CN103933582A - Application of Let-7 family miRNA (micro-ribonucleic acid) in preparing medicament for treating nerve regeneration related diseases - Google Patents
Application of Let-7 family miRNA (micro-ribonucleic acid) in preparing medicament for treating nerve regeneration related diseases Download PDFInfo
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Abstract
The invention discloses let-7 family miRNA (micro-ribonucleic acid) as well as a modulator thereof or a chemically-modified let-7 family miRNA and a modulator thereof in preparing a medicament for treating nerve regeneration related diseases which refer to peripheral nerve injury, central nerve injury, optic nerve injury or brain injury diseases, wherein the let-7 family miRNA is selected from miR-98, let-7a, let-7b, let-7c, let-7d, let-7e, let-7f, let-7g and let-7i with nucleotide sequences as shown in SEQ ID NO.1-9. The let-7 family miRNA can be used for directly regulating expression of NGF (nerve growth factor) in a targeted manner, enriching research of regulating effect of miRNA on nerve regeneration, and is beneficial to research and development of applying the Let-7 family miRNA as a novel treatment target point for regulating NGF.
Description
Technical field
The present invention relates to field of biology, specifically field of gene, the more particularly application of the miRNA of Let-7 family in the medicine of preparation treatment neuranagenesis relevant disease.
Background technology
Nerve injury is common clinical example, and sickness rate is and increases year by year trend.Neurotrophic factor (neurotrophic factor, NTF) be the peptide molecule that neurocyte is played to special dietary effect, not only participate in regulating neuronic growth and maturation, and in Adult Nervous System, also bring into play effect, there is the apoptosis after neuronal damage that suppresses to grow up, promote neuranagenesis, regulate the functions such as synaptic plasticity and transmission mediator.There is report NTF all bringing into play important regulating action biology in fetal development, cell differentiation, neuranagenesis, wound healing, immunomodulating and tumor generation etc. aspect many.NGF (nerve growth factor) confirms the earliest and studies to obtain neurotrophic factor the most clearly, be produced by neuron, innerv target tissue or glial cell can promotion maincenter and the reactive protein of peripheral nervous differentiation, growth and survival, neural growth and normal physiological function maintains and nerve injury after regeneration in play an important role.Nerve growth factor (NGF) in regeneration microenvironment has the effect of significantly short neuranagenesis, therefore external source adds or intramuscular injection NGF treatment nerve injury has obtained research widely, it is long apart from Sciatic that our seminar also attempts adding NGF to repair in artificial nerve graft, obtain good result, but NGF is difficult for passing through blood-nerve barrier, the biological activity half-life is shorter, and long-term a large amount of medication meeting causes toxic and side effects, although injection site myalgia and hyperpathia etc. have caused NGF to have good reparation neuranagenesis effect in clinical practice, but still difficulty is applied to clinical so far, therefore develop and a kind ofly can regulate and control the use that medicine that NGF expresses substitutes NGF and have potential using value.
MiRNA (microRNA, microRNA) is the non-coding microRNA of the about 22nt of a class length, is processed generation by its precursor molecule under the effect of enzyme, is extensively present in organism.At animal body, miRNA, by the expression of the many genes of post-transcriptional control and albumen, is attached on specific said target mrna and is regulated said target mrna translation or degraded target gene mRNA by nucleic acid array complementation, is a kind of molecule that plays negative regulation effect.Studies show that at present miRNA has participated in the physiological process such as organism growth, differentiation, growth, immunne response, and its expression and functional disorder may cause tumor to occur, the multiple pathological phenomenon such as autoimmune disease and viral infection, miRNA is also reported by people gradually at neural regulating and controlling effect.Basis and clinical research show, miRNA is still important regulatory factor in normal physiological processes in lysis.Different from the past have or the adjusting feature of nothing, and miRNA quantitatively regulates target gene.This feature is that antagonist and the agonist of the powerful regulating action of the mono-target spot of mi RNA or some molecules is incomparable.This has shown the huge prospect of miRNA aspect clinical practice.
The miRNA of let-7 family is one group of sequence height homology, and intimate miRNA, comprises miR-98, let-7a, let-7b, let-7c, let-7d, let-7e, let-7f, let-7g, let-7i.The miRNA of let-7 family high conservative between species, some member is methylated, the regulation and control of posttranscriptional modification and Lin28 gene, also regulate the target genes such as RAS, HMGA2, CDC25A, CDK6, FAS, CASP3 simultaneously, closely bound up with multiple biological phenomena and physiological process in life.
Existing bibliographical information the relevant function of the miRNA of let-7 family be mainly included in regulating action, regulating action to apoptosis and the regulating action in growth course of tumor in occurring.The relevant report that does not also regulate up to now NGF about miRNA, also lacks clearly and understands in the function in neuranagenesis field for the miRNA of let-7 family.Therefore, there is exigence this area for the novel targets and the means that regulate NGF.
Summary of the invention
This research confirms the directly expression of Targeted-control NGF of the miRNA of let-7 family first, inquire into first the regulating and controlling effect of the miRNA of let-7 family in nerve injury regenerative process, enrich the regulating and controlling effect research of miRNA to neuranagenesis, contributed to the research and development of the miRNA of Let-7 family as the new treatment target spot of regulation and control NGF.
The object of this invention is to provide the miRNA of let-7 family in the application in the medicine of preparation treatment neuranagenesis relevant disease.
The concrete technical scheme of the present invention is as follows:
The invention provides the miRNA of let-7 family and regulator thereof or the purposes in the medicine of preparation treatment neuranagenesis relevant disease through the let-7 of the chemical modification miRNA of family and regulator thereof, the relevant disease of described treatment neuranagenesis refers to peripheral nerve injury, central nervous system injury, optic nerve injury or brain injury disease, the described let-7 miRNA of family is selected from miR-98, let-7a, let-7b, let-7c, let-7d, let-7e, let-7f, let-7g, let-7i, its nucleotide sequence is as shown in SEQ ID NO.1-9.
Above-mentioned regulator is selected from inhibitor or the synergist of the described let-7 miRNA of family, the anti sense nucleotide sequence of the preferred let-7 miRNA of family of inhibitor; Synergist is the miRNA of let-7 family simulation nucleic acid preferably.
The simulation nucleic acid of miRNA is the endogenous miRNA of simulation organism, uses the method for chemosynthesis synthetic, can strengthen the function of endogenous miRNA.And miRNA suppresses the special inhibitor for specific target miRNA in cell that nucleic acid is chemical modification, use in the present invention the antisense sequences of former RNA sequence.
The miRNA of synthetic has been successfully applied to reticent expection expression of target gene and functional study thereof.MiRNA simulation nucleic acid can further strengthen the silence effect of interior miRNAs, reduces the expression of intracellular protein, carries out gain-of-function Journal of Sex Research.On the contrary, use the synthetic miRNA of method of chemosynthesis to suppress nucleic acid, special targeting suppresses single miRNA molecule, can weaken the reticent effect of interior miRNAs to specific gene, improve expressing quantity, carry out afunction Journal of Sex Research, can be used to screen miRNA target spot, the miRNA of a certain gene expression of screening regulation and control, screening affects the miRNA of cell development process.MiRNA simulation nucleic acid and the inhibition nucleic acid of chemosynthesis have become the useful tool of the function of research animal-plant gene family, and are also a kind of New Policies for the treatment of of cancer and clinical research.
The design of miRNA simulation nucleic acid and inhibitor can adopt this area routine techniques.The synthetic commercialization of miRNA simulation nucleic acid and inhibitor, can buy from each biotech firm at present.For example, the simulation nucleic acid of the present invention miRNA used and suppress nucleic acid from Rui Bo bio tech ltd, Guangzhou.Sequence analysis shows to have at least 1/3 human gene relevant to miRNA regulation and control.The function of research gene normally knocks out it from genome, then observes the variation before and after knocking out, but in the functional study of miRNA, research worker generally adopts the expression that strengthens or weaken this miRNA to identify its function.
The present invention is taking miR-98 and let-7d as example, and its inhibitor can be selected from following inhibition nucleic acid:
MiR-98 suppresses nucleic acid (strand): 5 ' AACAAUACAACUUACUACCUCA3 ' SEQ ID NO.10
Let-7d suppresses nucleic acid (strand): 5 ' AACUAUGCAACCUACUACCUCU3 ' SEQ ID NO.11
Taking miR-98 and let-7d as example, its synergist can be selected from Imitating nucleic acid:
MiR-98 simulation nucleic acid (two strands): 5 ' UGAGGUAGUAAGUUGUAUUGUU3 ' SEQ ID NO.12
3’ACUCCAUCAUUCAACAUAACAA5’SEQ?ID?NO.13
Let-7d simulation nucleic acid (two strands): 5 ' AGAGGUAGUAGGUUGCAUAGUU3 ' SEQ ID NO.14
3’UCUCCAUCAUCCAACGUAUCAA5’。SEQ?ID?NO.15
Purposes of the present invention, further, the miRNA of let-7 family and regulator thereof or through the let-7 of the chemical modification miRNA of family and regulator thereof in the time carrying out administration as medicine, can pass through in-vitro transfection cell, then by transfectional cell transplanting injury place performance repair.Glial cell or stem cell that described cell is selected from nerve injury place are induced to differentiate into glial cell.Transfection method is this area routine techniques, concrete operation example is as follows: the Scs of getting primary culture purified, utilize the let-7 simulation nucleic acid that Lipofectamine RNAiMAX (invitrogen) synthesized design or suppress nucleic acid and proceed to cell, 96 orifice plate simulation nucleic acid consumptions are 4pmol, suppressing nucleic acid consumption is 8pmol, 24 orifice plate simulation nucleic acid consumptions are 25pmol, and suppressing nucleic acid consumption is 50pmol.Or by described miRNA and regulator thereof or directly deliver medicine to injury region through miRNA and the regulator thereof of chemical modification, administering mode is conventional route of administration, includes but not limited to direct injection, venous transfusion, oral target administration etc.
Although miRNA simulation nucleic acid and inhibitor can therapeutic ground targeting miRNA functions, but while being directly used in body, easily be subject to attack the degraded of nuclease, may cause activity decreased, therefore for fear of the generation of above-mentioned situation or the effect that improves miRNA simulation nucleic acid and inhibitor, can further improve stability, effect and/or the toxicity of miRNA and inhibitor or synergist by chemical modification.Chemical modification of the present invention comprises thio-modification, methoxyl group modification, fluoro modification, cholesterol modification, Cy3 and Cy5 labelling, biotin modification, amido modified, DNP modifies, one or more in alkynyl-modified.
Under currently available technology, there is the multiple method that RNA is modified, as carried out chemical modification to RNA sequence with reference to the disclosed technical scheme of Chinese patent 201080035109, or can directly buy from business-like biotech company.In the present invention, the miRNA of chemical modification and regulator thereof transfer to Guangzhou Rui Bo biotech firm synthetic.
The preferred let-7 miRNAantagomir of family of the miRNA of let-7 family regulator or the agomir of the chemical modification adopting in the present invention.
Antagomir is according to the ripe body sequential design of microRNA, through the little RNA of strand of special marking and chemical modification, is to be specifically designed to the efficient blocker that suppresses endogenous microRNA.Agomir is the little RNA of two strands through special marking and chemical modification, regulates the biological function of target gene by simulating endogenic miRNA.
Antagomir and agomir chemical modification structures are as follows:
Antogimir carries out cholesterol modification to the 3 ' end that suppresses nucleic acid, two sulfo-backbone modifications of 5 ' end, and four sulfo-backbone modifications of 3 ' end, full chain methoxyl group is modified.
Agomir modifies at antisense strand, and 3 ' end carries out cholesterol modification, two sulfo-backbone modifications of 5 ' end, and the full chain methoxyl group of four sulfo-backbone modifications of 3 ' end is modified.
The present invention is taking Let-7d miRNA as example,
Let-7d agomir (two strands):
5’AGAGGUAGUAGGUUGCAUAGUU3’?SEQ?ID?NO.14
3’chol-s-s-s-s-(mU)(mC)(mU)(mC)(mC)(mA)(mU)(mC)(mA)(mU)(mC)(mC)(mA)(mA)(mC)(mG)(mU)(mA)(mU)(mC)(mA)(mA)-s-s5’?SEQ?ID?NO.16
Let-7d?antagomir：
5’s-s-(mA)(mA)(mC)(mU)(mA)(mU)(mG)(mC)(mA)(mA)(mC)(mC)(mU)(mA)(mC)(mU)(mA)(mC)(mC)(mU)(mC)(mU)-s-s-s-s-chol3’?SEQ?ID?NO.17
Wherein, chol is that cholesterol is modified, s is that thio-modification, m are the modification that methylates.
Antagomir and agomir tool have the following advantages:
1, compare with mimics with common miRNA inhibitor, antagomir and agomir and cell membrane affinity are higher, and cell transfecting experiment transfection reagent consumption significantly reduces.
2, be particularly suitable for the experiment of animal body internal interference. and in experiment, there is higher stability and inhibition in vivo, can adopt the administration of the various ways such as systemic injection or local injection, easy and simple to handle.
3, can be enriched in target cell, realize the stable interference of efficient specificity
4, suppress the persistent period long, at least reach week age, the longest sustainable 5-6 week of interference effect.
On the other hand, the present invention relates to a kind of method that body NGF of regulation and control expresses, by the above-mentioned let-7 miRNA of family and regulator thereof or through the let-7 of the chemical modification miRNA of family and regulator in-vitro transfection cell thereof, then by transfectional cell transplanting injury place, regulation and control NGF expresses; Or by the above-mentioned let-7 miRNA of family and regulator thereof or directly deliver medicine to injury region through the let-7 of the chemical modification miRNA of family and regulator thereof, regulation and control NGF expresses.
The let-7 miRNA of family of the present invention is except can being directly used for the treatment of neuranagenesis relevant disease as medicine, can also serve as the target spot of medicine, by with the effect of medicine, raise or suppress the expression of body miRNA, and then further regulate and control the expression of body NGF and play a role.
The present invention has studied the regulating and controlling effect of the miRNA of let-7 family in nerve injury regenerative process, in concrete an enforcement, the miRNA of let-7 family, by the expression of regulation and control target gene NGF, regulates and controls neuranagenesis thereby affected the propagation of glial cell schwann cell in neuranagenesis, migration, the secretion of NGF and the growth of aixs cylinder.
In the present invention one and the result of study of multiple embodiment show, the directly expression of Targeted-control NGF of the miRNA of let-7 family, translate the expression and secretion that reduces NGF by inhibition, and the inhibitor of Let-7 can promote the expression and secretion of NGF, thereby further affect axon growth.Thus, can utilize the miRNA of let-7 family the regulating action of NGF to be regulated and controled to the expression and secretion of NGF, treatment nerve injury, replaces or improves the side effect of NGF in clinical practice, as the alternative means treatment neuranagenesis of regulation and control NGF.
Advantage of the present invention: due to the existence of blood brain and blood-nerve barrier, a lot of medicines can not enter in nerve and play a role, and miRNA is because molecule is less, can pass through blood brain and blood-nerve barrier; Although have been found that in addition NGF has well short neuranagenesis effect, should not apply with clinical due to its side effect, can effectively avoid the side effect of NGF by the expression and secretion of regulation and control NGF.
Brief description of the drawings
The direct targeting NGF3 ' of Fig. 1 Let-7miRNA UTR.(A) NGF3 ' UTR mates with Let-7miRNA family to distinguish and analyses and clone structure; (B) the two reporter gene analyses (* * p<0.01) of luciferase.
Fig. 2 Let-7miRNA suppresses the translation of NGF.(A) Let-7d and miR-98 do not have a significant effect to NGF mRNA; (B) Let-7d and miR-98 have obviously affected the protein expression (* * p<0.01) of NGF.
Fig. 3 Let-7miRNA suppresses the propagation (* 0.01<p<0.05, * * p<0.01) of Scs.
Fig. 4 NGF Knockdown can reverse the short multiplication effect (* * p<0.01) of Anti-Let-7miRNA, shows that the propagation that let-7miRNA family affects schwann cell plays a role by NGF.
Fig. 5 Let-7miRNA suppresses the migration (* * p<0.01) of Scs.
Fig. 6 NGF Knockdown can reverse the short migration effect (* * p<0.01) of Anti-Let-7miRNA, shows that the migration that let-7miRNA family affects schwann cell plays a role by NGF.
Fig. 7 Let-7miRNA can suppress the secretion (* * p<0.01) of schwann cell NGF.
Fig. 8 Let-7miRNA can suppress axon growth after transforming schwann cell.
Fig. 9 Let-7miRNA can suppress the migration (* * p<0.01) of schwann cell in vivo.
Figure 10 Let-7miRNA can suppress the growth (* * p<0.01) of aixs cylinder in vivo.
Detailed description of the invention
Concrete steps of the present invention are described by the following examples, but not limited by embodiment.
The term that used in the present invention, except as otherwise noted, generally has the implication that those of ordinary skill in the art understand conventionally.
Below in conjunction with specific embodiment comparable data, the present invention is described in further detail.Should be understood that these embodiment just in order to demonstrate the invention, but not limit the scope of the invention by any way.
In following examples, various processes and the method do not described in detail are conventional methods as known in the art.
In embodiment, miR-98 suppresses nucleotide sequence as shown in SEQ ID NO.10, let-7d suppresses nucleotide sequence as shown in SEQ ID NO.11, miR-98 simulation nucleic acid (two strands) sequence is as shown in SEQ ID NO.12-13, and let-7d simulation nucleic acid (two strands) sequence is as shown in SEQ ID NO.14-15.Let-7d agomir (two strands) sequence is as shown in SEQ ID NO.14 and 16.
Determining of embodiment 1Let-7miRNA target gene.
By the downstream (schematic diagram as shown in Figure 1A) that comprises Let-7 and mate 3 ' of district-UTR with its target gene NGF and be building up to pLUC luciferase reporter gene, build reporter plasmid, the ACC in coupling district is sported to UGG simultaneously, build mutant plasmid as negative control.The pLUC luciferase reporter gene that comprises NGF3 '-UTR (the 24 every hole of orifice plate 0.6ug plasmid) building and Let-7d or miR-98 simulation nucleic acid (the every hole 25pmol of 24 orifice plate simulates nucleic acid) are proceeded to 293T cell jointly, after 24 hours, receive cell, measure reporter gene.Result as shown in Figure 1B, is compared Con group, and the uciferase activity of excessively expressing Let-7d and miR-98 simulation nucleic acid is obviously suppressed; And the mating after district suddenlyd change of Let-7miRNA and NGF3 '-UTR, crossing the uciferase activity of expressing Let-7d and miR-98 simulation nucleic acid does not have significant change.This shows, the miRNA of Let-7 family can match with NGF3 ' UTR district Seed Sequences and the directly expression of Targeted-control NGF specifically.
The impact that embodiment 2Let-7miRNA expresses NGF
Adopt RT-PCR and Western Blot to determine mRNA level and the protein level of NGF.PCR primer is NGF-forword:5 ' CCAAGGACGCAGCTTTCTATC3 ' (SEQ ID NO.18); NGF-reverse:5 ' CTGTGTCAAGGGAATGCTGAAG3 ' (SEQ ID NO.19).System is 94 DEG C of degeneration 40 seconds, anneals 30 seconds for 60 DEG C, and 72 DEG C are extended 45 seconds.As shown in Figure 2 A, compared with Con group, the NGF mRNA level of Let-7d and miR-98 (simulation nucleic acid) does not have significant change, and compared with Anti-Con group, the NGF mRNA level of Anti-Let-7d and Anti-miR-98 (inhibition nucleic acid) does not have significant change yet; As shown in Figure 2 B, compared with Con group, the NGF protein level of Let-7d and miR-98 obviously reduces, and compared with Anti-Con group, the NGF protein level of Anti-Let-7d and Anti-miR-98 obviously increases.This shows, the mechanism of the reticent NGF of the miRNA of let-7 family is to be translated instead of caused the degraded of NGF mRNA by inhibition.
The regulating and controlling effect research of embodiment 3Let-7miRNA to Scs.
(1) cell is drawn materials and is cultivated: primary Scs, get rat sciatic nerve, and with collagenase and trypsinization, add with DMEM that 10% hyclone is cultivated and 100U/ml penicillin-streptomycin is cultivated, 37 DEG C, 5%CO
2hatch (list of references B.Yu, et al. is early stage in injury of sciatic nerve, miR-182 suppresses propagation and migration (the miR-182inhibits Schwann cell proliferation and migration by targeting FGF9and NTM of schwann cell by targeting FGF9 and NTM, respectively at an early stage following sciatic nerve injury) [J] .Nucleic Acids Res, 2012,40:10356-65.).
(2) cell transfecting: with simulation nucleic acid and the inhibition nucleic acid of Lipofectamine RNAiMAX (Invitrogen company) transfection miR-98, let-7d, and contrast, the primary Scs of transfection, 96 orifice plate mimic consumptions are 4pmol, inhibitor consumption is 8pmol, 24 orifice plate mimic consumptions are 25pmol, and inhibitor consumption is 50pmol, and culture fluid is serum-free medium Opti-MEM.After 6-8h, add the complete medium containing 10% serum, then breed respectively, move and secrete experiment.
Let-7miRNA suppresses Scs propagation.EDU measures cell proliferation (Guangzhou Rui Bo biotech firm test kit).Con group is compared in Fig. 3 demonstration, and Let-7d and miR-98 group obviously suppress Scs propagation; Meanwhile, compare Anti-Con group, Anti-Let-7d and miR-98 group have all obviously promoted the propagation of Scs.This shows, the miRNA of Let-7 family inhibition Scs propagation.
Let-7miRNA suppresses Scs propagation by NGF.Adopt NGF knockdown to see and can reverse Anti-Let-7d and the facilitation of Anti-miR-98 to Scs propagation.NGF siRNA (the every hole 50pmol of 24 orifice plate) and let-7 and miR-98 are suppressed to nucleic acid (Anti-Let-7d or Anti-miR-98, the every hole 50pmol of 24 orifice plate) and be jointly transfected into Scs with Lipofectamine RNAiMAX transfection reagent.Result as shown in Figure 4, is compared Anti-con group, and Anti-Let-7d and Anti-miR-98 have obviously promoted Scs propagation, and NGF knock down can reverse the short multiplication effect of Anti-Let-7d and Anti-miR-98.This shows that the miRNA of Let-7 family has suppressed the propagation of Scs by target gene NGF.
Let-7miRNA suppresses Scs migration.Transwell measures cell migration (Guangzhou Rui Bo biotech firm test kit).The coated Fibronectin of lower chamber surface of Transwell cell, the cell after being coated with can be placed on 4 DEG C and preserve 30d.Collect the Scs after transfection 24h, adjusting cell density by DMEM culture medium is 3 × 10
5individual/mL, 100 μ L cell suspension in chamber on transwell.Lower chamber adds the complete medium of 500 μ L containing 10% hyclone.The purple dyeing of cellar culture 24h post crystallization, the rear Leica DC300F of dyeing is just putting microscope and is observing and take pictures, and chooses at random 10 visual field counting cells numbers.Finally, crystal violet is eluted completely with 33% glacial acetic acid, eluent can be in microplate reader 570nm, survey its OD value, indirectly reflect migrating cell number.Con group is compared in Fig. 5 demonstration, and Let-7d and miR-98 group obviously suppress Scs migration; Meanwhile, compare Anti-Con group, Anti-Let-7d and miR-98 group have all obviously promoted the migration of Scs.This shows, the migration of the miRNA of Let-7 family inhibition Scs.
Let-7miRNA suppresses Scs migration by NGF.Adopt NGF knockdown to see and can reverse Anti-Let-7d and the facilitation of Anti-miR-98 to Scs migration.NGF siRNA (the every hole 50pmol of 24 orifice plate) and let-7 and miR-98 are suppressed to nucleic acid (Anti-Let-7d or Anti-miR-98, the every hole 50pmol of 24 orifice plate) and be jointly transfected into Scs with Lipofectamine RNAiMAX transfection reagent.As shown in Figure 6, compare Anti-con group, Anti-Let-7d and miR-98 have obviously promoted Scs migration, and NGF knock down can reverse the short migration effect of Anti-Let-7d and miR-98.This shows that the miRNA of Let-7 family has suppressed the migration of Scs by target gene NGF.
Let-7miRNA suppresses NGF secretion.ELISA measures NGF secretion (millipore test kit).Each sample does three secondary holes, reads sample at 450nm light absorption value in microplate reader, calculates sample reagent concentration according to standard curve.Let-7 analogies and inhibitor are proceeded to the Scs of former culture, ELISA experiment finds that let-7 analogies can obviously suppress NGF secretion, Anti-let-7 can obviously promote NGF secretion (Fig. 7), and this shows that the miRNA of le7-7 family can suppress NGF secretion.
The Scs of Let-7miRNA transfection has suppressed axon growth.The Scs of the former culture of let-7d analogies and inhibitor transfection is planted in to 1.0mm Transwell cell, DRG neuron is planted on transwell plank, cultivates altogether, after 24 hours, with Anti-NF200 immunofluorescence label DRG neuron, fluorescence microscope is taken pictures.Found that, let-7 analogies can obviously suppress axon growth, and Anti-let-7 can obviously promote axon growth (Fig. 8), and this Scs that shows the miRNA of Let-7 family transfection has suppressed axon growth.
Embodiment 4Let-7miRNA in vivo microenvironment suppresses Scs migration and axon growth.
Let-7miRNA prepares sciatic nerve from disconnected wound model (5mm is damaged), by the silica gel tube bridge joint of diameter 1.0mm from breakthrough mouth, by let-7d the agomir ((let-7d modifying through cholesterol, improve the stability of miRNA, and do not need transfection reagent, can be directly used in cell and zoopery) and matrgel (BD Biosciences) 1:1 after mixing, be expelled in silica gel tube, at 7 and 10 days, put to death rat, get silica gel tube, Anti-S100 immunohistochemical staining, measures the migration distance of Scs from damage near-end.Fig. 9 is presented at 7 and 10 days, and let-7d has obviously suppressed the migration of Scs, and this shows the miRNA of Let-7 family microenvironment inhibition Scs migration in vivo.Use Anti-NF200 immunohistochemical staining, measure the growth distance of aixs cylinder from damage near-end.Figure 10 is presented at 7 and 10 days, and let-7d has obviously suppressed the growth distance of aixs cylinder, and this shows the miRNA of the Let-7 family growth of microenvironment inhibition aixs cylinder in vivo.
Discuss
Due to the existence of blood brain and blood-nerve barrier, a lot of medicines can not enter in nerve and play a role, and miRNA is because molecule is less, can pass through blood brain and blood-nerve barrier; Although have been found that in addition NGF has well short neuranagenesis effect, should not apply with clinical due to its side effect, can effectively avoid the side effect of NGF by the expression and secretion of regulation and control NGF.Above-mentioned studies confirm that, the directly expression of Targeted-control NGF of the miRNA of let-7 family, translate the expression and secretion that reduces NGF, and the inhibitor of Let-7 can promote the expression and secretion of NGF, thereby further affect axon growth by inhibition.Thus, can utilize the miRNA of let-7 family the regulating action of NGF to be regulated and controled to the expression and secretion of NGF, treatment nerve injury, replaces or improves the side effect of NGF in clinical practice, as the alternative means treatment neuranagenesis of regulation and control NGF.
Claims (10)
- The miRNA of 1.let-7 family and regulator thereof or through the let-7 of the chemical modification miRNA of family and regulator thereof the purposes in the medicine of preparation treatment neuranagenesis relevant disease, the relevant disease of described treatment neuranagenesis refers to peripheral nerve injury, central nervous system injury, optic nerve injury or brain injury disease, the described let-7 miRNA of family is selected from miR-98, let-7a, let-7b, let-7c, let-7d, let-7e, let-7f, let-7g, let-7i, its nucleotide sequence is as shown in SEQ ID NO.1-9.
- 2. purposes as claimed in claim 1, is characterized in that described regulator is selected from inhibitor or the synergist of the described let-7 miRNA of family.
- 3. purposes as claimed in claim 1, is characterized in that, described inhibitor is the anti sense nucleotide sequence of the miRNA of let-7 family.
- 4. purposes as claimed in claim 1, described synergist is the miRNA of let-7 family simulation nucleic acid.
- 5. purposes as claimed in claim 1, is characterized in that by by the described let-7 miRNA of family and regulator thereof or through the let-7 of the chemical modification miRNA of family and regulator in-vitro transfection cell thereof, then by transfectional cell transplanting injury place performance repair; Or, by described miRNA and regulator thereof or directly deliver medicine to injury region through miRNA and the regulator thereof of chemical modification.
- 6. purposes as claimed in claim 5, is characterized in that described chemical modification comprises thio-modification, methoxyl group modification, fluoro modification, cholesterol modification, Cy3 and Cy5 labelling, biotin modification, amido modified, DNP modifies, one or more in alkynyl-modified.
- 7. purposes as claimed in claim 1, the miRNA of the let-7 family regulator that it is characterized in that described chemical modification is the miRNA antagomir of let-7 family or agomir.
- 8. purposes as claimed in claim 7, is characterized in that the described let-7 miRNA antagomir of family or agomir, has sequence as shown in SEQ ID NO.16-17.
- 9. one kind regulates and controls the method that NGF expresses, it is characterized in that by one of claim 1-8 described let-7 miRNA of family and regulator thereof or through the let-7 of the chemical modification miRNA of family and regulator in-vitro transfection cell thereof, then by transfectional cell transplanting injury place, promote or suppress NGF and express; Or, by described miRNA and regulator thereof or directly deliver medicine to injury region through miRNA and the regulator thereof of chemical modification, promote or suppress NGF and express.
- The miRNA of 10.let-7 family is the purposes in the medicine of preparation treatment neuranagenesis relevant disease as drug target, the relevant disease of described treatment neuranagenesis refers to peripheral nerve injury, central nervous system injury, optic nerve injury or brain injury disease, and the described let-7 miRNA of family is selected from miR-98, let-7a, let-7b, let-7c, let-7d, let-7e, let-7f, let-7g, let-7i, its nucleotide sequence is as shown in SEQ ID NO.1-9.
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Cited By (3)
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| CN106466486A (en) * | 2015-08-18 | 2017-03-01 | 中国人民解放军第二军医大学 | Application in preparation anti-gastric cancer medicament for the miR-133 small molecule nucleic acid drug |
| CN106474549A (en) * | 2016-11-21 | 2017-03-08 | 南通大学 | The structure of the novel tissue tissue-engineered nerve of MicroRNA gene mediated and its application in reparation neurologic defect |
| CN109172595A (en) * | 2018-08-21 | 2019-01-11 | 昆明理工大学 | The new application of Microrna let-7g |
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| CN101675165A (en) * | 2006-12-08 | 2010-03-17 | 奥斯瑞根公司 | The function of LET-7 Microrna and target |
| WO2013007874A1 (en) * | 2011-07-12 | 2013-01-17 | Mart Saarma | A transgenic animal comprising a deletion or functional deletion of the 3'utr of an endogenous gene. |
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| CN101675165A (en) * | 2006-12-08 | 2010-03-17 | 奥斯瑞根公司 | The function of LET-7 Microrna and target |
| WO2013007874A1 (en) * | 2011-07-12 | 2013-01-17 | Mart Saarma | A transgenic animal comprising a deletion or functional deletion of the 3'utr of an endogenous gene. |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106466486A (en) * | 2015-08-18 | 2017-03-01 | 中国人民解放军第二军医大学 | Application in preparation anti-gastric cancer medicament for the miR-133 small molecule nucleic acid drug |
| CN106474549A (en) * | 2016-11-21 | 2017-03-08 | 南通大学 | The structure of the novel tissue tissue-engineered nerve of MicroRNA gene mediated and its application in reparation neurologic defect |
| WO2018090542A1 (en) * | 2016-11-21 | 2018-05-24 | 南通大学 | Construction of microrna gene-mediated novel tissue engineered nerve and applications thereof in repairing nerve defect |
| CN106474549B (en) * | 2016-11-21 | 2019-05-03 | 南通大学 | The novel tissue tissue-engineered nerve of MicroRNA gene mediated constructs and its in the application for repairing neurologic defect |
| EP3417887A4 (en) * | 2016-11-21 | 2020-03-25 | Nantong University | Construction of microrna gene-mediated novel tissue engineered nerve and applications thereof in repairing nerve defect |
| US10639399B2 (en) | 2016-11-21 | 2020-05-05 | Nantong University | Construction of MicroRNA gene-mediated novel tissue engineered nerve and applications thereof in repairing nerve defect |
| AU2017362105B2 (en) * | 2016-11-21 | 2020-10-22 | Nantong University | Construction of microRNA gene-mediated novel tissue engineered nerve and applications thereof in repairing nerve defect |
| AU2017362105B9 (en) * | 2016-11-21 | 2020-11-05 | Nantong University | Construction of microRNA gene-mediated novel tissue engineered nerve and applications thereof in repairing nerve defect |
| CN109172595A (en) * | 2018-08-21 | 2019-01-11 | 昆明理工大学 | The new application of Microrna let-7g |
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