Sirna for inhibiting LOC157273 gene expression in liver cells, composition and use

Abstract

To overcome the problem in the prior art that a siRNA applied to a human liver LOC157273 gene cannot be directly applied to a primate T2DM experiment in a clinical trial, a siRNA for inhibiting LOC157273 gene expression in liver cells is provided. The siRNA comprises a sense strand and an antisense strand corresponding to the sense strand, wherein the sense strand is selected from "sequence 1", and the antisense strand is selected from "sequence 2", or a siRNA having 80% or more homology with sequences of the sense strand and the antisense strand. In addition, also provided are a composition and a use of the siRNA for inhibiting LOC157273 gene expression in liver cells. The provided siRNA for inhibiting LOCI57273 gene expression in liver cells can silence LOC157273 genes in both human liver cells and primate liver cells, solving the problem in the prior art that a siRNA applied to a human liver LOC157273 gene cannot be directly applied to a primate T2DM experiment in a clinical trial, verifying the efficacy and pharmacokinetic effects of therapeutic drugs, thereby promoting the development of new drugs for the treatment of T2DM.

Description

siRNA, composition and application for inhibiting LOC157273 gene expression in liver cells Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an siRNA for inhibiting LOC157273 gene expression in liver cells, a composition and an application thereof. Background Art

[0002] Type 2 Diabetes Mellitus (T2DM) is a complex multi-gene inherited disease caused by the combined effects of genetic and environmental factors. Currently, due to insufficient understanding of its etiology and pathogenesis, existing intervention methods are still mainly focused on controlling blood sugar levels. Oral hypoglycemic drugs and insulin therapy are generally used, especially insulin, which is a common choice for the late-stage treatment of T2DM. Glycogen synthesis is an important way for insulin to lower blood sugar levels. When blood sugar levels are high due to eating, insulin can activate AKT, which then phosphorylates and inactivates glycogen synthase kinase (GSK3), thereby reducing the phosphorylation level of glycogen synthase (GS) and increasing its activity, promoting glycogen production, and thereby inhibiting glucose synthesis. Insulin can have a good blood sugar control effect in the initial stage, but in the later stages, the body gradually develops resistance to insulin, the efficacy decreases, and it cannot achieve a good therapeutic effect.

[0003] Growing evidence indicates that lncRNA plays an important role in chronic non-infectious diseases. Related research reports have shown that LOC157273 (8p23.1) is involved in regulating blood sugar and blood cholesterol. In patients with type 2 diabetes, the protein PPP1R3B acts as a target subunit of protein phosphatase, and its closest variant, rs4841132, is only 175 kb away. When LOC157273 is silenced, it can negatively regulate the protein PPP1R3B, thereby regulating (inhibiting) glucose synthesis. This target can be used to synthesize siRNA for RNA interference treatment of T2DM and develop new therapeutic drugs for T2DM.

[0004] The existing technology has siRNA applied to human liver LOC157273, but it is not suitable for large-animal experiments before the clinical application of new T2DM drugs. Large-animal experiments are the main experimental subjects for preclinical drug evaluation. Primates, as important research subjects in large-animal experiments, have partial but not complete homology with human genetic material. Therefore, siRNA applied to human liver LOC157273 cannot be directly used in primate experiments in clinical trials, resulting in difficulty in verifying its efficacy and pharmacokinetic effects, thereby affecting the development of new drugs for the treatment of type 2 diabetes.

[0005] Summary of the Invention

[0006] Aiming at the problem that the siRNA applied to human liver LOC157273 gene in the existing technology cannot be directly applied to primate T2DM experiments in clinical trials, an siRNA is provided to inhibit the expression of LOC157273 gene in liver cells.

[0007] The technical solutions adopted by the present invention to solve the above technical problems are as follows:

[0008] The present invention provides an siRNA for inhibiting the expression of the LOC157273 gene in liver cells, comprising a sense chain and an antisense chain corresponding to the sense chain, wherein the sense chain is selected from "sequence 1" and the antisense chain is selected from "sequence 2", comprising a sense chain and an antisense chain corresponding to the sense chain, wherein the sense chain is selected from "sequence 1" and the antisense chain is selected from "sequence 2", or an siRNA having more than 80% homology with the sequences of the sense chain and the antisense chain.

[0009] Optionally, two deoxyribonucleotides dT are added to the 3' end of the sense strand of the siRNA and / or the 3' end of the antisense strand of the siRNA to form a single-stranded hanging structure.

[0010] In one aspect, the present invention provides a composition for inhibiting the expression of LOC157273 gene in liver cells, comprising the siRNA, or an expression system capable of expressing the siRNA.

[0011] Optionally, the composition further comprises a carrier for delivering the siRNA.

[0012] Optionally, the carrier comprises a liposome.

[0013] Optionally, the composition further comprises a pharmaceutical active ingredient having a preventive or therapeutic effect on diabetes.

[0014] Optionally, the active pharmaceutical ingredient is encapsulated in the liposome carrier.

[0015] Optionally, the composition further comprises a pharmaceutically acceptable excipient.

[0016] Optionally, the auxiliary materials include one or more of a buffer, an emulsifier, a suspending agent, a stabilizer, a preservative, an excipient, a coagulant, a dispersant and a defoaming agent.

[0017] In another aspect, the present invention provides a use of a composition in preparing a drug for treating diabetes.

[0018] Optionally, the diabetes comprises type II diabetes.

[0019] The present application provides siRNAs for inhibiting the expression of the LOC157273 gene in liver cells. The sense and antisense strands comprise nucleotide sequences targeting the LOC157273 gene in human and primate liver cells. The siRNAs are designed for non-completely homologous portions of the human and animal LOC157273 genes, enabling the siRNAs to simultaneously silence the LOC157273 gene in both human and primate liver cells. This addresses the problem in the prior art that siRNAs applied to the human liver LOC157273 gene cannot be directly applied to primate T2DM experiments in clinical trials, and makes it more convenient to target the same gene in different species in T2DM new drug experiments. In addition, the siRNAs provided herein silence the LOC157273 gene, which is located upstream of the protein PP1R3B and is not expressed in a long non-coding gene. Compared with directly overexpressing the target gene, this siRNA has fewer byproducts and side effects. The present application provides a basis for the treatment of T2DM and the clinical application of new drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIG1 is a graph showing the effect of siRNA transfection in HepG2 cells for 24 hours provided by the present invention;

[0021] FIG2 is a graph showing the effect of siRNA provided by the present invention on HepG2 cells transfected for 48 hours;

[0022] FIG3 is a graph showing the effect of siRNA provided by the present invention on the expression of PPP1R3B protein in primary monkey hepatocytes after 96 hours of transfection;

[0023] FIG4 is a graph showing the effect of siRNA provided by the present invention on LOC157273 gene silencing in primary monkey hepatocytes after 96 hours of transfection;

[0024] FIG5 is a qPCR detection diagram of the silencing effect of the siRNA provided by the present invention on the LOC157273 gene in HepG2 cells transfected; (M Marker, B Blank, co human-monkey shared siRNA)

[0025] FIG6 is a qPCR detection diagram of the expression of PPP1R3B protein by transfection of siRNA provided by the present invention in primary monkey hepatocytes; (M Marker, B Blank, co human-monkey shared siRNA) DETAILED DESCRIPTION

[0026] In order to make the technical problems, technical solutions and beneficial effects solved by the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0027] The present invention provides an siRNA for inhibiting the expression of the LOC157273 gene in liver cells, comprising a sense chain and an antisense chain corresponding to the sense chain, wherein the sense chain is selected from "sequence 1" and the antisense chain is selected from "sequence 2", the sense chain is selected from "sequence 1" and the antisense chain is selected from "sequence 2", or an siRNA having more than 80% homology with the sequences of the sense chain and the antisense chain.

[0028] Specifically, the siRNA provided in the present application inhibits the expression of the LOC157273 gene in liver cells, and the sense chain and antisense chain include nucleotide sequences targeting the LOC157273 gene in human liver cells and primate liver cells. Compared with the previously disclosed siRNA that only targets the expression of the LOC157273 gene in human liver cells, the siRNA provided in the present application is designed to target the non-homologous parts of the LOC157273 gene in humans and primates (cynomolgus macaques), so that the siRNA can simultaneously silence the LOC157273 gene in human liver cells and primary hepatocytes of cynomolgus macaques, thereby solving the problem that the siRNA applied to the human liver LOC157273 gene in the prior art cannot be directly applied to primate T2DM experiments in clinical trials, thereby verifying the efficacy and pharmacokinetic effects of the therapeutic drug. That is, the siRNA provided in the present application provides a basis for the treatment of T2DM and the clinical application of new drugs, thereby promoting the development of new drugs for the treatment of T2DM.

[0029] In addition, the siRNA provided in the present application silences the LOC157273 gene, which is located upstream of the protein PP1R3B and is not expressed in a long non-coding gene. Compared with directly overexpressing the target gene, it has fewer byproducts and side effects.

[0030] In this application, the primate liver cells selected are primary crab-eating macaque liver cells, mainly because these cells have high cell viability, strong adhesion ability, and obvious polarization morphology after recovery in various experimental tests, and are therefore widely used in drug research and development.

[0031] In some embodiments, two deoxyribonucleotides dT are added to the 3' end of the sense strand of the siRNA and / or the 3' end of the antisense strand of the siRNA to form a single-stranded hanging structure.

[0032] Specifically, the siRNA sequence is chemically synthesized, and two overhanging dTdTs are added to the 3' end to enhance the stability of the siRNA.

[0033] Another embodiment of the present invention provides a siRNA targeting method for inhibiting LOC157273 gene expression in liver cells, comprising the following steps:

[0034] Design and synthesize siRNA;

[0035] Liposome-encapsulated siRNA;

[0036] siRNA transfection into HepG2 and primary monkey hepatocytes;

[0037] Target gene targeting detection.

[0038] Specifically, the siRNA synthesis includes: obtaining the human liver LOC157273 gene sequence from the Genebank database, designing using siDirect siRNA software, selecting siRNAs that simultaneously meet the three algorithms for effective siRNA selection by Ui Tei, Reynolds, Amarzguioui et al. as candidates, selecting siRNAs with a GC content of 50%, and aligning the siRNAs with the human LOC157273 sequence included in the Genebank database using the NCBI BLAST function to design the following siRNAs, wherein the sense strand sequence is identical to the target sequence designed on the target gene (U replaces T), and the antisense strand sequence is completely complementary to the target sequence (U replaces T):

[0039] The nucleotide sequence of the sense strand is:

[0040] 5'AUCAAUGCCCGUAACACCC3'

[0041] The nucleotide sequence of the antisense strand is:

[0042] 3'UAGUUACGGGCAUUGUGGG5'

[0043] The siRNA sequence was chemically synthesized and two overhanging dTdT residues were added to the 3' end to enhance the stability of the siRNA;

[0044] The liposome LNP encapsulation of siRNA specifically comprises the following steps: dissolving DLin-MC3-DMA, DSPC, cholesterol, and DMG-PEG2000 in an organic phase at a molar ratio of 50:10:38.5:1.5, dissolving siRNA in sterile water, and injecting the two phase solutions into two inlet channels of a microfluidic preparation system respectively, and completing the encapsulation of nucleic acid lipid nanoparticles through rapid mixing of the two phases;

[0045] The siRNA-encapsulated LNPs were transfected into HepG2 and cynomolgus monkey primary hepatocytes, respectively. Six-well plates were taken and cultured at a rate of 200,000 cells before transfection. The cell fusion reached 70% on the second day. The culture medium was replaced before transfection. Twelve sterile centrifuge tubes were prepared, and 250 ul of DMEM culture medium without serum and antibiotics were added to each of 6 tubes. 10 ul of the synthesized nanoliposome LNP mixture was added to 6 tubes. For the remaining 6 tubes, 100 pmol of the corresponding siRNA was added to each of 2 tubes, including siRNA No. 1, siRNA No. 2, Positive, and Negative. The mixture was gently mixed and allowed to stand for 5 minutes. The culture medium containing siRNA was mixed with the culture medium containing LNPs, allowed to stand for 20 minutes, and then 250 ul of the culture medium per well was evenly added to the culture dish. The cultures were cultured for 24 hours and 48 hours, respectively, and the culture medium was replaced 6 hours after transfection.

[0046] RNA was extracted from the cells cultured after transfection using the TRIzol method at 24 and 48 h, respectively, and reagents were added according to the reagents provided by the kit provided by Guangzhou Ruibo Company, and cDNA was obtained by PCR under a temperature of 42°C for 15 min and 85°C for 5 s.

[0047] Synthesized cDNA was used for qPCR. The reverse-transcribed cDNA was diluted half and then amplified using TransStart Top Green qPCR SuperMix. The reaction conditions were: 20 μl reaction system, 1 μl cDNA template was added to each system.

[0048] Then, according to the instructions of the reverse transcription kit provided by Guangzhou Ruibo, reagents were added to set up a two-step qPCR, and 40 cycles were performed at 94°C for 30 seconds, 94°C for 5 seconds, and 60°C for 30 seconds.

[0049] Using ACTIN as an internal control, the following are the ACTIN and LOC157273 primer sequences used in the qPCR reaction:

[0050] Another embodiment of the present invention provides a composition for inhibiting the expression of LOC157273 gene in liver cells, comprising the siRNA.

[0051] The components in the composition can specifically inhibit the replication and transcription of the mRNA of the LOC157273 gene in liver cells, thereby exerting a therapeutic effect.

[0052] In some embodiments, the composition further comprises a vector for delivering the siRNA, or an expression system for the siRNA.

[0053] Specifically, drug delivery systems are engineering technologies used for the targeted delivery and / or controlled release of therapeutic agents. Currently, vector delivery technology is combined with RNAi technology to form a new gene drug delivery system that can effectively deliver siRNA into the body. Nanosized particles can aggregate around diseased cells by enhancing penetration and retention effects. siRNA delivery vectors typically use nanoparticles, nanomicelles, nanohydrogels, and polymers as drug or gene carriers, and can also bond targeting molecules such as antibodies, nucleic acid aptamers, targeting peptides, or folic acid to the surface, allowing them to enter target cells through interaction with the cell surface.

[0054] In some embodiments, the carrier comprises a liposome.

[0055] Specifically, the siRNA can be administered to cells by various methods known to those skilled in the art, including encapsulation in liposomes, by iontophoresis, or by incorporation into other carriers, such as biodegradable polymers, hydrogels, cyclodextrin poly (lactic-co-glycolic acid) (PLGA) and PLCA microspheres, biodegradable nanocapsules and bioadhesive microspheres, or by protein-like carriers; in one embodiment of the present invention, the liposomes include DLin-MC3-DMA, DSPC, cholesterol and DMG-PEG2000, and the molar ratio of DLin-MC3-DMA, DSPC, cholesterol and DMG-PEG2000 is 50:10:38.5:1.5;

[0056] As an effective and ionizable cationic liposome, DLin-MC3-DMA has a unique pH-dependent charge-variable property: it is positively charged under acidic conditions and electrically neutral under physiological pH conditions. Furthermore, the positive charge of DLin-MC3-DMA cationic liposomes can increase the escape of particles from lysosomes in the body, improving transfection efficiency while making them less susceptible to phagocytosis by macrophages. Utilizing these properties, DLin-MC3-DMA can form a liposome / siRNA complex with negatively charged siRNA through electrostatic interaction, resulting in high transfection efficiency and low toxicity.

[0057] The cholesterol in the liposome regulates the fluidity of the phospholipid bilayer membrane, reduces membrane permeability, and reduces drug leakage. At the same time, it can maintain a certain flexibility of the lipid membrane, enhance the ability of the liposome vesicle to resist changes in external conditions, and has a certain protective effect on the oxidation of phospholipids.

[0058] The DSPC often acts on liposome microbubble contrast agents and drug lipid microspheres. When DSPC acts on membrane materials, it is mostly used in liposome drugs and has good stability.

[0059] The DMG-PEG2000 is used to prepare liposomes for siRNA transfection, which can improve transfection efficiency. In addition, DMG-PEG2000 can also be used to prepare lipid nanoparticles for the in vivo delivery of oral plasmid DNA. DMG-PEG 2000 can improve the mucus permeability and delivery efficiency of nanoparticles.

[0060] In some embodiments, the composition further comprises a pharmaceutically active ingredient having a preventive or therapeutic effect on diabetes.

[0061] Specifically, the active ingredients of the drug used to prevent or treat diabetes include one or more insulins (e.g., Humulin, Novolin, NovoLog, FlexPen, Fiasp, Apidra, Humalog, Humulin N, Novolin N, Tresiba, Levemir, Lantus, Toujeo, NovoLog Mix 70 / 30, Humalog Mix 75 / 25, Humalog Mix 50 / 50, Humulin 70 / 30, Novolin 70 / 30, Ryzodeg, etc.), amylin analogs or pramlintide (e.g., SymlinPen 120 and SymlinPen60), alpha-glucosidase inhibitors (e.g., acarbose (Precose) and miglitol (Glyset)), biguanides (e.g., metformin-alogliptin (Kazano), metformin-canagliflozin (Invokamet), metformin-dapagliflozin (Xigduo XR), metformin-empagliflozin (Synjardy), metformin-glipizide, metformin-glyburide (Glucovance), metformin-linagliptin (Jentadueto), metformin-pioglitazone (Actoplus), metformin-repaglinide (PrandiMet), metformin-rosiglitazone (Avandamet), metformin-saxagliptin (Kombiglyze XR), and metformin-sitagliptin (Janumet), dopamine agonists (e.g., bromocriptine (Cycloset)), dipeptidyl peptidase 4 (DPP-4) inhibitors (e.g., alogliptin (Nesina), alogliptin-metformin (Kazano), alogliptin-pioglitazone (Oseni), linagliptin (Tradjenta), linagliptin-empagliflozin (Glyxambi), linagliptin-metformin (Jentadueto), saxagliptin (Onglyza), saxagliptin-metformin (Kombiglyze XR), sitagliptin (Januvia), sitagliptin-metformin (Janumet), and Janumet XR), sitagliptin, and simvastatin (Juvisync)), glucagon-like peptide-1 receptor agonists (e.g., albiglutide (Tanzeum), dulaglutide (Trulicity), exenatide (Byetta), extended-release exenatide (Bydureon), liraglutide (Victoza), and semaglutide (Ozempic)), meglitinib (e.g.,Nateglinide (Starlix), repaglinide (Prandin), and repaglinide-metformin (Prandimet)), sodium glucose transporter (SGLT) 2 inhibitors (for example, dapagliflozin (Farxiga), dapagliflozin-metformin (Xigduo XR), canagliflozin (Invokana), canagliflozin-metformin (Invokamet), empagliflozin (Jardiance), empagliflozin-linagliptin (Glyxambi), empagliflozin-metformin (Steglatro)), sulfonylureas (for example, glimepiride (Amaryl), glimepiride-pioglitazone (Duetact), glimepiride-rosiglitazone (Avandaryl), gliclazide, glipizide (Glucotrol), glipizide- Metformin (Metaglip), glibenclamide (DiaBeta, Glynase, Micronase), glibenclamide-metformin (Glucovance), chlorpropamide (Diabinese), tolazamide (Tolinase), and tolbutamide (Orinase, Tol-Tab)), thiazolidinediones (e.g., rosiglitazone (Avandia), rosiglitazone-glimepiride (Avandaryl), rosiglitazone-metformin (Amaryl M), pioglitazone (Actos), pioglitazone-alogliptin-glimepiride (Duetact), and pioglitazone-metformin (Actoplus Met, Actoplus Met XR)), aspirin, and the like, and pharmaceutically acceptable salts or acids of any of the foregoing.

[0062] In some embodiments, the pharmaceutically active ingredient is encapsulated in the liposome carrier.

[0063] Specifically, the active pharmaceutical ingredient in the composition for inhibiting LOC157273 gene expression in liver cells is encapsulated in a liposome carrier for drug delivery, so that the composition containing siRNA is efficiently transported, drug degradation is avoided, and the occurrence of toxic side effects is reduced.

[0064] In some embodiments, the composition further comprises a pharmaceutically acceptable excipient.

[0065] In some embodiments, the excipients include one or more of a buffer, an emulsifier, a suspending agent, a stabilizer, a preservative, an excipient, a coagulant, a dispersant, and a defoaming agent.

[0066] In addition to providing shape, acting as a carrier, and improving stability, the excipients in the composition also have important functions such as solubilization, dissolution assistance, and sustained and controlled release.

[0067] The embodiment of the present invention provides the use of the siRNA or the composition in the preparation of a drug for treating diabetes.

[0068] In one embodiment, the diabetes comprises type II diabetes.

[0069] The siRNA is used to treat patients with T2D and diabetic complications such as diabetic dyslipidemia, impaired or inadequate glucose tolerance, impaired fasting glucose, metabolic acidosis, ketosis, appetite regulation, obesity, and other diabetes-related complications, including diabetic neuropathy, diabetic retinopathy, and nephropathy; hyperlipidemia, including hypertriglyceridemia, hypercholesterolemia, and postprandial hyperlipidemia; atherosclerosis, and hypertension. The present invention is further illustrated by the following examples.

[0070] Example 1

[0071] This example is used to illustrate an siRNA disclosed in the present invention for inhibiting the expression of the LOC157273 gene in liver cells, comprising the following steps:

[0072] 1. Design and synthesis of siRNA

[0073] Based on the human liver LOC157273 gene sequence obtained from the Genebank database, siRNAs were designed using siDirect siRNA software. siRNAs that met the three algorithms for effective siRNA selection developed by Ui Tei, Reynolds, and Amarzguioui et al. were selected as candidates. siRNAs with a GC content of 50% were then selected. The siRNAs were then aligned with the human LOC157273 sequence in the Genebank database using the NCBI BLAST function. The following siRNAs were designed, in which the sense strand sequence was identical to the designed target sequence on the target gene (U substituted for T), and the antisense strand sequence was completely complementary to the target sequence (U substituted for T):

[0074] The nucleotide sequence of the sense strand is:

[0075] 5'AUCAAUGCCCGUAACACCC3'

[0076] The nucleotide sequence of the antisense strand is:

[0077] 3'UAGUUACGGGCAUUGUGGG5'

[0078] The siRNA sequence was chemically synthesized and two overhanging dTdT residues were added to the 3' end to enhance the stability of the siRNA;

[0079] 2. Liposome-encapsulated siRNA

[0080] The liposome LNP encapsulation of siRNA specifically includes the following steps: dissolving DLin-MC3-DMA, DSPC, cholesterol, and DMG-PEG2000 in an organic phase at a molar ratio of 50:10:38.5:1.5, dissolving siRNA in sterile water, and injecting the two phase solutions into two inlet channels of a microfluidic preparation system respectively, and completing the encapsulation of nucleic acid lipid nanoparticles through rapid mixing of the two phases;

[0081] 3. siRNA transfection into HepG2 and cynomolgus monkey primary hepatocytes

[0082] The siRNA-encapsulated LNPs were transfected into HepG2 and cynomolgus monkey primary hepatocytes, respectively. A six-well plate was taken and cultured at 200,000 cells before transfection. The cell fusion reached 70% on the second day. The culture medium was replaced before transfection. Twelve sterile centrifuge tubes were prepared, and 250ul of DMEM culture medium without serum and antibiotics were added to each of 6 tubes. 10ul of the synthesized nanoliposome LNP mixture was added to 6 tubes. For the remaining 6 tubes, 100pmol of the corresponding siRNA was added to each 2 tubes, including siRNA No. 1, siRNA No. 2, Positive and Negative corresponding siRNAs, respectively. The mixture was gently mixed and allowed to stand for 5 minutes. The culture medium containing siRNA was mixed with the culture medium containing LNPs and allowed to stand for 20 minutes. Other conditions were the same and no liposome-encapsulated siRNA was added as a blank control group (Blank). 250ul of the content was evenly added to the culture dish per well, and the culture medium was replaced 6 hours after transfection, and then cultured for 24 hours and 48 hours respectively.

[0083] IV. qPCR Verification of LOC157273 Gene Silencing Effect

[0084] RNA was extracted from the cells cultured after transfection using the TRIzol method at 24 and 48 h, respectively, and reagents were added according to the reagents provided by the kit provided by Guangzhou Ruibo Company, and cDNA was obtained by PCR under a temperature of 42°C for 15 min and 85°C for 5 s.

[0085] Synthesized cDNA was used for qPCR. The reverse-transcribed cDNA was diluted half and then amplified using TransStart Top Green qPCR SuperMix. The reaction conditions were: 20 μl reaction system, 1 μl cDNA template was added to each system.

[0086] Then, according to the instructions of the reverse transcription kit provided by Guangzhou Ruibo, reagents were added to set up a two-step qPCR, and 40 cycles were performed at 94°C for 30 seconds, 94°C for 5 seconds, and 60°C for 30 seconds.

[0087] Using ACTIN as an internal control, the following are the ACTIN and LOC157273 primer sequences used in the qPCR reaction:

[0088] Comparative Example 1

[0089] This comparative example 1 is used to illustrate an siRNA disclosed in the present invention for inhibiting the expression of the LOC157273 gene in liver cells, and includes most of the steps in Example 1, except that:

[0090] The H-siRNA nucleotide sequence targets the LOC157273 gene of human liver cells.

[0091] Performance Testing

[0092] As shown in Figures 1 and 2, Figures 1 and 2 show the results of transfection of the siRNA provided in the present application into HepPG2 cells for 24 hours and 48 hours. It can be seen from the detection results of Figure 1 that by using the siRNA provided in the present application to silence the LOC157273 gene in HepG2 cells, the expression of the LOC157273 gene can be effectively reduced. Compared with the H-siRNA in Comparative Example 1, it is proved that the siRNA provided in the present application is also suitable for silencing the human LOC157273 gene.

[0093] As shown in Figure 3, the siRNA provided by the present application was transfected into primary monkey hepatocytes. Compared with the blank control group (Blank), the siRNA of the present application promoted the high expression of PPP1R3B protein. The PPP1R3B protein can regulate the production of liver glycogen. The PPP1R3B protein binds to the dephosphorylated glycogen synthase and promotes the synthesis of liver glycogen. Therefore, when the PPP1R3B protein is highly expressed, the glucose in the blood sugar can be reduced to achieve the purpose of lowering blood sugar.

[0094] In Figure 4, the LOC157273 gene in primary monkey hepatocytes was silenced using the siRNA provided by the present application. It can be seen that the expression level of the LOC157273 gene was reduced. A large number of studies have shown that the LOC157273 gene negatively regulates the PPP1R3B protein, that is, when the expression level of the LOC157273 gene is reduced, the corresponding expression level of the PPP1R3B protein is increased, and glycogen synthesis is increased. Therefore, the results of Figures 3 and 4 are consistent, proving from different angles that the siRNA provided by the present application can act on the LOC157273 gene in human HepPG2 cells and primary monkey hepatocytes at the same time.

[0095] Figures 5 to 6 are the results of qPCR electrophoresis after transfection, which further verified the expression of the LOC157273 gene and PPP1R3B protein by the siRNA of the present application in HepPG2 cells and primary monkey hepatocytes, respectively. The results are consistent with the detection results of Figures 3 to 4 in the quantitative experiment; this shows that the siRNA of the present application can simultaneously silence the LOC157273 gene in human liver cells and primate liver cells, solving the problem that the siRNA applied to the human liver LOC157273 gene in the prior art cannot be directly applied to primate T2DM experiments in clinical trials.

[0096] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. An siRNA for inhibiting the expression of LOC157273 gene in liver cells, characterized in that: It comprises a sense strand and an antisense strand corresponding to the sense strand, wherein the sense strand is selected from "sequence 1", the antisense strand is selected from "sequence 2", or an siRNA having more than 80% homology with the sense strand and the antisense strand.

2. The siRNA for inhibiting the expression of LOC157273 gene in liver cells according to claim 1, characterized in that: Two deoxyribonucleotides dT are added to the 3' end of the sense strand of the siRNA and / or the 3' end of the antisense strand of the siRNA to form a single-stranded hanging structure.

3. A composition for inhibiting the expression of LOC157273 gene in liver cells, characterized in that: Comprising the siRNA according to any one of claims 1 to 2; Or an expression system capable of expressing the siRNA according to any one of claims 1 to 2.

4. The composition for inhibiting the expression of LOC157273 gene in liver cells according to claim 3, characterized in that The composition also includes a carrier for delivering the siRNA.

5. The composition for inhibiting the expression of LOC157273 gene in liver cells according to claim 4, characterized in that The carrier includes a liposome.

6. The composition for inhibiting the expression of LOC157273 gene in liver cells according to claim 4, characterized in that The composition also includes a pharmaceutical active ingredient having a preventive or therapeutic effect on diabetes.

7. The composition for inhibiting the expression of LOC157273 gene in liver cells according to claim 6, characterized in that The active pharmaceutical ingredient is encapsulated in the liposome carrier.

8. The composition for inhibiting the expression of LOC157273 gene in liver cells according to claim 4, characterized in that The composition further comprises pharmaceutically acceptable excipients, which include one or more of a buffer, an emulsifier, a suspending agent, a stabilizer, a preservative, an excipient, a coagulant, a dispersant and a defoaming agent.

9. Use of the siRNA according to any one of claims 1 to 2 or the composition according to any one of claims 3 to 8 in the preparation of a drug for treating diabetes.

10. Use of siRNA according to claim 9 in preparing a drug for treating diabetes, characterized in that: The diabetes mellitus includes type II diabetes mellitus.

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