CN105079808B - MiR590 purposes and its related drugs - Google Patents

Abstract

The present invention relates to biological technical field, and in particular to miR590 purposes and its related drugs.The invention firstly discloses miR590 in EMT external models and In vivo model it is all on a declining curve, EMT processes during renal fibrosis can effectively be suppressed by being overexpressed miR590.Present invention research shows that miR590 plays a part of suppressing EMT processes by suppressing TGF β R2, is provided the foundation for renal fibrosis research.

Description

MiR590 purposes and its related drugs

Technical field

The present invention relates to biological technical field, and in particular to miR590 purposes and its related drugs.

Background technology

Kidney is one of most important organ in mammal body, is played an important role in animal body, its main work( The internal discarded object of excretion can be presented as, maintain the function such as cylinder electrolyte balance and secretion EPO.Therefore renal function once by Compensatory more than itself to damage, series of physiological and pathological dysfunction just occurs in body, until ultimately resulting in renal function Exhaustion.In most kidney troubles, the fibrosis of kidney is almost present in during all kidney troubles.Its feature Property performance mainly excessive extrtacellular matrix deposition between cell and cell, so as to affect glomerulus and renal tubule Fibrosis or hardening.For this pathologic process of fibrosis, Most scholars tend to a kind of sight of approval both at home and abroad at present Point is that epithelial cell plays an important role to transition of mesenchymal cells (i.e. EMT) during renal fibrosis, and prevents EMT Preventing and treating of the process to renal fibrosis may have important clinical value.But there have which molecule to be risen during EMT to be important Effect is also imperfectly understood.With small molecule microrna discovery and its illustrating for mechanism, researcher has found large quantities of Microrna molecules take part in EMT processes.

MiRNAs is that molecule size is about 22-25nt non-coding RNA sequences malo greatly, and its main function is By influenceing target gene mRNA degraded or translation with reference to the UTR of target gene 3 '.This micromolecular was found first from 1993 Since RNA, the research for miRNA, which is presented, breaks out formula growth.It is mainly based upon in kidney research field on miRNA research Conditionity knocks out Drosa or Dicer mouse model.By the technology it is found that miRNAs organism various physiological diseases All play and act on during reason, development, differentiation as embryo, the increment apoptosis of cell, metabolism, the migration of tumour etc..And MiRNA is also in the starting stage in the research of the related pathologic process of renal fibrosis, and many related molecular events do not have also Illustrate completely.

TGF β R2 are the molecules that a comparison is crucial in TGF signal beta paths.It is formed as cell-membrane receptor and TGF β R1 Heterodimer receives extraneous signal stimulus so as to activate TGF β/SMAD signal paths.TGF β/SMAD signal paths participate in biology Internal various pathological processes, wherein just including EMT.There are some researches show TGF β R2 can be as EMT rush before Enter the factor and play in various pathological processes to act on, the regulator of its upstream is miR655.

And functions of the miR590 in kidney fibrosis does not have document report still.

The content of the invention

The defects of in order to overcome in the prior art, the invention provides miR590 genes renal tissue fibrotic processes In effect.

The present invention is achieved by the following technical solutions：

The first aspect of the present invention, there is provided a kind of miR590 genes of separation are preparing or screened renal fibrosis treatment Purposes in medicine or preparation.

The miR590 genes by separation include two sides for preparing or screening renal fibrosis medicine or preparation The content in face：First, it is applied to prepare renal fibrosis curative using miR590 genes as the action target of medicine or preparation Thing or preparation；Second, it is applied to screening renal fibrosis curative using miR590 genes as the action target of medicine or preparation Thing or preparation.

It is described to be applied to prepare renal fibrosis medicine using miR590 genes as the action target of medicine or preparation Or preparation specifically refers to：The target that miR590 genes are acted on as medicine or preparation, to develop for renal fibrosis cell Medicine or preparation, so as to improve the expression of the intracellular miR590 genes of renal fibrosis.

It is described to be applied to screening renal fibrosis medicine using miR590 genes as the action target of medicine or preparation Or preparation specifically refers to：The target that miR590 genes are acted on as medicine or preparation, is carried out to medicine or preparation Screening, the medicine of miR590 gene expressions can be promoted to treat drug candidate as renal fibrosis to find.Such as gene, resist Body medicine, small-molecule drug etc. can be using miR590 genes and its albumen as effective object.For example, the present invention is by kidney fiber Change and pcDNA3.1-miR590 is transfected in cell to be overexpressed miR590.

Further, the medicine or preparation play the work for the treatment of renal fibrosis by promoting miR590 expressions With.

Further, the medicine or preparation play a part for the treatment of renal fibrosis by being overexpressed miR590.

Further, it is fine to play treatment kidney by the EMT processes during suppressing renal fibrosis by the miR590 The effect of dimensionization.

The EMT processes refer to the process of epithelial cell to transition of mesenchymal cells.

Further, the miR590 plays a part of suppressing EMT processes by targeting in TGF β R2.

Preferably, the miR590 with the UTR of TGF β R2 3 ' by being combined and targeting in TGF β R2.

Preferably, the miR590 plays a part of suppressing EMT paths by suppressing TGF β R2 expressions.

The second aspect of the present invention, there is provided a kind of renal fibrosis medicine or preparation, the medicine or preparation lead to The miR590 genes for crossing separation prepare or screened acquisition, and the medicine or preparation contain the overexpression miR590 of effective dose drug effect Composition.

The renal fibrosis medicine or preparation include but is not limited to：It is nucleic acid molecules, carbohydrate, lipid, small Molecular chemistry medicine, antibody medicine, polypeptide, albumen or interference slow virus.

The nucleic acid includes but is not limited to：ASON, double-stranded RNA (dsRNA), ribozyme, endoribonuclease SiRNA (esiRNA) or short hairpin RNA (shRNA) prepared by III.

The amount of application of the medicine or preparation is enough transcriptions or translation for improving miR590 genes, or is improved enough The expression of miR590 albumen or the dosage of activity.So that the expression of miR590 genes maintains the level that can suppress EMT processes.

The third aspect of the present invention provide the miR590 genes of separation a kind of prepare renal fibrosis diagnostic medicine or Purposes in preparation.

It is described to be used to miR590 genes prepare renal fibrosis diagnostic medicine or preparation, refer to miR590 gene expressions Product is applied to the preparation of renal fibrosis diagnostic medicine or preparation as a renal fibrosis diagnosis index.

Research is found：Expression quantity of the miR590 genes in renal fibrosis cell is substantially less than normal kidney cell.Carry Show that miR590 genes may play a significant role in the occurrence and development of renal fibrosis；The expression of miR590 genes may As the mark of renal fibrosis wound diagnosis.

The fourth aspect of the present invention, there is provided a kind of renal fibrosis diagnostic kit, the kit include：Specificity Expand the primer of miR590 transcripts or the anti-miR590 of specificity antibody.

The beneficial effects of the present invention are：

Obvious EMT phenomenons really be present and along with miR590's in fibrotic processes the invention firstly discloses kidney Lower, on the other hand we have found that TGF β R2 are probably miR590 potential target gene by bioinformatics software.In vitro Dual-Luciferase gene reporter assay present invention demonstrates the possibility that miR590 and TGF β R2 have interaction, and using real When quantitative fluorescent PCR and western blot experiments confirm in people's proximal tubular epithelial cells (i.e. HK2) to interstitial cell turn The downward that miR590 can cause TGF β R2 to express is overexpressed during change, and suppresses its EMT process.Then done by siRNA Disturb experiment to find, the influence of TGF β R2 downward to the EMT of HK2 cells is identical with overexpression miR590.It is overexpressed in cell During TGF β R2, HK2 cell EMT phenomenons can be reversed partly, and this volume of data illustrates that miR590 influences the upper of HK2 cells Skin interstitial process may be worked by TGF β R2, and then participate in progression of fibrosis.

Brief description of the drawings

Fig. 1：MiR590 conservative Analysis schematic diagrames.

Fig. 2：TGF β R2 3, UTR conservative Analysis schematic diagram.

Fig. 3：The EMT induced in HK2 cells with TGFB1.

Fig. 4：In the checking of mRNA level in-site inside and outside EMT models.

Fig. 5：In the checking of the external EMT models of protein level.

Fig. 6：Expression declines in the EMT models of miR590 in vivo and in vitro.

Fig. 7：The histology checking of renal fibrosis model.

Fig. 8：In the checking of the external EMT models of protein level.

Fig. 9：MiR590 is overexpressed efficiency checking.

Figure 10：MiR590 influences (mRNA) to HK2 cells EMT.

Figure 11：MiR590 influences (protein) to HK2 cells EMT.

Figure 12：MiR590 influences (protein) to HK2 cells EMT.

Figure 13：MiR590 is to TGF β R2 3, UTR action sites analysis schematic diagram.

Figure 14：MiR590 is to TGF β R2 3, UTR effect experiments.

Figure 15：Protein level miR590 influences on TGF β R2.

Figure 16：SiRNA perturbation technique silence TGF β R2 efficiency.

Figure 17：SiRNA silences TGFBR2 influences (mRNA) to HK2 cells EMT.

Figure 18：SiRNA silences TGF β R2 influence (protein level) to HK2 cells EMT.

Figure 19：Be overexpressed TGF β R2 influences on HK2 cells EMT.

Embodiment

The present invention has found miR590 in EMT external models and In vivo model first by in-depth study extensively It is on a declining curve.Then, the inventors found that and be overexpressed miR590 can effectively suppress epithelial cell marker bases The expression of cause and promotion interstitial cell marker gene upregulations.Downstream key molecule involved by it is TGF β R2, because MiR590 can influence TGF β R2 expression so as to influence EMT.

Illustrate embodiments of the present invention below by way of specific instantiation, those skilled in the art can be by this specification Disclosed content understands the further advantage and effect of the present invention easily.The present invention can also pass through specific realities different in addition The mode of applying is embodied or practiced, the various details in this specification can also be based on different viewpoints with application, without departing from Various modifications or alterations are carried out under the spirit of the present invention.

Before the specific embodiment of the invention is further described, it should be appreciated that protection scope of the present invention is not limited to down State specific specific embodiment；It is also understood that the term used in the embodiment of the present invention is specific specific in order to describe Embodiment, the protection domain being not intended to be limiting of the invention；In description of the invention and claims, unless in text Explicitly point out in addition, singulative "one", " one " and " this " include plural form.

When embodiment provides number range, it should be appreciated that except non-invention is otherwise noted, two ends of each number range Any one numerical value can be selected between point and two end points.Unless otherwise defined, in the present invention all technologies for using and Scientific terminology is identical with the meaning that those skilled in the art of the present technique are generally understood that.Except used in embodiment specific method, equipment, Outside material, according to grasp of the those skilled in the art to prior art and the record of the present invention, it can also use and this Any method, equipment and the material of the similar or equivalent prior art of method, equipment described in inventive embodiments, material come real The existing present invention.

Unless otherwise indicated, disclosed in this invention experimental method, detection method, preparation method using this technology lead Domain conventional molecular biology, biochemistry, chromatin Structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and The routine techniques of association area.These technologies existing perfect explanation in the prior art, for details, reference can be made to the MOLEC such as Sambrook μLAR CLONING：A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001；Ausubel etc., CURRENT PROTOCOLS IN MOLEC μ LAR BIOLOGY, John Wiley＆Sons, New York, 1987 and periodic updates；the series METHODS IN ENZYMOLOGY, Academic Press, San Diego；Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998；METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999；With METHODS IN MOLEC μ LAR BIOLOGY, Vol.119, Chromatin Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc..

Conservative Analysis of the miR590 of embodiment 1 between each kind

In order to whether analyze miR590 nucleotide sequence between species with well-conserved.By people, mouse, macaque and black The miR590 nucleotide sequences of orangutan, gorilla etc. are analyzed, to wherein H.sapiens, M.musculus, Macaca Mulatta and Pan troglodytes, Gorilla gorilla etc. primary miR590 have found after having done comparison analysis Primary miR590 have well-conserved (see Fig. 1) between species.

Conservative Analysis of the UTR of 2 TGF β R2 of embodiment 3 ' between each species

Whether have to analyze the UTR of TGF β R2 3 ' between species it is well-conserved, by people, rat, mouse, dog, big As, the UTR nucleotide sequences of TGF β R2 3 ' of zebra fish are analyzed, to wherein H.sapiens, M.musculus, Rat, dog, The elephant UTR of TGF β R2 3 ' University of California Santa Cruz genome database (UCSC) hierarchial-cluster analysis have been done, the UTR of TGF β R2 3 ' have well-conserved (see Fig. 2) between showing each species.

The miR590 of embodiment 3 is reduced in the EMT models that TGF β 1 are induced

In order to study the relation between miR590 and EMT, we simulate EMT processes, TGF β 1 with TGF β 1 in vitro After (10ng/ml) acts on HK2 cells 48h, significant change occurs for its morphology, from similar round to star-like change (see Fig. 3), and Us, which are detected, from mRNA level in-site and protein level can be found that E-cadherin is decreased obviously, laminin, a-SMA, collagen Obvious to rise (see Fig. 4,5), this sequence of events all shows that HK2 cells live through EMT processes.We also enter to miR590 afterwards Detection is gone, has as a result shown that miR590 expressions in HK2 cell EMT models are decreased obviously (see Fig. 6).

The miR590 of embodiment 4 is reduced in the EMT models that UUO is simulated

Because downward trend is presented during the EMT that TGF β 1 are induced in miR590, whether present in animal body same Individual trend.Then it is fine so as to reach kidney using unilateral ostruction technology simulate the model of uronephrosis in Mice Body for we Dimensionization, for ligation after 2 weeks, masson dyeing and HE dyeing show that obvious destruction and tissue fibrosis phenomenon occur for Renal Structure (see Fig. 7).And detect us from mRNA level in-site and protein level and can be found that E-cadherin is decreased obviously, laminin, a- SMA, collagen substantially rise (see Fig. 4,8), and a series of this fact all illustrates EMT mistakes occur during renal fibrosis Journey.We are also detected to miR590 afterwards, as a result show that miR590 expressions in UUO are decreased obviously (see Fig. 6).

The miR590 of embodiment 5 suppresses the EMT of epithelial cell

Because miR590 shows as downward trend in EMT models, then miR590 and EMT is causality or companion With being related to that we are not very clear.Whether then we are overexpressed miR590 in HK2 cells, see it to the EMT of epithelial cell Have an impact.(see Fig. 9) after Successful transfection pcDNA3.1-miR590 in HK2 cells, mRNA or protein level prompting E- Cadherin substantially rises, laminin, a-SMA, and collagen is decreased obviously (see Figure 10,11,12), opposite with EMT processes. This explanation miR590 may be played an important role during EMT.

The regulatable target genes of Bioinformatics Prediction miR590 of embodiment 6

Due to miR590 in the HK2 epithelial cells EMT that TGFB1 is induced and in the renal fibrosis model of UUO mouse all The trend reduced is shown as, and is overexpressed miR590 in HK2 to cause E-cadherin, laminin, a-SMA, The change of the EMT marker genes such as collagen, generally develop towards suppression EMT directions.So we guess that miR590 exists EMT plays a role.In order to know that miR590 is worked by that key factor, we have used miRDB, PicTar, These three softwares of and TargetScan predict miR590 target gene simultaneously, and which is related to EMT, takes the common factor of three software We guess that TGF β R2 are most likely to be potential target sites of the miR590 during EMT.The miR590 and UTR of TGF β R2 3 ' make With site as shown in the figure (see Figure 13).

The miR590 of embodiment 7 can be directly in conjunction with TGF β R2 3 '-UTR

By bioinformatic analysis above, TGF β R2 3 ' UTR are quite conservative in mammal.In order to demonstrate,prove Bright miR590 can directly with TGF β R2 3 ' UTR direct interactions, the miR590 of mouse is cloned into carrier by us On pcDNA3.1, and the TGF β R2 of mouse 3 ' UTR are cloned into the carrier pcDNA3.1- with firefly luciferase On Luciferase.Then pcDNA3.1, pcDNA3.1-miRNA 590, and pcDNA3.1-Luc-TGF β R2-UTR are total to 293T cells are transfected, do the experiment of Dual-Luciferase reporter, miR590 can substantially suppress the activity of luciferase (see figure 14)。

For the site of further clear and definite miR590 and TGF β R2 3 '-UTR direct interactions, we are to predicting Seed Sequences have carried out point mutation (see Figure 13), do the discovery of Dual-Luciferase reporter after Seed Sequences mutation again, mutation Seed Sequences can cause luciferase activity be increased significantly for Seed Sequences are unmutated, but still less than pair According to group (see Figure 14).In summary, the 3 '-UTR that miR590 can be directly with TGF β R2 in 293T cells are acted on, and it is made It is exactly the Seed Sequences that predict with site.We are overexpressed pcDNA3.1-miR590 in HK2 cells afterwards, TGF β after 48h R2 on a declining curve this of expression can be proven (see Figure 15) from western experiments.

Embodiment 8 suppresses EMT progress using siRNA technologies in people's proximal tubular epithelial cells HK2 silences TGFBR2

We have subscribed targeting TGF β R2 siRNA from Guangzhou Rui Bo companies, detects siRNA silence efficiency first, turns After contaminating HK2 cells, western blot experiments prove that siRNA can effectively silence TGF β R2 (Figure 16).In order to confirm silence TGF Whether β R2 can have an impact to EMT processes, we using QPCR and western blot detection E-cadherin, laminin, A-SMA and collagen.As a result prove that silence TGF β R2 can suppress the EMT (Figure 17,18) of HK2 cells.

Embodiment 9 is overexpressed TGF β R2 and promotes EMT processes

Because TGF β R2 silence can suppress EMT, that be overexpressed TGF β R2 have what influence to the process, then we Structure pCMV-tag2B-TGF β R2 plasmids are simultaneously transfected in HK2 cells, and E-cadherin is substantially lowered after 48h, laminin, a- SMA, collagen substantially rise, identical with EMT processes.Therefore confirm TGF β R2 really during EMT from the pros and cons Play an important role.

Embodiment above is to illustrate embodiment disclosed by the invention, can not be interpreted as the limit to the present invention System.In addition, in various modifications and invention listed herein method, composition change, do not departing from the scope of the present invention Be obvious for those skilled in the art on the premise of spirit.Although a variety of specific of the present invention has been combined Preferred embodiment has carried out specific description to the present invention, it is to be understood that, the present invention should not be limited only to these specific embodiments. In fact, various, obvious modification should all include to obtain invention for those skilled in the art as described above Within the scope of the invention.

Claims (7)

1. purposes of the miR590 genes of separation in preparing or screening renal fibrosis medicine or preparation.

2. purposes according to claim 1, it is characterised in that the medicine or preparation are by promoting miR590 expressions To play a part for the treatment of renal fibrosis.

3. purposes according to claim 2, it is characterised in that the miR590 passes through during suppressing renal fibrosis EMT processes come play a part of treat renal fibrosis.

4. purposes according to claim 3, it is characterised in that the miR590 is played by targeting in TGF β R2 Suppress the effect of EMT processes.

5. purposes according to claim 4, it is characterised in that the miR590 with the UTR of TGF β R2 3 ' by being combined and target To acting on TGF β R2.

6. purposes according to claim 4, it is characterised in that the miR590 is risen by suppressing TGF β R2 expressions To the effect for suppressing EMT paths.

7. purposes of the miR590 genes of separation in renal fibrosis diagnostic medicine or preparation is prepared.