WO2011029404A1 - 调节RANTES表达的miR-125a、其组合物及其用途 - Google Patents

调节RANTES表达的miR-125a、其组合物及其用途 Download PDF

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WO2011029404A1
WO2011029404A1 PCT/CN2010/076787 CN2010076787W WO2011029404A1 WO 2011029404 A1 WO2011029404 A1 WO 2011029404A1 CN 2010076787 W CN2010076787 W CN 2010076787W WO 2011029404 A1 WO2011029404 A1 WO 2011029404A1
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mir
expression
rantes
cell
tissue
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沈南
唐元家
赵遐
曲波
崔慧娟
王树军
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中国科学院上海生命科学研究院
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • 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
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
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    • A61P37/00—Drugs for immunological or allergic disorders
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q2600/00—Oligonucleotides characterized by their use
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    • C—CHEMISTRY; METALLURGY
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • miR-125a for regulating RANTES expression composition thereof and use thereof
  • the present invention relates to miR-125a which modulates RANTES expression, compositions thereof and uses thereof. Background technique
  • SLE Systemic lupus erythematosus
  • RANTES is a typical member of the CC subfamily inflammatory chemokine, which induces the infiltration of various immune cells such as T cells, dendritic cells and eosinophils into the inflammation site, and plays an important role in organ inflammation. [18-20 ]. In pathological conditions, it can enhance cytotoxicity, recruit immune cells, enhance inflammatory processes, and aggravate arthritis, atopic dermatitis, nephritis, colitis and other diseases [21].
  • RANTES expression levels in tissues are associated with disease activity and can serve as biomarkers for disease activity. More noteworthy is the receptor for RANTES.
  • CCR5 is a co-receptor for HIV entry into target cells and plays an important role in HIV fusion and entry into target cells.
  • RANTES has become a hot spot for the development of new drugs such as chronic inflammation, alleviation of transplant rejection, and HIV infection [22-27].
  • MicroRNAs are non-coding protein single-stranded small-molecule RNAs of about 21-25 nt in length that are endogenously expressed in plants and animals. They bind to specific target mRNAs by complementary complementation of nucleic acid sequences. mRNA translation or degradation of target mRNA is a molecule that plays a negative role in post-transcriptional regulation [3, 4], involved in a series of important processes in life, including development, cell proliferation, apoptosis, differentiation, and viral infections and cancer. A variety of biological effects [5-7].
  • miRNAs are a major breakthrough in scientific research in recent years, not only contributing to human self-regulation of genes. The section mechanism is more comprehensive and profound, and it also provides new ideas for disease occurrence, prevention and treatment research. Numerous studies have shown that miRNAs are involved in all aspects of the regulation of the immune system, including innate immunity, adaptive immune response, immune cell development, etc. [8-10], fine-tuning the immune system. However, the relationship between miRNA and SLE inflammatory response has not been reported so far. Summary of the invention
  • miR-125a was down-regulated in SLE patients, and in vitro experiments revealed that overexpression of miR-125a in T cells reduced the expression level of the inflammatory chemokine RANTES.
  • miR-125a expression signal can be used as an important biomarker for clinical diagnosis of SLE; and may serve as a new target for drug intervention.
  • the specific intervention level of miR-125a can be developed into a new therapeutic method to regulate tissues and cells.
  • the RANTES expression brings new hope for the treatment of chronic inflammation including lupus nephritis, arthritis, and alleviation of transplant rejection.
  • a first aspect of the invention relates to the use of miR-125a for the preparation of a medicament for the treatment of a inflammatory chemokine RANTES mediated disease.
  • the disease is selected from the group consisting of chronic inflammation, transplant rejection, and HIV infection.
  • the disease is systemic lupus erythematosus, lupus nephritis or arthritis.
  • the substance is selected from the group consisting of: a pharmaceutical composition comprising a miR-125a precursor, a mature or an analog thereof; an miR-125a expression agonist; a pharmaceutical composition comprising a miR-125a expression agonist; An expression vector expressing a miR-125a precursor, a mature body or an analog thereof; and a pharmaceutical composition comprising the expression vector.
  • the miR-125a expression agonist is selected from the group consisting of lipopolysaccharide and phytohemagglutinin.
  • the substance is a pharmaceutical composition comprising an miR-125a expression agonist.
  • a second aspect of the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a miR-125a precursor, a mature body, an miR-125a expression agonist, and/or an expression vector expressing a miR-125a mature, and pharmaceutically acceptable Carrier.
  • the pharmaceutical composition comprises a miR-125a mature.
  • the pharmaceutical composition comprises a miR-125a expression agonist. In a specific embodiment, the pharmaceutical composition comprises an expression vector that expresses the miR-125a mature.
  • the miR-125a expression agonist is selected from the group consisting of lipopolysaccharide and phytohemagglutinin.
  • the invention also provides a method of modulating expression of an inflammatory chemokine RANTES in a tissue or cell in vitro, the method comprising the steps of:
  • the miR-125a expression agonist is selected from the group consisting of lipopolysaccharide and phytohemagglutinin.
  • the modulation is inhibition of RANTES expression in the tissue or cell.
  • the present invention also provides a method of screening for a substance capable of inhibiting the expression of the inflammatory chemokine RANTES, the method comprising the steps of:
  • the tissue or the cell is administered with a miR-125a precursor, a mature body, an miR-125a expression agonist, and/or an expression vector expressing the miR-125a mature body without adding a candidate substance;
  • the method further includes:
  • the expression of KLF13 and/or RANTES in the tissue or cell is detected; and the test result is compared with the test result of step (2) to determine a substance capable of inhibiting the expression of the inflammatory chemokine RANTES.
  • the present invention also includes those obtained by the above screening methods. DRAWINGS
  • Figure 1 shows that the expression of miR-125a in peripheral blood mononuclear cells of 55 SLE patients and normal controls was significantly decreased (P ⁇ 0.001).
  • Figure 2 shows the results of human primary T lymphocytes transfected with miR-125a and control miRNA in vitro, and activated with Phytohemagglutinin (PHA-P) 8 hours later.
  • Figure A Primary T lymphocytes overexpressed miR-125a, 7 days after PHA activation, the expression of miR-125a was detected by RT-PCR.
  • Panel B shows that after 7 days, the difference in RNATES levels in the supernatant is detected, and Panel C shows the difference in expression of KLF13 at the mRNA level.
  • FIG. 3 shows that miR-125a inhibits endogenous KLF13 expression.
  • Panel A shows that overexpression of miR-125a in Hela cells inhibits the expression of endogenous KLF13 in Hela cells, whereas overexpression of other miRNAs (miR-98) does not affect KLF13 expression. Conversely, the expression of miR-125a was inhibited in Hela cells, and the expression of endogenous KLF13 was increased in Hela cells.
  • Panel B shows that miR-125a inhibits the expression of KLF13 in a concentration-dependent manner.
  • Panel C shows inhibition of miR-125a expression, and KLF13 expression is also increased in a concentration-dependent manner.
  • Figure 4 shows the luciferase reporter gene method and point mutation technique verification miR-125a directly acts on
  • KLF13 3 UTR.
  • A Bioinformatics prediction of KLF13 3, UTR binding to miR-125a 2 sites, clone KLF13 3'UTR 3 fragments to psi-CHECK-TM2, named Kl, ⁇ 2, ⁇ 3, where Kl and K2 are predicted In combination with the miR-125a site, K3 does not bind to the miR-125a site.
  • B Hela cells were co-transfected with K1 and miR-125a, and Kl luciferase activity was significantly decreased compared with control miRNA. When K1 and miR-98 were co-transfected, the K1 luciferase activity did not change significantly compared with the control miRNA.
  • K1 luciferase activity was significantly increased compared to control miRNA.
  • the K1 luciferase activity did not change significantly compared with the control miRNA.
  • miR-98 was used as a non-related miRNA control.
  • C Hela cells were co-transfected with K2 and miR-125a, and Kl luciferase activity was significantly decreased compared with control miRNA. Co-transfection of K2 and miR-125a inhibitors, K2 luciferase activity was significantly increased compared to control miRNA.
  • D Hela cells were co-transfected with K1 and miR-125a.
  • the K1 luciferase activity decreased in a concentration-dependent manner with miR-125a.
  • E indicates that the decrease in K2 luciferase activity is concentration-dependent with miR-125a.
  • F Hela cells were co-transfected with miR-125a and K1 mutants or k2 mutants, and the K1 mutant or k2 mutant luciferase activity did not change significantly compared to the control miRNA. Hela cells were co-transfected with K3 and miR-125a, and K3 luciferase activity was not significantly different from that of the control miRNA.
  • the present invention relates to the treatment of RANTES mediated diseases.
  • RANTES is a typical member of the C-C subfamily inflammatory chemokine, which can aggravate arthritis, atopic dermatitis, nephritis, colitis and other diseases [21].
  • RANTES receptor CCR5 is a co-receptor for HIV entry into target cells and plays an important role in HIV fusion and entry into target cells.
  • RANTES has become a new drug development for chronic inflammation, alleviation of transplant rejection, and HIV infection.
  • the "RANTES-mediated diseases” described in the present invention may include various chronic inflammation, mitigation of transplant rejection, HIV infection and the like.
  • the "RANTES-mediated disease” of the present invention includes arthritis, atopic dermatitis, nephritis, colitis, systemic lupus erythematosus, lupus nephritis, etc. [28-31].
  • the method of the present invention for treating a RANTES mediated disease comprises overexpressing miR-125a in a subject.
  • the precursor sequence of miR-125a is UGCCAGUCUCUAGGUCCCUGAGACCCUUUAA
  • the mature sequence of miR-125a is: UCCCUGAGACCCUUUAACCUGUGA (SEQ ID NO: 18).
  • a method of overexpressing miR-125a comprises: administering an expression agonist of miR-125a to promote expression thereof; and/or transfecting a miR-125a mature or an expression vector expressing a miR-125a mature body into a subject cell such that It is expressed in this cell.
  • Methods of transfection are well known in the art.
  • methods for introducing foreign DNA into mammalian cells can be broadly classified into two broad categories, biological and physicochemical.
  • Commonly used in physicochemical methods are calcium phosphate method, microinjection, electroporation, liposome-mediated transfection, and gene delivery technology using a polymer polymer as a carrier.
  • the biological method mainly uses the virus as a carrier to introduce foreign DNA into the cells by means of virus infection, and the retrovirus and adenovirus transfection systems are most commonly used.
  • the miR-125a of the invention is transfected using the LipofectamineaTM 2000 liposome transfection method.
  • Substances that promote expression of miR125a in cells include LPS (lipopolysaccharide) [32] and the like. Bazzoni F et al [32] found that LPS (lipopolysaccharide) stimulated the expression of miR-125a in primary monocytes significantly increased within 8 hours. This article is incorporated herein by reference. In addition, our experiments also found that PHA-P stimulated primary T cells 3-5 days later, miR-125a expression was significantly increased.
  • the vector pSuper.gfp/neo can be purchased from OligoEngine, and the miR-125a precursor fragment is PCR-derived from the human genome.
  • the primers used include the upstream primer CGTAGATCTGCCCCTCCCGATAT and the downstream primer ATGCTCGAGGTCAGGTTTCAGTT.
  • the plasmid vector and the PCR fragment were digested, and the restriction sites were Bgl 2 and Xho l.
  • the T4 ligase is ligated to the PCR fragment and the plasmid is digested. After the ligation product is transformed into competent E. coli, the plasmid insert sequence is sequenced, and the purified plasmid is amplified to obtain an expression vector containing miR-125a.
  • the "transfection of miR-125a or an expression vector containing miR-125a” also includes an analog which transfects miR-125a or an expression vector containing the same.
  • an analog of miR-125a refers to a substitution, deletion or addition of one or several bases in the sequence of miR-125a and is still capable of binding from position +247 to position +255 and/or position +1616 of KLF13 Short RNA sequence to position +1622.
  • Such analogs also include miRIDIANTM (designed and synthesized by Dharmacon), a microRNA mimetic that is a chemically modified double-stranded RNA nucleotide molecule.
  • miR- 125a mimetic sequence (double stranded): 5' UCCCUGAGACCCUUUAACCUGUGA 3'
  • microRNA inhibitor is a chemically modified single-stranded RNA nucleotide molecule that enhances the expression of endogenous microRNAs.
  • Control mimics and inhibitor sequences are designed based on nematode miRNAs, ensuring no homology by aligning with all human, mouse and rat genomic sequences and miRBase sequence database miRNA sequences; thus serving as human, mouse and rat Negative experimental control product for cells. It can be used as a target-free mimetic with no sequence-specific effects in miRNA experiments, and can effectively distinguish between mimetic activity and background effect. Designed and synthesized by Dharmacon.
  • miR-125a inhibitor sequence (single strand): 5'UCACAGGUUAAAGGGUCUCAGGGA 3'.
  • the invention also relates to the use of miR-125a for the preparation of a medicament for the treatment of a RANTES mediated disease.
  • the miR-125a comprises an analog thereof.
  • the substance comprises a pharmaceutical composition comprising a miR-125a precursor, a mature body or an analog thereof; an miR-125a expression agonist; a pharmaceutical composition comprising a miR-125a expression agonist; An expression vector for expressing a miR-125a mature or an analog thereof; and a pharmaceutical composition comprising the expression vector.
  • the substance also includes materials suitable for administration by different modes of delivery, such as those suitable for administration by transfection.
  • the pharmaceutical composition of the present invention may contain a miR-125a precursor, a mature body or an analog thereof, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition of the present invention may further comprise an expression agonist of miR-125a expression or an expression vector expressing a miR-125a precursor, a mature body or an analog thereof, and a pharmaceutically acceptable carrier.
  • compositions of the present invention also contain one or more other preparations dissolved or dispersed in a pharmaceutically acceptable carrier or excipient.
  • pharmaceutically acceptable refers to molecular entities and compositions that, when used in animals, such as humans, do not produce side effects, allergies or other adverse effects.
  • Those skilled in the art will be aware of the preparation of pharmaceutical compositions containing at least one polypeptide, and in some embodiments one or more other active ingredients, as disclosed herein, for example, see Remington Drugs. Science, 18th ed., Mack Printing Company, 1990 (incorporated in this article).
  • administration to animals may be understood to be consistent with sterile, pyrogen-free, overall safety and purity standards.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (eg, antibacterial, antifungal agents;), isotonic agents, Absorbing retardants, salts, preservatives, drugs, drug stabilizers, binders, excipients, disintegrators, lubricants, sweeteners, flavoring agents, dyes, and the like, and combinations thereof, which are As known to the ordinarily skilled artisan (see, for example, Remington's Pharmaceutical Sciences, 18th ed., Mack Printing Company, 1990, pages 1289-1329, incorporated herein by reference); It is considered to be useful in therapeutic or pharmaceutical compositions, except in conventional carriers which are incompatible with the active ingredient.
  • composition of the invention is determined by physical and physiological factors such as body weight, severity of the disease, type of disease to be treated, original and common therapeutic measures, subject's specific disease and route of administration. .
  • the physician responsible for administration will determine the concentration of the active ingredient in the composition and the appropriate dosage of the individual subject.
  • the pharmaceutical compositions may contain, for example, at least about 0.001% by weight of active ingredient.
  • the pharmaceutical composition may contain, for example, 0.01 to 99.9% by weight, 0.01 to 50% by weight, 0.01 to 10% by weight, and the like, of the polypeptide of the present invention.
  • the concentration of the polypeptide in the administered pharmaceutical composition may range from 0.01 to 5 mM, such as from 0.01 to 3 mM, 0.05 to 1 lm.
  • the mode of administration is conventional and can be determined by the clinician based on the specific circumstances of the patient. For example, it can be injected directly into the lateral ventricle or the subarachnoid space.
  • compositions of the present invention may contain various antioxidants to prevent oxidation of one or more components.
  • Preservatives can be used to prevent the action of microorganisms, such as various antibacterial and antifungal agents, including but not limited to p-hydroxyphenylpropionates (such as methyl p-hydroxyphenylpropionate, propyl p-hydroxyphenylpropionate), chlorine Butanol, phenol, sorbic acid, thimerosal or a combination thereof.
  • the carrier can be a solvent or dispersion medium including, but not limited to, water, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.;), lipids (such as triglycerides, vegetable oils, liposomes) and combinations thereof.
  • polyols e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.
  • lipids Such as triglycerides, vegetable oils, liposomes
  • a coating such as lecithin
  • a carrier such as a liquid polyol or a lipid dispersion
  • a surfactant such as hydroxypropylcellulose
  • composition of the present invention can be formulated by methods conventional in the art.
  • composition must be stable under the conditions of manufacture and storage to prevent contamination by microorganisms such as bacteria and fungi. It should be noted that endotoxin contamination should be kept to a minimum and within safe levels, for example less than 0.5 ng/mg protein.
  • the present invention also provides a method of regulating the expression of inflammatory chemokine RANTES in tissues or cells in vitro, and a method of screening for a substance capable of inhibiting the expression of the inflammatory chemokine RANTES.
  • a method of screening for a substance capable of inhibiting the expression of the inflammatory chemokine RANTES In the case of knowing the role of miR-125a in the expression of the inflammatory chemokine RANTES and the expression of KLF13, it is not difficult in the art to regulate the expression of inflammatory chemokine RANTES in tissues or cells in vitro, and This principle screens for substances that inhibit the expression of the inflammatory chemokine RANTES. The adjustment includes up- and down-down (suppression).
  • the tissue or cell includes any expression that can be expressed by itself
  • Tissues or cells of KLF13 and/or RANTES are well known in the art.
  • the invention also includes a kit comprising miR-125a or a mature thereof, a coding sequence thereof, an expression vector comprising the coding sequence, and the like, optionally including various suitable for transfecting the active ingredient Reagents into the cells and instructions.
  • a kit comprising miR-125a or a mature thereof, a coding sequence thereof, an expression vector comprising the coding sequence, and the like, optionally including various suitable for transfecting the active ingredient Reagents into the cells and instructions.
  • miRNAs reverse transcription primers and Real-time PCR reaction probes were purchased directly from ABI (Applied Biosystems).
  • the target gene quantification primers used ribosomal protein L13a (RPL13A) as an internal reference gene, and the full-length cDNA of human KLF13 gene was searched in the NCBI database.
  • the primers designed to amplify the template by Oligo 6.71 software are as follows:
  • KLF13 sense strand 5 CCGCAGAGGAAGCACAA3, (SEQ ID NO: 1), antisense strand 5'CTTCTTCTCGCCCGTGT3' (SEQ ID NO: 2);
  • RANTES sense strand 5 AGTCGTCTTTGTCACCCGAAA3, (SEQ ID NO: 3), antisense strand 5, AGCTCATCTCCAAAGAGTTGATGTAC3, (SEQ ID NO: 4).
  • the RPL13A primer was derived from the sequence reported by the Quantitative PCR Primer Database (QPPD) website, sense strand 5 'CCTGGAGGAGAAGAGGAAAGAGA3 ' (SEQ ID NO: 5), antisense strand 5 ' TTGAGGACCTCTGTGT ATTTGTC AA3 ' (SEQ ID NO: 6) , for Shanghai Company synthesis.
  • QPPD Quantitative PCR Primer Database
  • MMLV 0.2uK 10 X Buffer 0.3 ul, RNase inhibitor 0.02 ul, dd3 ⁇ 4O (no RNase) 0.45 ul, primer lul and RNA lul, reverse transcription reaction conditions were 16 V, 30 min; 42 ° C, 30 min; 85 ° C, 5 min.
  • Real-time PCR Real-time fluorescent quantitative PCR was performed on an ABI Prism 7900 sequencer (Applied Biosystems).
  • Kl, ⁇ 2, ⁇ 3 Three fragments of the 3'UTR of the KLF13 gene are amplified by genomic DNA.
  • the cleavage sites Not I and Xho I were inserted into the psi-check plasmid (purchased from Promega) (named Kl, ⁇ 2, ⁇ 3).
  • Kl and ⁇ 2 contain a KLF13 gene 3'UTR and miR-125a binding site gene fragment, and all plasmids were sequenced and identified.
  • primers obtained from Promega
  • K1 upstream bow 5'CCGCTCGAGAGCAGCCCCACCATCAG3' (SEQ ID NO: 7) ; downstream primer 5, ATTTGCGGCCGCCGGGCAAACTCGGAACT3, (SEQ ID NO: 8);
  • K2 upstream primer 5 CCGCTCGAGCTGTGTGAGCGGCTGTG3, (SEQ ID NO: 9); downstream bow I substance 5'ATTTGCGGCCGCTCCCACCACAGCAGAAAC3' (SEQ ID NO: 10);
  • Mutant Kl, ⁇ 2 vector ligation fragment Four-base sequence complementary to mir-125a, using the following primers:
  • upstream primer 5 'GGGGATTGGGTGGACTGTCCGTATCTTGCCAGA GA3' SEQ ID NO: 15
  • downstream primer 5 TCTCTGGCAAGATACGGACAGTCC ACCCAATCCCC3' (SEQ ID NO: 16).
  • Human primary mononuclear cells are derived from healthy blood donors in the Shanghai Blood Center.
  • Human peripheral blood T lymphocytes were positively sorted by magnetic bead method (Miltenyi Biotec CD3 MicroBeads), cultured in 1640 nutrient solution of 10% calf serum, and T lymphocytes were stimulated with PHA (sigma) 20 ⁇ ⁇ / ⁇ 1 for 7 days. .
  • Hela cells were obtained from the American Collection of Cultures (ATCC), purchased from the Institute of Cell Research, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, and cultured in DMEM supplemented with 10% calf serum plus 100 U/ml penicillin-streptomycin nutrient solution, 37 Incubate at °C 5% CO 2 and change solution once every 2 to 3 days. 6.
  • ATCC American Collection of Cultures
  • DMEM fetal bovine serum
  • penicillin-streptomycin nutrient solution 37 Incubate at °C 5% CO 2 and change solution once every 2 to 3 days.
  • Hela cells were cultured in 96-well plates. When cells were fused at 80%, the KLF13 3'UTR reporter vector and miR-125a matured were co-transfected with LipofectamineaTM 2000 liposome (Invitrogen). The cells were transfected for 24 hours. The dual luciferase reporter assay system detects luciferase activity. The KLF13 3'UTR mutant was transfected and tested for luciferase activity in the same manner.
  • Transfection was carried out according to the interferin siRNA transfection reagent (polyplus) method.
  • the MiR-125a mimic and inhibitor and the corresponding negative control were from Thermo SCIENTIFIC Dharmacon.
  • T lymphocytes of miR-125a mimic and control miRNA mimic were transfected respectively. After 7 days of PHA activation, the level of RANTES in the supernatant of the cells was detected by ELISA (Xitang Bio). 9. Data analysis
  • the expression level of miR-125a in SLE patients is significantly lower than that of normal people.
  • miR-125a regulates T cell production of inflammatory chemokines RANTES
  • miR-125a directly negatively regulates the expression of KLF13
  • the effect of miR-125a on the luciferase activity of the ⁇ 3 vector was not significant (see Figure 4F).
  • the effect of miR-125a on the luciferase activity of Kl and ⁇ 2 vectors was concentration dependent (see Figure 4D and ⁇ ⁇ ).
  • the site of miR-125a on the K1 and ⁇ 2 vectors was mutated by site-directed mutagenesis, and then the effect of miR-125a on luciferase activity was analyzed. It was found that the effect of miR-125a on Kl and ⁇ 2 mutants was not obvious (see Figure 4F).
  • miRNA plays an important role in the occurrence and development of many diseases in humans. Unlike previous regulatory features with or without miRNAs, miRNAs regulate the target genes quantitatively. This feature is a powerful regulation of siRNA single target or unmatched by some molecular antagonists and agonists. This demonstrates the great prospects of miRNAs for clinical applications.
  • the present application found that miR-125a is down-regulated in SLE patients, and in vitro experiments have found that overexpression of miR-125a in T cells can reduce the expression level of the inflammatory chemokine RANTES.
  • miR-125a expression signal can be used as an important biomarker for clinical diagnosis of SLE; and may serve as a new target for drug intervention. The specific intervention level of miR-125a can be developed into a new therapeutic method to regulate tissues and cells.
  • the RANTES expression brings new hope for the treatment of chronic inflammation including lupus nephritis, arthritis, and alleviation of transplant rejection. references
  • Li, Q. J. et al. miR 181a is an intrinsic modulator of T cell sensitivity and selection. Cell 129, 147-161 (2007).
  • MIP-1 - as the major HIV- suppre s si vef actor s produced by CD8_ T cells. 1995. Science 270: 1811-1815.

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Description

调节 RANTES表达的 miR-125a、 其组合物及其用途 技术领域
本发明涉及调节 RANTES表达的 miR-125a、 其组合物及其用途。 背景技术
系统性红斑狼疮（systemic lupus erythematosus, SLE)是一种常见的累及多脏 器的慢性炎症性自身免疫病， 其临床表现复杂多样， 累积全身多系统的炎症反应， 但其病因复杂， 发病的分子机制尚不明确， 炎症是其主要的病理改变 [1]。 目前治 疗主要是糖皮质激素、免疫抑制剂等非特异性炎症抑制剂，尚缺乏特异的治疗手段， 无法从根本上提高疾病防治水平 [2]。
目前已有大量的相关研究报道炎症性趋化因子在 SLE 发病中发挥重要作用 [11-14]， 特别是在肾炎发病中发挥重要作用。 RANTES是一种典型的 C-C亚族成 员炎症性趋化因子， 诱导 T 细胞、 树突状细胞、 嗜酸性细胞等多种免疫细胞向炎 症部位浸润，在器官炎症反应中发挥重要作用 [18-20]。在病理情况下可以增强细胞 毒性、 招募免疫细胞、 增强炎症过程， 加重关节炎、 异位性皮炎、 肾炎、 结肠炎等 疾病 [21]。 多项研究表明 RANTES 在组织中表达水平与疾病的活动性相关， 可以 作为疾病活动的生物标志物。 更值得关注的是 RANTES的受体 CCR5是 HIV进入 靶细胞的辅助受体， 在 HIV病毒融合和进入靶细胞过程中起重要作用。 RANTES 作为药物靶点， 已成为慢性炎症、减轻移植排斥反应、 HIV感染等疾病新药研发的 热点 [22-27]。
微小 RNA(microRNA， miRNA)是动植物体内普遍存在的内源性表达的一类长 度约 21— 25nt的非编码蛋白质单链小分子 RNA， 通过核酸序列互补性结合到特定 的靶 mRNA上来调节靶 mRNA翻译或降解靶 mRNA，是一种起转录后负调控作用 的分子 [3， 4],参与生命过程中的一系列重要进程，包括发育、 细胞增殖、 凋亡、 分 化以及病毒感染和癌症等多种生物学作用 [5-7]。
miRNAs是近年来科学研究的一项重大突破，不仅有助于人类对基因的自身调 节机制更全面深刻地认识， 也为疾病发生、 预防及治疗研究提供新的思路。众多研 究表明 miRNA参与了免疫系统调节作用的各个环节， 包括固有免疫、 适应性免疫 反应， 免疫细胞发育等 [8-10]， 对免疫系统进行精细的调节。 但是 miRNA与 SLE 炎症反应的关系至今还未有相关报道。 发明内容
本申请人发现 miR-125a在 SLE患者中表达降低， 体外实验发现在 T细胞中 过表达 miR-125a可以降低炎症性趋化因子 RANTES的表达水平。 miR-125a表达 信号可作为 SLE临床诊断的一个重要生物标志物； 并可能作为一个新的药物干预 靶点， 特异性干预 miR-125a的表达水平可发展为新的治疗手段， 进而调节组织、 细胞的 RANTES表达， 为治疗包括狼疮肾炎、 关节炎等慢性炎症、 减轻移植排斥 反应等疾病带来新的希望。
因此， 本发明第一方面涉及 miR-125a在制备治疗炎症性趋化因子 RANTES 介导的疾病用的物质中的用途。
在一个具体实施方式中，所述疾病选自慢性炎症、移植排斥反应和 HIV感染。 在一个具体实施方式中， 所述疾病是系统性红斑狼疮、 狼疮肾炎或关节炎。 在一个具体实施方式中， 所述物质选自： 含有 miR-125a前体、 成熟体或其类 似物的药物组合物； miR- 125a表达激动剂； 含有 miR-125a表达激动剂的药物组合 物； 表达 miR-125a前体、 成熟体或其类似物的表达载体； 和含所述表达载体的药 物组合物。
在一个具体实施方式中，所述 miR-125a表达激动剂选自脂多糖和植物血球凝 聚集素。
在一个具体实施方式中， 所述物质为含有 miR-125a表达激动剂的药物组合 物。
本发明第二方面提供一种药物组合物，该组合物含有 miR-125a前体、成熟体、 miR-125a表达激动剂、和 /或表达 miR-125a成熟体的表达载体， 以及药学上可接受 的运载体。
在一个具体实施方式中， 所述药物组合物含有 miR-125a成熟体。
在一个具体实施方式中， 所述药物组合物含有 miR-125a表达激动剂。 在一个具体实施方式中， 所述药物组合物含有表达 miR-125a成熟体的表达载 体。
在一个具体实施方式中，所述 miR-125a表达激动剂选自脂多糖和植物血球凝 聚集素。
本发明还提供一种体外调节组织或细胞的炎症性趋化因子 RANTES表达的方 法， 所述方法包括以下步骤：
( 1 ) 提供所述组织或细胞； 和
(2) 向所述组织或细胞施与 miR-125a前体、 成熟体、 miR- 125a表达激动剂、 和 /或表达 miR-125a成熟体的表达载体， 从而调节所述组织或细胞中的 RANTES 表达。
在一个具体实施方式中，所述 miR-125a表达激动剂选自脂多糖和植物血球凝 聚集素。
在一个具体实施方式中，所述调节为抑制所述组织或细胞中的 RANTES表达。 本发明还提供一种筛选能抑制炎症性趋化因子 RANTES 的表达的物质的方 法， 该方法包括以下步骤：
( 1 ) 提供组织或细胞；
(2)作为试验组， 向所述组织或细胞施与 miR-125a前体、成熟体、 miR- 125a 表达激动剂、 和 /或表达 miR-125a成熟体的表达载体， 和候选物质； 和
(3 )作为对照组， 向所述组织或细胞施与 miR-125a前体、成熟体、 miR-125a 表达激动剂、 和 /或表达 miR-125a成熟体的表达载体， 而未加入候选物质；
(4)检测试验组和对照组中 KLF13和 /或 RANTES的表达， 其中， 与对照组 相比， 试验组中能使所述组织或细胞中的 KLF13和 /或 RANTES的表达降低的候 选物质确定为能抑制炎症性趋化因子 RANTES的表达的物质。
在一个具体实施方式中， 所述方法还包括：
(5 ) 向所述组织或细胞施与 miR-125a前体、 成熟体、 miR- 125a表达激动剂、 和 /或表达 miR-125a成熟体的表达载体， 和脂多糖或植物血球凝聚集素； 检测此 组织或细胞中的 KLF13和 /或 RANTES的表达； 和将检测结果与步骤 （2) 的检测 结果进行比较， 以确定能抑制炎症性趋化因子 RANTES的表达的物质。
本发明也包括由上述筛选方法筛选得到的物质。 附图说明
图 1显示， 55个 SLE患者和正常对照组外周血单个核细胞 miR-125a表达量 明显下降 (P<0.001)。
图 2显示人原代 T淋巴细胞在体外分别转染 miR-125a和对照 miRNA, 8小时 后， 用植物血球凝聚集素 (Phytohemagglutinin, PHA-P)激活所得的结果。 图 A: 原代 T淋巴细胞过表达 miR-125a， PHA激活 7天后， RT-PCR检测 miR-125a的表 达。图 B:表示 7天后，检测细胞上清 RNATES水平差异，图 C表示检测细胞 KLF13 在 mRNA水平表达差异。
图 3显示 miR-125a抑制内源性 KLF13表达。 图 A表示在 Hela细胞过表达 miR-125a， Hela细胞内源性 KLF13表达受抑制， 而过表达其它 miRNA (miR-98) 则不影响 KLF13的表达。 反之， 在 Hela细胞抑制 miR- 125a表达， Hela细胞内源 性 KLF13表达增高。 图 B表示 miR-125a浓度依赖性抑制 KLF13的表达。 图 C表 示抑制 miR-125a的表达， KLF13表达增高也呈浓度依赖性。
图 4 显示荧光素酶报告基因方法和点突变技术验证 miR-125a 直接作用于
KLF13 3，UTR。 （A)生物信息学预测 KLF13 3，UTR与 miR-125a结合的 2个位点， 克隆 KLF13 3'UTR 3个片段至 psi-CHECK-TM2， 命名 Kl、 Κ2、 Κ3， 其中 Kl和 K2预测有结合 miR-125a位点， K3没有结合 miR- 125a位点。 （B)Hela细胞共转染 K1和 miR-125a， Kl荧光素酶活性与对照 miRNA相比， 明显下降。 K1与 miR-98 共转染则 K1荧光素酶活性与对照 miRNA相比无明显变化。共转染 K1和 miR-125a 抑制物， K1荧光素酶活性与对照 miRNA相比， 明显上升。 K1与 miR-98共转染 则 K1荧光素酶活性与对照 miRNA相比无明显变化。 miR-98作为非相关 miRNA 对照。（C)Hela细胞共转染 K2和 miR-125a， Kl荧光素酶活性与对照 miRNA相比， 明显下降。共转染 K2和 miR-125a抑制物， K2荧光素酶活性与对照 miRNA相比， 明显上升。 （D)Hela细胞共转染 K1和 miR-125a， 与对照 miRNA相比， K1荧光素 酶活性下降与 miR-125a呈浓度依赖性。（E)表示 K2荧光素酶活性下降与 miR-125a 呈浓度依赖性。 （F)Hela细胞共转染 miR-125a和 K1突变体或 k2突变体， K1突变 体或 k2突变体荧光素酶活性与对照 miRNA相比， 无明显变化。 Hela细胞共转染 K3和 miR-125a， K3荧光素酶活性与对照 miRNA相比， 无明显变化。 具体实施方式
本发明涉及 RANTES介导的疾病的治疗。
RANTES是一种典型的 C-C亚族成员炎症性趋化因子， 它可加重关节炎、 异 位性皮炎、 肾炎、 结肠炎等疾病 [21]。 此外， RANTES的受体 CCR5是 HIV进入 靶细胞的辅助受体， 在 HIV病毒融合和进入靶细胞过程中起重要作用。 RANTES 作为药物靶点， 已成为慢性炎症、减轻移植排斥反应、 HIV感染等疾病新药研发的 ¾¾点。
因此， 本发明所述的 "RANTES 介导的疾病"可包括各种慢性炎症、 减轻移 植排斥反应、 HIV感染等疾病。 在具体实施方式中， 本发明所述的 "RANTES 介 导的疾病"包括关节炎、 异位性皮炎、 肾炎、 结肠炎、 系统性红斑狼疮、 狼疮肾炎 等 [28-31]。
本发明治疗 RANTES介导的疾病的方法包括过表达对象体内的 miR-125a。 miR-125a 的前体序列为 UGCCAGUCUCUAGGUCCCUGAGACCCUUUAA
UGGCC ( SEQ ID NO: 17) ；
miR- 125a的成熟体序列为： UCCCUGAGACCCUUUAACCUGUGA ( SEQ ID NO: 18) 。
过表达 miR-125a的方法包括： 给予 miR- 125a的表达激动剂， 以促进其表达； 和 /或转染 miR-125a成熟体或表达 miR-125a成熟体的表达载体到对象细胞中， 以 使其在该细胞中表达。
转染的方法是本领域周知的。 目前， 将外源 DNA导入哺乳动物细胞的方法大 致可分为两大类，即生物方法和物理化学方法。物理化学方法中常用的有磷酸钙法、 显微注射、 电穿孔、脂质体介导的转染， 以及以高分子聚合物为载体的基因传递技 术。 生物方法则主要是以病毒作为载体,通过病毒感染的方式将外源 DNA 导入到 细胞中， 其中以逆转录病毒及腺病毒转染系统最为常用。在本发明一个优选实施例 中， 使用 LipofectamineaTM2000脂质体转染方法转染本发明的 miR-125a。
促进 miR125a在细胞内表达的物质包括 LPS (脂多糖） [32]等。 Bazzoni F 等 [32]发现 LPS (脂多糖） 剌激原代单核细胞 miR-125a表达在 8小时内明显增高。 本文将该文以引用的方式纳入参考。 此外， 我们实验也发现 PHA-P剌激原代 T细 胞 3-5天后， miR-125a表达明显增高。
构建含有 miR-125a的表达载体也是本领域周知的。 例如， 可从 OligoEngine 公司购得载体 pSuper.gfp/neo， 从人基因组中 PCR得到 miR-125a前体片段， 使用 的 引 物包括上游 引 物 CGTAGATCTGCCCCTCCCGATAT ， 下游 引 物 ATGCTCGAGGTCAGGTTTCAGTT。 双酶切质粒载体和 PCR 片段， 酶切位点为 Bgl 2 和 Xho l。 T4连接酶连接 PCR片段和酶切质粒。 连接产物转化感受态大肠 杆菌后， 测序质粒插入序列， 扩增纯化质粒， 从而可获得含有 miR-125a的表达载 体。
本发明所述 "转染 miR-125a 或含有 miR-125a 的表达载体" 也包括转染 miR-125a的类似物或含有该类似物的表达载体。 在本文中， miR- 125a的类似物指 在 miR-125a的序列中取代、 缺失或添加一个或几个碱基且仍能够结合 KLF13第 +247位到第 +255位和 /或第 +1616位到第 +1622位的短 RNA序列。 本领域技术人 员知晓哪个位置上的突变能产生仍能与 LKF13的所述位置特异性结合的 miR-125a 类似物。 这样的类似物也包括 miRIDIAN™ (Dharmacon公司设计并合成） ， 它是 一种 microRNA模拟物， 是经化学修饰的双链 RNA核苷酸分子。
miR- 125a模拟物序列 (双链)： 5' UCCCUGAGACCCUUUAACCUGUGA 3'
3 AGGGACUCUGGGAAAUUGGACACU 5' microRNA抑制物是化学修饰的提高其稳定性的单链 RNA核苷酸分子， 用于 抑制内源性 microRNA的表达。对照模拟物和抑制物序列基于线虫 miRNA而设计， 通过与所有的人、小鼠和大鼠基因组序列以及 miRBase序列数据库 miRNA序列进 行比对确保无同源性； 从而作为人、 小鼠和大鼠细胞的阴性实验对照产品。 在 miRNA实验中可作为无序列特异性效应的无靶模拟物， 可将模拟物活性和本底效 应有效区分。 均为 Dharmacon公司设计并合成。
miR-125a抑制物序列（单链） ： 5'UCACAGGUUAAAGGGUCUCAGGGA 3'。 本发明也涉及 miR-125a在制备治疗 RANTES介导的疾病用的物质中的用途。 在一个具体实施方式中， 所述 miR-125a包括其类似物。
在一个具体实施方式中， 所述物质包括含有 miR- 125a前体、 成熟体或其类似 物的药物组合物； miR- 125a表达激动剂；含有 miR-125a表达激动剂的药物组合物； 表达 miR-125a成熟体或其类似物的表达载体； 和含所述表达载体的药物组合物。 所述物质也包括适用于通过不同递送方式给予的物质，例如适用于通过转染给予的 物质。
本发明的药物组合物可含有 miR-125a前体、 成熟体或其类似物， 和药学上可 接受的运载体。 本发明的药物组合物还可含有 miR-125a 表达激动剂或表达 miR-125a前体、 成熟体或其类似物的表达载体， 和药学上可接受的运载体。
本发明药物组合物中还含有溶于或分散于药学上可接受的运载体或赋形剂中 的一种或多种其它制剂。 短语"药学上可接受的"是指当用于动物时， 例如人， 不 会产生副作用、过敏或其它不良反应的分子实体和组合物。通过本文所公开的内容， 本领域技术人员将会知道含有至少一种多肽、在某些实施方式中还含有一种或多种 其它活性成分的药物组合物的制备， 例如见 《雷明顿药物科学》 第 18 版， Mack Printing Company, 1990 (纳入本文参考文献） 。 另外， 对动物 (；如人)给药， 可以理 解的是制品应符合无菌、 无致热原， 总体安全和纯度标准。
本文使用的 "药学上可接受的运载体"包括任何和所有的溶剂、 分散介质、 包衣剂、 表面活性剂、 抗氧化剂、 防腐剂 (如抗菌剂， 抗真菌剂；)、 等渗剂、 吸收延 缓剂、 盐类、 防腐剂、 药物、 药物稳定剂、 粘合剂、 赋形剂、 崩解剂、 润滑剂、 增 甜剂、 调味剂、 染料等物质和它们的组合， 这是本领域普通技术人员知道的 (见例 如， 《雷明顿药物科学》 第 18版， Mack Printing Company, 1990， 1289-1329页， 纳入本文参考文献；)。 除了与活性成分不相容的常规运载体外， 认为均可用于治疗 或药物组合物中。
给予患病动物本发明组合物的实际剂量由物理和生理因素， 如体重、 疾病严 重性、待治疗疾病的类型、 原有和共同的治疗措施、受试者的特发病和给药途径所 决定。 负责给药的医生将决定组合物中活性成分的浓度和受试者个体的合适剂量。
某些实施方式中， 药物组合物可含有， 例如至少约 0.001重量％的活性成分。 在其它实施方式中， 药物组合物可含有例如 0.01-99.9 重量％、 0.01-50 重量％、 0.01-10重量％等的本发明多肽。 在一个具体实施方式中， 给予的药物组合物中多 肽的浓度可为 0.01-5mM， 例如 0.01-3mM、 0.05-lmM。 给药的方式是常规的， 可 由临床医师根据患者的具体情况来确定。例如，可直接注射入侧脑室或蛛网膜下腔。
本发明药物组合物可含有各种抗氧化剂以防止一种或多种组分的氧化。 此外 可用防腐剂来预防微生物的作用，如各种抗菌和抗真菌剂，包括但不仅限于对羟基 苯丙酸酯 (如甲基对羟基苯丙酸酯、丙基对羟基苯丙酸酯)、氯丁醇、苯酚、 山梨酸、 硫柳汞或其组合。
在该组合物是液体形式的实施方式中， 运载体可以是溶剂或分散介质， 包括 但不限于:水、 多元醇 (如甘油、 丙烯二醇、 液态聚乙二醇等；)、 脂质 (如甘油三酯、 植物油、 脂质体)和它们的组合。 例如， 可通过采用包衣如卵磷酯； 通过用运载体 如液体多元醇或脂分散维持所需颗粒大小； 用表面活性剂如羟丙基纤维素； 或这些 方法的组合来维持适当的流动性。许多情况下， 优选包含等渗剂如糖、氯化钠或其 组合。
可采用本领域常规的方法配置本发明的药物组合物。
该组合物在制备和贮存条件下必须稳定， 防止微生物如细菌和真菌的污染。 需知应将内毒素的污染控制到最低， 处在安全水平内， 例如低于 0.5ng/mg蛋白质。
本发明还提供一种体外调节组织或细胞的炎症性趋化因子 RANTES表达的方 法， 以及筛选能抑制炎症性趋化因子 RANTES 的表达的物质的方法。 在知晓 miR-125a在炎症性趋化因子 RANTES的表达以及 KLF13 的表达中的作用的情况 下， 本领域技术不难在此基础上体外调节组织或细胞的炎症性趋化因子 RANTES 表达， 以及根据此原理筛选能抑制炎症性趋化因子 RANTES的表达的物质。 所述 调节包括上调和下调（抑制） 。 上述方法中， 所述组织或细胞包括任何本身能表达
KLF13和 /或 RANTES的组织或细胞， 这是本领域周知的。
本发明也包括一种试剂盒， 其含有 miR-125a或其成熟体、 其编码序列、 含所 述编码序列的表达载体等活性成分，任选地包括各种适用于将所述活性成分转染入 细胞中的试剂以及指导说明书。 本发明的实施除非另外说明， 将使用本领域技术人员已知的化学、 生物化学、 重组 DNA技术和免疫学的常规方法。 这些技术在文献中有完整的解释。 参见， 如 《基础免疫学》 （Fundamental Virology) ， 第二版， 第 I和 II 卷 (B.N.Fields 和 D.M.Knipe编）； 《实验免疫学手册》 （Handbook of Experimental Immunology) , 第 I-IV卷 (D.M.Weir和 C.C.Blackwell编， Blackwell Scientific Publications); T.E. Creighton , 《蛋白质： 结构和分子特性》 （Proteins: Structures and Molecular properties)(W.H. Freeman and Company, 1993)； A.L. Lehninger , 《生物化学》 (Biochemistry) (Worth Publishers, Inc.最新版)； Sambrook等， 《分子克隆： 实验 室手册》 （Molecular Cloning: a Laboratory Manual) ,第二版， 1989; 《酶学方法》 (Methods in Engymology) (S.Colowick禾口 N. Kaplan编， Academic Press, Inc.)。 一. 实验方法及材料
1. 研究对象
SLE患者共 55例， 均为上海仁济医院风湿免疫科门诊与病房病人， 诊断均符 合美国风湿病学会 SLE分类标准推荐的 11项标准中的至少 4 项； 正常对照组 40 例， 来自上海市瑞金医院体检正常人群。 各组性别和年龄差异无统计学意义， 研究 组与对照组每人采外周静脉 ACD抗凝血 5ml。
2. 引物和探针设计
miRNAs 逆转录引物和 Real-time PCR 反应探针直接从 ABI ( Applied Biosystems公司） 购买获得。 靶基因定量引物以核糖体蛋白 L13a (RPL13A) 作为 内参照基因，检索 NCBI数据库査到人类 KLF13基因 cDNA全长序列,以 Oligo 6.71 软件设计扩增模板的引物如下：
KLF13 正义链 5，CCGCAGAGGAAGCACAA3， ( SEQ ID NO: 1 ) ， 反义链 5'CTTCTTCTCGCCCGTGT3' ( SEQ ID NO: 2) ；
RANTES正义链 5，AGTCGTCTTTGTCACCCGAAA3， ( SEQ ID NO: 3 ) ， 反 义链 5，AGCTCATCTCCAAAGAGTTGATGTAC3， ( SEQ ID NO: 4) 。
引物设计好后通过 BLAST分析 (www.ncbi.nlm.nih.gov/BLAST；)避免扩增的目 的片段序列存在非特异性， 由上海生工公司合成。 RPL13A引物来源于 Quantitative PCR Primer Database ( QPPD ) 网 站 所 报 导 序 列 ， 正 义 链 5 'CCTGGAGGAGAAGAGGAAAGAGA3 ' ( SEQ ID NO: 5 ) ， 反 义 链 5 ' TTGAGGACCTCTGTGT ATTTGTC AA3 ' (SEQ ID NO: 6) ， 为上海生工公司合 成。
3. 实时荧光定量 PCR (Real-time PCR) 应用酚氯仿法抽提 Trizol (Invitrogen 公司产品） 总 RNA， 得到的 RNA用毛 细管电泳 （NanoDrop Specthophotometer) 鉴定其完整性， 紫外分光光度计测定其 浓度。 总 RNA—部分用做 oligo dT逆转录， 使用 PrimeScript RT reagent Kit逆转 录酶试剂盒（Takala公司产品）逆转录为 cDNA; 另一部分用做 miRNAs特异性引 物逆转录， 加样体系为 dNTP 0.03ul、 MMLV 0.2uK 10 X Buffer 0.3 ul、 RNase 抑 制剂 0.02ul、 dd¾O(无 RNase) 0.45ul、 引物 lul和 RNA lul， 逆转录反应条件为 16V， 30min; 42°C， 30min; 85°C， 5min。 实时荧光定量 PCR (Real-time PCR) 在 ABI Prism7900测序仪（Applied B iosystems公司）上进行实时荧光定量 PCR操 作。
4. 报告基因载体和突变质粒构建
KLF13基因 3'UTR的 3个片段， 由基因组 DNA扩增。酶切位点 Not I 和 Xho I插入 psi-check质粒 （购于 Promega公司） （命名 Kl， Κ2， Κ3 ) 。 Kl、 Κ2包含 KLF13基因 3'UTR与 miR-125a的结合位点基因片段，所有质粒都测序鉴定。使用 以下引物：
K1上游弓 I物 5'CCGCTCGAGAGCAGCCCCACCATCAG3'(SEQ ID NO:7);下 游引物 5，ATTTGCGGCCGCCGGGCAAACTCGGAACT3，(SEQ ID NO: 8)；
K2上游引物 5，CCGCTCGAGCTGTGTGAGCGGCTGTG3，(SEQ ID NO: 9);下 游弓 I物 5'ATTTGCGGCCGCTCCCACCACAGCAGAAAC3'(SEQ ID NO: 10)；
K3上游引物 5，CCGCTCGAGTCTGCTGTGGTGGGAACT3，)SEQ ID NO: 11)； 下游引物 5，ATTTGCGGCCGCTGGAGGCTGTTTCAGGAG3，(SEQ ID NO: 12)； 点突变系统使用 KOD Plus酶 （TOYOBO公司） 。 突变 Kl、 Κ2载体连接片 段与 mir-125a互补结合的 4个碱基序列， 使用以下引物：
K1突变体上游弓 I物 5 'TGTTGAACCCCCTTTCTCTCCCATGGACACGTTTC3 ' ( SEQ ID NO: 13 ) ； 下游弓 I物 5 ' GAAACGTGTCC ATGGGAGAGAAAGGG GGTTCAACA3' ( SEQ ID NO: 14) ；
k3 突变体上游引物 5 'GGGGATTGGGTGGACTGTCCGTATCTTGCCAGA GA3' SEQ ID NO: 15 ) ； 下游引物 5，TCTCTGGCAAGATACGGACAGTCC ACCCAATCCCC3' ( SEQ ID NO: 16) 。 5. 细胞分选和培养
人原代单个核细胞 （PBMC ) 来自上海市血液中心健康献血者。 人外周血 T 淋巴细胞用磁珠法 （Miltenyi Biotec CD3 MicroBeads) 阳性分选出， 培养于 10%小 牛血清的 1640营养液中， T淋巴细胞用 PHA ( sigma) 20μδ/ηι1剌激 7天。 Hela细 胞来源于来源于美国菌种保藏中心 （ATCC ) ， 购至中国科学院上海生命科学研究 院细胞所， 培养于 10%小牛血清的 DMEM加 100U/ml青霉素 -链霉素营养液中， 37°C 5% CO2孵育， 2〜3天换液一次。 6. 荧光素酶报告基因系统和突变系统
Hela细胞培养于 96孔板， 细胞 80%融合时， 用 LipofectamineaTM2000脂质 体 （Invitrogen公司） 转染方法共转染 KLF13 3'UTR报告基因载体和 miR-125a的 成熟体， 转染 24小时后用双荧光酶报告基因分析系统检测荧光素酶活性。 KLF13 3'UTR突变体用同样的方法转染和检测荧光素酶活性。
7. 转染人原代 T淋巴细胞
按照 interferin siRNA transfection reagent(polyplus公司）说明书方法进行转染。 MiR-125a 成熟体 (mimic)及抑制体 (inhibitor)及相应阴性对照 (control)来自 Thermo SCIENTIFIC Dharmacon公司。
8. 酶联免疫吸附实验 （ELISA)
分别转染 miR-125a mimic和对照 miRNA mimic的 T淋巴细胞， PHA活化 7 天后， 用 ELISA法 （西塘生物） 检测细胞上清 RANTES水平。 9. 数据分析
分析各孔 CT值， 选取 CT值位于可信区间范围内的样本 （不包括 CT值大于 33的样本） ， 计算出标化后的 2 (- Δ A CT ) 值， 即代表该样本该基因初始拷贝数 的数量。 第一批表达数据首先运用 SAM 2.20 软件 （Stanford University , http://www-stat.stanford.edu/~tibs/SAM/)分析，然后将表达有差异的数据导入 HCE 3.0软件 (University of Maryland http://www.cs.umd.edu/hcil/hce/) 作聚类分析。 后 一批扩大样本数据用 Graph Pad 4.03软件（ GraphPad Software公司）进行分析， 两 组独立样本数据比较应用非参数 Mann-Whitney检验， 相关分析应用 Spearman相 关性检验。 P值小于 0.05代表有统计学意义。 二. 结果
1. miR-125a在 SLE患者中的表达水平明显低于正常人
我们应用 Real-time PCR方法对 SLE 患者和正常对照组外周血 156个成熟 miRNAs表达水平差异进行了研究， 发现有 42个 miRNAs表达水平在 SLE患者中 明显低于正常人， 其中包括 miR-125a。 进一步扩大样本检测 55个 SLE病人和 40 个健康对照者外周血单个核细胞 miR-125a， 结果证实其表达水平在 SLE患者中显 著低于正常对照组， 具有统计学意义 （P<0.001， 见图 1 ) 。
2. miR-125a能调节 T细胞产生炎症性趋化因子 RANTES
为探索 miR-125a的生物学功能， 我们利用生物信息学软件 Target Scan, miRanda, PicTar等多个软件 （来自于网上数据库
http：〃 www.targetscan.org/http：〃 www.microrna.org/ http://pictar.bio.nyu. edu/ ) 同日寸予页 测 miR-125a的靶基因。生物信息学分析发现，一种 T淋巴细胞激活晚期促 RANTES 分泌的主要转录因子 KLF13是 miR-125a的靶基因。 我们检测 SLE患者血清中 RANTES的水平，发现 SLE患者 RANTES的水平明显高于正常对照组（9300±2955 vs. 3971 ± 1003 pg/ml,p<0.05 ) ， 这与文献报道的一致 [11-14]。 SLE患者中 miR-125 表达不足可能是 RANTES异常升高的原因之一。
为探索 miR-125a在 T细胞分泌 RANTES过程中的作用，我们在原代培养的 T 淋巴细胞中过表达 miR-125a， 分析 miR-125a对 PHA活化 T细胞分泌 RANTES的 影响。 研究结果显示， miR-125a能明显抑制 T细胞分泌 RANTES (见图 2B；)。 我们 同时检测了 KLF13的表达， 发现过表达 miR-125a能显著抑制 KLF13的表达 （见 图 4C)。
3. miR-125a直接负向调节 KLF13的表达 我们利用功能缺少和功能获得的策略研究 miR-125a对 KLF13 的调节作用， 发现： 在 Hela细胞系中过表达 miR-125a能明显抑制 KLF13的表达， 反之， 抑制 miR-125a的表达， KLF13表达则升高。 miR-125a对 KLF13的调节具有浓度依赖性
(见图 3 ) 。
4. 生物信息学分析显示 KLF13 3'UTR序列上有两个 miR-125a的作用位点 (+247〜+255和 +1616〜+1622) 。 我们分别将含有这两段序列的 UTR片段克隆到 报告基因载体中， 得到克隆的载体 K1和 K2， 同时也克隆不含 miR-125a作用位点 的序列， 得到载体 K3 (见图 4A) 。 荧光素酶报告基因系统证实， 过表达 miR-125a 能抑制 Kl、 Κ2载体荧光素酶活性。 反之， 抑制 miR-125a功能， Kl、 Κ2载体荧 光素酶活性则升高 （见图 4Β和 C) 。 miR-125a对 Κ3载体荧光素酶活性的影响不 显著 (；见图 4F) 。 miR-125a对 Kl、 Κ2载体荧光素酶活性的影响呈浓度依赖性 （见 图 4D禾 Π Ε) 。利用定点突变将 Kl、 Κ2载体上的 miR-125a的作用位点突变掉， 然 后分析 miR-125a对荧光素酶活性的影响， 发现 miR-125a对 Kl、 Κ2突变体的影响 不明显 (；见图 4F) 。 这些研究结果充分表明 miR-125a能直接调节 KLF13的表达。 三. 讨论
miRNA作为一种调控分子， 在人类多种疾病的发生和发展过程都有着重要作 用。不同于以往的有或无的调节特点， miRNA主要是在数量上对靶基因进行调节。 这一特点是 siRNA单靶点强大的调节作用或一些分子的拮抗剂和激动剂所无法比 拟的。这展示了 miRNA在临床应用方面的巨大前景。本申请发现 miR-125a在 SLE 患者中表达降低， 体外实验发现在 T细胞中过表达 miR-125a可以降低炎症性趋化 因子 RANTES的表达水平。 miR-125a表达信号可作为 SLE临床诊断的一个重要生 物标志物； 并可能作为一个新的药物干预靶点， 特异性干预 miR-125a的表达水平 可发展为新的治疗手段， 进而调节组织、 细胞的 RANTES表达， 为治疗包括狼疮 肾炎、 关节炎等慢性炎症、 减轻移植排斥反应等疾病带来新的希望。 参考文献
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Claims

权 利 要 求 书
1. miR-125a在制备治疗炎症性趋化因子 RANTES介导的疾病用的物质中的 用途。
2. 如权利要求 1所述的用途， 其特征在于， 所述疾病选自慢性炎症、 移植排 斥反应和 HIV感染。
3. 如权利要求 1所述的用途， 其特征在于， 所述疾病是系统性红斑狼疮、 狼 疮肾炎或关节炎。
4. 如权利要求 1 所述的用途， 其特征在于， 所述物质选自： 含有 miR-125a 前体、 成熟体或其类似物的药物组合物； miR- 125a表达激动剂； 含有 miR-125a表 达激动剂的药物组合物； 含 miR-125 a前体、 成熟体或其类似物的表达载体； 和含 所述表达载体的药物组合物。
5. 如权利要求 4所述的用途， 其特征在于， 所述 miR- 125a表达激动剂选自 脂多糖和植物血球凝聚集素。
6. 一种药物组合物， 其特征在于， 该组合物含有 miR-125a前体、 成熟体、 miR-125a表达激动剂、和 /或表达 miR-125a成熟体的表达载体， 以及药学上可接受 的运载体。
7. 如权利要求 6所述的药物组合物， 其特征在于， 所述 miR-125a表达激动 剂选自脂多糖和植物血球凝聚集素。
8. 一种体外调节组织或细胞的炎症性趋化因子 RANTES 表达的方法， 其特 征在于， 所述方法包括以下步骤：
( 1 ) 提供所述组织或细胞； 和
(2) 向所述组织或细胞施与 miR-125a前体、 成熟体、 miR- 125a表达激动剂、 和 /或表达 miR-125a成熟体的表达载体， 从而调节所述组织或细胞中的 RANTES 表达。
9. 一种筛选能抑制炎症性趋化因子 RANTES 的表达的物质的方法， 该方法 包括以下步骤：
( 1 ) 提供组织或细胞；
(2)作为试验组， 向所述组织或细胞施与 miR-125a前体、成熟体、 miR- 125a 表达激动剂、 和 /或表达 miR-125a的表达载体， 和候选物质； 和
(3 )作为对照组， 向所述组织或细胞施与 miR-125a前体、成熟体、 miR-125a 表达激动剂、 和 /或表达 miR-125a成熟体的表达载体， 而未加入候选物质；
(4)检测试验组和对照组中 KLF13和 /或 RANTES的表达， 其中， 与对照组 相比， 试验组中能使所述组织或细胞中的 KLF13和 /或 RANTES的表达降低的候 选物质确定为能抑制炎症性趋化因子 RANTES的表达的物质。
10. 如权利要求 9所述的方法， 其特征在于， 所述方法还包括：
(5 ) 向所述组织或细胞施与 miR-125a前体、 成熟体、 miR- 125a表达激动剂、 和 /或表达 miR-125a成熟体的表达载体， 和脂多糖或植物血球凝聚集素； 检测此 组织或细胞中的 KLF13和 /或 RANTES的表达； 和将检测结果与步骤 （2) 的检测 结果进行比较， 以确定能抑制炎症性趋化因子 RANTES的表达的物质。
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