequence encoding the human RNF125 gene was inserted into the multiple cloning site region of the vector backbone to construct and fusion-express SEAP luciferase (secreted human placental alkaline phosphatase). Mice were injected using the hydrodynamic high-pressure method (HDI) to construct a human transgenic mouse model. The transposon plasmid and the helper plasmid were mixed to an appropriate concentration and injected into the tail vein at a volume of 8-10% of the mouse body weight. The injection was completed within 6-8 seconds, so that each model mouse was simultaneously injected with 50 μg of the transposon plasmid and the helper plasmid. Blood was collected from the mouse tail vein on days 3, 7, 14, and 21 after injection, and the expression level of RNF125 in the human transgenic mice was detected by measuring the fluorescence expression level of SEAP in the serum.

[0240] 2. Target gene silencing effect of human siRNA in a human RNF125 overexpression mouse model

[0241] The modification method for human siRNA is as follows: among the 21 nucleotides of the sense strand, two thiolate modifications are made at the 5' end, positions 9, 10, and 11 are fluorinated, and the remaining nucleotides are methoxy-modified, and the 3' end is coupled with GalNAc ([Gal-6]s[Gal-6]s[Gal-6]); among the 23 nucleotides of the antisense strand, two thiolate modifications are made at the 5' end, positions 2, 5, 14, and 16 are fluorinated, two thiolate modifications are made at the 3' end, and the remaining nucleotides are methoxy-modified; see Table 5 for details:

[0242] Note: m indicates methoxy modification of the left nucleotide, f indicates fluorination modification of the left nucleotide, s indicates phosphorothioate linkage between the left and right nucleotides; ms indicates simultaneous modification of m and s, and mf indicates simultaneous modification of f and s.

[0243] Model mice with consistent SEAP expression levels were selected to detect the knockdown efficiency of different siRNAs. Each GalNAc-modified siRNA and siCtrl were administered subcutaneously behind the neck at 3 mpk. Five mice were administered each sequence. Blood was collected on the 14th day after administration, and the serum was separated. The SEAP fluorescence signal intensity was detected to determine the silencing effect of different siRNAs on the target gene mRNA. The target gene mRNA expression levels were ranked from low to high: see Table 6.

[0244] Table 6:

[0245] Example 12

[0246] In this example, three naked sequences, siRNF125-84, siRNF125-2, and siRNF125-73, were selected from Table 6 and subjected to four different nucleotide modifications (see Table 7). The silencing effects of different modifications on target genes in a human transgenic mouse model were compared.

[0247] Table 7:

[0248] In the above table, the lowercase letter g in modification method 4 indicates that the left side is ethylene glycol nucleic acid.

[0249] Wherein, modification method 1 is shown in Example 11; methods 2-4 are as follows:

[0250] Method 2: Among the 21 nucleotides of the sense strand, two 5'-end thiolation modifications are performed, and positions 9, 10, and 11 are fluorinated. The remaining nucleotides are alternatingly methoxylated and fluorinated. The 3'-end is thiolated with two modifications, and the 3'-end is coupled with GalNAc. The 23 nucleotides of the antisense strand are alternatingly methoxylated and fluorinated, with two 5'-end thiolation modifications and two 3'-end thiolation modifications.

[0251] Method 3: Among the 21 nucleotides of the sense strand, there are two thio-modified nucleotides at the 5' end, fluorinated nucleotides at positions 7, 9, 10, and 11, and methoxy-modified nucleotides at the remaining positions. There are two thio-modified nucleotides at the 3' end, and the 3' end is coupled with GalNAc. Among the 23 nucleotides of the antisense strand, there are fluorinated nucleotides at positions 2, 6, 8, 9, 15, and 17, and methoxy-modified nucleotides at the remaining positions. There are two thio-modified nucleotides at the 5' end and two thio-modified nucleotides at the 3' end.

[0252] Method 4: Among the 21 nucleotides of the sense chain, there are 2 thio-modified nucleotides at the 5' end, fluorinated modifications at positions 7, 9, 10, and 11, and methoxy modifications at the remaining positions. There are 2 thio-modified nucleotides at the 3' end, and the 3' end is coupled with GalNAc. Among the 23 nucleotides of the antisense chain, positions 2, 14, and 16 are fluorinated, position 7 is ethylene glycol nucleic acid, and the remaining positions are methoxy modified. There are 2 thio-modified nucleotides at the 5' end and 2 thio-modified nucleotides at the 3' end.

[0253] Using the human RNF125 overexpression mouse model constructed in Example 11, each modified siRNA and siCtrl were administered subcutaneously behind the neck at 3 mpk, and 5 mice were administered each sequence. Blood was collected on the 14th day after administration, serum was separated, and the SEAP fluorescence signal intensity was detected to determine the silencing effect of different siRNAs on the target gene mRNA. The target gene mRNA expression levels were ranked from low to high (see Table 8), where the lower the target gene mRNA expression level, the higher the silencing efficiency of the siRNA modification on human RNF125.

[0254] Table 8:

[0255] As shown in the table above, nucleotide modification of the naked siRNA sequence using different modification methods altered the silencing efficiency of the modified product against the human RNF125 gene in the mouse model. Modification method 4, in particular, significantly enhanced the silencing effect of the modified product on the target gene RNF125. This also indicates that once the siRNA sequence targeting RNF125 is determined, even different nucleic acid modification methods can effectively silence the target gene. Modification of the naked siRNA sequence can improve delivery efficacy, facilitate in vivo administration, and reduce the degradation of nucleic acid drugs in the body.

[0256] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. Application of RNF125 inhibitors in the preparation of drugs for preventing or treating obesity and related complications.

2. The use according to claim 1, characterized in that: The RNF125 inhibitor can produce any of the following changes in metabolic levels: (1) inhibiting obesity caused by a high-fat diet in mice; (2) inhibiting fat accumulation caused by a high-fat diet; (3) inhibiting glucose tolerance and insulin resistance caused by a high-fat diet and thus preventing hyperglycemia and hyperlipidemia; (4) inhibiting liver fatty degeneration caused by a high-fat diet.

3. The use according to claim 1, characterized in that: The RNF125 inhibitor is any protein molecule, nucleic acid molecule, small molecule compound, viral particle or any combination of two or more of the foregoing that can reduce the activity or expression level of the RNF125 protein; alternatively, the RNF125 inhibitor is any protein molecule, nucleic acid molecule, small molecule compound, viral particle, gene editing kit or any combination of two or more of the foregoing that can knock out, knock down or silence the RNF125 gene or protein.

4. siRNA targeting and inhibiting RNF125, characterized in that: Any one of the following siRNAs: siRNA formed by annealing two single strands shown in SEQ ID No.9-10; siRNA formed by annealing two single strands shown in SEQ ID NO.41-42; siRNA formed by annealing two single strands shown in SEQ ID NO.43-44; siRNA formed by annealing two single strands shown in SEQ ID NO.111-112; siRNA formed by annealing two single strands shown in SEQ ID NO.1-2; siRNA formed by annealing two single strands shown in SEQ ID NO.37-38; siRNA formed by annealing two single strands shown in SEQ ID NO.85-86; siRNA formed by annealing two single strands shown in SEQ ID NO.205-206; siRNA formed by annealing two single strands shown in SEQ ID NO.3-4; siRNA formed by annealing two single strands shown in SEQ ID NO.87-88; siRNA formed by annealing two single strands shown in SEQ ID NO.45-46; siRNA formed by annealing two single strands shown in SEQ ID NO.5-6; siRNA formed by annealing two single strands shown in SEQ ID NO.55-56; siRNA formed by annealing two single strands shown in SEQ ID NO.159-160; siRNA formed by annealing two single strands shown in SEQ ID NO.23-24; siRNA formed by annealing two single strands shown in SEQ ID NO.29-30; siRNA formed by annealing two single strands shown in SEQ ID NO.175-176; siRNA formed by annealing two single strands shown in SEQ ID NO.11-12; siRNA formed by annealing two single strands shown in SEQ ID NO.33-34; siRNA formed by annealing two single strands shown in SEQ ID NO.17-18; siRNA formed by annealing two single strands shown in SEQ ID NO.93-94; siRNA formed by annealing two single strands shown in SEQ ID NO.49-50; siRNA formed by annealing two single strands shown in SEQ ID NO.35-36; siRNA formed by annealing two single strands shown in SEQ ID NO.75-76; siRNA formed by annealing two single strands shown in SEQ ID NO.79-80; siRNA formed by annealing two single strands shown in SEQ ID NO.109-110; siRNA formed by annealing two single strands shown in SEQ ID NO.153-154; siRNA formed by annealing two single strands shown in SEQ ID NO.15-16; siRNA formed by annealing two single strands shown in SEQ ID NO.131-132; siRNA formed by annealing two single strands shown in SEQ ID NO.59-60; siRNA formed by annealing two single strands shown in SEQ ID NO.51-52; siRNA formed by annealing two single strands shown in SEQ ID NO.167-168; siRNA formed by annealing two single strands shown in SEQ ID NO.133-134; siRNA formed by annealing two single strands shown in SEQ ID NO.77-78; siRNA formed by annealing two single strands shown in SEQ ID NO.219-220; siRNA formed by annealing two single strands shown in SEQ ID NO.19-20; siRNA formed by annealing two single strands shown in SEQ ID NO.7-8; siRNA formed by annealing two single strands shown in SEQ ID NO.13-14; siRNA formed by annealing two single strands shown in SEQ ID NO.27-28; siRNA formed by annealing two single strands shown in SEQ ID NO.165-166; siRNA formed by annealing two single strands shown in SEQ ID NO.201-202; siRNA formed by annealing two single strands shown in SEQ ID NO.145-146; siRNA formed by annealing two single strands shown in SEQ ID NO.123-124; siRNA formed by annealing two single strands shown in SEQ ID NO.195-196; siRNA formed by annealing two single strands shown in SEQ ID NO.61-62; siRNA formed by annealing two single strands shown in SEQ ID NO.207-208; siRNA formed by annealing two single strands shown in SEQ ID NO.39-40; siRNA formed by annealing two single strands shown in SEQ ID NO.83-84; siRNA formed by annealing two single strands shown in SEQ ID NO.69-70; siRNA formed by annealing two single strands shown in SEQ ID NO.107-108; siRNA formed by annealing two single strands shown in SEQ ID NO.47-48; siRNA formed by annealing two single strands shown in SEQ ID NO.125-126; siRNA formed by annealing two single strands shown in SEQ ID NO.25-26; siRNA formed by annealing two single strands shown in SEQ ID NO.199-200; siRNA formed by annealing two single strands shown in SEQ ID NO.31-32; siRNA formed by annealing two single strands shown in SEQ ID NO.171-172; siRNA formed by annealing two single strands shown in SEQ ID NO.135-136; siRNA formed by annealing two single strands shown in SEQ ID NO.143-144; siRNA formed by annealing two single strands shown in SEQ ID NO.89-90; siRNA formed by annealing two single strands shown in SEQ ID NO.21-22; siRNA formed by annealing two single strands shown in SEQ ID NO.97-98; siRNA formed by annealing two single strands shown in SEQ ID NO.151-152; siRNA formed by annealing two single strands shown in SEQ ID NO.221-222; siRNA formed by annealing two single strands shown in SEQ ID NO.105-106; siRNA formed by annealing two single strands shown in SEQ ID NO.57-58; siRNA formed by annealing two single strands shown in SEQ ID NO.129-130; siRNA formed by annealing two single strands shown in SEQ ID NO.229-230; siRNA formed by annealing two single strands shown in SEQ ID NO.141-142; siRNA formed by annealing two single strands shown in SEQ ID NO.155-156; siRNA formed by annealing two single strands shown in SEQ ID NO.147-148; siRNA formed by annealing two single strands shown in SEQ ID NO.65-66; siRNA formed by annealing two single strands shown in SEQ ID NO.67-68; siRNA formed by annealing two single strands shown in SEQ ID NO.189-190; siRNA formed by annealing two single strands shown in SEQ ID NO.193-194; siRNA formed by annealing two single strands shown in SEQ ID NO.53-54; siRNA formed by annealing two single strands shown in SEQ ID NO.73-74; siRNA formed by annealing two single strands shown in SEQ ID NO.213-214; siRNA formed by annealing two single strands shown in SEQ ID NO.181-182; siRNA formed by annealing two single strands shown in SEQ ID NO.101-102; siRNA formed by annealing two single strands shown in SEQ ID NO.139-140; siRNA formed by annealing two single strands shown in SEQ ID NO.91-92; siRNA formed by annealing two single strands shown in SEQ ID NO.197-198; siRNA formed by annealing two single strands shown in SEQ ID NO.99-100; siRNA formed by annealing two single strands shown in SEQ ID NO.137-138; siRNA formed by annealing two single strands shown in SEQ ID NO.71-72; siRNA formed by annealing two single strands shown in SEQ ID NO.127-128; siRNA formed by annealing two single strands shown in SEQ ID NO.121-122; siRNA formed by annealing two single strands shown in SEQ ID NO.115-116; siRNA formed by annealing two single strands shown in SEQ ID NO.215-216; siRNA formed by annealing two single strands shown in SEQ ID NO.173-174; siRNA formed by annealing two single strands shown in SEQ ID NO.103-104; siRNA formed by annealing two single strands shown in SEQ ID NO.177-178; siRNA formed by annealing two single strands shown in SEQ ID NO.95-96; siRNA formed by annealing two single strands shown in SEQ ID NO.231-232; siRNA formed by annealing two single strands shown in SEQ ID NO.209-210; siRNA formed by annealing two single strands shown in SEQ ID NO.119-120; siRNA formed by annealing two single strands shown in SEQ ID NO.149-150; siRNA formed by annealing two single strands shown in SEQ ID NO.81-82; siRNA formed by annealing two single strands shown in SEQ ID NO.203-204; siRNA formed by annealing two single strands shown in SEQ ID NO.157-158; siRNA formed by annealing two single strands shown in SEQ ID NO.163-164; siRNA formed by annealing two single strands shown in SEQ ID NO.217-218; siRNA formed by annealing two single strands shown in SEQ ID NO.191-192; siRNA formed by annealing two single strands shown in SEQ ID NO.187-188; siRNA formed by annealing two single strands shown in SEQ ID NO.169-170; siRNA formed by annealing two single strands shown in SEQ ID NO.113-114; siRNA formed by annealing two single strands shown in SEQ ID NO.117-118; siRNA formed by annealing two single strands shown in SEQ ID NO.63-64; siRNA formed by annealing two single strands shown in SEQ ID NO.161-162; siRNA formed by annealing two single strands shown in SEQ ID NO.223-224; siRNA formed by annealing two single strands shown in SEQ ID NO.185-186; siRNA formed by annealing two single strands shown in SEQ ID NO.211-212; siRNA formed by annealing two single strands shown in SEQ ID NO.215-216; siRNA formed by annealing two single strands shown in SEQ ID NO.227-228; siRNA formed by annealing two single strands shown in SEQ ID NO.183-184; The siRNA is formed by annealing two single strands shown in SEQ ID NO. 179-180.

5. A drug for preventing or treating obesity and related complications, wherein the active ingredient of the drug is an RNF125 inhibitor, characterized in that: The RNF125 inhibitor is any protein molecule, nucleic acid molecule, small molecule compound, viral particle or any combination of two or more of the foregoing that can reduce the activity or expression level of the RNF125 protein; alternatively, the RNF125 inhibitor is any protein molecule, nucleic acid molecule, small molecule compound, viral particle, gene editing kit or any combination of two or more of the foregoing that can knock out, knock down or silence the RNF125 gene or protein.

6. The drug according to claim 5, characterized in that The percentage of the RNF125 inhibitor in the medicine is 1-99%.

7. The drug according to claim 5 or 6, characterized in that The RNF125 inhibitor is any one of the following siRNAs: siRNA formed by annealing two single strands shown in SEQ ID No.9-10; siRNA formed by annealing two single strands shown in SEQ ID NO.41-42; siRNA formed by annealing two single strands shown in SEQ ID NO.43-44; siRNA formed by annealing two single strands shown in SEQ ID NO.111-112; siRNA formed by annealing two single strands shown in SEQ ID NO.1-2; siRNA formed by annealing two single strands shown in SEQ ID NO.37-38; siRNA formed by annealing two single strands shown in SEQ ID NO.85-86; siRNA formed by annealing two single strands shown in SEQ ID NO.205-206; siRNA formed by annealing two single strands shown in SEQ ID NO.3-4; siRNA formed by annealing two single strands shown in SEQ ID NO.87-88; siRNA formed by annealing two single strands shown in SEQ ID NO.45-46; siRNA formed by annealing two single strands shown in SEQ ID NO.5-6; siRNA formed by annealing two single strands shown in SEQ ID NO.55-56; siRNA formed by annealing two single strands shown in SEQ ID NO.159-160; siRNA formed by annealing two single strands shown in SEQ ID NO.23-24; siRNA formed by annealing two single strands shown in SEQ ID NO.29-30; siRNA formed by annealing two single strands shown in SEQ ID NO.175-176; siRNA formed by annealing two single strands shown in SEQ ID NO.11-12; siRNA formed by annealing two single strands shown in SEQ ID NO.33-34; siRNA formed by annealing two single strands shown in SEQ ID NO.17-18; siRNA formed by annealing two single strands shown in SEQ ID NO.93-94; siRNA formed by annealing two single strands shown in SEQ ID NO.49-50; siRNA formed by annealing two single strands shown in SEQ ID NO.35-36; siRNA formed by annealing two single strands shown in SEQ ID NO.75-76; siRNA formed by annealing two single strands shown in SEQ ID NO.79-80; siRNA formed by annealing two single strands shown in SEQ ID NO.109-110; siRNA formed by annealing two single strands shown in SEQ ID NO.153-154; siRNA formed by annealing two single strands shown in SEQ ID NO.15-16; siRNA formed by annealing two single strands shown in SEQ ID NO.131-132; siRNA formed by annealing two single strands shown in SEQ ID NO.59-60; siRNA formed by annealing two single strands shown in SEQ ID NO.51-52; siRNA formed by annealing two single strands shown in SEQ ID NO.167-168; siRNA formed by annealing two single strands shown in SEQ ID NO.133-134; siRNA formed by annealing two single strands shown in SEQ ID NO.77-78; siRNA formed by annealing two single strands shown in SEQ ID NO.219-220; siRNA formed by annealing two single strands shown in SEQ ID NO.19-20; siRNA formed by annealing two single strands shown in SEQ ID NO.7-8; siRNA formed by annealing two single strands shown in SEQ ID NO.13-14; siRNA formed by annealing two single strands shown in SEQ ID NO.27-28; siRNA formed by annealing two single strands shown in SEQ ID NO.165-166; siRNA formed by annealing two single strands shown in SEQ ID NO.201-202; siRNA formed by annealing two single strands shown in SEQ ID NO.145-146; siRNA formed by annealing two single strands shown in SEQ ID NO.123-124; siRNA formed by annealing two single strands shown in SEQ ID NO.195-196; siRNA formed by annealing two single strands shown in SEQ ID NO.61-62; siRNA formed by annealing two single strands shown in SEQ ID NO.207-208; siRNA formed by annealing two single strands shown in SEQ ID NO.39-40; siRNA formed by annealing two single strands shown in SEQ ID NO.83-84; siRNA formed by annealing two single strands shown in SEQ ID NO.69-70; siRNA formed by annealing two single strands shown in SEQ ID NO.107-108; siRNA formed by annealing two single strands shown in SEQ ID NO.47-48; siRNA formed by annealing two single strands shown in SEQ ID NO.125-126; siRNA formed by annealing two single strands shown in SEQ ID NO.25-26; siRNA formed by annealing two single strands shown in SEQ ID NO.199-200; siRNA formed by annealing two single strands shown in SEQ ID NO.31-32; siRNA formed by annealing two single strands shown in SEQ ID NO.171-172; siRNA formed by annealing two single strands shown in SEQ ID NO.135-136; siRNA formed by annealing two single strands shown in SEQ ID NO.143-144; siRNA formed by annealing two single strands shown in SEQ ID NO.89-90; siRNA formed by annealing two single strands shown in SEQ ID NO.21-22; siRNA formed by annealing two single strands shown in SEQ ID NO.97-98; siRNA formed by annealing two single strands shown in SEQ ID NO.151-152; siRNA formed by annealing two single strands shown in SEQ ID NO.221-222; siRNA formed by annealing two single strands shown in SEQ ID NO.105-106; siRNA formed by annealing two single strands shown in SEQ ID NO.57-58; siRNA formed by annealing two single strands shown in SEQ ID NO.129-130; siRNA formed by annealing two single strands shown in SEQ ID NO.229-230; siRNA formed by annealing two single strands shown in SEQ ID NO.141-142; siRNA formed by annealing two single strands shown in SEQ ID NO.155-156; siRNA formed by annealing two single strands shown in SEQ ID NO.147-148; siRNA formed by annealing two single strands shown in SEQ ID NO.65-66; siRNA formed by annealing two single strands shown in SEQ ID NO.67-68; siRNA formed by annealing two single strands shown in SEQ ID NO.189-190; siRNA formed by annealing two single strands shown in SEQ ID NO.193-194; siRNA formed by annealing two single strands shown in SEQ ID NO.53-54; siRNA formed by annealing two single strands shown in SEQ ID NO.73-74; siRNA formed by annealing two single strands shown in SEQ ID NO.213-214; siRNA formed by annealing two single strands shown in SEQ ID NO.181-182; siRNA formed by annealing two single strands shown in SEQ ID NO.101-102; siRNA formed by annealing two single strands shown in SEQ ID NO.139-140; siRNA formed by annealing two single strands shown in SEQ ID NO.91-92; siRNA formed by annealing two single strands shown in SEQ ID NO.197-198; siRNA formed by annealing two single strands shown in SEQ ID NO.99-100; siRNA formed by annealing two single strands shown in SEQ ID NO.137-138; siRNA formed by annealing two single strands shown in SEQ ID NO.71-72; siRNA formed by annealing two single strands shown in SEQ ID NO.127-128; siRNA formed by annealing two single strands shown in SEQ ID NO.121-122; siRNA formed by annealing two single strands shown in SEQ ID NO.115-116; siRNA formed by annealing two single strands shown in SEQ ID NO.215-216; siRNA formed by annealing two single strands shown in SEQ ID NO.173-174; siRNA formed by annealing two single strands shown in SEQ ID NO.103-104; siRNA formed by annealing two single strands shown in SEQ ID NO.177-178; siRNA formed by annealing two single strands shown in SEQ ID NO.95-96; siRNA formed by annealing two single strands shown in SEQ ID NO.231-232; siRNA formed by annealing two single strands shown in SEQ ID NO.209-210; siRNA formed by annealing two single strands shown in SEQ ID NO.119-120; siRNA formed by annealing two single strands shown in SEQ ID NO.149-150; siRNA formed by annealing two single strands shown in SEQ ID NO.81-82; siRNA formed by annealing two single strands shown in SEQ ID NO.203-204; siRNA formed by annealing two single strands shown in SEQ ID NO.157-158; siRNA formed by annealing two single strands shown in SEQ ID NO.163-164; siRNA formed by annealing two single strands shown in SEQ ID NO.217-218; siRNA formed by annealing two single strands shown in SEQ ID NO.191-192; siRNA formed by annealing two single strands shown in SEQ ID NO.187-188; siRNA formed by annealing two single strands shown in SEQ ID NO.169-170; siRNA formed by annealing two single strands shown in SEQ ID NO.113-114; siRNA formed by annealing two single strands shown in SEQ ID NO.117-118; siRNA formed by annealing two single strands shown in SEQ ID NO.63-64; siRNA formed by annealing two single strands shown in SEQ ID NO.161-162; siRNA formed by annealing two single strands shown in SEQ ID NO.223-224; siRNA formed by annealing two single strands shown in SEQ ID NO.185-186; siRNA formed by annealing two single strands shown in SEQ ID NO.211-212; siRNA formed by annealing two single strands shown in SEQ ID NO.215-216; siRNA formed by annealing two single strands shown in SEQ ID NO.227-228; siRNA formed by annealing two single strands shown in SEQ ID NO.183-184; The siRNA is formed by annealing two single strands shown in SEQ ID NO. 179-180.

8. The drug according to claim 7, characterized in that The RNF125 inhibitor is a modified product obtained by performing nucleotide modification on the antisense strand and the sense strand of any of the siRNAs; the nucleotide modification includes one or a combination of methoxy modification, fluoro modification, GalNAc coupling modification, thio modification, and ethylene glycol.

9. The drug according to claim 8, characterized in that The modification method is any of the following: Method 1: Among the 21 nucleotides of the sense chain, there are 2 thio-modified nucleotides at the 5' end, fluorinated nucleotides at the 9th, 10th and 11th positions, methoxy-modified nucleotides at the remaining positions, and GalNAc coupling at the 3' end; among the 23 nucleotides of the antisense chain, there are 2 thio-modified nucleotides at the 5' end, fluorinated nucleotides at the 2nd, 5th, 14th and 16th positions, 2 thio-modified nucleotides at the 3' end, and methoxy-modified nucleotides at the remaining positions; Method 2: Among the 21 nucleotides of the sense chain, 2 thio-modified nucleotides are modified at the 5' end, and the 9th, 10th and 11th positions are modified with fluorine respectively. The nucleotides at the remaining positions are modified with methoxy and fluorine alternately in sequence, and 2 thio-modified nucleotides are modified at the 3' end, and the 3' end is coupled with GalNAc; the 23 nucleotides of the antisense chain are modified with methoxy and fluorine alternately in sequence, and 2 thio-modified nucleotides are modified at the 5' end and 2 thio-modified nucleotides at the 3' end; Method 3: Among the 21 nucleotides of the sense chain, there are 2 thio-modified nucleotides at the 5' end, fluorinated nucleotides at the 7th, 9th, 10th, and 11th positions, methoxy-modified nucleotides at the other positions, 2 thio-modified nucleotides at the 3' end, and GalNAc coupled at the 3' end; among the 23 nucleotides of the antisense chain, the 2nd, 6th, 8th, 9th, 15th, and 17th positions are fluorinated, and methoxy-modified nucleotides at the other positions, 2 thio-modified nucleotides at the 5' end, and 2 thio-modified nucleotides at the 3' end; Method 4: Among the 21 nucleotides of the sense chain, there are 2 thio-modified nucleotides at the 5' end, fluorinated modifications at the 7th, 9th, 10th and 11th positions, methoxy modifications at the remaining positions, 2 thio-modified nucleotides at the 3' end, and GalNAc coupled at the 3' end; of the 23 nucleotides of the antisense chain, the 2nd, 14th and 16th positions are fluorinated, the 7th position is ethylene glycol nucleic acid, methoxy modifications at the remaining positions, 2 thio-modified nucleotides at the 5' end, and 2 thio-modified nucleotides at the 3' end.

10. Application of RNF125 gene or protein as a target in constructing a model for treating obesity.

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