WO2024164974A1 - 与miR-146a功能类似的小核酸及其应用 - Google Patents

与miR-146a功能类似的小核酸及其应用 Download PDF

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WO2024164974A1
WO2024164974A1 PCT/CN2024/075767 CN2024075767W WO2024164974A1 WO 2024164974 A1 WO2024164974 A1 WO 2024164974A1 CN 2024075767 W CN2024075767 W CN 2024075767W WO 2024164974 A1 WO2024164974 A1 WO 2024164974A1
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mir
mice
dss
small nucleic
nucleic acid
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彭长庚
朱冯婷
于昕
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同济大学
上海市第一康复医院(上海市杨浦区老年医院)
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing

Definitions

  • the present invention belongs to the field of biomedicine technology, and in particular relates to a class of small nucleic acids with similar functions to miR-146a or their pharmaceutical salts, chemical modifications, and their use in preparing drugs for treating miR-146a-related diseases or drugs for relieving pain.
  • IBD Inflammatory bowel disease
  • UC ulcerative colitis
  • CD Crohn’s disease
  • the main clinical manifestations are diarrhea, abdominal pain, mucus in the stool, and bloody and purulent stools; they are often accompanied by weight loss and fatigue, and they often recur.
  • Current studies believe that the disease is caused by the interaction of multiple factors such as genetic susceptibility, external environmental factors, immune disorders, intestinal flora disorders, and intestinal mucosal barrier damage.
  • the drugs currently used to treat inflammatory bowel disease mainly include salicylic acid preparations, glucocorticoids, immunosuppressants, and new biological agents.
  • the current drugs for the treatment of inflammatory bowel disease are not effective enough and have obvious side effects.
  • There is a clinical need for better new drugs for the treatment of inflammatory bowel disease Inflammatory factors that increase during enteritis can cause abdominal pain (visceral pain), so inhibiting inflammation (such as anti-inflammatory drugs) can relieve pain.
  • genes involved in inflammatory bowel disease such as cytokine family (IL-6, IL-1a, IL-1b, IL-12, etc.), chemokine family cluster (Cxcl2, Cxcl3, Cxcl5), matrix metalloproteinase family (MM3, MM8, MM10, MM9) and extracellular matrix genes.
  • cytokine family IL-6, IL-1a, IL-1b, IL-12, etc.
  • Cxcl2, Cxcl3, Cxcl5 chemokine family cluster
  • matrix metalloproteinase family MM3, MM8, MM10, MM9
  • extracellular matrix genes There are 84 genes (gene family clusters) in the IBD KEGG pathway.
  • the ideal drug is to be able to systematically regulate most of the genes in the pathological mechanism of inflammatory bowel disease and cover multiple signaling pathways.
  • miR-146a can negatively regulate the immune response to LPS by inhibiting NF-KB, TRAF6 and IRAK [Zhao, JLet al. NF-kappaB dysregulation in microRNA-146a-deficient mice drives the development of myeloid malignancies. Proceedings of the National Academy of Sciences of the United States of America 108, 9184-9189, doi: 10.1073/pnas.1105398108 (2011).
  • Boldin, MPet al. miR-146a is a significant brake on autoimmunity, myeloproliferation, and cancer in mice. The Journal of experimental medicine 208, 1189-1201, 2011.], but there are contrary reports on the function of miR-146a in inflammatory bowel disease. Wu et al.
  • TNBS 2,4,6-trinitrobenzenesulfonic acid
  • YQ MiR-146a regulates the development of ulcerative colitis via mediating the TLR4/MyD88/NF- ⁇ B signaling pathway.
  • Obesity is known to cause metabolic disorders, type 2 diabetes, hypertension, heart disease, fatty liver, gallbladder disease, cardiovascular and cerebrovascular diseases, cancer (breast cancer, colon cancer and endometrial cancer), joint disease, hypercholesterolemia and other diseases. It has been reported that the expression of miR-146a-5p is reduced in type 2 diabetic mice and is associated with type 2 diabetes [Rasoulinejad, S.A., Akbari, A. & Nasiri, K. Interaction of miR-146a-5p with oxidative stress and inflammation in complications of type 2 diabetes mellitus in male rats: Anti-oxidant and anti-inflammatory protection strategies in type 2 diabetic rats. inopathy.
  • miR-146a is increased in white adipose tissue of high-fat fed C57BL/6J mice, which is positively correlated with the development of obesity [Chartoumpekis, D.V.et al. Differential expression of microRNAs in adipose tissue after long-term high-fat diet-induced obesity in mice. PloS one 7, e34872, 2012.].
  • Taiki Sanada et al. reported that tail vein injection of miR-146a could reduce the volume of adipocytes in high-fat fed mice, but did not reduce the weight of adipose tissue [Sanada, T. et al. Anti-inflammatory effects of miRNA-146a induced in adipose and periodontal tissues. Biochemistry and biophysics reports 22, 100757, 2020.].
  • the present invention analyzes the mechanism of action of miR-146a in miR-146a-related diseases and discovers new miR-146a-like small nucleic acid molecules that can treat enteritis and reduce visceral fat, and uses them or their pharmaceutical salts to treat enteritis, inflammatory bowel disease, obesity-related diseases or pain-related diseases.
  • the present invention analyzes the mechanism of action of miR-146a in miR-146a-related diseases, and proves that small nucleic acids with similar functions to miR-146a play an important role in treating enteritis, inflammatory bowel disease, obesity-related diseases or pain-related diseases.
  • the first aspect of the present invention provides a small nucleic acid with similar function to miR-146a or a pharmaceutically acceptable salt or chemically modified product thereof, wherein the structure of the small nucleic acid with similar function to miR-146a is selected from at least one of the following structures:
  • the chemical modification is selected from 2-methoxy modification, 2-fluoro substitution, 2-deoxy modification, and locked nucleic acid modification;
  • the second aspect of the present invention provides the use of the above-mentioned small nucleic acid having similar function to miR-146a or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating miR-146a-related diseases or a drug for alleviating pain.
  • the miR-146a-related diseases include enteritis, inflammatory bowel disease, inflammation, obesity and obesity-related diseases.
  • the drug contains the small nucleic acid having similar function to miR-146a or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the small nucleic acid with similar function to miR-146a is selected from one of PL11 to PL22, or at least one of the small nucleic acids PL-11 to PL-18 similar to miR-146a-5p, or at least one of the small nucleic acids PL-19 to PL-2 similar to miR-146a-3p, or a combination of the above.
  • the dosage of the small nucleic acid having similar function to miR-146a or a pharmaceutically acceptable salt thereof is 0.01 ng/kg-10 mg/kg.
  • the small nucleic acid having similar function to miR-146a or a pharmaceutically acceptable salt thereof can be prepared into a pharmaceutical preparation.
  • the dosage form of the pharmaceutical preparation is selected from at least one of powder, tablet, granule, capsule, solution, emulsion, suspension, aerosol and injection.
  • the present invention has the following advantages and beneficial effects:
  • the present invention prepares miR-146a knockout mice, uses dextran sulfate sodium salt (DSS) to induce wild-type mice and miR-146 knockout mice into IBD models, and finds that compared with wild-type mice, the enteritis phenotype of miR-146a knockout mice is very serious, with severe blood in the stool, weight loss and mucus in the stool. Consistent with the phenotype, RNA sequencing finds that DSS induces more gene upregulation in miR-146a knockout mice than in wild-type mice, and the upregulated genes in miR-146a knockout mice cover about 67% of the upregulated genes in wild-type mice.
  • DSS dextran sulfate sodium salt
  • miR-146a regulates the vast majority of IBD-related genes induced by DSS, 97% of which are greater than 6 times and 90% of which are greater than 4 times, including numerous cytokines, chemokines and matrix metalloproteinases, and the upregulated multiples of these genes are also increased.
  • knocking out miR-146a did not affect the body weight of mice, but increased the fat content of mice (epididymal fat and perirenal fat).
  • RNA sequencing found that miR-146a regulated the known adipogenesis-related Prkcdbp, Ube3a, Scd1, Fasn, Mogat2, Pm20d1, Alox15, Prkag3 and Sesns genes.
  • miR-146a knockout mice are prone to severe inflammatory bowel disease, proving that miR-146a has the function of inhibiting inflammatory bowel disease, and for the first time revealed that miR-146a regulates 90% of the genes in the inflammatory bowel disease-related gene network, making it a very good target; and designed and synthesized new miR-146a-related small nucleic acids or their pharmaceutical salts, which can significantly treat inflammatory bowel disease through oral and intraperitoneal injection.
  • the small nucleic acid similar to miR-146a or its pharmaceutically acceptable salt provided by the present invention can be used to treat inflammation, enteritis, inflammatory bowel inflammation and obesity, as well as obesity-related metabolic disorders, type 2 diabetes, hypertension, heart disease, fatty liver, gallbladder disease, cardiovascular and cerebrovascular diseases, cancer (breast cancer, colon cancer and endometrial cancer), joint diseases, hypercholesterolemia and other diseases.
  • the chemically modified small nucleic acid with similar function to miR-146a prepared by the present invention can enhance the stability of the small nucleic acid in vivo.
  • FIG1 is a schematic diagram of the preparation of miR-146a knockout mice and a diagram of gene identification results
  • FIG2 is a schematic diagram showing the result that miR-146a knockout mice are more sensitive to DSS than wild-type mice;
  • FIG3 is a schematic diagram showing that DSS induces up-regulation of more genes in the colon of miR-146a knockout mice
  • FIG4 is a schematic diagram of the protein functional interaction network regulated by miR-146a in DSS-induced upregulation
  • FIG5 is a schematic diagram showing the results of miR-146a-induced intestinal disease in miR-146a -/- mice induced by small nucleic acids PL-18+PL-19 with similar functions to miR-146a;
  • FIG6 is a schematic diagram showing the results that small nucleic acid PL-18+PL-19, which has similar functions to miR-146a, significantly alleviates DSS-induced enteritis in wild-type mice;
  • Figure 7 shows that the small nucleic acid PL-18+PL-19, which has similar functions to miR-146a, significantly alleviates DSS-induced wild-type mice Schematic diagram of the results of intestinal inflammation;
  • FIG8 is a schematic diagram showing the results of the inhibition of IBD-related gene expression by small nucleic acid PL-18+PL-19, which has similar functions to miR-146a;
  • FIG9 is a schematic diagram showing the results of increased visceral fat in miR-146a -/- mice.
  • FIG10 is a schematic diagram showing the results that small nucleic acids similar to miR-146a, PL-18+PL-19 and PL-19 alone can reduce visceral fat in miR-146a -/- mice;
  • FIG. 11 is a schematic diagram of the gene network of miR-146a inhibiting adipogenesis.
  • a Guide RNA sequence was designed and synthesized. The sequence is shown in Table 1. Then, Cas9 and Guide RNA (gRNA) were injected into the fertilized eggs of C57BL/6N mice, and the fertilized eggs were implanted into the uterus of pseudopregnant mice. The F0 generation mice born were identified by PCR using two pairs of primers (F1&R1; F1&R2.
  • F1 5’-AAGGGAAGGATTGAACATGACACA-3’ (SEQ ID NO.3), R1: 5’-TTATTGCCTCTCTACAAGGACCTG-3’ (SEQ ID NO.4), R2: 5’-ACCATCAATAGCAGAGATGACTGG-3’ (SEQ ID NO.5)) ( Figure 1 A and B).
  • the positive F0 generation The mice were mated with wild-type (WT) mice to obtain F1 mice.
  • the F1 mice were identified by PCR and sequencing, and mice with knockout of miR-146a gene (AGCUCUGAGAACUGAAUUCCAUGGGUUAUAUCAAUGUCAGACCUGUGAAAUUCAGUUCUUCAGCU (SEQ ID NO.6)) were obtained.
  • the knockout gene fragment was 1467bp long ( Figure 1C), and did not affect the gene sequence of Gm12148, the gene closest to miR-146a.
  • Figure 1 is a schematic diagram of the preparation of miR-146a knockout mice and a diagram of the gene identification results, wherein A is a schematic diagram of the miR-146a wild-type allele and knockout allele, and the positions of the three primers F1, R1 and R2 for gene identification; B is the PCR gene identification result, the primer pair F1&R1 amplifies the wild-type allele (PCR product 400bp), and the primer pair F1&R2 amplifies the miR-146 knockout allele (PCR product 742bp) and the wild-type allele (PCR product 2209bp); C is Sanger sequencing showing that the miR-146a knockout mouse has knocked out a 1467bp DNA fragment containing the miR-146a gene.
  • A is a schematic diagram of the miR-146a wild-type allele and knockout allele, and the positions of the three primers F1, R1 and R2 for gene identification
  • B is the PCR gene identification result, the primer pair F1&R1 amp
  • DSS dextran sodium sulfate
  • DSS 2% DSS
  • the time when DSS was first given was recorded as the start time of the experiment.
  • the mice were weighed one day before and every day after DSS was given, and the feces and blood in the stool of the mice were scored.
  • the scoring criteria were as follows: after the start of the experiment, the weight, stool characteristics and blood in the stool of each mouse were recorded every day, and the total score of the above indicators of the mouse was used to evaluate the inflammatory activity index (DAI) of the mouse [Bang, B. & Lichtenberger, L. M. Methods of Inducing Inflammatory Bowel Disease in Mice. Current protocols in pharmacology 72, 5.58.51-55.58.42, 2016.].
  • DAI inflammatory activity index
  • Weight change was scored as follows: 0 points for a weight loss of ⁇ 1%, 1 point for a weight loss of 1% ⁇ 5%, 2 points for a weight loss of 5% ⁇ 10%, 3 points for a weight loss of 10% ⁇ 20%, and 4 points for a weight loss of >20%.
  • FIG. 2 is a schematic diagram of the results that miR-146a knockout mice are more sensitive to DSS than wild-type mice
  • A is a schematic diagram of the changes in body weight of DSS-treated and untreated female wild-type (WT) and miR-146a knockout mice before and after treatment
  • B is a schematic diagram of the changes in body weight of DSS-treated and untreated male wild-type and miR-146a knockout mice before and after treatment
  • C is a schematic diagram of the fecal scoring results of DSS-treated and untreated female wild-type and miR-146a knockout mice
  • D is a schematic diagram of the fecal scoring results of DSS-treated and untreated male wild-type and miR-146a knockout mice
  • E is a schematic diagram of the tail photographs of DSS-treated female wild-type and miR-146a knockout mice
  • F is a schematic diagram of the tail photographs of DSS-treated male wild-type and miR-146a knockout mice
  • RNA sequencing reveals that miR-146a inhibits about 90% of DSS-induced IBD genes
  • a and B are volcano diagrams of gene expression changes in the colon of wild-type mice and miR-146a knockout mice induced by DSS, respectively;
  • C is the number of genes that were upregulated 6-fold, 4-fold, and 2-fold in the intestine of wild-type mice and miR-146a knockout mice induced by DSS compared with untreated wild-type mice, when p ⁇ 0.01, and the number of genes that were upregulated (overlapping) in both wild-type mice and miR-146a knockout mice.
  • D is when p ⁇ 0.01, the threshold is reduced from 6-fold to 2-fold, and miR-146a regulates about 67% of the genes induced by DSS in the colon of wild-type mice.
  • E is the number of IBD-related genes that were upregulated 6-fold, 4-fold, and 2-fold in the intestine of wild-type mice and miR-146a knockout mice induced by DSS, when p ⁇ 0.01, and the number of genes related to inflammatory bowel disease that were upregulated (overlapping) in both wild-type mice and miR-146a knockout mice.
  • F is when p ⁇ 0.01, the threshold value is reduced from 6-fold to 2-fold, and miR-146a regulates about 90% of the genes related to inflammatory bowel disease induced by DSS in the colon of wild-type mice.
  • RNA sequencing results showed that DSS induced upregulation of 479 genes and downregulation of 512 genes in the colon of wild-type mice (p ⁇ 0.01) ( Figure 3A), and DSS induced upregulation of 1465 genes and downregulation of 791 genes in the colon of miR-146a knockout mice.
  • Figure 4 is a schematic diagram of the protein function interaction network induced by DSS, wherein A is the protein function interaction network of genes up-regulated greater than 10 times in the colon of wild-type mice induced by DSS and p ⁇ 0.01, and the gray-filled ones are IBD-related genes; B is the gene function interaction network of genes up-regulated greater than 10 times in the colon of miR-146a -/- mice induced by DSS, and the gray-filled ones are IBD-related genes; C is the IBD gene function interaction network of genes up-regulated greater than 45 times in the colon of wild-type mice induced by DSS and p ⁇ 0.01; D is the IBD gene function interaction network of genes up-regulated greater than 45 times in the colon of miR-146a -/- mice induced by DSS and p ⁇ 0.01.
  • Examples 2 and 3 indicate that miR-146a plays a very important function in IBD and is a very important target.
  • the 3'UTR of the human TRAF6 gene containing the miR-146a binding site was cloned to the BamHI site of PGL4.17[Luc2/Neo] (downstream of the luciferase gene) to construct a luciferase reporter plasmid PGL4.17-miR-146a-Response that responds to miR-146a. Because the natural microRNA sequence is easily degraded and difficult to be orally absorbed, the natural sequence needs to be modified to resist nuclease degradation and promote oral absorption, but the modification of nucleotides may reduce the activity of microRNA.
  • a batch of chemically modified (including 2-methoxy modification, 2-fluoro substitution, 2-deoxy modification, and locked nucleic acid modification) small nucleic acids with similar functions to miR-146a were designed and synthesized.
  • HEK293 cells were seeded into 24-well culture plates at a density of 1 ⁇ 10 5 cells per well. Twelve hours after seeding, 50 ng of a single small nucleic acid with similar functions to miR-146a, 100 ng of PGL4.17-Luc2-miR-146a-Response plasmid, and 5 ng of SV40-Renilla plasmid were co-transfected into HEK293 cells using Lipofectamine 2000 (Invitrogen, USA).
  • the control group was only transfected with 100 ng of PGL4.17-Luc2-miR-146a-Response plasmid and 5 ng of SV40-Renilla plasmid. 36 hours after transfection, the ratio of firefly luciferase activity to Renilla luciferase activity was detected using a dual luciferase assay kit (Promega, USA), and the ability of small nucleic acids with similar functions to miR-146a to inhibit firefly luciferase activity was calculated (the ratio of firefly luciferase activity to Renilla luciferase activity in the group transfected with small nucleic acids with similar functions to miR-146a was divided by the ratio of firefly luciferase activity to Renilla luciferase activity in the group not transfected with small nucleic acids with similar functions to miR-146a).
  • Example 5 Treatment of inflammatory bowel disease in miR-146a -/- mice with small nucleic acids with similar functions to miR-146a
  • mice 2-4 month old miR-146a -/- male and female mice were randomly divided into three groups: an untreated control group (13 mice), a saline-treated DSS model group (13 mice), and a DSS model group (13 mice) treated with small nucleic acid PL-18 + PL-19 with similar functions to miR-146a.
  • the three groups of mice were fasted for 24 hours after the body weight was measured, and 2% DSS was added to the drinking water, which was recorded as the start time of the experiment.
  • the mice were weighed one day before and every day after DSS administration, and the feces and blood in the stool of the mice were scored (reference embodiment 2).
  • small nucleic acid PL-18 (0.04ng per gram of body weight) + PL-19 (0.08ng per gram of body weight) was gavage and intraperitoneal injection once, and then administered once every other day.
  • DSS was stopped. After scoring on the 9th day, all mice were killed by dislocation of the neck, and the colon was dissected and its length was measured.
  • FIG5 is a schematic diagram of the results of miR-146a-induced intestinal disease in miR-146a -/- mice induced by small nucleic acids PL-18+PL-19, which have similar functions to miR-146a
  • A is a graph showing the weight changes of miR-146a -/- male and female mice in the untreated control group, the saline-treated DSS model group, and the PL-18+PL-19-treated DSS model group
  • B is a graph showing the weight changes of miR-146a-/- male and female mice in the untreated control group, the saline-treated DSS model group, and the PL-18+PL-19-treated DSS model group
  • C is a picture of the tails of mice in the untreated control group, saline-treated DSS model group and PL-18+PL-19-treated DSS model group
  • D is the disease activity index of mice in the untreated control group, saline-treated DSS model group and PL-18+PL-19-treated DSS model group
  • E is the colon length of mice in the untreated control group, saline-treated DSS model group and PL-18+PL-19-treated DSS model group
  • F is the ratio of colon length to body weight of mice in the untreated control group, saline-treated DSS model group and PL-18+PL-19-treated DSS model group.
  • n 13, 13, 13 *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, ****p ⁇ 0.0001, analyzed by two-way ANOVA and T-test.
  • Example 6 Treatment of inflammatory bowel disease in wild-type mice with small nucleic acids with similar functions to miR-146a
  • mice 7-week-old wild-type C57BL/6 male mice (Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd.) were randomly divided into four groups: untreated control group (5 mice), DSS group (9 mice), saline-treated DSS group (8 mice), and DSS model group treated with small nucleic acid PL-18+PL-19 with similar functions to miR-146a (9 mice).
  • the four groups of mice were fasted for 24 hours after the body weight was measured, and 2.5% DSS was added to the drinking water, which was recorded as the start time of the experiment.
  • the mice were weighed one day before and every day after DSS administration, and the feces and blood in the feces of the mice were scored (refer to Example 2).
  • small nucleic acid PL-18 (0.0065ng per gram of body weight) + PL-19 (0.00325ng per gram of body weight) was intraperitoneally injected once, and then administered once every other day, for a total of three times.
  • the DSS concentration was reduced to 2%, and on the sixth day of the experiment, DSS was stopped. All mice were scored on day 7 and then sacrificed by cervical dislocation. The colon was dissected and its length was measured.
  • Figure 6 is a schematic diagram of the results that the small nucleic acid PL-18+PL-19 with similar functions to miR-146a significantly alleviated DSS-induced enteritis in wild-type mice, wherein A is that the body weight of the DSS model group mice treated with PL-18+PL-19 was no different from that of the DSS group treated with normal saline, B is that the fecal score of the DSS model group mice treated with PL-18+PL-19 was significantly lower than that of the DSS group and DSS group mice treated with normal saline, C is that the disease activity index of the DSS model group mice treated with PL-18+PL-19 was significantly lower than that of the DSS group and DSS group mice treated with normal saline; D is a representative colon length photo of each group; E is that the colon length of the DSS model group mice treated with PL-18+PL-19 was significantly longer than that of the DSS group and DSS group mice treated with normal saline; F is that the DSS model group mice treated with
  • FIG. 7 is a schematic diagram of the results that small nucleic acids PL-18+PL-19 with similar functions to miR-146a significantly alleviate intestinal inflammation induced by DSS in wild-type mice, wherein A is a representative HE staining image of intestinal sections of mice in the water and solvent treatment groups, B is a representative HE staining image of intestinal sections of mice in the DSS and solvent treatment groups, C is a representative HE staining image of intestinal sections of mice in the DSS and small nucleic acid treatment groups, D is an enlarged image of the framed area in Figure A, E is an enlarged image of the framed area in Figure B, F is an enlarged image of the framed area in Figure C, and G is a statistical graph of rectal inflammation scores.
  • A is a representative HE staining image of intestinal sections of mice in the water and solvent treatment groups
  • B is a representative HE staining image of intestinal sections of mice in the DSS and solvent treatment groups
  • C is a representative HE staining image of intestinal sections of
  • FIG. 8 is a schematic diagram of the results of small nucleic acid PL-18+PL-19, which has similar functions to miR-146a, inhibiting the expression of IBD-related genes
  • A is an immunofluorescence staining image of MMP3, MMP8, MMP10, IL1A, IL1B, IL6, CXCL2, CXCL3, S100A8, S100A9, TRAF6, p65, p-p65 and IRAK1 in the intestinal sections of mice in the water and solvent treatment group, the DSS and solvent treatment group, and the DSS and small nucleic acid treatment group
  • B is a statistical graph of the expression levels of MMP3, MMP8, MMP10, IL1A, IL1B, IL6, CXCL2, CXCL3, S100A8, S100A9, TRAF6, p65, p-p65 and IRAK1 obtained by immunofluorescence staining analysis.
  • Example 7 miR-146a knockout mice are obese mice
  • miR-146a -/- mice may have increased visceral fat, so six wild-type male mice aged 4-7 months and nine miR-146a -/- male mice aged 4-7 months were taken, weighed and killed by cervical dislocation, and then the epididymal fat and perinephric fat were dissected and weighed.
  • Figure 9 is a schematic diagram of the results of increased visceral fat in miR-146a -/- mice, wherein A is a schematic diagram of the body weight of 4-7 month old wild-type mice and miR-146a -/- mice; B is a schematic diagram of the results that the epididymal fat weight of miR-146a -/- mice is significantly higher than that of wild-type mice; C is a schematic diagram of the results that the ratio of epididymal fat weight to body weight of miR-146a -/- mice is significantly higher than that of wild-type mice.
  • n 6, 9, *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, ****p ⁇ 0.0001, analyzed by T test.
  • mice 4-7 month old miR-146a -/- male mice were divided into three groups: 9 in the saline treatment group, 5 in the PL-19 (0.08 ng/g) small nucleic acid treatment group, and 5 in the PL-18 (0.16 ng/g) + PL-19 (0.08 ng/g) small nucleic acid treatment group.
  • Small nucleic acids were administered by intraperitoneal injection, once on the first, fifth, and tenth days.
  • the control group of mice was injected with an equal volume of saline. The mice were weighed every day, and the mice were killed by cervical dislocation on the 12th day of the experiment, and the visceral fat tissue of the mice was dissected.
  • Figure 10 is a schematic diagram of the results that small nucleic acids PL-18+PL-19 and PL-19 alone, which have similar functions to miR-146a, can reduce visceral fat in miR-146a -/- mice
  • A is a schematic diagram of the body weight of 4-7 month old miR-146a -/- mice before and after treatment (saline-treated control group miR-146a -/- , PL-19-treated miR-146a -/- , PL-19+PL-18-treated miR-146a -/- );
  • B is a schematic diagram of the epididymal fat weight of untreated and treated miR-146a -/- mice;
  • C is a schematic diagram of the ratio of epididymal fat weight to body weight of untreated and treated miR-146a -/- mice;
  • D is a schematic diagram of the perirenal fat weight of untreated and treated miR-146a -/- mice;
  • E is a schematic diagram of the ratio
  • RNA sequencing reveals miR-146a regulates the adipogenic gene network
  • the visceral fat of wild-type mice and miR-146a -/- mice was lysed with Trizol (Invitrogen, USA), and the total RNA of the visceral fat was extracted according to the instructions and then sent to a sequencing company (Shanghai Bio-Tech Co., Ltd.) for sequencing.
  • FIG11 is a schematic diagram of the gene network for miR-146a to inhibit adipogenesis.
  • RNA sequencing analysis results showed that 148 genes were up-regulated more than 2-fold in the epididymal fat of miR-146a knockout mice, with p ⁇ 0.01.
  • the up-regulated genes were analyzed using the String database, and the results showed that among the 131 proteins, Prkcdbp, Ube3a, Scd1, Fasn, Mogat2, Pm20d1, Alox15, Prkag3, and Sesns were known to be related to fat metabolism and lipogenesis (Figure 11). This suggests that the 131 protein genes may be target genes for miR-146a to regulate lipogenesis.

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Abstract

一种与miR-146a功能类似的小核酸或其药用盐，以及所述与miR-146a功能类似的小核酸或其药用盐在制备治疗肠炎、炎症性肠病、炎症、肥胖和肥胖相关的疾病的药物或缓解疼痛的药物中的应用。

Description

与miR-146a功能类似的小核酸及其应用 技术领域
本发明属于生物医药技术领域，特别是涉及一类与miR-146a功能类似的小核酸或其药用盐、化学修饰物及其在制备治疗miR-146a相关疾病的药物或缓解疼痛的药物中的应用。
背景技术
炎症性肠病(Inflammatory bowel disease，IBD)是一种多基因参与的复杂的慢性非特异性肠道炎症性疾病，根据发病特征不同，主要分为溃疡性结肠炎(Ulcerative colitis，UC)和克罗恩病(Crohn’s disease，CD)两种类型[Taganov,K.D.,Boldin,M.P.,Chang,K.J.&Baltimore,D.NF-kappaB-dependent induction of microRNA miR-146,an inhibitor targeted to signaling proteins of innate immune responses.Proceedings of the National Academy of Sciences of the United States of America 103，12481-12486，(2006).]。主要临床表现为腹泻、腹痛、黏液便及脓血便；常伴有消瘦乏力等，多反复发作。目前研究认为该病由遗传易感性、外界环境因素、免疫失调、肠道菌群紊乱及肠黏膜屏障损伤等多种因素交互作用引起。目前用于治疗炎症性肠病的药物主要有水杨酸类制剂、糖皮质激素类、免疫抑制剂类及新型生物制剂类等。目前治疗炎症性肠病的药物有效率不足、且副作用明显，临床需要更好的治疗炎症性肠病的新药。肠炎时升高的炎症因子会引起腹痛(内脏痛)，因此抑制炎症(比如抗炎药)能缓解疼痛。
参与炎症性肠病的基因众多(超600个)，比如细胞因子家族(IL-6,IL-1a,IL-1b,IL-12等)，趋化因子家簇(Cxcl2,Cxcl3,Cxcl5)，基质金属蛋白酶家族(MM3,MM8,MM10,MM9)和细胞外基质基因。IBD KEGG pathway中就有84个基因(基因家簇)。理想的药物是能系统性地调控炎症性肠病的病理机制中的大多数基因，并且需要涵盖多个信号通路。
已报道miR-146a可以通过抑制NF-KB、TRAF6和IRAK来负调节对LPS的免疫反应[Zhao,J.L.et al.NF-kappaB dysregulation in microRNA-146a-deficient mice drives the development of myeloid malignancies.Proceedings of the National Academy of Sciences of the United States of America 108,9184-9189,doi:10.1073/pnas.1105398108(2011).Boldin,M.P.et al.miR-146a is a significant brake on autoimmunity,myeloproliferation,and cancer in mice.The Journal of experimental medicine 208,1189-1201,2011.]，但miR-146a在炎症性肠病中的功能有相反的报道，Wu等报道用包含病毒载体过表达的miR-146a的外泌体能够缓解2,4,6-trinitrobenzenesulfonic acid(TNBS)诱导的大鼠肠炎[Wu,H.et al.Extracellular vesicles containing miR-146a attenuate experimental colitis by targeting TRAF6and IRAK1.International  immunopharmacology 68,204-212,(2019).]；而Wang等报道miR-146a的抑制剂能够缓解TNBS诱导的大鼠肠炎[Wang,J.P.,Dong,L.N.,Wang,M.,Guo,J.&Zhao,Y.Q.MiR-146a regulates the development of ulcerative colitis via mediating the TLR4/MyD88/NF-κB signaling pathway.European review for medical and pharmacological sciences 23,2151-2157,2019.]。因此，miR-146a到底是促进还是抑制肠炎和炎症，需要进一步的研究确认。而且目前未知miR-146a是否能调控肠炎相关基因网络中的大多数基因和多个信号通路。
已知肥胖可能导致代谢紊乱、二型糖尿病、高血压、心脏病、脂肪肝、胆囊疾病、心脑血管疾病、癌症(乳腺癌、结肠癌和子宫内膜癌)、关节病变、高胆固醇症等疾病。已报道miR-146a-5p在二型糖尿病小鼠中的表达降低，与二型糖尿病相关[Rasoulinejad,S.A.,Akbari,A.&Nasiri,K.Interaction of miR-146a-5p with oxidative stress and inflammation in complications of type 2diabetes mellitus in male rats:Anti-oxidant and anti-inflammatory protection strategies in type 2diabetic retinopathy.Iranian journal of basic medical sciences 24,1078-1086,2021.]，也有报道miR-146a在高脂喂养C57BL/6J小鼠的白色脂肪组织中升高，与肥胖的发展正相关[Chartoumpekis,D.V.et al.Differential expression of microRNAs in adipose tissue after long-term high-fat diet-induced obesity in mice.PloS one 7,e34872,2012.]。2020年Taiki Sanada等报道尾静脉注射miR-146a能减少高脂饲养的小鼠的脂肪细胞的体积，但没有减少脂肪组织的重量[Sanada,T.et al.Anti-inflammatory effects of miRNA-146a induced in adipose and periodontal tissues.Biochemistry and biophysics reports 22,100757,2020.]。
因此，基于以上背景，本发明通过分析miR-146a在miR-146a相关疾病的作用机理进行分析，发现了可以治疗肠炎和降低内脏脂肪的新的miR-146a类似的小核酸分子，并将其或其药用盐用于治疗肠炎、炎症性肠病、肥胖相关的疾病或疼痛相关的疾病。
发明内容
本发明对miR-146a在miR-146a相关疾病的作用机理进行分析，证明miR-146a功能类似的小核酸在治疗肠炎、炎症性肠病、肥胖相关的疾病或疼痛相关的疾病中发挥重要作用。
为了实现上述目的，本发明采用的技术方案如下：
本发明的第一方面提供了一种与miR-146a功能类似的小核酸或其药用盐、化学修饰物，所述与miR-146a功能类似的小核酸的结构选自以下结构的至少一种：
所述化学修饰选自2-甲氧基修饰、2-氟取代、2-脱氧修饰、锁核酸修饰；


本发明的第二方面提供了上述与miR-146a功能类似的小核酸或其药用盐在制备治疗miR-146a相关疾病的药物或缓解疼痛的药物中的应用。
优选地，所述miR-146a相关疾病包括肠炎、炎症性肠病、炎症、肥胖和肥胖相关的疾病。
优选地，所述药物是以所述与miR-146a功能类似的小核酸或其药用盐作为活性成分。
优选地，所述与miR-146a功能类似的小核酸选自PL11至PL22中的一种，或与miR-146a-5p类似的小核酸PL-11至PL-18中的至少一种，或与miR-146a-3p类似的小核酸PL-19至PL-2中的至少一种，或上述几种的组合。
优选地，所述与miR-146a功能类似的小核酸或其药用盐的使用剂量为0.01ng/kg-10mg/kg。
优选地，所述与miR-146a功能类似的小核酸或其药用盐可制成药物制剂。
优选地，所述药物制剂的剂型选自粉剂、片剂、颗粒剂、胶囊剂、溶液剂、乳剂、混悬剂、气雾剂、注射剂中的至少一种。
与现有技术相比，本发明具有以下优点和有益效果：
本发明制备获得miR-146a敲除小鼠，用葡聚糖硫酸钠(Dextran Sulfate Sodium Salt，DSS)诱导野生型小鼠和miR-146敲除小鼠成为IBD模型，发现与野生型小鼠相比，miR-146a敲除小鼠肠炎表型非常严重，有严重的便血、体重下降和黏液便。与表型相吻合，RNA测序发现DSS在miR-146a敲除小鼠中比在野生型小鼠中诱导更多的基因上调，而且miR-146a敲除小鼠中上调的基因覆盖野生型小鼠上调基因的67％左右，miR-146a调控DSS诱导的IBD相关基因中的绝大多数基因，大于6倍中的97％和大于4倍中的90％，包含众多细胞因子、趋化因子和基质金属蛋白酶，这些基因上调的倍数也增大。同时，研究发现敲除miR-146a不影响小鼠的体重，但增加小鼠脂肪含量(附睾脂肪和肾周围脂肪)，RNA测序发现miR-146a调控已知的脂肪生成相关的Prkcdbp，Ube3a，Scd1，Fasn，Mogat2，Pm20d1，Alox15，Prkag3和Sesns基因。
本发明发现miR-146a敲除小鼠易得严重的炎症性肠病，证明miR-146a是抑制炎症性肠病的功能，首次揭示miR-146a调控炎症性肠病相关基因网络中90％的基因，是一个非常好的靶点；并设计和合成新的与miR-146a的小核酸或其药用盐，经口服和腹腔注射能显著治疗炎症性肠病。同时发现敲除miR-146a不影响小鼠的体重，但增加小鼠脂肪含量(附睾脂肪和肾周围脂肪)；腹腔注射与miR-146a类似的小核酸或其药用盐能显著降低内脏脂肪的量。因此，本发明提供的与miR-146a类似的小核酸或其药用盐可以用于治疗炎症、肠炎、炎症性肠炎和肥胖，以及肥胖相关的代谢紊乱、二型糖尿病、高血压、心脏病、脂肪肝、胆囊疾病、心脑血管疾病、癌症(乳腺癌、结肠癌和子宫内膜癌)、关节病变、高胆固醇症等疾病，除此之外，本发明制备获得的化学修饰的与miR-146a功能类似的小核酸，可增强小核酸在体内的稳定性。
附图说明
图1为miR-146a敲除小鼠的制备示意图和基因鉴定结果图；
图2为miR-146a敲除小鼠比野生型小鼠对DSS更敏感的结果示意图；
图3为DSS诱导miR-146a敲除小鼠结肠中更多基因的表达上调的示意图；
图4为miR-146a调控DSS诱导上调的蛋白质功能互作网络示意图；
图5为与miR-146a功能类似的小核酸PL-18+PL-19缓解DSS诱导的miR-146a-/-小鼠的肠病结果示意图；
图6为与miR-146a功能类似的小核酸PL-18+PL-19显著缓解DSS诱导的野生型小鼠的肠炎的结果示意图；
图7为与miR-146a功能类似的小核酸PL-18+PL-19显著缓解DSS诱导的野生型小鼠的 肠道炎症的结果示意图；
图8为与miR-146a功能类似的小核酸PL-18+PL-19抑制IBD相关基因表达的结果示意图；
图9为miR-146a-/-小鼠内脏脂肪增多的结果示意图；
图10为与miR-146a类似的小核酸PL-18+PL-19和单用PL-19能减少miR-146a-/-小鼠的内脏脂肪的结果示意图；
图11为miR-146a抑制脂肪生成的基因网络示意图。
具体实施方式
为了更清楚地说明本发明，下面结合优选实施例对本发明做进一步的说明。本领域技术人员应当理解，下面所具体描述的内容是说明性的而非限制性的，不应以此限制本发明的保护范围。
实施例1制备miR-146a基因敲除小鼠
为了敲除miR-146a基因，设计合成Guide RNA序列，序列见表1所示，然后利用Cas9和Guide RNA(gRNA)注射到C57BL/6N小鼠的受精卵中，受精卵种植到假孕小鼠子宫中。出生的F0代小鼠利用两对引物(F1&R1；F1&R2。F1：5’-AAGGGAAGGATTGAACATGACACA-3’(SEQ ID NO.3)，R1:5’-TTATTGCCTCTCTACAAGGACCTG-3’(SEQ ID NO.4)，R2:5’-ACCATCAATAGCAGAGATGACTGG-3’(SEQ ID NO.5))做PCR鉴定(图1中A和B)，阳性的F0代小鼠与野生型(WT)小鼠交配繁殖得到F1代小鼠，F1代小鼠经PCR鉴定和测序鉴定，得到敲除miR-146a基因(AGCUCUGAGAACUGAAUUCCAUGGGUUAUAUCAAUGUCAGACCUGUGAAAUUCAGUUCUUCAGCU(SEQ ID NO.6))的小鼠，敲除基因片段长1467bp(图1中C)，不影响离miR-146a最近的基因Gm12148的基因序列。
表1：miR-146a敲除小鼠的Guide RNA序列表
结果如图1所示，图1为miR-146a敲除小鼠的制备示意图和基因鉴定结果图，其中，A为miR-146a野生型等位基因和敲除等位基因示意图，基因鉴定的三条引物F1，R1和R2的位置；B为PCR基因鉴定结果，引物对F1&R1扩增野生型等位基因(PCR产物400bp)，引物对F1&R2扩增miR-146敲除等位基因(PCR产物742bp)和野生型等位基因(PCR产物2209bp)；C为Sanger测序显示miR-146a敲除小鼠敲除了含miR-146a基因在内的1467bp的DNA片段。
实施例2 miR-146a敲除小鼠炎症性肠病研究
为了研究敲除miR-146a是否促进炎症性肠病，利用葡聚糖硫酸钠(DSS)诱导野生型C57BL/6N小鼠和miR-146a敲除小鼠成为炎症性肠病模型。10只2-6月龄雌性C57BL/6N小鼠(浙江维通利华实验动物技术有限公司)、11只2-6月龄雌性miR-146a敲除小鼠；11只2-6月龄雄性C57BL/6N小鼠(浙江维通利华实验动物技术有限公司)和10只2-6月龄雄性miR-146敲除小鼠各分为两组，禁食24小时后，一组饮水中加2％DSS(MP Biomedicals)处理7天，另一组为对照组，不给2％DSS处理，开始给DSS的时间记为实验的起始时间。在给DSS之前的一天和之后每天称量小鼠的体重，并对小鼠的粪便和便血进行评分。评分标准为：实验开始后每天记录每只小鼠的体重、粪便性状及便血情况，将小鼠的上述指标的评分总和用以评估小鼠的炎症活动指数(disease activity index，DAI)[Bang,B.&Lichtenberger,L.M.Methods of Inducing Inflammatory Bowel Disease in Mice.Current protocols in pharmacology 72,5.58.51-55.58.42,2016.]。
体重变化评分为：体重下降≤1％为0分，1％<体重下降≤5％计1分，5％<体重下降≤10％记为2分，10％<体重下降≤20％记为3分，体重下降>20％记为4分。
粪便性状评分为：0分＝粪便正常，2分＝粪便松散，4分＝水样稀便。
便血情况评分为：0分＝无血便，2分＝轻度血便，4分＝大量出血。
结果如图2所示，图2为miR-146a敲除小鼠比野生型小鼠对DSS更敏感的结果示意图，其中，A为DSS处理和未处理的雌野生型(WT)和miR-146a敲除小鼠的体重在处理前后的变化结果示意图，B为DSS处理和未处理的雄野生型和miR-146a敲除小鼠的体重在处理前后的变化结果示意图；C为DSS处理和未处理的雌野生型和miR-146a敲除小鼠的粪便评分结果示意图；D为DSS处理和未处理的雄野生型和miR-146a敲除小鼠的粪便评分结果示意图；E为DSS处理的雌野生型和miR-146a敲除小鼠的尾巴照片结果示意图，F为DSS处理的雄野生型和miR-146a敲除小鼠的尾巴照片结果示意图，DSS处理敲除小鼠的尾巴上有明显血迹，血迹从尾巴根部至箭头所指处；G为DSS处理和未处理的雌野生型和miR-146a敲除小鼠的疾病活动指数结果示意图，H为DSS处理和未处理的雄野生型和miR-146a敲除小鼠的疾病活动指数结果示意图；I为代表性的DSS处理和未处理的雌野生型和miR-146a敲除小鼠的结肠长度结果示意图，J为代表性的DSS处理和未处理的雄野生型和miR-146a敲除小鼠的结肠长度结果示意图；K为代表性的DSS处理和未处理的雌野生型和miR-146a敲除小鼠的结肠长度和结肠长度与体重的比值结果示意图，L为代表性的DSS处理和未处理的雄野生型和miR-146a敲除小鼠的结肠长度和结肠长度与体重的比值结果示意图。雌鼠n＝5，5，5，6，雄鼠n＝5，5，6，5，*p<0.05，**p<0.01，***p<0.001，****p<0.0001，采用双因素方差分和T检验分析。
实验结果显示，miR-146a敲除雌鼠和雄鼠在DSS处理4天时体重开始下降，而DSS处理的雌性野生型小鼠体重在第7天才下降，DSS处理的雄性野生型小鼠的体重未下降(图2A和B)。与DSS处理的野生型小鼠相比，DSS处理的miR-146a敲除小鼠体重显著降低(图2A和B)。DSS处理的miR-146a敲除雌鼠和野生型小鼠的粪便评分有显著差异，但雄鼠没有(图2C和D)；DSS处理的miR-146a敲除小鼠有严重的便血，雌雄miR-146a敲除小鼠的尾巴上都有明显的血迹(图2D和E)。因此，DSS处理的miR-146a敲除小鼠的疾病活动指数显著高于DSS处理的野生型小鼠的疾病活动指数(图2G和H)。在DSS处理第七天时安乐死小鼠，解剖结肠并测量其长度和重量，发现DSS处理的野生型雌鼠的结肠长度显著短于未处理的野生型雌鼠的结肠长度，DSS处理的miR-146a敲除雌鼠的结肠比DSS处理的野生型雌鼠的更短(图2I)，而且其肠子长度与体重的比值也更低(图2I)；DSS处理的野生型雄鼠的结肠长度与未处理的野生型雄鼠的结肠长度没有显著差异，但DSS处理的miR-146a敲除雄鼠的结肠长度显著短于DSS处理的野生型雄鼠的(图2J)结肠长度，而且其肠子长度与体重的比值也低于DSS处理的野生型雄鼠的比值(图2J)。综上，实施例2的结果说明miR-146a敲除小鼠对DSS更敏感，更易被DSS诱导成严重的肠病，有严重的便血和体重下降。
实施例3 RNA测序揭示miR-146a抑制90％左右的DSS诱导的IBD基因
为了研究miR-146a调控肠炎的分子机制全貌，未处理的野生型小鼠、DSS处理的野生型小鼠和DSS处理的miR-146a敲除小鼠的结肠组织用Trizol(Invitrogen，USA)裂解，根据说明书抽提结肠组织的total RNA，然后送给测序公司(上海伯豪生物技术有限公司)测序。
结果如图3所示，A和B分别为DSS诱导的野生型小鼠和miR-146a敲除小鼠结肠中基因表达变化的火山示意图；C为与未处理的野生型小鼠相比，当p<0.01，DSS诱导野生型小鼠和miR-146a敲除小鼠肠中上调6倍、4倍、2倍的基因数目，以及在野生型小鼠和miR-146a敲除小鼠中都上调(重叠)的基因数目。D为当p<0.01，阈值从6倍降到2倍，miR-146a都调控DSS在野生型小鼠结肠中诱导基因中的约67％。E为与未处理的野生型小鼠相比，当p<0.01，DSS诱导野生型小鼠和miR-146a敲除小鼠肠中上调6倍、4倍、2倍的IBD相关的基因数目，以及在野生型小鼠和miR-146a敲除小鼠中都上调(重叠)的炎症性肠病相关的基因数目。F为当p<0.01，阈值从6倍降到2倍，miR-146a都调控DSS在野生型小鼠结肠中诱导炎症性肠病相关的基因中的90％左右。
RNA测序结果显示，DSS诱导野生型小鼠结肠中479个基因上调和512个基因下调(p<0.01)(图3A)，DSS诱导miR-146a敲除小鼠结肠中1465个基因上调和791个基因下 调(p<0.01)(图3B)。进一步分析发现：在p<0.01时，上调倍数从6倍、4倍到2倍，DSS处理的miR-146a敲除小鼠结肠中上调的基因都明显多于DSS处理的野生型小鼠(图3C)，并且阈值从6倍降到2倍，miR-146a都调控DSS在野生型小鼠结肠中诱导基因中的约67％(在6倍时为71％，4倍时为67％，2倍时为64％)(图3D)；而且，DSS诱导miR-146a敲除小鼠结肠中上调6倍、4倍、2倍的IBD相关的基因数目明显比DSS诱导野生型小鼠结肠中上调的IBD相关的基因数目多，上调倍数从6倍降到2倍，miR-146a都调控DSS在野生型小鼠结肠中诱导IBD相关的基因中的90％左右(在6倍时为97％，4倍时为90％，2倍时为80％)(图3E和F)。
当设定p<0.01，上调倍数大于10倍时，70个基因在DSS处理的野生型小鼠结肠中上调，381个基因在DSS处理的miR-146a敲除小鼠结肠中上调。结果如图4所示，图4为DSS诱导上调的蛋白质功能互作网络示意图，其中，A为DSS诱导野生型小鼠结肠中基因上调大于10倍，且p<0.01的蛋白质功能互作网络，灰色填充的是IBD相关基因；B为DSS诱导miR-146a-/-小鼠结肠中基因上调大于10倍，且p<0.01的基因功能互作网络，灰色填充的是IBD相关基因；C为DSS诱导野生型小鼠结肠中上调大于45倍，且p<0.01的IBD基因功能互作网络；D为DSS诱导miR-146a-/-小鼠结肠中基因上调大于45倍，且p<0.01的IBD基因功能互作网络。
利用String database分析发现，DSS处理的野生型小鼠和miR-146a敲除小鼠上调10倍以上基因的功能互作网络图(图4A和B)；DSS处理的野生型小鼠结肠中上调45倍的IBD相关基因有5个(图4C)，miR-146a敲除小鼠结肠中上调45倍的IBD相关基因有54个(图4D)，包含众多细胞因子、趋化因子和基质金属蛋白酶。这些结果表明miR-146a通过抑制DSS诱导的IBD相关基因网络中90％左右的基因来抑制IBD。
进一步研究发现，DSS诱导野生型小鼠结肠中上调2倍的基因中有11个基因(Il17a，Lcn2，Gpr84，Mmp9，Spp1，Nos2，Il1rl1，Ltb4r1，Cd14，Tnf，Tac1)是促进疼痛的基因，这些基因参与肠炎性疼痛；而miR-146a敲除小鼠结肠中上调2倍的基因中除了以上11个基因之外，还有额外的27个促疼痛的基因(Prok2，Il6，Osm，Ptgs2，C5ar1，Egr1，Gfap，Chl1，Adcyap1，Trpa1，Tacr1，Nos1，Nos3，Map3k8，Ptges，Tlr2，Tlr5，Il1r1，Scn9a，Ptger2，Mapk10，Lama4，Per1，Ptgs1，Il6st，Tnfrsf1a，P2ry2)。这些结果说明miR-146a抑制肠炎相关的疼痛基因；miR-146a敲除小鼠在DSS诱导成炎症性肠病后，有比野生小鼠更严重的疼痛。
实施例4与miR-146a功能类似的小核酸筛选
实施例2和3的数据表明，miR-146a在IBD中起非常重要的功能，是非常重要的靶点。 克隆人TRAF6基因的含miR-146a结合位点的3’UTR至PGL4.17[Luc2/Neo]的BamHI位点(luciferase基因的下游)，构建成一个响应miR-146a的荧光素酶报告质粒PGL4.17-miR-146a-Response。因为天然的microRNA序列容易降解和不易口服吸收，所以需要对天然序列进行修饰以抵抗核酸酶的降解和促进口服吸收，但核苷酸的修饰可能会降低microRNA的活性。设计和合成了一批化学修饰(包括2-甲氧基修饰、2-氟取代、2-脱氧修饰、锁核酸修饰)的与miR-146a功能类似的小核酸，按1×105个HEK293细胞每孔的密度接种细胞至24-孔培养板中，接种12小时后利用Lipofectamine 2000(Invitrogen,USA)把50ng的单条与miR-146a功能类似的小核酸和100ng的PGL4.17-Luc2-miR-146a-Response质粒和5ng的SV40-Renilla质粒共转至HEK293细胞中，对照组只转染100ng的PGL4.17-Luc2-miR-146a-Response质粒和5ng的SV40-Renilla质粒。转染36小时后用dual luciferase assay kit(Promega，USA)检测萤火虫荧光素酶活性与海肾荧光素酶活性的比值，并计算与miR-146a功能类似的小核酸抑制萤火虫荧光素酶活性的能力大小(转染与miR-146a功能类似的小核酸组的萤火虫荧光素酶活性与海肾荧光素酶活性的比值除以未转染与miR-146a功能类似的小核酸组的萤火虫荧光素酶活性与海肾荧光素酶活性的比值)。经筛选发现12条(PL-11～PL-22)与miR-146a功能类似的小核酸能显著抑制PGL4.17-Luc2-miR-146a-Response质粒中萤火虫荧光素酶的表达，见表2所示。
表2
与miR-146a功能类似的小核酸的结构如下所示：



与miR-146a功能类似的小核酸PL-11～PL-22的钠盐，如PL-18和PL-19的钠盐结构如下所示：
实施例5与miR-146a功能类似的小核酸治疗miR-146a-/-小鼠的炎症性肠病
2-4月龄的miR-146a-/-雌雄小鼠随机分为三组：不处理的对照组(13只)、生理盐水处理的DSS模型组(13只)、与miR-146a功能类似的小核酸PL-18+PL-19治疗的DSS模型组(13只)。三组小鼠测量体重后禁食24小时，开始在饮水中添加2％DSS，此时记为实验的起始时间。在给DSS之前的一天和之后每天称量小鼠的体重，并对小鼠的粪便和便血进行评分(参考实施列2)。在给DSS 24小时后灌胃和腹腔注射小核酸PL-18(0.04ng每克体重)+PL-19(0.08ng每克体重)一次，之后每隔一天给药一次。在实验的第7天，停止给DSS。所有小鼠在第9天评分后，脱颈处死，解剖结肠，测量其长度。
结果如图5所示，图5是与miR-146a功能类似的小核酸PL-18+PL-19缓解DSS诱导的miR-146a-/-小鼠的肠病结果示意图，其中，A为不处理的对照组、生理盐水处理的DSS模型组和PL-18+PL-19治疗的DSS模型组的miR-146a-/-雌雄小鼠的体重变化图；B为不处理的对 照组、生理盐水处理的DSS模型组和PL-18+PL-19治疗的DSS模型组的小鼠的粪便评分；C为不处理的对照组、生理盐水处理的DSS模型组和PL-18+PL-19治疗的DSS模型组的小鼠的尾巴图，生理盐水处理的DSS模型组小鼠的尾巴上有明显血迹，血迹从尾巴根部至箭头所指处，PL-18+PL-19治疗的DSS模型组的小鼠尾巴上无血迹；D为不处理的对照组、生理盐水处理的DSS模型组和PL-18+PL-19治疗的DSS模型组的小鼠的疾病活动指数；E为不处理的对照组、生理盐水处理的DSS模型组和PL-18+PL-19治疗的DSS模型组的小鼠的结肠长度；F为不处理的对照组、生理盐水处理的DSS模型组和PL-18+PL-19治疗的DSS模型组的小鼠的结肠长度与体重的比值。n＝13，13，13*p<0.05，**p<0.01，***p<0.001，****p<0.0001，采用双因素方差分析和T检验分析。
实验结果显示：与生理盐水处理的DSS模型组相比，与miR-146a功能类似的小核酸PL-18+PL-19治疗的DSS模型组能显著减少小鼠体重的下降(图5A)，显著减少小鼠的粪便评分(图5B)，减少miR-146a-/-小鼠的便血(图5C)和降低DSS诱导的肠病活动指数(图5D)。而且，PL-18+PL-19能显著增加DSS处理的miR-146a-/-小鼠的结肠长度(图5E)和增加其结肠长度与体重的比值(图5F)。这些结果说明与miR-146a功能类似的小核酸具有治疗炎症性肠病和消炎的作用。
实施例6与miR-146a功能类似的小核酸治疗野生型小鼠的炎症性肠病
7周龄的野生型C57BL/6雄小鼠(浙江维通利华实验动物技术有限公司)随机分为四组：未处理的对照组(5只)、DSS组(9只)、生理盐水处理的DSS组(8只)、与miR-146a功能类似的小核酸PL-18+PL-19治疗的DSS模型组(9只)。四组小鼠测量体重后禁食24小时，开始在饮水中添加2.5％DSS，此时记为实验的起始时间。在给DSS之前的一天和之后每天称量小鼠的体重，并对小鼠的粪便和便血进行评分(参考实施列2)。在给DSS 24小时后腹腔注射小核酸PL-18(0.0065ng每克体重)+PL-19(0.00325ng每克体重)一次，之后每隔一天给药一次，共给药三次。在实验的第2天，DSS浓度降低为2％，在实验的第6天，停止给DSS。所有小鼠在第7天评分后，脱颈处死，解剖结肠，测量其长度。
结果如图6所示，图6为与miR-146a功能类似的小核酸PL-18+PL-19显著缓解DSS诱导的野生型小鼠的肠炎的结果示意图，其中，A为PL-18+PL-19治疗的DSS模型组小鼠的体重与生理盐水处理的DSS组相比无差异，B为PL-18+PL-19治疗的DSS模型组小鼠的粪便评分显著低于生理盐水处理的DSS组和DSS组小鼠的评分，C为PL-18+PL-19治疗的DSS模型组小鼠的疾病活动指数显著低于生理盐水处理的DSS组和DSS组小鼠的疾病活动指数；D为各组代表性结肠长度照片；E为PL-18+PL-19治疗的DSS模型组小鼠的结肠长度显著长于生理盐水处理的DSS组和DSS组小鼠的结肠长度；F为PL-18+PL-19治疗的DSS 模型组小鼠的结肠长度与体重的比值显著大于生理盐水处理的DSS组和DSS组小鼠的结肠长度与体重的比值。n＝5，9，8，9，*p<0.05，**p<0.01，***p<0.001，****p<0.0001，采用双因素方差分析和T检验分析。
实验结果显示：PL-18+PL-19治疗的DSS模型组小鼠的粪便评分和疾病活动指数都显著低于生理盐水处理的DSS组和DSS模型组(图6B和C)，而且PL-18+PL-19治疗的DSS模型组小鼠的结肠长度和结肠长度与体重的比值都比生理盐水处理的DSS组和DSS模型组的好。这些结果再次说明与miR-146a功能类似的小核酸能治疗炎症性肠病和消炎。
同时对经过与miR-146a功能类似的小核酸处理的DSS诱导的IBD小鼠的肠切片做HE染色、显微镜拍照和病理评分，结果如图7所示，图7为与miR-146a功能类似的小核酸PL-18+PL-19显著缓解DSS诱导的野生型小鼠的肠道炎症的结果示意图，其中，A为水和溶剂处理组小鼠肠切片HE染色代表图，B为DSS和溶剂处理组小鼠肠切片HE染色代表图，C为DSS和小核酸处理组小鼠肠切片HE染色代表图，D为A图中框住区域的放大图，E为B图中框住区域的放大图，F为C图中框住区域的放大图，G为直肠炎症评分统计图。结果表明，与水和溶剂处理的对照组相比，溶剂处理的DSS模型组有严重肠炎，而小核酸处理的DSS组小鼠的肠炎有显著的缓解。
进一步的免疫荧光染色结果如图8所示，图8为与miR-146a功能类似的小核酸PL-18+PL-19抑制IBD相关基因表达的结果示意图，其中，A为水和溶剂处理组、DSS和溶剂处理组和DSS和小核酸处理组的小鼠肠切片的MMP3,MMP8,MMP10,IL1A,IL1B,IL6,CXCL2,CXCL3,S100A8,S100A9,TRAF6,p65,p-p65和IRAK1的免疫荧光染色图，B为免疫荧光染色分析得到的MMP3,MMP8,MMP10,IL1A,IL1B,IL6,CXCL2,CXCL3,S100A8,S100A9,TRAF6,p65,p-p65和IRAK1的表达水平的统计图。结果表明野生型小鼠肠中MMP3,MMP8,MMP10,IL1A,IL1B,IL6,CXCL2,CXCL3,S100A8,S100A9,TRAF6,p65,p-p65和IRAK1的表达被DSS诱导显著升高，使用与miR-146a功能类似的小核酸处理后这些基因的表达显著降低，从而缓解了肠炎。
实施例7 miR-146a敲除小鼠是肥胖小鼠
在实施例2中注意到miR-146a-/-小鼠内脏脂肪可能增多，于是取4-7月龄的6只野生型雄鼠和4-7月龄的9只miR-146a-/-雄鼠，称量体重后脱颈处死，然后解剖附睾脂肪和肾周围脂肪、称重。
结果如图9所示，图9是miR-146a-/-小鼠内脏脂肪增多的结果示意图，其中，A为4-7月龄野生型小鼠和miR-146a-/-小鼠的体重示意图；B为miR-146a-/-小鼠附睾脂肪重量显著高于野生型小鼠的结果示意图，C为miR-146a-/-小鼠附睾脂肪重量与体重的比重显著高于野生 型小鼠的结果示意图；D为miR-146a-/-小鼠肾周围脂肪重量显著高于野生型小鼠的结果示意图，E为miR-146a-/-小鼠肾周围脂肪重量与体重的比重显著高于野生型小鼠的结果示意图。n＝6，9，*p<0.05，**p<0.01，***p<0.001，****p<0.0001，采用T检验分析。
统计发现，虽然野生型小鼠和miR-146a-/-小鼠的体重没有统计学上的显著差异(图9A)，但miR-146a-/-小鼠的附睾脂肪重量显著高于野生型小鼠的重量(图9B)，而且其附睾脂肪重量与体重的比值也比野生型小鼠的高(图9C)。miR-146a-/-小鼠的肾周围脂肪以及肾周围脂肪重量与体重的比值都显著高于野生型小鼠(图9D)。
实施例8与miR-146a功能类似的小核酸降低小鼠的脂肪量
为了测试与miR-146a功能类似的小核酸是否能降低miR-146a-/-小鼠的内脏脂肪量，4-7月龄miR-146a-/-雄鼠分为三组，生理盐水处理组9只、PL-19(0.08ng/g)小核酸处理组5只，PL-18(0.16ng/g)+PL-19(0.08ng/g)小核酸处理组5只。小核酸经腹腔注射给药，在第一天、第五天和第十天各给药一次。对照组小鼠注射等体积的生理盐水。每天称量小鼠的体重，小鼠在实验的第12天脱颈处死，解剖小鼠的内脏脂肪组织。
结果如图10所示，图10为与miR-146a功能类似的小核酸PL-18+PL-19和单用PL-19能减少miR-146a-/-小鼠的内脏脂肪的结果示意图，其中，A为4-7月龄miR-146a-/-小鼠在处理前后的体重(生理盐水处理的对照组miR-146a-/-，PL-19处理的miR-146a-/-，PL-19+PL-18处理的miR-146a-/-)结果示意图；B为未处理和处理组的miR-146a-/-小鼠附睾脂肪重量示意图；C为未处理和处理组的miR-146a-/-小鼠附睾脂肪重量与体重的比重结果示意图；D为未处理和处理组的miR-146a-/-小鼠肾周围脂肪重量示意图；E为未处理和处理组的miR-146a-/-小鼠肾周围脂肪重量与体重的比重结果示意图。n＝9，5，5，*p<0.05，**p<0.01，采用T检验分析。
结果显示，小核酸处理前后三组小鼠的体重都没有显著差异(图10A)，与生理盐水处理组相比，联用PL-18和PL-19小核酸能显著降低miR-146a-/-小鼠的附睾脂肪与体重的比例(图10B和C)；单用PL-19或联用PL-18和PL-19都能显著著降低miR-146a-/-小鼠的肾周围脂肪重量(图10D)和肾周围脂肪重量与体重的比例(图10E)。这些结果说明与miR-146a功能类似的小核酸能显著降低脂肪，从而治疗肥胖和肥胖相关疾病。
实施例9 RNA测序揭示miR-146a调控脂肪生成基因网络
为了研究miR-146a调控脂肪生成的分子机制，野生型小鼠和miR-146a-/-小鼠的内脏脂肪用Trizol(Invitrogen，USA)裂解，根据说明书抽提内脏脂肪的total RNA，然后送给测序公司(上海伯豪生物技术有限公司)测序。
结果如图11所示，图11为miR-146a抑制脂肪生成的基因网络示意图。脂肪组织的 RNA测序分析结果显示：miR-146a敲除小鼠的附睾脂肪中有148个基因上调倍数大于2倍，p<0.01。利用String database分析上调的基因，结果显示131个蛋白质中已知Prkcdbp，Ube3a，Scd1，Fasn，Mogat2，Pm20d1，Alox15，Prkag3和Sesns与脂肪代谢和脂肪生成相关(图11)。这表明131个蛋白质基因可能是miR-146a调控脂肪生成的靶基因。
以上所述仅是本发明的较佳实施例而已，并非对本发明作任何形式上的限制，虽然本发明已以较佳实施例揭露如上，然而并非用以限定本发明，任何熟悉本专利的技术人员在不脱离本发明技术方案范围内，当可利用上述提示的技术内容作出些许更动或修饰为等同变化的等效实施例，但凡是未脱离本发明技术方案的内容，依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化与修饰(氘代等修饰)，均仍属于本发明方案的范围内。

Claims (8)

  1. 一种与miR-146a功能类似的小核酸或其药用盐、化学修饰物，其特征在于，所述与miR-146a功能类似的小核酸的结构选自以下结构的至少一种：
    所述化学修饰选自2-甲氧基修饰、2-氟取代、2-脱氧修饰、锁核酸修饰；






  2. 一种如权利要求1所述的与miR-146a功能类似的小核酸或其药用盐在制备治疗miR-146a相关疾病的药物或缓解疼痛的药物中的应用。
  3. 如权利要求2所述的应用，其特征在于，所述miR-146a相关疾病包括肠炎、炎症性肠病、炎症、肥胖和肥胖相关的疾病。
  4. 如权利要求2-3任一项所述的应用，其特征在于，所述药物是以所述与miR-146a功能类似的小核酸或其药用盐作为活性成分。
  5. 如权利要求2-3任一项所述的应用，其特征在于，所述与miR-146a功能类似的小核酸选自PL11至PL22中的一种，或与miR-146a-5p类似的小核酸PL-11至PL-18中的至少一种，或与miR-146a-3p类似的小核酸PL-19至PL-2中的至少一种，或上述几种的组合。
  6. 如权利要求2-3任一项所述的应用，其特征在于，所述与miR-146a功能类似的小核酸或其药用盐的使用剂量为0.01ng/kg-10mg/kg。
  7. 如权利要求2-3任一项所述的应用，其特征在于，所述与miR-146a功能类似的小核酸或其药用盐可制成药物制剂。
  8. 如权利要求7所述的应用，其特征在于，所述药物制剂的剂型选自粉剂、片剂、颗粒剂、胶囊剂、溶液剂、乳剂、混悬剂、气雾剂、注射剂中的至少一种。
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