CN116492470A

CN116492470A - Application of miR-148a-5p in treatment of diabetic nephropathy

Info

Publication number: CN116492470A
Application number: CN202310673606.0A
Authority: CN
Inventors: 朱瑞阳; 张政; 孙艳; 廖晓辉; 刘含登; 彭睿
Original assignee: Chongqing Medical University
Current assignee: Chongqing Medical University
Priority date: 2023-06-08
Filing date: 2023-06-08
Publication date: 2023-07-28

Abstract

The invention discloses an application of miR-148a-5p expression promoter in preparation of a medicament for preventing and treating diabetic nephropathy, and an application of miR-148a-5p serving as a target in screening of the medicament for preventing and treating diabetic nephropathy. The homing of BMSCs plays an important role in DKD, key miRNAs and action mechanisms for regulating DKBMIMS migration are researched, and DKD is found to be capable of down-regulating miR-148a-5p expression to inhibit BMSCs migration, and Wnt5a is a key effector molecule for regulating DKBMUMS migration by miR-148a-5 p. The completion of the research of the invention clarifies a new molecular action mechanism of DKBMSCs migration, provides a new target and treatment strategy for DKD relief in the future, provides a new direction for DKD treatment, and has important scientific research significance and clinical application prospect.

Description

Application of miR-148a-5p in treatment of diabetic nephropathy

Technical Field

The invention relates to the technical field of biological medicines, in particular to application of miR-148a-5p in treatment of diabetic nephropathy.

Background

Currently, the incidence of diabetes mellitus (diabetes mellitus, DM) is increasing worldwide and is becoming a major public health problem. Diabetic nephropathy (diabetic kidney disease, DKD) is a major cause of end-stage kidney disease, closely related to the morbidity and mortality of DM. In recent 10 years, the prevalence and incidence of DKD in China are remarkably increased, which seriously affects the life and health of human beings. During the progression of DKD, glomerular basement membrane thickening, mesangial matrix expansion, glomerular hyperfiltration and tubular interstitial fibrosis are changes in kidney morphology and ultrastructural development involving multiple intricate pathophysiological pathways, therefore, research into molecular processes controlling DKD development and identification of new therapeutic targets is crucial, and new therapeutic technologies to prevent, stop, treat and reverse DKD still need to be explored. DKD is a complex physiological process involving various cells and molecules, whereas bone marrow mesenchymal stem cells (bone marrow stromal stem cells, BMSCs) are one of the most important cells involved in them, which have the ability to migrate directionally to sites of inflammation, ischemia and injury, and differentiate into bone, cartilage and fat cells, involved in injury repair.

One study conducted in 2006 reports that BMSCs can increase insulin-producing beta cells in DM mice and reduce mesangial thickening and macrophage infiltration, which for the first time demonstrates the possibility of MSCs to treat DKD, and that improving homing ability of MSCs may be key to its therapeutic effect. Since then, more and more research progress has made MSCs a viable option for DKD. MSCs homing is a multi-step process mediated by specific molecular interactions. Although specific mechanisms of BMSCs homing have been studied since the 70 s of the 20 th century, many aspects of this process remain unknown and require further confirmation. In summary, homing of BMSCs has a positive impact on treatment of DKD. In recent years, the use of the repair actions of BMSCs, other stem cells and cell derivatives to reduce glomerulosclerosis, interstitial fibrosis, tubular interstitial inflammation and oxidative stress in damaged kidneys has become a hotspot in clinical DKD research. However, targeting BMSCs to damaged tissues such as kidneys is inefficient and has limited availability, and therefore, it is necessary to find an effective way to promote homing of BMSCs, so that endogenous or exogenous BMSCs migrate to the damaged site more effectively, which is beneficial to recovery and treatment of DKD.

Microribonucleic acid (miRNA) is a small molecule endogenous RNA, can be output from a cell nucleus through posttranscriptional regulation of gene expression, acts as a short-chain mature miRNA with the length of 18-24 nucleotides, and is specifically bound to the 3' UTR region of target mRNA so as to inhibit translation or degradation of the target mRNA and participate in posttranscriptional gene expression regulation. Since mirnas are key mediators of cellular homeostasis, deregulation can cause cell and organ damage. In recent years, a great deal of research shows that miRNA is involved in regulating proliferation and inflammation of mesangial cells, podocyte injury and tubular injury in DKD, which indicates that the miRNA and the development of DKD are closely related. In addition, mirnas are involved in the regulation of almost all cellular processes, and current research suggests that they can regulate the migration, differentiation and metabolic processes of a variety of cells, including changes in cell function in DKD environments. For example, in a high sugar environment, miR-29b can influence the functions of proliferation, activity, apoptosis, migration, invasion and the like of placenta trophoblast cells through negative regulation and control of a downstream target gene HIF 3A; upregulation of miR-125b-5p to inhibit HIF-1α/SP 1-mediated Robo4 expression can significantly improve cellular function of human retinal pigment epithelial cells in DM conditions, including restoration of reduced cell viability, reduced intercellular permeability, and reduced cell migration capacity. Meanwhile, more and more researches prove that miRNA has an effect in BMSCs, and the up-regulated miR-100-5p participates in pathogenesis of non-traumatic femoral head necrosis by inhibiting migration and osteogenic differentiation of the BMSCs; STAT3/miR-211/STAT5A signal pathway plays a key role in the migration process of BMSCs, and the migration capacity of MSCs is enhanced by over-expressing miR-211, so that the treatment effect of MSCs transplantation is promoted to improve the cardiac function after myocardial infarction. However, few studies report how mirnas regulate BMSCs migration in DKD environments, so the role and mechanism of mirnas in DKD by BMSCs need further exploration.

The Wnt family consists of 19 different Wnt ligands that are involved in regulating various metabolic activities of cells, such as cell proliferation, differentiation, migration, and apoptosis. Wnt signaling pathways include canonical Wnt/beta-catenin signaling pathway, planar cell polarity pathway, wnt/Ca ²⁺ Pathways, intracellular pathways that regulate spindle-directed pathways and asymmetric cell division. Two general categories are known: the canonical Wnt signaling pathway, which relies on β -catenin; a non-canonical Wnt signaling pathway that is not related to β -catenin. Wnt5a is widely studied in the Wnt family, which can regulate organ development and cellular function through Wnt/β -catenin canonical and atypical Wnt signaling pathways, respectively. Previous studies have shown that Wnt5a promotes the development of kidney diseases, such as DKD. Whereas the Wnt/β -catenin signaling pathway plays an important role in DKD pathogenesis, a number of studies have shown that specific blockade of the Wnt/β -catenin pathway can prevent DKD progression, including elimination of proliferation of mesangial cells and expression of extracellular matrix. Thus, blocking the activation of Wnt/β -catenin signaling pathway is an important strategy to prevent DKD. In addition, in human umbilical mesenchymal stem cells, proliferation and migration of skin cells in vitro can be influenced by regulating and controlling Wnt/beta-catenin channels, and the method has great significance in skin wound healing treatment strategies. Previous studies have also demonstrated that the Wnt/β -catenin signaling pathway is involved in the inhibition of miR-124 chemotactic migration of MSCs, directing migration of MSCs to hepatocyte growth factor secreted by damaged and inflammatory areas. However, BMSCs of interaction mechanism of miRNA and Wnt/beta-catenin under DKD environment are not reported.

Disclosure of Invention

The invention researches key miRNA and action mechanisms for regulating and controlling DKD BMSCs migration, and discovers that DKD can down regulate the expression of miR-148a-5p to inhibit BMSCs migration, and Wnt5a is a key effector molecule for regulating and controlling DKD BMSCs migration by miR-148a-5 p. Based on the above, the invention protects the following technical scheme:

application of miR-148a-5p expression promoter in preparation of medicines for preventing and treating diabetic nephropathy.

The miR-148a-5p expression promoter is miR-148a-5p micrometers.

The invention also protects the application of miR-148a-5p serving as a target in screening of drugs for preventing and treating diabetic nephropathy.

The medicament promotes the expression of miR-148a-5 p.

In the above arbitrary application technical scheme, the sequence of the miR-148a-5p is shown as SEQ ID NO. 9.

In any of the above use protocols, the increased expression of miR-148a-5p promotes homing of BMSCs.

In any of the above application schemes, the increase of miR-148a-5p expression upregulates the expression of DKD BMSCs migration related factors PAK1 and MMP 9.

In any of the above application technical schemes, miR-148a-5p regulates the migration of BMSCs in DKD through targeting Wnt5 a-mediated Wnt signaling pathway, and miR-148a-5p plays a negative regulation role on Wnt5a.

In any of the above usage technical schemes, miR-148a-5p acts on DKD BMSCs through a Wnt/beta-catenin classical signal pathway mediated by a target gene Wnt5a.

The research route of the invention is as follows:

primary BMSCs are extracted from C57BL/6J male mice, cultured until the third generation, identified according to international stem cell standards, and DKD BMSCs model is constructed by simulating DKD microenvironment. The scratch experiment proves that the migration capacity of BMSCs in DKD is obviously reduced compared with that of normal BMSCs.

Through chip data analysis and qRT-PCR experiments, miR-148a-5p is verified to be an important factor involved in regulating and controlling DKD BMSCs, and the expression in the DKD BMSCs is abnormally reduced.

Research on the influence of miR-148a-5p on migration of DKD BMSCs, we construct mimics and inhibitors of miR-148a-5p, and detect change of cell migration ability after successful transfection of BMSCs, and the result proves that miR-148a-5p can promote migration of BMSCs.

In order to explore a specific mechanism of miR-148a-5p for regulating DKD BMSCs migration, wnt5a is verified to be an important target of miR-148a-5p through raw signal target gene prediction, a double luciferase reporter gene and a western blot. Wnt5a is an important protein on the Wnt canonical signaling pathway, which plays an important role in a variety of kidney diseases including DKD. Specific blockade of the Wnt/β -catenin pathway can prevent DKD progression, including elimination of mesangial cell proliferation and ECM expression in glomeruli. DKD can lead to excessive activation of Wnt/β -catenin signaling pathway, leading to apoptosis and epithelial mesenchymal transition of podocytes and tubule epithelium, and leading to kidney injury and fibrosis. Thus, blocking the activation of Wnt/β -catenin signaling pathway is an important strategy to prevent DKD. We confirm that the expression of Wnt5a and beta-catenin in DKD BMSCs is abnormally up-regulated through qRT-PCR and western blot. After the over-expression plasmid and siRNA of Wnt5a are successfully constructed and transfected, verification is carried out through western blot, and the fact that in CONBMSCs, the migration capacity of BMSCs is obviously reduced after Wnt5a is over-expressed, and the migration capacity of BMSCs is obviously enhanced after Wnt5a is knocked down in DKD BMSCs is found. Co-transfecting miR-148a-5p and Wnt5a, we found that Wnt5a could reverse the effect of miR-148a-5p on migration ability. The above suggests that miR-148a-5p regulates DKD BMSCs migration through Wnt5 a/beta-catenin.

The beneficial effects of the invention are as follows:

the homing of BMSCs plays an important role in DKD, key miRNAs and action mechanisms for regulating DKD BMSCs migration are researched, and DKD is found to be capable of down regulating miR-148a-5p expression to inhibit BMSCs migration, and Wnt5a is a key effector molecule for regulating DKD BMSCs migration by miR-148a-5 p. The completion of the research of the invention clarifies a new molecular action mechanism of DKD BMSCs migration, provides a new target and treatment strategy for DKD relief in the future, provides a new direction for DKD treatment, and has important scientific research significance and clinical application prospect.

Drawings

FIG. 1 shows 33 differentially expressed miRNAs associated with DKD MSCs.

FIG. 2 is the expression of miR-148a-5P in CON BMSCs and DKD BMSCs (< 0.01).

FIG. 3 shows qRT-PCR detection of miR-148a-5P chemicals and inhibitor transfection efficiency (P < 0.01).

Fig. 4 is the effect on migration ability of BMSCs after overexpression or knock-down of miR-148a-5P (< 0.01).

Fig. 5 is the effect on the migratory capacity of BMSCs after overexpression or knock-down of miR-148a-5P (< 0.01).

FIG. 6 is the expression profile of BMSCs-associated migration factors MMP9 and PAK1 after transfection of miR-148a-5P micrometers or inhibitors (P < 0.01).

FIG. 7 is a predicted result of miR-148a-5p target gene.

FIG. 8 is a schematic representation of miR-148a-5p binding to Wnt5a 3' UTR.

Fig. 9 is miR-148a-5P binding to Wnt5a targeting (< 0.01; ns, no significance).

Fig. 10 is the expression profile of Wnt5a in CON BMSCs and DKD BMSCs (< 0.01).

Fig. 11 is miR-148a-5P targeted regulation of Wnt5a (< 0.01).

Fig. 12 is Wnt5a overexpression and silencing transfection efficiency (×p < 0.01).

Fig. 13 is the effect on the migratory capacity of BMSCs after overexpression or knock-down of Wnt5a (< 0.01).

Fig. 14 is the change in the migratory capacity of BMSCs after overexpression or knock-down of Wnt5a (< 0.01).

Fig. 15 is the expression profile of BMSCs-related migration factors MMP9 and PAK1 after overexpression or knock-down of Wnt5a (< 0.01).

Fig. 16 is the effect of Wnt5a reversible miR-148a-5P on BMSCs-related migration factors MMP9 and PAK1 (< 0.01).

Detailed Description

The invention is further illustrated, but is not limited, by the following examples.

The experimental methods in the following examples are conventional methods unless otherwise specified.

Example 1 extraction, culture and identification of Primary mesenchymal Stem cells

1 Experimental materials and reagents

1.1 major reagents

1.2 preparation of reagents

(1) DMEM/F12 complete medium: to 455mL of DMEM/F12 medium, 50mL of fetal bovine serum and 5mL of diabody were added, and the whole medium was prepared as 10% FBS and stored at 4 ℃.

(2) DKD microenvironment medium: DKD micro-environment medium containing 0.25mg AGEs, 1mg LPS and 2.25mg glucose was added to simulate high sugar, high AGEs and inflammatory states on the basis of DMEM/F12 complete medium with 10% FBS.

1.3 laboratory animals

C57BL/6J male mice of 6-8 weeks old, grade SPF, were supplied by the university of Chongqing medical laboratory animal center. All procedures were agreed by the ethical committee of Chongqing medical university (approval number: 2022113).

2 Experimental methods

2.1 extraction and culture of primary BMSCs

Male C57BL/6J mice of 6-8 weeks old were sacrificed, the femur and tibia were rapidly removed, the epiphyses on both sides were cut after removal of superficial muscles and periosteum, the bone marrow cavity was repeatedly flushed with a 1mL syringe until blunted, the flushing fluid was collected and centrifuged for 5min, and the supernatant was discarded. The single cell suspension was prepared by gently beating with DMEM/F12 complete medium, transferred to a cell flask, and cultured in a cell incubator at 37 ℃ with 5% co 2. Cell growth and morphological changes were monitored daily under an inverted microscope and the medium was changed after 48 hours. When cells were fused to 80%, the cell was fused to 80% at 1:2, when cells were passaged to passage 3 for subsequent experiments, the cells cultured in a simulated normal environment were designated as the CON BMSCs group.

2.2 flow cytometry detection of surface antigens

Digesting and collecting BMSCs to be detected, and adjusting the cell concentration to be 3 multiplied by 10 ⁶ . A1.5 mL EP tube was taken and labeled FITC-CD34, FITC-CD45, FITC-CD29, FITC-CD90 and FITC-Control groups, respectively. 100 mu L of cell suspension and 2 mu L of corresponding fluorescent antibody are added into each EP tube, incubated for 30min in a dark place and then detected by a machine.

2.3BMSCs osteogenesis Induction experiments

BMSCs to be tested were washed with PBS and then digested with pancreatin and passaged into six well plates for growth. After about 80% fusion of cells, 2mL of C57BL/6J mouse osteoinductive fluid was added and the fluid was changed every 2 days. After about 28 days, PBS was washed, 4% paraformaldehyde was fixed for 30min, and alizarin red was stained for 30min, and photographed under an inverted microscope for observation.

2.4BMSCs adipogenic Induction experiment

BMSCs to be tested were washed with PBS and then digested with pancreatin and passaged into six well plates for growth. After cells were approximately 80% confluent, 2mL of lipogenic induction solution was added to C57BL/6J mice, and the solution was changed every 2 days. After about 28 days, PBS is used for cleaning, 4% paraformaldehyde is fixed for 30min, and then oil red O staining solution is used for staining for 30min, and photographing and observation are carried out under an inverted microscope.

2.5 DKD-like environmental culture

When BMSCs were transferred to the third generation, the BMSCs were cultured in DMEM/F12 complete medium with 5. Mu.g/mL AGE, 2. Mu.g/mL LPS and 4.5. Mu.g/mL glucose to simulate DKD-like microenvironments of high glucose, high AGEs and inflammatory states, under which the cultured cells were designated as DKD BMSC group.

2.6 scratch test

(1) BMSCs were seeded into 6-well plates with uniform horizontal lines drawn in advance on the back.

(2) After BMSCs grow to be full, drawing vertical lines by using a 200 mu L gun head to manufacture scratches;

(3) The medium was discarded, the cell debris was washed with PBS, and 2mL of serum-free DMEM/F12 medium was added.

(4) Photographic observation was performed under an inverted microscope at 0h and 24h, and Image J software measured the distance between scratches of each group of BMSCs, and analyzed the BMSCs migration distance.

2.7 statistical analysis

Statistical analysis was performed using SPSS 21.0 and GraphPad Prism 6.0, and data are expressed as mean ± standard deviation. the t-test is used for comparison of the averages of two samples, and the one-factor analysis of variance is used for comparison of multiple sets of averages. P <0.05 indicates that the result is statistically significant.

3 results

3.1 Primary culture and morphological observations of BMSCs

After the primary BMSCs are extracted and cultured for 24 hours, a large number of floating cells and impurities are visible under a microscope, after 48 hours, the primary liquid exchange is carried out, a small number of adherent cells are mostly round or oval, part of the adherent cells are shuttle-shaped, and after the culture is continued for 3 days, a large number of adherent cells are aggregated and grow, and the cell body is transparent. When the cells were transferred to the third generation, the BMSCs exhibited a long fusiform shape with uniform morphology. 3.2 flow cytometry characterization of BMSCs

The results of detecting the third generation BMSCs surface markers by flow cytometry show that in the BMSCs, the positive expression rates of CD29 and CD90 are 99.96% and 99.31% respectively, high expression is shown, the expression rates of CD45 and CD34 are 7.71% and 1.73% respectively, obvious low expression is shown, and the expression rates are consistent with the surface antigen expression standard specified by the International therapeutic Association. 3.3BMSCs osteogenesis adipogenesis induced osteogenesis induced liquid can be dyed into orange after 28 days of induction, alizarin red is combined with osteoid to form concentric dark red calcium nodules, which indicates that BMSCs differentiate into bone-like cells; after 21 days of culture of the adipogenic induction solution, a large number of lipid droplets can be observed under a microscope, and part of cells can be stained in punctate orange, which indicates that BMSCs are differentiated into fat-like cells. 3.4DKD BMSCs model construction

And (3) performing DKD-like micro-environment culture on the BMSCs to construct a DKD BMSCs model. And (3) detecting the migration capacity of cells after culturing for 24 hours, 48 hours and 72 hours respectively in the CON BMSCs group and DKD-like microenvironment by a scratch experiment. The results showed that DKD BMSCs group had significantly reduced migration capacity compared to CON BMSCs group. In addition, the mobility of BMSCs is most remarkably reduced when DKD-like microenvironment is cultivated for 48 hours. The results prove that the DKD BMSCs model is successfully constructed and used for subsequent experiments.

Conclusion 4

In the embodiment, BMSCs are simply, stably and efficiently separated by adopting an adherent cell culture method for subsequent study. The separated BMSCs are in a form that spindle-shaped cells are gathered to be in a vortex shape or beam shape and are arranged in parallel, the morphology is uniform, the cells are mutually fused into pieces, the three-dimensional effect is good, and the polarity is good. Flow cytometry is carried out on cells cultured to the third generation to detect cell surface antigen markers, and the results show that BMSCs express CD29 and CD90 at high levels and CD45 and CD34 at low levels, and an osteogenic and adipogenic experiment shows that the cells can be induced to form osteogenic like cells and adipocytes, and the characteristics of stem cells are met. The scratch experiment is used for detecting migration capacity of the CON BMSCs group and the DKD BMSCs group under different culture time, and a DKD BMSCs cell model is successfully constructed and used for subsequent experiments.

Example 2 MiR-148a-5p regulates migration of bone marrow mesenchymal Stem cells of diabetic nephropathy

1 Experimental materials and reagents

The main experimental reagents in this example are as follows:

and (3) preparation of a reagent:

(1) TBST: adding ddH2O into TBS powder, dissolving, adding Tween-20, mixing, and storing at 4deg.C.

(2) Sealing liquid: the blocked protein powder was dissolved with TBST.

Experimental animals: as in example 1.

2 Experimental methods

2.1 acquisition of chip information

Chip GSE217711 was obtained from the United states bioinformatic center website (National Center for Biotechnology Information-Gene Expression Omnibus, NCBI-GEO) database as miRNA-seq data from fat-derived MSCs of DKD patients and control groups. The microarray platform of GSE217711 was GPL20301 Illumina HiSeq 4000 (Homo sapiens), comprising 25 sample data with 21 DKDs and 4 healthy controls.

2.2 screening of differential miRNAs

The difference in miRNA expression levels between DKD group and healthy control group was compared using the "limma" R language package. Screening was performed using P less than 0.05 as a screening criteria.

2.3 extraction of Total RNA

The method for extracting total RNA of the cells to be extracted adopts a conventional method.

2.4 reverse transcription of RNA

The extracted total RNA is firstly subjected to reverse transcription reaction after removing genome DNA, and the reaction system is as follows:

u6 reverse transcription reaction uses the downstream Primer of U6 to replace RT Primer Mix;

after being gently blown and evenly mixed, the mixture is centrifuged, and the reaction program of the PCR instrument is as follows: preserving at 37deg.C for 15min, 85deg.C for 5s, and 4deg.C.

2.5qRT-PCR reactions

The qRT-PCR reaction system was as follows (30. Mu.L system):

the solution is added into enzyme-free eight-linked tubes after being mixed evenly, 10 mu L of each hole is provided with three compound holes, and the sample is centrifuged instantaneously after the sample is loaded. Setting a reaction program: pre-denaturation at 95℃for 3min; denaturation at 95℃for 5s, annealing at 60℃for 30s, extension at 72℃for 30s,40 cycles. Use 2 ^-ΔΔct And (5) calculating a method. All experiments were performed at least three times.

The primer sequences used are shown in Table 1:

TABLE 1 primer sequences

2.6 Cell transfection of miR-148a-5p micrometers and inhibitor

(1) BMSCs in the logarithmic phase were evenly inoculated at appropriate density into six well plates containing complete medium prior to transfection.

(2) After each group of BMSCs grew to about 80%, the BMSCs were replaced with serum-free DMEM/F12 medium.

(3) The autoclaved 1.5mL EP tube was taken and 250. Mu.L of serum-free medium and 10. Mu.LLipofectamine were added ^TM 2000, standing at room temperature for 5min.

(4) A new 1.5mL EP tube was added with 250. Mu.L of serum-free medium and 10. Mu.L of miR-148a-5pmimics or miR-148a-5p inhibitors or micrometers NC or inhibitors NC, and the mixture was allowed to stand at room temperature for 5min after uniform mixing.

(5) Mixing the reagents obtained in the steps (3) and (4), lightly blowing and uniformly mixing, and standing at room temperature for 20min.

(6) Cells to be transfected are added to the prepared transfection reagent and serum-free medium and placed in an incubator for incubation. After 5-6h, the medium was replaced with DMEM/F12 complete medium.

2.7 Transwell cell migration experiment

(1) Each group of BMSCs was centrifuged at 800g for 5min after digestion with pancreatin, the supernatant was discarded, and after addition of serum-free medium, the mixture was blown up evenly.

(2) Cell count was performed to adjust the cell count of each group of BMSCs to 1X 10 ⁴ /mL。

(3) 500. Mu.L of complete medium was added to the lower chamber of the chamber, 200. Mu.L of single cell suspension was added to the upper chamber, and each well was plated with cells uniformly and then placed in a cell incubator for 48 hours and removed.

(4) After fixing 4% paraformaldehyde for 30min, the crystal violet dye is used for dyeing for 30min.

(5) And (3) cleaning the cell with PBS, gently wiping the surface of the bottom membrane of the cell with a cotton swab, air-drying, and then placing the cell under an inverted microscope for observation, photographing and counting.

2.8 scratch test

As in example 1.

2.9 extraction of Total protein

Conventional methods are employed.

2.10 protein concentration determination

Conventional methods are employed.

2.11 Western blot

(1) And (3) glue preparation: separating gel and concentrating gel with proper concentration are selected according to molecular weight, absolute ethyl alcohol is used for pressing gel, and a comb is carefully inserted to avoid bubble generation. And after the gel is completely solidified, assembling the gel in an electrophoresis tank, and adding electrophoresis buffer solution.

(2) The comb was gently pulled out and each well was loaded at 10-20. Mu.g and 5. Mu.L protein Marker.

(3) And (3) switching on a power supply, carrying out constant-voltage 80V electrophoresis, and when the protein Marker reaches the separation gel, regulating the voltage to 120V, and stopping electrophoresis when the protein Marker reaches the bottom of the separation gel.

(4) Cutting PVDF film of proper size in advance, marking, placing in methanol for 32s, ddH ₂ O for 3min, and then completely soaking in the transfer membrane liquid for standby.

(5) The gel was carefully removed and the glass plate removed and the portion containing the protein of interest was excised. And sequentially placing 2 layers of sponges, 2 sheets of filter paper and 2 layers of sponges from top to bottom in a sandwich mode, then assembling, and adding an electrotransfer liquid until the sponges are completely immersed. Note that no bubbles can be generated between the gel and the membrane.

(6) The ice bath film transfer is carried out at a constant current of 250mA, and the film transfer time depends on the molecular weight of the protein.

(7) And (5) taking out the strip after the completion, putting the strip into a sealing liquid prepared in advance, and sealing the strip on a shaking table at room temperature for 2-3 hours.

(8) After blocking, the primary antibody was incubated, and the strips were placed in diluted antibodies (dilution ratio 1:800 for beta-actin, 1:500 for MMP9, and 1:1000 for PAK 1) and incubated overnight at 4 ℃.

(9) The strips were removed and the primary antibody recovered and stored at-20℃for repeated use. Wash vigorously 3 times with TBST on a shaker for 10min each.

(10) The secondary antibodies were incubated and TBST diluted (1:5000) with the secondary antibodies of the corresponding species. Incubating for 90min at room temperature on a shaking table,

(11) TBST was vigorously washed 3 times for 10min each.

(11) And finally, developing, mixing the solution A and the solution B in the ECL chemiluminescence kit according to the ratio of 1:1, and dripping a proper amount of the luminescent solution on the strip and completely covering. The gel imager is exposed and photographed, and the Image Lab software performs gray scale analysis on the strips.

2.12 statistical analysis

As in example 1.

3 results

3.1DKD differential miRNA

In this chip we selected, 583 mirnas were totally identified with the 0 sample deleted, of which 152 were differentially expressed (fig. 1A). The average expression reads were greater than 350 for the remaining 33 differential mirnas after filtering using the algorithm of the "limma" R language package and screening according to P <0.05 (fig. 1B).

3.2 determination of target miRNAs

The search was performed according to NCBI Gene, and these 33 mirnas expressed in human samples, 20 of which were also expressed in mice (table 2). Of the mirnas expressed in all of these 20 mice, miR-148a-5p was down-regulated in DKD patient samples and fold differences were most pronounced. Finally, miR-148a-5p miRNA is selected as a target point for further research. The sequence of miR-148a-5p is shown in SEQ ID NO. 9: 5'-AAAGUUCUGAGACACUCCGACU-3'.

TABLE 2 20 miRNAs expressed in both human and mouse

3.3 expression level of miR-148a-5p in BMSCs

To study the expression of miR-148a-5p in BMSCs, we used qRT-PCR to detect the expression level of miR-148a-5p in both the normal and DKD groups. The results showed a significant decrease in expression in DKD BMSCs (P < 0.01) compared to the CON BMSCs group, as shown in fig. 2.

3.4 transfection efficiency of miR-148a-5p micrometers and inhibitor

To explore the effect of over-expression or silencing miR-148a-5p on cell migration, we used BMSCs transfected with miR-148a-5p micrometers and inhibitor respectively as experimental groups, BMSCs transfected with micrometers NC and inhibitor NC as control groups, and qRT-PCR determined the expression level of miR-148a-5p among the groups to verify transfection efficiency. The results show that in CON BMSCs, compared with inh-NC groups, the expression of miR-148a-5p in cells after inh-miR-148a-5p transfection is obviously reduced; in DKD BMSCs, expression was significantly enhanced after transfection of miR-148a-5pmimics compared to the miR-NC group (FIG. 3). The above results indicate that miR-148a-5p overexpression and knock-down were successfully performed.

3.5Transwell cell migration experiments to detect the influence of miR-148a-5p on BMSCs migration

To explore the effect of miR-148a-5p on BMSCs migration, we applied a Transwell chamber migration assay to detect changes in BMSCs migration ability after over-expression or knockdown of miR-148a-5 p. The result shows that in the CON BMSCs group, after miR-148a-5p is knocked down, the number of migration cells is obviously reduced; in DKD BMSCs, cell numbers increased significantly after over-expression of miR-148a-5p (FIG. 4).

3.6 scratch assay to detect the influence of miR-148a-5p on BMSCs migration

Next, we applied scratch experiments to detect, and the results showed that the relative migration distance of BMSCs after silencing miR-148a-5p was significantly reduced compared to inh-NC group, while the relative migration distance after overexpressing miR-148a-5p was significantly increased compared to miR-NC group (fig. 5).

3.7western blot detection of influence of miR-148a-5p on BMSCs-related migration factor

Further, we analyzed protein expression of migration related factors MMP9 and PAK1 in BMSCs by western blot experiments. The results show that knockdown of miR-148a-5p inhibited the expression levels of MMP9 and PAK1, while overexpression of miR-148a-5p promoted protein expression of MMP9 and PAK1 (FIG. 6). The results suggest that miR-148a-5p promotes migration ability of BMSCs and has an important effect on DKD BMSCs.

Conclusion 4

The experiment verifies that miR-148a-5p is probably an important biological factor involved in regulation and control of DKD BMSCs through chip analysis and qRT-PCR. Furthermore, miR-148a-5p micrometers and inhibitor plasmids are constructed, and after transfection, the fact that the over-expression of miR-148a-5p can promote migration of BMSCs is found, and the migration capacity of BMSCs is reduced due to knocking down. Taken together, we demonstrate that miR-148a-5p can play a role as an important target in DKD BMSCs migration.

Example 3, miR-148a-5p Regulation of DKD BMSCs migration by Wnt5a

1 Experimental materials and reagents

The same as in examples 1 and 2.

2 Experimental methods

2.1 double luciferase assay

2.1.1 Experimental group

(1) PC (positive control) +mic NC group

(2) PC+mimics miR-148a-5p group

(3) pmiRGLO+mics NC group

(4) pmiRGLO+mimics miR-148a-5p group

(5) pmiRGLO-Wnt5 aWT+mics NC group

(6) pmiRGLO-Wnt5aWT+mimics miR-148a-5p group

(7) pmiRGLO-Wnt5 aMUT+mics NC group

(8) pmiRGLO-Wnt5aMUT+mimics miR-148a-5p group

2.1.2 Experimental procedure

(1) 293T cells in log phase were seeded in 96-well plates and when cell density was around 60%, wnt5aWT, wnt5aMUT, mimcs NC and miR-148a-5p were transfected into cells, each group was plated with 3 duplicate wells.

(2) After transfection for 6 hours, fresh culture medium is changed, and after 48 hours of culture, the culture solution is discarded to collect cells for detection.

(3) 5 XPLB was diluted to 1 XPLB with distilled water, added in an amount of 100. Mu.L per well of a 96-well plate, the cells were blown with a pipette, and after being slowly shaken on a shaking table at room temperature for 15min, the cell lysate was sucked into a 1.5mL EP tube, centrifuged at 12000g for 10min at 4℃and the supernatant was taken.

(4) To a 96-well plate, 100. Mu.L of LuciferaseAssay Reagent II (LAR II) working solution was added.

(5) 20 mu L of cell lysate is added, the mixture is blown and mixed for 2 to 3 times by a liquid transfer device, and the value Firefly luciferase is measured and recorded, and the value is taken as an internal reference.

(6) 100. Mu.L Stop was added&And (5) measuring and recording Renilla luciferase value after the pipette is blown and evenly mixed, namely the report gene luminescence value.

2.2 Total RNA extraction from cells

As in example 2.

2.3 reverse transcription of RNA

As in example 2.

2.4qRT-PCR reactions

As in example 2.

Primer sequences are shown in table 3:

TABLE 3 primer sequences

2.5 extraction of Total protein

As in example 2.

2.6 protein concentration determination

As in example 2.

2.7Western blot

As in example 2.

2.8 vector construction

The overexpression plasmid of Wnt5a (OE-Wnt 5 a) and the control plasmid (pcDNA3.1) were constructed from Ruibo organisms and identified by sequencing. siRNA for knock-down of Wnt5a was designed and synthesized by the qinghao, the sequences are shown in table 4:

TABLE 4 Table 4

2.9Transwell cell migration experiments

As in example 2.

2.10 scratch test

As in example 2.

2.11 statistical analysis

As in example 2.

3 results

3.1miR-148a-5p targets binding Wnt5a

To investigate the potential mechanism of miR-148a-5p to regulate DKD BMSCs migration, we predicted the potential target genes of miR-148a-5p using the bioinformatics website Targetscan 7.2 (http:// www.targetscan.org/vet_72 /), starbase (http:// www.starbase.sysu.edu.cn /) and mirDB (http:// www.mirdb.org /), and crossed the resulting target genes to find the target genes of miR-148a-5p that affect BMSCs migration in DKD. As shown in FIG. 7, a total of 13 genes were found to be predicted in all of these 3 databases. Then, these 13 genes were searched for documents on the literature database PubMed using "DKD", "MSCs", "mapping" as the subject word, and finally we focused on Wnt5a. Previous literature studies report that Wnt5a mediated Wnt canonical signaling pathway plays an important role in cell migration, while Wnt5a is abnormally high expressed in DKD, however whether Wnt5a has a regulatory role in DKD BMSCs is unknown. We then further predicted binding site status of miR-148a-5p and Wnt5a by Targetscan 7.2 (fig. 8) and constructed luciferase reporter plasmids containing Wnt5a cDNA wild-type (Wnt 5 aWT) or binding sequence mutants (Wnt 5 aMUT) using a dual luciferase assay to detect Wnt5a luciferase activity in 293T cells. The results showed that miR-148a-5p micrometers significantly reduced the luciferase activity of Wnt5aWT, whereas miR-148a-5p micrometers had no effect on Wnt5a MUT (FIG. 9). The results suggest that miR-148a-5p has a targeting binding relationship with Wnt5a.

3.2 negative control of Wnt5a by miR-148a-5p in DKD BMSCs

We analyzed the expression of Wnt5a mRNA in DKD BMSCs using qRT-PCR, and the results showed that Wnt5a mRNA in DKD BMSCs was significantly enhanced compared to CON BMSCs (fig. 10A). Meanwhile, western blot detects the protein content of Wnt5a in CON BMSCs and DKD BMSCs, and the result trend is also that the content in DKD is remarkably high-expressed (figure 10B), which suggests that Wnt5a is an important factor affecting BMSCs in DKD environment and is opposite to the expression trend of miR-148a-5 p. The result of detecting the expression of the beta-catenin on the Wnt5a and the Wnt classical signal path mediated by the Wnt5a after the overexpression or the knock-down of the miR-148a-5p through western blot shows that the expression of the Wnt5a and the beta-catenin is inhibited when the miR-148a-5p is highly expressed, and the knock-down of the miR-148a-5p can promote the expression of the Wnt5a and the beta-catenin (figure 11). The result suggests that miR-148a-5p negatively regulates Wnt5 a/beta-catenin in DKD BMSCs.

3.3Wnt5a overexpression and knockdown efficiency verification

To clarify the specific role of Wnt5a in DKD, we constructed Wnt5a overexpression vectors, while designing siRNA sequences. The CON BMSCs and DKD BMSCs were transfected with the overexpression plasmid and siRNA, respectively, and it was verified by qRT-PCR that Wnt5a overexpression plasmid was effective in upregulating Wnt5a expression (fig. 12A). Meanwhile, 3 siRNAs can effectively knock down Wnt5a, wherein the inhibition effect of siWnt5a-1 on Wnt5a expression is most remarkable (figure 12B), so that siWnt5a-1 is used for subsequent experiments and is named as siWnt5a. The above results indicate that the overexpression and knock-down construction of Wnt5a was successful.

3.4Transwell cell migration experiments to examine the effects of Wnt5a on BMSCs migration

To explore the effect of Wnt5a on BMSCs migration, we used a Transwell chamber migration assay to detect changes in BMSCs migration capacity after overexpression or knock-down of Wnt5a. The results showed that the number of migratory cells was significantly reduced in the CON BMSCs group after Wnt5a overexpression, whereas the number of cells was significantly increased in the DKD BMSCs group after Wnt5a silencing (fig. 13).

3.5 scratch experiments to detect the effect of Wnt5a on BMSCs migration

Next, we used a scratch test for detection. The results showed that the relative migration distance of BMSCs after Wnt5a overexpression was significantly reduced compared to the control, while the relative migration distance of BMSCs after Wnt5a silencing was significantly increased compared to the control (fig. 14).

3.6western blot detection of Wnt5a effects on BMSCs-related migration factor

Next, we use western blot to detect the protein expression changes of the migration related factors MMP9, PAK1 of BMSCs after over-expression or knockdown of Wnt5a. The results showed that in CON BMSCs, after overexpression of Wnt5a, the expression of migration related factors MMP9, PAK1 protein was decreased, whereas in DKD environment-cultured BMSCs, after knock-down of Wnt5a, the expression of migration related factors MMP9, PAK1 was increased (fig. 15). The above results suggest that Wnt5a, which is highly expressed in DKD, inhibits the ability of BMSCs to migrate.

3.7miR-148a-5p regulates BMSCs related migration factors through Wnt5 a/beta-catenin, the interaction between miR-148a-5p and Wnt5a exists, and protein expression changes of BMSCs migration related factors MMP9 and PAK1 are detected through western blot. The results show that miR-148a-5 pininhibitor reduces MMP9 and PAK1 expression in CON BMSCs, but siWnt5a can reverse the effect; also in the DKD context, BMSCs overexpressing Wnt5a reversed the regulation of migration related factors MMP9, PAK1 protein expression by miR-148a-5p micrometers (FIG. 16). The results suggest that miR-148a-5p regulates migration of BMSCs through Wnt5 a/beta-catenin in DKD.

Conclusion 4

And verifying that miR-148a-5p can target and regulate Wnt5a through bioinformatics prediction, double-luciferase experiments and qRT-PCR. In DKD BMSCs, the expression trend of Wnt5a and miR-148a-5p is opposite and abnormally increased. Further, a Transwell cell migration experiment, a scratch experiment and a western blot experiment show that Wnt5a inhibits the migration of BMSCs. The migration factor after co-transfection of miR-148a-5p and Wnt5a is detected by Western Blot, and the effect of the Wnt5a on the migration factor can be reversed by miR-148a-5 p. Namely, miR-148a-5p regulates the migration of BMSCs in DKD by targeting Wnt5 a-mediated Wnt signaling pathway.

Claims

Application of miR-148a-5p expression promoter in preparation of medicine for preventing and treating diabetic nephropathy.
2. Use according to claim 1, characterized in that: the miR-148a-5p expression promoter is miR-148a-5pmimics.
Application of miR-148a-5p as target in screening of drugs for preventing and treating diabetic nephropathy.
4. Use according to claim 3, characterized in that: the medicament promotes the expression of miR-148a-5 p.
5. Use according to claim 1 or 3, characterized in that: the sequence of miR-148a-5p is shown in SEQ ID NO. 9.
6. Use according to claim 1 or 3, characterized in that: increased miR-148a-5p expression promotes homing of BMSCs.
7. Use according to claim 6, characterized in that: the increase of miR-148a-5p expression up-regulates the expression of DKBMIMS migration related factors PAK1 and MMP 9.
8. Use according to claim 6, characterized in that: miR-148a-5p regulates the migration of BMSCs in DKD by targeting Wnt5 a-mediated Wnt signaling pathway, and miR-148a-5p plays a role in negative regulation of Wnt5a.
9. Use according to claim 6, characterized in that: miR-148a-5p acts on DKBMIMS through a Wnt/beta-catenin classical signaling pathway mediated by a target gene Wnt5a.