Application of miR-184-3p in preparation of medicine for improving diabetes related tissue damage repair

Abstract

The invention belongs to the technical field of biological medicine, and particularly discloses application of miR-184-3p in preparation of medicine for improving diabetes related tissue damage repair. The miR-184-3p has a nucleotide sequence as shown in SEQ ID NO. 1 (sequence identifier number 1). Researches find that miR-184-3p can regulate diabetic wound anti-inflammatory factors and play a role in wound repair, down-regulate the expression of inflammatory factors IFN-gamma, up-regulate the expression level of anti-inflammatory cytokines IL-4 and IL-13, especially regulate diabetic wound excessive inflammatory response and promote wound healing, and can be used for treating diabetic wounds. The miR-184-3p can be used as an active component for diabetes wound inflammation resistance or development of pharmaceutical preparations for promoting related tissue damage repair, and has a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to the application of miR-184-3p in the preparation of a drug for improving the repair of diabetes-related tissue damage. Background Art

[0002] Skin wound repair is a complex process that involves the coordinated action of multiple cells to regulate inflammation, proliferation, and remodeling. The key points of healing are from early inflammation to the subsequent proliferation stage. Once the skin is damaged, the body will immediately initiate a cascade of precisely regulated wound repair processes to restore the skin's barrier function and maintain the stability of the internal environment as quickly as possible. However, the regeneration process of traumatic skin is easily affected by its own disease and the external environment, leading to chronic inflammation, difficult wound healing or slow healing. Diabetes is a chronic metabolic disease that affects multiple systems throughout the body, and its prevalence is increasing year by year. Diabetic wounds are characterized by long-lasting inflammation and poor angiogenesis. The chronic inflammatory state of diabetic patients hinders the normal healing process, resulting in delayed wound closure and increased risk of infection.

[0003] miRNAs are a class of 18-25nt single-stranded endogenous non-coding RNA molecules that are widely involved in the physiological or pathological processes of cell proliferation, differentiation, aging, apoptosis, and various diseases. Existing studies have shown that many specific miRNAs are enriched in EPSCs-derived exosomes, including miR-16, let-7a, miR-425-5p, and miR-142-3p. These specific RNAs reduce scar formation by reducing the expression of TGF-β1 in dermal fibroblasts and inhibiting myofibroblast differentiation. In addition, studies have shown that miRNAs can also play an important role in the healing of skin wounds and the pathogenesis of chronic inflammatory wounds, but there are currently no reports on the use of miR-184-3p for wound repair treatment. Summary of the invention

[0004] In view of the deficiencies in the prior art, the present application proposes the use of miR-184-3p in the preparation of drugs or preparations for promoting tissue damage repair, so that it may be used in new drug preparations for the skin, nerve and blood vessel repair treatment of chronic wounds in the future.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] In the first aspect, the present invention provides an application of a miRNA in the preparation of a drug or preparation for promoting tissue damage repair, which can promote wound healing. The miRNA is miR-184-3p, and its nucleotide sequence is shown in SEQ ID NO.1, specifically TGGACGGAGAACTGATAAGGGT.

[0007] Preferably, the tissue damage includes skin trauma, burns, chronic wound healing, radiation ulcers, skin cell repair and regeneration, etc.

[0008] Further preferably, the tissue damage is diabetes-related tissue damage.

[0009] In a second aspect, the present invention provides a use of a reagent for promoting the expression of miR-184-3p or enhancing the function of miR-184-3p in the preparation of a drug for promoting tissue damage repair.

[0010] Preferably, the tissue damage is diabetes-associated tissue damage.

[0011] Preferably, the reagent includes AgomiR-184-3p, a chemically modified form of miR-184-3p, a mimic of miR-184-3p, or a vector carrying miR-184-3p.

[0012] In a third aspect, the present invention provides a pharmaceutical composition for promoting tissue damage repair, the drug comprising miR-184-3p or an agent that promotes miR-184-3p expression or enhances miR-184-3p function, and pharmaceutically acceptable excipients, and the miR-184-3p nucleotide sequence is shown in SEQ ID NO.1.

[0013] Preferably, the reagent includes AgomiR-184-3p, a chemically modified form of miR-184-3p, a mimic of miR-184-3p, or a vector carrying miR-184-3p.

[0014] Preferably, the pharmaceutically acceptable excipient is selected from one or more of a solvent, a solubilizer, a wetting agent, an antioxidant, an antibacterial agent, a chelating agent, and a surfactant.

[0015] In a fourth aspect, the present invention provides the use of the miR-184-3p in the preparation of anti-inflammatory drugs, wherein the nucleotide sequence of the miR-184-3p is shown in SEQ ID NO.1.

[0016] Preferably, anti-inflammatory means being used to regulate excessive inflammatory response in diabetic wounds.

[0017] Preferably, the anti-inflammatory effect refers to up-regulating the expression level of cytokines IL-4 and IL-13, and down-regulating the expression level of pro-inflammatory factor IFN-γ.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention provides the application of miR-184-3p in promoting tissue damage repair, especially significantly increasing the proliferation, migration and scratch repair ability of keratinocytes in a high-sugar environment, and reducing the inflammatory response of wounds by down-regulating the expression of inflammatory factor IFN-γ and up-regulating the expression of anti-inflammatory cytokines IL-4 and IL-13, providing a new approach for non-cell therapy in the inflammatory stage. The miR-184-3p mimic treatment animal model has been used to verify its effect of promoting diabetic wound healing, and has good application prospects and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Relative expression level of miR-184-3p (a) and OD value (b) reflecting cell proliferation in keratinocytes transfected with miR-184-3p mimic under negative control (NC), normal glucose (NG, 6 mM) or high glucose (HG, 30 mM) culture conditions; migration number of HUVECs cells (c) and scratch repair rate (d) in each group; values ​​are mean ± SEM (n = 3); *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0021] Figure 2 The figure is a graph showing the wound healing curves over time for each group of wound healing treatments in the diabetic mouse model.

[0022] Figure 3 These are the detection results of the expression levels of the pro-inflammatory factor IFN-γ and the anti-inflammatory cytokines IL-4 and IL-13 in the wound tissue. DETAILED DESCRIPTION

[0023] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] Unless otherwise specified, the experimental methods used in the examples of the present invention are all conventional methods; the materials, reagents, etc. used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0025] In the absence of special instructions, the miR-184-3p and the like listed in the context of the present invention are microRNA types known in the art, and those skilled in the art are capable of obtaining molecular biological information related to miR-184-3p and the like from conventional databases, such as sequences, etc., wherein the nucleotide sequence of miR-184-3p used in the present invention is TGGACGGAGAACTGATAAGGGT. At the same time, in order to facilitate the development of in vivo and in vitro experiments, the present invention selects the commonly used molecular biological tool miR-184-3p mimic in the art to simulate the endogenous miR-184-3p in the organism to enhance the function of endogenous miR-184-3p. The miR-184-3p mimic can be purchased through commercial channels according to experimental requirements, or it can be designed and synthesized based on the sequence of miR-184-3p. The miR-184-3p mimic and the negative control miR-NCmimic used in the present invention are both commercially available (purchased from Guangzhou Ruibo Biotechnology Co., Ltd.) and used according to the product instructions.

[0026] Example 1 Effect of miR-184-3p on proliferation, migration and wound repair of tissue repair-related cells

[0027] 1. Cell proliferation assay

[0028] The experimental method for detecting cell proliferation in the present invention adopts CCK-8 method. Keratinocytes were inoculated in 96-well plates (5×10 3 / well), after the cells adhered to the wall and were cultured for 4 hours, transfection was performed according to the instructions of the Ruibo transfection reagent, and the miR-184-3p expression group (miR-184-3p mimic group) and the blank control group (miR-NC mimic group) were upregulated. After 48 hours of transfection, the original culture medium was discarded, and 100 microliters were added to each well according to the ratio of culture medium: CCK-8 reagent of 9:1. The culture plate was placed in a cell culture incubator and incubated for 2 hours. The OD value was detected using a multifunctional microplate reader, and the data were expressed as Relative OD Value (OD value of the experimental group / OD value of the control group).

[0029] The results are as follows Figure 1 As shown in a, the present invention sets normal glucose (NG, 6mM) and high glucose (HG, 30mM) culture conditions, and transfects miR-184-3p expression group (miR-184-3p mimic group) and blank control group (miR-NC mimic group) respectively. qRT-PCR is used to detect the content of miR-184-3p in keratinocytes of each group. Compared with the transfection NC group, the content of miR-184-3p in the miR-184-3p mimic transfection group is significantly increased (see Figure 1 a).

[0030] The CCK-8 results showed that ( Figure 1 b), the OD value of the HG+NC group was significantly lower than that of the NG+NC group, that is, the cell proliferation of the HG+NC group was significantly inhibited under high glucose environment. Compared with the NG+NC group, the OD value of the NG+miR-184-3p mimic group increased, but the difference was not significant. However, the OD value of the HG+miR-184-3p mimic group was significantly higher than that of the HG+NC group, indicating that upregulating miR-184-3p can promote the proliferation of keratinocytes, especially in a high glucose environment.

[0031] 2. Cell migration assay

[0032] The present invention adopts the Trans-well method to detect cell migration ability. A trans-well plate with a filter membrane pore size of 8 μm is inserted into a 24-well plate as the upper chamber, and keratinocytes are then inoculated at a cell density of 1×10 4 After 4 hours of inoculation in the upper chamber, transfection was performed according to the instructions of the Ruibo transfection reagent to upregulate the miR-184-3p expression group (miR-184-3p mimic group) and the blank control group (miR-NC mimic group), and then fresh culture medium containing 10% FBS was added to the lower chamber of the 24-well plate. Cultured at 37°C for 24 hours, sterile PBS was added for washing after the culture, and then the cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet for 20 minutes. Finally, after washing the upper chamber with PBS and removing non-migrated cells, the cells that migrated to the lower chamber were retained. The migration was recorded by taking pictures with an inverted microscope, and the number of migrated cells was calculated by Image J software, and then statistical analysis was performed.

[0033] Trans-well experiment results show (such as Figure 1 c), the cell migration of HG+NC group was significantly lower than that of NG+NC group, that is, the cell migration of HG+NC group was significantly inhibited under high glucose environment. The cell migration of NG+miR-184-3p mimic group was significantly increased compared with NG+NC group, and the cell migration of HG+miR-184-3p mimic was significantly increased compared with HG+NC group, indicating that upregulation of miR-184-3p can promote keratinocyte migration.

[0034] 3. Cell scratch test

[0035] Experimental method: Keratinocytes were cultured in DMEM / F12 medium containing 10% fetal bovine serum and 1% antibiotics (100 U / mL penicillin, 100 U / mL streptomycin) at 37°C and 5% CO 2 Keratinocytes were seeded in 24-well plates (2×10 5 / well), when the cell density reached 60%, transfection was performed according to the instructions of Ribo transfection reagent to upregulate the miR-184-3p expression group (miR-184-3p mimic group) and the blank control group (miR-NC mimic group). When the keratinocytes formed a confluent monolayer, a 200 μL sterile pipette tip was used to scratch the cell monolayer to simulate a wound. After washing the cell debris with PBS, 500 μL of serum-free medium was added to the transfection group and the blank group and placed at 37°C, 5% CO 2 Continue to culture for 24 h.

[0036] The results are as follows Figure 1 As shown in Figure d, upregulating the expression of miR-184-3p can significantly promote the scratch repair of keratinocytes. In summary, compared with NC treatment under NG culture conditions, HG culture conditions inhibited cell proliferation, migration and scratch repair ability. Transfection of miR-184-3p mimic enhanced the proliferation, migration and scratch repair ability of cells, and these effects were more obvious under HG than NG culture conditions. The miR-184-3p molecule can significantly improve the biological function of keratinocytes.

[0037] Example 2 Effect of miR-184-3p on promoting diabetic wound repair

[0038] The present invention purchases male C57BL / 6 mice of about 6-8 weeks old and 22g, and prepares a diabetic mouse model by intraperitoneal injection of streptozotocin (STZ). The mice need to fast for 12h before each injection, and STZ powder is weighed according to the number and weight of mice, and a STZ injection solution with a concentration of 2μg / μL is prepared using a citrate buffer solution with a pH of 4.2-4.5. The injection dose adopted in this experiment is 50μg / g, once a day, and continuously injected for 5 days. The volume of a single STZ injection solution is calculated according to the weight of each mouse, and a sterile syringe is used to draw the corresponding volume of injection solution for intraperitoneal injection. Eating is resumed after injection. One week after all injections are completed, the fasting blood sugar is measured by the tail-cutting blood sampling method. Fasting blood sugar is greater than 16.7mmol / L, indicating that the diabetic mouse model is successfully established.

[0039] All mice included in the diabetic group had fasting blood glucose greater than 16.7 mmol / L and were prepared for skin wound experiments. All experimental procedures complied with relevant international regulations outlined in the Guide for the Care and Use of Laboratory Animals. This study was approved by the Institutional Animal Ethics Committee for animal experiments. Before the formal experiment began, the mice were fed adaptively for two weeks and then divided into groups according to the purpose of the experiment. Each group had at least 6 mice. The incision was prepared with electric scissors and 70% ethanol. The mice were fed normally during the wound creation and treatment. This experiment was conducted in two groups: (1) The first group was control mice: divided into the AgomiR-184-3p injection group (Ctrl miR-184-3p mimic) and the NC (miR-NC mimic) group (Ctrl NC); (2) The second group was diabetic model mice, divided into the AgomiR-184-3p injection group (DM miR-184-3p mimic) and the NC (miR-NC mimic) group (DM NC). 5 μl of 50 mmM injection solution was injected at 6 points equidistant from the wound edge, and subcutaneous injection was performed every other day. The wound healing progress was measured every two days, and the wound diameter was measured in Photoshop to calculate the closure rate. The mice were humanely killed, and the wound tissues were collected and preserved according to the experimental requirements. The expression of cytokines L-4, L-13 and IFN-γ was detected by qRT-PCR.

[0040] The results are as follows Figure 2 First, we evaluated the overall healing speed of the wound. The wound healing curve results showed that the healing rate of the AgomiR-184-3p treatment group was significantly improved compared with the NC group, whether it was normal mice or diabetic mice. Among them, the wound size of the diabetic group injected with AgomiR-184-3p was smaller than that of other groups at each time point, and the degree of damage was significantly reduced, which promoted the healing of the wound. The wound repair rate after 7 days of treatment could reach 89.8%, which was better than the healing effect of the Ctrl miR-184-3p mimic group.

[0041] Under normal circumstances, appropriate inflammatory response is conducive to pathogen elimination and tissue repair, while excessive inflammatory response may lead to tissue damage. The results of detecting the expression of inflammatory factor indicators during wound healing show that (see Figure 3 ), compared with the control group, the expression level of the pro-inflammatory factor IFN-γ in the diabetic group injected with AgomiR-184-3p was decreased, and the expression levels of the anti-inflammatory cytokines IL-4 and IL-13 were increased, which effectively reduced the excessive inflammatory response of the diabetic wound surface, thereby enabling the wound healing of diabetic patients to be completed in an orderly and rapid manner.

[0042] Obviously, the above embodiments of the present invention are only examples for clearly illustrating the technical solution of the present invention, and are not intended to limit the specific implementation methods of the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the claims of the present invention shall be included in the protection scope of the claims of the present invention.

Claims

1. Use of miR-184-3p in the preparation of a drug or preparation for promoting tissue damage repair, characterized in that: The nucleotide sequence of miR-184-3p is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: The tissue damage repair includes skin trauma, burns, chronic wound healing, radiation ulcers, and skin cell repair and regeneration.

3. The use according to claim 1, characterized in that: The tissue damage refers to tissue damage caused by diabetes.

4. The use of miR-184-3p in the preparation of anti-inflammatory drugs, characterized in that: The nucleotide sequence of miR-184-3p is shown in SEQ ID NO.

1.

5. The use according to claim 4, characterized in that: The anti-inflammatory effect refers to inhibiting the excessive inflammatory response of diabetic wounds.

6. The use according to claim 5, characterized in that: The anti-inflammatory effect refers to up-regulating the expression levels of cytokines IL-4 and IL-13, and down-regulating the expression level of the pro-inflammatory factor IFN-γ.

7. A pharmaceutical composition for promoting tissue damage repair, characterized in that: The drug includes miR-184-3p or an agent that promotes the expression of miR-184-3p or enhances the function of miR-184-3p, and pharmaceutically acceptable excipients. The miR-184-3p nucleotide sequence is shown in SEQ ID NO.

1.

8. The pharmaceutical composition according to claim 7, characterized in that: The reagents include AgomiR-184-3p, a chemically modified form of miR-184-3p, a mimic of miR-184-3p, or a vector carrying miR-184-3p.

9. The pharmaceutical composition according to claim 7, characterized in that: The tissue damage refers to tissue damage associated with diabetes.

10. The pharmaceutical composition according to claim 7, characterized in that: The pharmaceutically acceptable excipient is selected from one or more of a solvent, a solubilizer, a wetting agent, an antioxidant, an antibacterial agent, a chelating agent, and a surfactant.

Images