Use of miR-4488 for preparing a drug for preventing and or treating premature ovarian failure

Abstract

The present invention relates to the field of biomedicine technology, and specifically to the use of miR-4488 for preparing a drug for preventing and / or treating premature ovarian failure. When miRNA-4488 is loaded into a carrier, particularly stem cell-derived extracellular vesicles, it significantly promotes cell proliferation and migration, exhibits a significant anti-inflammatory effect in inflammatory cell models, alleviates ovarian granulosa cell damage, increases cellular reactive oxygen species levels, and protects mitochondria, thereby preventing and / or treating premature ovarian failure. Furthermore, it does not have the side effects of, for example, hormonal drugs.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular, to use of miR-4488 in preparing a drug for preventing and or treating premature ovarian failure. Background Art

[0002] Premature ovarian failure (POF) is a common gynecological condition characterized by amenorrhea, infertility, low estrogen levels, high gonadotropin levels, and a lack of mature follicles. It is more common in women under 40, but its onset is trending toward younger women. Chemotherapy is a common treatment for tumors, but it can cause severe damage to various organs. Studies have shown that chemotherapy drugs can cause premature ovarian failure, manifested by ovarian granulosa cell apoptosis, increased cellular oxidative stress, and tissue inflammation. Premature ovarian failure is primarily treated with hormonal medications, which have significant side effects, and currently no better treatments exist. Studies have shown that extracellular vesicles (EVs) derived from mesenchymal stem cells exhibit immune regulation and tissue repair capabilities similar to those of stem cells, and are considered promising for widespread application in regenerative medicine.

[0003] Therefore, there is a need in the art for a drug for preventing and / or treating premature ovarian failure, which has the advantages of small side effects and good efficacy. Summary of the Invention

[0004] In view of this, one of the objects of the present invention is to provide the use of miR-4488 for preparing a drug for preventing and / or treating premature ovarian failure. Another object of the present invention is to provide a drug for preventing and / or treating premature ovarian failure.

[0005] In a first aspect, the present invention provides a use of miR-4488 for preparing a drug for preventing and or treating premature ovarian failure.

[0006] Furthermore, the nucleotide sequence of miR-4488 is shown in SEQ ID NO.1.

[0007] Furthermore, the miR-4488 may also be a mimic of miR-4488.

[0008] Furthermore, the miR-4488 may also be miRNA-4488 isolated from other cells or extracellular vesicles.

[0009] Furthermore, the miR-4488 is located in a vector.

[0010] Furthermore, the carrier is an extracellular vesicle; further, the extracellular vesicle is an extracellular vesicle derived from stem cells.

[0011] In some specific embodiments, the stem cell-derived extracellular vesicles can be bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells, placental mesenchymal stem cells, or induced pluripotent stem cells.

[0012] Furthermore, the miR-4488 is located in the extracellular vesicles by mixing the extracellular vesicles with miRNA-4488 and performing transfection.

[0013] Furthermore, the transfection is electrotransfection.

[0014] Furthermore, the concentration of miR-4488 is: during electrotransfection, the final concentration of miR-4488 is 30nM-1000nM.

[0015] In some specific embodiments, the conditions of electrotransfection are: 200 V, 1000 μs, and an interval of 784 ms.

[0016] Furthermore, the drug has at least one or more of the following effects:

[0017] (a) Promote cell proliferation and migration;

[0018] (b) inhibiting inflammatory responses;

[0019] (c) Promote the proliferation of ovarian granulosa cells;

[0020] (d) reduce the level of reactive oxygen species that damage cells;

[0021] (e) protect mitochondria; or

[0022] (f) Reduced apoptosis of ovarian granulosa cells.

[0023] In the present invention, the term "prevention and / or treatment" means preventing and / or delaying the occurrence of a disease or normalizing it.

[0024] In the present invention, the term "miR-4488" may refer to miR-4488 or a mimetic thereof known in the prior art.

[0025] Furthermore, the premature ovarian failure is caused by the use of drugs. Still further, the premature ovarian failure is caused by the use of cisplatin.

[0026] In a second aspect, the present invention provides a drug for preventing and or treating premature ovarian failure, comprising: miR-4488 and a carrier.

[0027] Furthermore, the nucleotide sequence of miR-4488 is shown in SEQ ID NO.1.

[0028] Furthermore, the miR-4488 may also be a mimic of miR-4488.

[0029] Furthermore, the miR-4488 may also be miRNA-4488 isolated from other cells or extracellular vesicles.

[0030] Furthermore, the carrier is an extracellular vesicle; further, the extracellular vesicle is an extracellular vesicle derived from stem cells.

[0031] In some specific embodiments, the stem cell-derived extracellular vesicles can be bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells, placental mesenchymal stem cells, or induced pluripotent stem cells.

[0032] The beneficial effects of the present invention are that when miRNA-4488 is loaded into a carrier, in particular, into extracellular vesicles derived from stem cells, it significantly promotes cell proliferation and migration, has a significant anti-inflammatory effect in an inflammatory cell model, can alleviate ovarian granulosa cell damage, increase cellular reactive oxygen levels, and protect mitochondria, thereby preventing and or treating premature ovarian failure. At the same time, it does not have the side effects of, for example, the therapeutic effects of hormonal drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Figure 1 shows the correlation between the properties of extracellular vesicles (EVs) and stem cell-derived EVs loaded with miRNA-4488 (EVs-miR4488) in the examples of the present invention. a) Transmission electron microscopy image of EVs; b) Western blot image of EV surface markers CD63 and TSG 101; c) Nanoparticle size distribution of EVs; d) Zeta potential distribution of EVs; and e) Electroporation efficiency of miRNA-4488.

[0034] Figure 2 Figure 1 shows the effects of EVs-miR4488 on cell proliferation in the examples of the present invention. Panel a shows live-dead staining of NIH3T3 cells at 24 and 48 hours. Panel b shows fluorescence quantification of panel a at 24 and 48 hours. Panel c shows the effects of EVs and EVs-miR4488 on NIH3T3 cell proliferation at 24 and 48 hours, respectively, as assessed by the CCK-8 assay.

[0035] Figure 3 Figure 2 shows the effect of EVs-miR4488 on NIH 3T3 cell migration in an example of the present invention. Figure a shows the cell migration effect at 0 h, 24 h, and 48 h under a microscope; Figure b shows the calculated cell migration area.

[0036] Figure 4Figures showing the ability of EVs-miR4488 to alleviate lipopolysaccharide-induced inflammation in this example. Figure a shows ELISA assay for TNF-α levels in cells; Figure b shows ELISA assay for IL-10 levels in cells; Figure c shows qRT-PCR assay for TNF-α levels in cells; and Figure d shows qRT-PCR assay for IL-1β levels in cells.

[0037] Figure 5 Figure 2 shows the effects of EVs-miR4488 on the proliferation of ovarian granulosa cells 24 hours after cisplatin-induced injury. Panel a shows the effects of EVs-miR4488 on ovarian granulosa cell proliferation at 24 and 48 hours, respectively. Panel b shows the effects of EVs-miR4488 on the proliferation of ovarian granulosa cells 24 and 48 hours after cisplatin-induced injury, respectively. Panel c shows the concentration of cisplatin used to investigate cell damage. Panel d shows the changes in reactive oxygen species levels in ovarian granulosa cells under a fluorescence microscope, and Panel e shows the fluorescence intensity statistical graph of Panel d.

[0038] Figure 6 Figures 1 and 2 show the endocytosis of EVs-miR4488 by normal and cisplatin-damaged mouse ovarian granulosa cells, as well as changes in estradiol secretion levels in mouse ovarian granulosa cells, as described in the examples of the present invention. Figure a shows the endocytosis of EVs-miR4488; Figure b shows the concentration of E2 in MOGC cell culture medium after 24 hours of treatment by ELISA; and Figure c shows the concentration of E2 in MOGC cell culture medium after 48 hours of treatment by ELISA.

[0039] Figure 7 Figure 1 shows the mitochondrial membrane potential of ovarian granulosa cells protected against cisplatin-induced damage by EVs-miR4488. Figure a shows a fluorescence microscopy image of mitochondrial membrane potential changes in ovarian granulosa cells induced by cisplatin and the protection of mitochondria by EVs-miR4488. Figure b shows the mean fluorescence intensity percentage.

[0040] Figure 8 Figure 1 shows how EVs-miR4488 reduces cisplatin-induced apoptosis in ovarian granulosa cells. Panel a shows the level of apoptosis in ovarian granulosa cells 24 hours after EVs-miR4488 treatment; panel b shows the level of apoptosis in ovarian granulosa cells 48 hours after EVs-miR4488 treatment.

[0041] Figure 9Figure 1 shows the effect of EVs-miR4488 on the expression of apoptotic factors in ovarian granulosa cells, as well as cell cycle and early apoptosis detection. Panel a shows the qRT-PCR results for the apoptotic factor Bax; panel b shows the qRT-PCR results for the apoptotic factor Bcl-2; and panel c shows the cell cycle and early apoptosis levels.

[0042] Figure 10 Figure 1 shows the mechanism of the regulatory pathways and upstream and downstream target genes involved in miR-4488 predicted by bioinformatics. Figure a shows the pathways regulated by miR-4488; Figure b shows the enrichment map of the upstream and downstream target genes predicted for miR-4488; and Figure c shows a bubble chart. DETAILED DESCRIPTION

[0043] Example 1: Preparation of extracellular vesicles

[0044] 1.1 Cell culture

[0045] Identified rat BMSCs were cultured in a serum-free medium containing α-MEM, glutamine, ribonucleosides, deoxyribonucleosides, 1% penicillin-streptomycin, and 10% fetal bovine serum at 37°C and 5% CO2.

[0046] 1.2 Isolation of extracellular vesicles

[0047] EVs were isolated from BMSC culture supernatants by ultracentrifugation. Conditioned medium was collected under sterile conditions for pretreatment and centrifuged at 300×g for 10 minutes, 2000×g for 10 minutes, and 10,000×g for 30 minutes at 4°C. The supernatant was then isolated by ultracentrifugation at 100,000×g for 90 minutes at 4°C and resuspended in cold PBS. The supernatant was filtered through a 0.22μm filter and stored at −80°C.

[0048] 1.3 Identification of extracellular vesicles

[0049] The structure of EVs was observed using a transmission electron microscope and electron microscope images were recorded. Figure 1 As shown in a, extracellular vesicles have a lipid bilayer structure and are spherical.

[0050] Western Blotting was used to identify the surface markers of BMSC extracellular vesicles. First, protein samples of BMSC and EVs were extracted. After preparing the protein electrophoresis gel, the sample was loaded and electrophoresis was started at 90 V. After the sample entered the separation gel, the voltage was changed to 110 V until the electrophoresis was completed. The gel was removed and prepared for transfer. After the transfer was completed, the membrane was blocked with 5% skim milk for 1 hour and then incubated with the primary antibody overnight. The next day, TSG101, CD63 and GAPDH secondary antibody dilutions were incubated separately. After using EBL developer, images of each membrane were collected, and the results are shown as follows. Figure 1 As shown in b, TSG101 and CD63 markers are expressed on the surface of extracellular vesicles.

[0051] Nanocoulter G nanocoulter size analyzer was used to measure the hydrated particle size and potential of EVs. Figure 1 As shown in Figures c and d, the average diameter of extracellular vesicles is about 60 nm and the potential is between -10 and -20 mV.

[0052] Example 2: EVs-miR4488 electroporation efficiency and efficacy

[0053] 2.1 Electrotransfection to load miRNA-4488 into EVs

[0054] The sequence of hsa-miRNA-4488 is: AGGGGGCGGGCUCCGGCG (SEQ ID NO. 1).

[0055] EVs (20 μg) and miRNA-4488 (50 nM, 100 nM, 200 nM) were mixed in 200 μL electrofection buffer and electroporated in a cuvette at 200 V, 1000 μs, and 784 ms intervals to obtain extracellular vesicles (EVs-miR) rich in miRNA-4488. Cy3-labeled miR-4488 was electroporated into EVs at concentrations of 50 nM, 100 nM, and 200 nM. Samples were collected and placed on a flow cytometer using the PE channel. The electroporation efficiency was 2.0: Figure 1 As shown in f.

[0056] 2.2 EVs loaded with miR-4488 significantly promoted cell proliferation

[0057] 1×10 4NIH 3T3 cells were seeded in a 96-well plate. After the cells adhered to the wall, EBEL, EVs, and EVs-miR4488 were added for cell culture. After 24 and 48 hours of culture, the live-dead cell ratio was determined by live-dead cell staining, and cell viability was detected using a CCK-8 kit to evaluate the effect of the drug on cell viability. EVs-miR4488 can significantly promote the proliferation of NIH 3T3 cells. Figure 2 shown.

[0058] 2.3 EVs loaded with miR-4488 significantly promoted cell migration

[0059] 2×10 4 NIH 3T3 cells were seeded on a 6-well plate and cultured in a 37°C incubator. When the cell density reached 80%, the culture medium was aspirated and the NIH3T3 monolayer was carefully scraped with a 200 μL sterile pipette tip to form a uniform cell-free area. The cells were then washed with PBS to remove cell debris, photographed under a microscope, and the cell migration area was subsequently calculated using Image J. EVs-miR4488 can significantly promote NIH 3T3 cell migration, such as Figure 3 shown.

[0060] 2.4 Effects of miR-4488-loaded EVs on inflammatory factors

[0061] First, an inflammatory cell model was constructed. 1.0×10 6 RAW 264.7 cells (mouse monocytes and macrophages) were seeded into six-well plates and induced with 1 μg / mL lipopolysaccharide for 4 hours. The culture medium was then discarded and replaced with DMEM complete medium containing EVs and EVs-miR4488. After 4 hours of incubation, the cells and supernatant were collected.

[0062] The expression levels of anti-inflammatory factor IL-10 and pro-inflammatory factor TNF-α in RAW264.7 cells of each group were detected by ELISA. The results of ELISA were as follows: Figure 4 a. Figure 4 b. The mRNA expression levels of inflammatory factors TNF-α and IL-1β in RAW264.7 cells of each group were detected by qRT-PCR. Figure 4 c. Figure 4d. NC represents normal cells, and LPS represents inflammatory cells stimulated with bacterial lipopolysaccharide. Compared with the LPS-induced group, EVs-miR4488 treatment increased IL-10 secretion and decreased TNF-α secretion after the addition of extracellular vesicles. Furthermore, expression of the inflammatory factors IL-1β and TNF-α was lower after EVs-miR4488 administration compared with the EVs group, indicating that miR4488 and stem cell-derived EVs have a synergistic anti-inflammatory effect.

[0063] Based on the above results, it can be seen that EVs-miR4488 significantly promotes cell proliferation and migration, and has a certain anti-inflammatory effect.

[0064] Example 3: Evaluation of the value of EVs-miR4488 in treating premature ovarian failure

[0065] Effect of EVs-miR4488 on cell viability after cisplatin injury

[0066] 1×10 4 Ovarian Granulosa Cells (OGC) were seeded in 96-well plates. After the cells adhered, cisplatin was added or not, followed by EVs and EVs-miR4488 for cell culture. After 24 and 48 hours of culture, cell viability was measured using the CCK-8 kit to evaluate the effects on cell viability. Figure 5 As shown in Figures 5a and 5b, both EVs and EVs-miR4488 significantly promoted OGC proliferation. However, in the cisplatin-injured group, the cell proliferation-promoting effect of EVs-miR4488 after 48 h of treatment was significantly higher than that of the EVs group, indicating that miR4488 and stem cell EVs jointly promoted cell proliferation.

[0067] Effect of EVs-miR4488 on Reactive Oxygen Species Levels

[0068] 1×10 6 OGCs were seeded in 6-well plates. After the cells adhered to the wall, cisplatin was added first, followed by EBEL, EVs, and EVs-miR4488 for cell culture. After 24 h of culture, DCFH-DA probes were loaded in situ and observed using a fluorescence microscope. Cells were collected and reactive oxygen species fluorescence was detected using a fluorescence spectrophotometer. Figure 5 As shown in Figures c and d, EVs-miR4488 can significantly reduce the level of reactive oxygen species in OGCs.

[0069] 3.2 Investigation of the endocytic effect of extracellular vesicles loaded with miR-4488

[0070] Take the OGC in the logarithmic growth period and calculate it by 1×10 6The density of each well was seeded in a six-well plate. After the cells adhered overnight, cisplatin was added or not, and then EVs-miR4488 was added. Six hours later, 4% paraformaldehyde was added to fix the cells, and then DAPI staining solution was used to stain the cells for 10 minutes. The EVs-miR4488 internalization was observed under an inverted fluorescence microscope. Figure 6 shown.

[0071] 3.4 Effect of EVs-miR4488 on protecting mitochondrial membrane potential

[0072] 1×10 6 OGCs were seeded in a 6-well plate. After the cells adhered to the wall, cisplatin was added first, followed by EVs and EVs-miR4488 for cell culture. After 24 h of culture, the culture medium was removed, the cells were washed once with PBS, and 1 mL of cell culture medium was added. The changes in mitochondrial membrane potential were observed by JC-1 staining. After staining, the cells were observed under a fluorescence microscope. Figure 7 As shown in a and b, EVs and EVs-miR4488 could significantly alleviate the decrease in mitochondrial membrane potential caused by cisplatin at 24 h.

[0073] 3.5 Effect of EVs-miR4488 on reducing cell apoptosis

[0074] 1×10 6 OGCs were seeded in 6-well plates. After the cells adhered to the wall, cisplatin was added first, followed by EVs and EVs-miR4488 for cell culture. After 24 h and 48 h of culture, the cells were digested and prepared into cell suspensions. The apoptosis level was detected by flow cytometry using Annexin V-FITC / propidium iodide staining. Figure 8 As shown in Figure 3, EVs-miR4488 can reduce the level of cell apoptosis.

[0075] 1×10 6 OGCs were seeded in 6-well plates. After the cells adhered to the wall, cisplatin was added first, followed by EVs and EVs-miR4488 for cell culture. After 24 hours of culture, the cells were collected, and the cell RNA was extracted. After reverse transcription, qRT-PCR was used to detect the expression of cell apoptosis factors Bax and Bcl-2. Figure 9 As shown in the figure, the expression of pro-apoptotic gene Bax was significantly decreased, while the expression of anti-apoptotic gene Bcl-2 was significantly increased.

[0076] 1×10 6OGCs were seeded in 6-well plates. After cells adhered, cisplatin was added, followed by EVs and EVs-miR4488. After 24 and 48 hours of culture, 50,000–100,000 resuspended cells were centrifuged at 1000 g for 5 minutes. The supernatant was discarded and the pellet was added to 1 ml of ice-cold 70% ethanol. Gently pipette to mix thoroughly and then fix at 4°C overnight. 0.5 ml of propidium iodide staining solution was added to each tube of cell sample. The cell pellet was slowly and thoroughly resuspended, incubated at 37°C in the dark for 30 minutes, and then stored at 4°C in the dark. Red fluorescence was then detected by flow cytometry at an excitation wavelength of 488 nm.

[0077] Example 4: Bioinformatics prediction of miR-4488 involved in regulatory pathway mechanism diagram and upstream and downstream target genes

[0078] 4.1 Bioinformatics prediction of miR-4488 involved in regulatory pathway mechanism diagram

[0079] Figure a is a diagram of the regulatory pathway mechanism of miR-4488 predicted by Funrich.

[0080] Figure b shows the target genes of miRNA-4488 predicted by TargetScan7.2 (targetscan.org) (4488 is the abbreviation of miRNA-4488)

[0081] Figure c is a bubble chart analyzing the involvement of miR-4488 in the regulation of reactive oxygen species or mitochondrial pathways. It is predicted that miRNA-4488 regulates mitochondrial organization through the regulation of mitochondrion organization pathway.

[0082] In summary, these results indicate that EVs-miR4488 can treat premature ovarian failure through the synergistic effect of miR-4488 and stem cell extracellular vesicles. It should be noted that according to the above embodiments of the present invention, those skilled in the art can fully realize the full scope of the independent claims and dependent rights of the present invention, and the implementation process and method are the same as the above embodiments; and the part not described in detail in the present invention belongs to the common technology in the art. However, the scope of protection of the present invention is not limited to this. Any changes or substitutions that can be easily thought of by any person familiar with the art within the technical scope disclosed in the present invention should be covered within the scope of protection of the present invention.

Claims

1. Use of miR-4488 for preparing a drug for preventing and / or treating premature ovarian failure caused by cisplatin, wherein: The nucleotide sequence of miR-4488 is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that The final concentration of miR-4488 is: 30nM-1000nM.

3. The use according to claim 1, characterized in that The miR-4488 is located in the vector.

4. The use according to claim 3, characterized in that The carrier is an extracellular vesicle.

5. The use according to claim 4, characterized in that The extracellular vesicles are stem cell-derived extracellular vesicles.

6. The use according to claim 5, characterized in that The miR-4488 is located in the extracellular vesicles by mixing the extracellular vesicles with miRNA-4488 and performing transfection.

7. The use according to any one of claims 1 to 6, characterized in that The drug has at least one or more of the following effects: (a) Promote cell proliferation and migration; (b) inhibiting inflammatory responses; (c) Promote the proliferation of ovarian granulosa cells; (d) reduce the level of reactive oxygen species that damage cells; (e) protect mitochondria; or (f) Reduced apoptosis of ovarian granulosa cells.

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