CN104846102A - Purpose and relevant medicine of miR-135 - Google Patents
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Abstract
The invention relates to the field of biotechnology, in particular to a purpose and relevant medicine of miR-135. The invention discloses the dependency of the miR-135 and the foot cell injury for the first time, and discloses the capability of overexpression miR-135 to cause foot cell injury for the first time, and the process is more similar to the human disease causing process. A foot cell injury model has the sufficient foot cell injury typical features, can well simulate the occurrence and the development of foot cells in the body, and belongs to an ideal model of foot cell injury base and clinic application study, so that the miR-135 can be well applied to the medicine for screening and treating the foot cell injury.
Description
Technical field
The present invention relates to biological technical field, be specifically related to purposes and the related drugs thereof of miR-135.
Background technology
As (18-25 Nucleotide) non-coding RNA that a class is little, microRNA, in the vital movement of various biology, plays vital regulating and controlling effect to the expression of gene.In Mammals, be found more than the sequence of 1000 kinds of microRNA, each microRNA can regulate and control multiple messenger RNA(mRNA) (mRNA), simultaneously each mRNA can regulate and control by hundreds and thousands of microRNA.Research is estimated, the protein coding gene more than 60% expresses the regulation and control by microRNA.
Nearest research finds in kidney disease, and microRNA can the expression of abnormal regulatory gene.Such as by activating vascular endothelial growth factor, miR-93 can promote the damage of renal glomerulus; By suppressing the expression of BCL2, miR-195 can promote podocyte apoptosis; Podocytes in Renal Tissue can be accelerated by lowering miR-30.In the mesangial cell of diabetes model, miR-192 can promote the formation of collagen and the formation of renal interstitial fibrosis; In diabetic nephropathy experimental model, miR-192, miR-216 and miR-217 can induce kidney mesangial cell loose.Dicer is necessary enzyme in microRNA ripening process, and in kidney cell, knock out Dicer specifically can cause renal lesions: can urinate and glomerular sclerosis by inducible protein as knocked out Dicer in podocyte specifically; Knock out Dicer can reduce messangial cell number and alleviate proximal convoluted tubule ischemical reperfusion injury.These results all confirm that microRNA plays indispensable effect in the developing of kidney disease, but concrete mechanism wherein is also furtherd investigate.
MiR-135 family guards at Mammals camber, and its family member comprises two i.e. miR-135a and miR-135b.At present some researchs find that miR-135 plays an important role in the developing of tumour as a kind of oncogene: as induced the generation of the rectum cancer; Improve nonsmall-cell lung cancer to the opposing of medicine; Promote invasion and attack and the growth of the rectum cancer.But also having some researchs to find the generation of miR-135a as a kind of cancer suppressor gene Tumor suppression: propagation and selectivity as suppressed kidney cancer cell kill Malignant glioma cells.MiR-135a can regulate the growth of midbrain and promote the fibrosis of diabetes kidney to also have some researchs to find in addition.Although there is so many research about miR-135 at present, miR-135 major part function is also not studied, the effect particularly in kidney disease (such as urinary podocytes).
Summary of the invention
In order to overcome defect of the prior art, the invention provides the novelty teabag of miR-135, and by miR-135 for the preparation of Podocytes in Renal Tissue diagnostic medicine or preparation, also miR-135 is used for inducing podocyte to damage, successfully constructs Podocytes in Renal Tissue model.
The present invention is achieved by the following technical solutions:
A first aspect of the present invention, the miR-135 gene providing a kind of separation is preparing the purposes in Podocytes in Renal Tissue diagnostic medicine or preparation.
Described by miR-135 gene for the preparation of Podocytes in Renal Tissue diagnostic medicine or preparation, refer to preparation miR-135 gene expression product being applied to podocyte diagnostic medicine as a Podocytes in Renal Tissue diagnosis index.
Research finds: the expression amount of miR-135 in damage podocyte is significantly higher than normal foot cell.Prompting miR-135 may play a significant role in the developing of Podocytes in Renal Tissue; The expression level of miR-135 gene may become the mark of Podocytes in Renal Tissue diagnosis.
A second aspect of the present invention, provide a kind of Podocytes in Renal Tissue diagnostic kit, described test kit comprises: the primer of specific amplification miR-135 transcript or the antibody of the anti-miR-135 of specificity.
A third aspect of the present invention provides the purposes of miR-135 in the medicine or preparation of preparation induction podocyte generation damage.
Preferably, described podocyte generation impaired performance is the expression level rise of desmin and snail in podocyte.
Preferably, described podocyte generation impaired performance is the expression level downward of nephrin, WT1 and E-cadherin in podocyte.
Preferably, described podocyte generation impaired performance is the rearrangement of the minimizing of podocyte skeleton or podocyte skeleton.
Further, described medicine or preparation play by expressing miR-135 the effect that damage occurs induction podocyte.
Further, described miR-135 plays by activating wnt signal path the effect that damage occurs induction podocyte.
Preferably, described miR-135 can activate wnt signal path and show as miR-135 activation Topflash activity.
Preferably, described miR-135 can activate wnt signal path and show as the expression that miR-135 promotes unphosphorylated activated β-catenin.
Preferably, described miR-135 can activate wnt signal path and shows as miR-135 and induce β-catenin to enter nucleus.
Further preferably, described miR-135 plays by suppressing the expression of GSK3 β the effect activating wnt signal path.
Preferably, described miR-135 suppresses mRNA and the protein expression level of GSK3 β by the 3'UTR of direct target GSK3 β.
A fourth aspect of the present invention, additionally provides miR-135 and is building the purposes in Podocytes in Renal Tissue model.
A fifth aspect of the present invention, additionally provides a kind of Podocytes in Renal Tissue model building method, comprises the following steps: the miR-135 of process LAN effective dose in podocyte; Obtain Podocytes in Renal Tissue model.
After process LAN miR-135 induces, the present inventor utilizes the index of Podocytes in Renal Tissue disease-related to verify Modling model success or not.Checking finds, after adopting process LAN miR-135 to induce, in podocyte, the expression level of desmin and snail raises; In podocyte, the expression level of nephrin, WT1 and E-cadherin is lowered; The rearrangement of the minimizing of podocyte skeleton or podocyte skeleton.These indexs comprehensive are known, successfully construct Podocytes in Renal Tissue model after the present inventor's process LAN miR-135 induces.
Successfully construct Podocytes in Renal Tissue model after process LAN miR-135 of the present invention induces, described model has many-sided application.
Such as, may be used for studying and set forth the pathogenesis on the cell of Podocytes in Renal Tissue disease and even molecular level.
Also can be applicable to the screening of the medicine for the treatment of Podocytes in Renal Tissue.
A sixth aspect of the present invention, additionally provide a kind of method of screening the drug candidate for the treatment of Podocytes in Renal Tissue, said method comprising the steps of: test drug candidate is applied to the Podocytes in Renal Tissue model built by preceding method, and with do not use the Podocytes in Renal Tissue model testing drug candidate and compare, cause Podocytes in Renal Tissue symptom to be improved after wherein using or the test compounds of curing is exactly the drug candidate for the treatment of Podocytes in Renal Tissue.
The above-mentioned drug candidate filtered out can form a screening storehouse, can carry out further cell experiment, experimentation on animals and/or clinical trial, to confirm the treatment Podocytes in Renal Tissue effect of described potential material further to these materials.
Because the present invention adopts process LAN miR-135 to induce, constructed model is more similar to the pathogenic process of the mankind, and thus more accurate than model in the past in the screening of medicine, efficiency is higher.
Beneficial effect of the present invention is:
(1) the present invention discloses the dependency of miR-135 and Podocytes in Renal Tissue first, high expression level is had in Podocytes in Renal Tissue case more than 90%, expression amount in damage podocyte: the expression amount >=3.0:1 in normal foot cell, the sensitivity that the inventive method is detecting, accuracy is all significantly better than existing diagnostic method.
(2) the present invention discloses process LAN miR-135 first and can damage by podocyte, is more similar to the pathogenic process of the mankind.
(3) Podocytes in Renal Tissue model of the present invention has the characteristic feature of Podocytes in Renal Tissue, can the developing of Podocytes in Renal Tissue in analogue body admirably, be the ideal model of Podocytes in Renal Tissue basis and clinical application research, thus the medicine of screening treatment Podocytes in Renal Tissue can be applied to well.
Accompanying drawing explanation
Fig. 1: miR-135 in Podocytes in Renal Tissue model expression change.
Fig. 2: miR-135 expresses change in podocyte (MPC5) damage model in vitro.
Fig. 3: miR-135 expresses change in FSGS patient clinical sample.
Fig. 4: miR-135 expresses impact to MPC5 cell relating gene-1 rna level, miR-NC:microRNA mimics negative control.
Fig. 5: miR-135 on the impact of MPC5 cell relating gene-1 protein expression, miR-NC:microRNA mimics negative control.
Fig. 6: miR-135 to MPC5 cytoskeleton distribution influence, NT: without any process; MiR-NC:microRNAmimics negative control, 10 μm, scale.
Fig. 7: in 293T and MPC5 cell, miR-135 promote the activity of Topflash report carrier.MiR-NC:microRNA mimics negative control; LiCl group is as positive control.
The expression of the unphosphorylated activated β-catenin of Fig. 8: miR-135 promotion, NC:microRNA mimics negative control; LiCl group is as positive control.
Fig. 9: miR-135 can induce β-catenin to enter nucleus, miR-NC:microRNA mimics negative control; LiCl group as positive control, 50 μm, scale.
Figure 10: bioinformatic analysis GSK3 β is the potential target gene of miR-135.
The 3'UTR conservative Analysis of Figure 11: GSK3 β.
Figure 12: GSK3 β 3'UTR vector construction schematic diagram.
: Figure 13: miR-135 can be incorporated into wild-type GSK3 β 3'UTR and suppresses luciferase active in 293T and MPC5 cell, miR-NC:microRNA negative control; The negative control of Inh-NC:microRNA inhibitor; Inh-135:miR-135 inhibitor.
Figure 14: miR-135 to the active unrestraint effect of the luciferase of mutagenicity GSK3 β 3'UTR report carrier, miR-NC:microRNA negative control.
Figure 15: the miR-135 rna expression that can suppress GSK3 β, miR-NC:microRNA negative control.
Figure 16: the miR-135 protein expression that can suppress GSK3 β, miR-NC:microRNA negative control.
Embodiment
The present invention, through extensive and deep research, utilizes different models, is studied the function of miR-135 gene in Podocytes in Renal Tissue process.Find unexpectedly, in the kidney sample of Podocytes in Renal Tissue model and FSGS patient, miR-135 specificity overexpression, miR-135 is relevant with Podocytes in Renal Tissue in prompting.The medicine designed according to people miR-135 gene and expression product thereof and diagnostic techniques, can be used for the Podocytes in Renal Tissue of the Diagnosis and Treat mankind.
In addition, in mouse podocytes system MPC5, process LAN miR-135 can cause podocyte impaired, successfully sets up Podocytes in Renal Tissue model.Described Podocytes in Renal Tissue model has the characteristic feature of Podocytes in Renal Tissue, can be used as model, simulates developing of Podocytes in Renal Tissue well.Meanwhile, described Podocytes in Renal Tissue model also can be applicable to the drug candidate screening choosing treatment Podocytes in Renal Tissue.Complete the present invention on this basis.
Below by way of specific specific examples, embodiments of the present invention are described, those skilled in the art the content disclosed by this specification sheets can understand other advantage of the present invention and effect easily.The present invention can also be implemented or be applied by embodiments different in addition, and the every details in this specification sheets also can based on different viewpoints and application, carries out various modification or change not deviating under spirit of the present invention.
Before further describing the specific embodiment of the invention, should be understood that protection scope of the present invention is not limited to following specific specific embodiments; It is also understood that the term used in the embodiment of the present invention is to describe specific specific embodiments, instead of in order to limit the scope of the invention; In specification sheets of the present invention and claims, unless explicitly pointed out in addition in literary composition, singulative " ", " one " and " this " comprise plural form.
When embodiment provides numerical range, should be understood that except non-invention is otherwise noted, between two end points of each numerical range and two end points, any one numerical value all can be selected.Unless otherwise defined, the same meaning that all technology used in the present invention and scientific terminology and those skilled in the art of the present technique understand usually.Except the concrete grammar used in embodiment, equipment, material, according to those skilled in the art to the grasp of prior art and record of the present invention, any method of prior art that is similar with the method described in the embodiment of the present invention, equipment, material or that be equal to, equipment and material can also be used to realize the present invention.
Unless otherwise indicated, disclosed in the present invention experimental technique, detection method, preparation method all adopt the routine techniques of the molecular biology of the art routine, biological chemistry, chromatin Structure and analysis, analytical chemistry, cell cultures, recombinant DNA technology and association area.These technology are existing in existing document improves explanation, specifically can see the MOLEC μ LAR CLONING:A LABORATORY MANUAL such as Sambrook, Second edition, Cold Spring HarborLaboratory Press, 1989and Third edition, 2001; Ausubel etc., CURRENT PROTOCOLS INMOLEC μ LAR BIOLOGY, John Wiley & Sons, New York, 1987and periodic updates; Theseries METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATINSTRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODSIN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), AcademicPress, San Diego, 1999; With METHODS IN MOLEC μ LAR BIOLOGY, Vol.119, ChromatinProtocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc.
Embodiment 1 is miR-135 up-regulated in Podocytes in Renal Tissue model
1.miR-135 up-regulated in Podocytes in Renal Tissue mouse model
For the effect of research miR-135 in Podocytes in Renal Tissue, first we construct mouse podocytes damage model with Zorubicin, and after tail vein injection Zorubicin, different time points puts to death mouse, and solution cuts kidney, isolated glomeruli.Then detect the expression change of its miR-135 with real-time fluorescence quantitative PCR, as shown in Figure 1, miR-135 expresses and raises for the 4th day after Zorubicin injection, within the 11 day, reaches peak value.
2.miR-135 up-regulated in Podocytes in Renal Tissue external model
For studying the effect of miR-135 in vitro in cell further, we are with the expression change detecting miR-135 in podocyte after the Zorubicin of different concns, process different time sections.As shown in Figure 2, the expression amount of miR-135 raises along with the increase of doxorubicin concentration and the time lengthening of process.
3.miR-135 is up-regulated in the kidney sample of FSGS patient
Next we further study the relation of miR-135 and Podocytes in Renal Tissue disease, we have collected the kidney sample of 3 routine FSGS and the clinical samples (in contrast) of 3 routine rupture kidneys, real-time quantitative PCR detects the changing conditions of miR-135 expression amount in these samples, result is as Fig. 3, in FSGS kidney sample, the expression of miR-135 is obviously raised.
Embodiment 2 process LAN miR-135 in mouse podocytes system MPC5 can cause podocyte impaired
1. process LAN miR-135 can cause the downward of the functional marker of upper mediation of Podocytes in Renal Tissue marker
After MPC5 transit cell dye miR-135mimics, the 48h cultivated, lysing cell extracts RNA and protein.As everyone knows, desmin and snail is the classical damage marker of podocyte two, and nephrin, E-cadherin and WT1 functional marker that is podocyte, changed by the expression of quantitative PCR and some genes involveds of western blot technology for detection podocyte, its detected result is as shown in Fig. 4 (rna level) and Fig. 5 (protein level), the up-regulated of desmin and snail, and the expression of nephrin, WT1 and E-cadherin declines.
The process LAN of 2.miR-135 can cause the arrangement disorder of podocyte skeleton
Nearest bibliographical information, the disorder of podocyte skeleton is along with the decline of function, so skeleton is also the common index passing judgment on Podocytes in Renal Tissue.Therefore we guess whether miR-135 also can cause the rearrangement of podocyte skeleton.Guess our process LAN miR-135 in podocyte according to this, then with the F-actin skeleton of Phalloidine dye podocyte, its result such as Fig. 6, miR-135 can make podocyte skeleton reduce and reset around cytolemma.
Embodiment 3miR-135 can activate wnt signal path
1.miR-135 the activity of Topflash report carrier can be activated
Fig. 4 and Fig. 5 shows the expression that miR-135 can promote desmin and snail, and these two genes are the target genes in wnt signal path downstream, therefore we guess whether miR-135 can activate wnt signal path, and the activation of bibliographical information wnt signal path causes Podocytes in Renal Tissue.According to these research, first we have studied the impact of miR-135 on Topflash activity in MPC5 and 293T cell, as shown in Figure 7, no matter in MPC5 cell or in 293T cell, miR-135 can activate Topflash activity to result, and LiCl is as positive control.
2.miR-135 can promote the expression of unphosphorylated activated β-catenin
In MPC5 cell, after process LAN miR-135,48h extracts total protein, the expression of unphosphorylated activated β-catenin is detected by the method for protein immunization imprinting, as Fig. 8, miR-135 can promote the expression of unphosphorylated activated β-catenin, LiCl is as positive control.
3.miR-135 induction β-catenin enters core
Mimics and the mimics control of process LAN miR-135 in the MPC5 cell cultivated, by the Asia location of cellular immunofluorescence technology for detection β-catenin in MPC5 after 48h.As shown in Figure 9, under normal circumstances, the wnt signal path of podocyte is in silence state, and β-catenin is important to be positioned in kytoplasm and the junction of cell and cell.When cell damage or when being in morbid state, wnt signal path activates, and β-catenin can enter nucleus and start the expression of downstream target gene.Experimental result display miR-135 can induce β-catenin to enter nucleus.
Embodiment 4miR-135 can suppress the expression of GSK3 β
1. Bioinformatics Prediction miR-135 can the 3'UTR of target GSK3 β
The concrete mechanism of wnt signal path is activated in order to study miR-135 further, we carry out bioinformatic analysis and prediction to wnt signal path and miR-135 by applying biological bioinformatics analysis software, found that miR-135 can target GSK3 β (wnt signal path suppress son), as shown in Figure 10.Figure 11 represents that the 3'UTR of GSK3 β is high conservative in Mammals.
2.GSK3 β report carrier builds
For confirming predicting the outcome of above-mentioned information biology, we predict binding site according to it, utilize molecule clone technology to construct wild-type reporter carrier and the mutagenicity report carrier of pCDNA3.1-luciferase-GSK3 β-3'UTR, and its schematic diagram is as Figure 12.
3.miR-135 can the 3'UTR of direct target GSK3 β
For the target gene whether checking GSK3 β is miR-135, we utilize the report carrier of above-mentioned wild-type in 293T and MPC5 cell, do the experiment of luciferase luciferase reporter, experimental result as shown in figure 13, no matter be that in 293T cell or in MPC5 cell, miR-135 can suppress the expression of luciferase.But when carrying out above-mentioned experiment with the carrier of sudden change, we find that miR-135 can not the expression of luciferase of mutation inhibiting report carrier, as Figure 14.These results confirm that miR-135 can the 3'UTR of direct target GSK3 β, and action site conforms to completely with Bioinformatics Prediction.
The expression of 4.GSK3 β can be suppressed by miR-135
In order to study the impact that miR-135 expresses GSK3 β further, we are inside MPC5 cell after process LAN miR-135, detect the mRNA of GSK3 β and the change of protein level, its result, as Figure 15 and Figure 16, therefrom comes to see that miR-135 can suppress the mRNA of GSK3 β and the expression of albumen significantly.
Embodiment 5 drug screening
The Podocytes in Renal Tissue model prepared with the method process LAN miR-135 in embodiment 2 is for study subject, and detect before and after drug candidate uses, Podocytes in Renal Tissue changes, thus judges that whether drug candidate is effective for treating Podocytes in Renal Tissue.
Test group: the model giving drug candidate;
Control group: the model not giving drug candidate.
If compared with control group, Podocytes in Renal Tissue obviously improves, then illustrate that this drug candidate can treat Podocytes in Renal Tissue.
Can utilize and suffer from Podocytes in Renal Tissue animal, to the above-mentioned drug candidate can treating Podocytes in Renal Tissue through screening, carry out further animal experiment, verify result for the treatment of further.
Above embodiment is in order to embodiment disclosed by the invention is described, can not be interpreted as limitation of the present invention.In addition, various amendment listed herein and invention in method, composition change, be apparent concerning those skilled in the art without departing from the scope and spirit in the present invention.Although in conjunction with multiple concrete preferred embodiment of the present invention to invention has been concrete description, should be appreciated that the present invention should not be only limitted to these specific embodiments.In fact, variously as above invention is obtained concerning apparent amendment those skilled in the art and all should comprise within the scope of the invention.
Claims (10)
1. the miR-135 gene be separated is preparing the purposes in Podocytes in Renal Tissue diagnostic medicine or preparation.
2. a Podocytes in Renal Tissue diagnostic kit, described test kit comprises: the primer of specific amplification miR-135 transcript or the antibody of the anti-miR-135 of specificity.
3.miR-135 the purposes in the medicine or preparation of preparation induction podocyte generation damage.
4. purposes according to claim 3, is characterized in that, described podocyte generation impaired performance is that the expression level of desmin and snail in podocyte raises; In podocyte, the expression level of nephrin, WT1 and E-cadherin is lowered; Podocyte generation impaired performance is the rearrangement of the minimizing of podocyte skeleton or podocyte skeleton.
5. purposes according to claim 3, is characterized in that, described medicine or preparation play by expressing miR-135 the effect that damage occurs induction podocyte.
6. purposes according to claim 5, is characterized in that, described miR-135 plays by activating wnt signal path the effect that damage occurs induction podocyte.
7. purposes according to claim 6, is characterized in that, described miR-135 plays by suppressing the expression of GSK3 β the effect activating wnt signal path.
8.miR-135 building the purposes in Podocytes in Renal Tissue model.
9. a Podocytes in Renal Tissue model building method, comprises the following steps: the miR-135 of process LAN effective dose in podocyte; Obtain Podocytes in Renal Tissue model.
10. one kind is screened the method for the drug candidate for the treatment of Podocytes in Renal Tissue, said method comprising the steps of: test drug candidate is applied to the Podocytes in Renal Tissue model built by preceding method, and with do not use the Podocytes in Renal Tissue model testing drug candidate and compare, cause Podocytes in Renal Tissue symptom to be improved after wherein using or the test compounds of curing is exactly the drug candidate for the treatment of Podocytes in Renal Tissue.
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| CN109266616A (en) * | 2018-08-21 | 2019-01-25 | 山西省人民医院 | A kind of humanization mouse podocytes model of stable expression AQP2 albumen and its construction method and application |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109266616A (en) * | 2018-08-21 | 2019-01-25 | 山西省人民医院 | A kind of humanization mouse podocytes model of stable expression AQP2 albumen and its construction method and application |
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