Use of a miR-30e-5p inhibitor as a medicament for treating thyroid eye disease

Abstract

The application discloses an application of a miR-30e-5p inhibitor as a medicine for treating thyroid eye disease, and relates to the technical field of a new application of the miR-30e-5p inhibitor. The miR-30e-5p inhibitor is used for preparing a medicine composition for treating thyroid eye disease. The miR-30e-5p inhibitor (miR-30e-5p-inh) can inhibit the activation of an AKT / mTOR signal path, and can exhibit a significant TED treatment effect in both cell and animal levels; the miR-30e-5p inhibitor can simultaneously resist inflammation, resist fibrosis and inhibit fat generation, and has a significant treatment effect on thyroid eye disease; the miR-30e-5p inhibitor can be locally administered to eyes, and has small systemic side effects.

Description

Technical Field

[0001] This invention relates to the technical field of novel applications of miR-30e-5p inhibitors, specifically the application of a miR-30e-5p inhibitor as a drug for treating thyroid eye diseases. Background Technology

[0002] Thyroid ophthalmopathy (TED), the most common extrathyroidal complication of Graves' disease, is characterized by orbital tissue inflammation, adipose tissue hyperplasia, increased hyaluronic acid secretion, extraocular muscle hypertrophy, and progressive tissue remodeling. This disease not only severely damages the patient's appearance and quality of life, but may also cause irreversible damage to the patient's visual function, resulting in significant clinical harm.

[0003] Currently, the first-line treatment for TED (tetanus antitoxin) is still mainly intravenous glucocorticoids, while second-line treatments include immunosuppressants and biologics. These treatments have several significant drawbacks: First, the response rate is limited, and some patients are insensitive to glucocorticoids and immunosuppressants, resulting in poor treatment outcomes. Second, adverse reactions are significant; long-term use of glucocorticoids can easily lead to systemic side effects such as osteoporosis, hyperglycemia, and gastrointestinal ulcers, while immunosuppressants may increase the risk of infection. Biologics, such as the IGF1-R inhibitor tetumab, can easily cause menstrual disorders, hearing loss, and muscle spasms. Third, the treatment cycle is long and the cost is high; the price of biologics remains high, placing a heavy financial burden on patients. Fourth, existing treatments mainly focus on symptom relief and cannot regulate the disease progression, resulting in a high relapse rate after discontinuation of medication, and patients' clinical treatment needs have not been fully met. Summary of the Invention

[0004] To address the aforementioned problems, specifically those raised in the background section, this invention proposes the application of a miR-30e-5p inhibitor as a treatment for thyroid eye diseases. The specific technical solution is as follows: The use of a miR-30e-5p inhibitor as a drug for treating thyroid eye disease, wherein the miR-30e-5p inhibitor is used to prepare a pharmaceutical composition for treating thyroid eye disease.

[0005] Preferably, the miR-30e-5p is hsa-miR-30e-5p.

[0006] Preferably, the pharmaceutical composition comprises an effective amount of an hsa-miR-30e-5p inhibitor and a pharmaceutically acceptable carrier.

[0007] Preferably, the pharmaceutical composition is an ophthalmic preparation, including eye drops, ophthalmic gels, subconjunctival injections, periorbital and intravenous injections, etc.

[0008] Preferably, the nucleic acid sequence of the miR-30e-5p inhibitor is an inhibitory sequence based on hsa-miR-30e-5p.

[0009] Preferably, the nucleic acid sequence of the miR-30e-5p inhibitor is as described in SEQ ID NO:1.

[0010] The beneficial technical effects of this invention are as follows: the miR-30e-5p inhibitor (miR-30e-5p-inh) exhibits significant TED therapeutic effects at both cellular and animal levels by inhibiting the activation of the AKT / mTOR signaling pathway; it can simultaneously have anti-inflammatory, anti-fibrotic, and fat-inhibiting effects, and has significant therapeutic effects on thyroid eye diseases, and has potential preventive and therapeutic effects on thyroid-associated optic neuropathy (DON); the administration method is flexible, and it can be administered locally to the eye with few systemic side effects. Attached Figure Description

[0011] Figure 1 The images show the appearance of the eyes of the thyroid ophthalmopathy mice constructed in this invention and healthy control mice. Figure 2 This is a comparison diagram of the changes in the appearance of the mouse's eyes before and after treatment in Example 3 of the present invention; Figure 3 The images show the retroorbital magnetic resonance images of healthy controls, miR-30e-5p inhibitor controls, and inhibitor groups of mice in this invention.

[0012] Figure 4 This is a comparison of the relative area of ​​retroorbital fat and retroorbital fibrosis in mice in Example 3 of the present invention. Detailed Implementation

[0013] Preferred embodiments of the present invention will now be described with reference to the accompanying drawings. Those skilled in the art should understand that these embodiments are merely illustrative of the technical principles of the present invention and are not intended to limit the scope of protection of the present invention.

[0014] Example 1: Construction of human miR-30e-5p lentiviral vector and cell transfection experiment 1.1 Construction of Lentiviral Vector Based on the mature human hsa-miR-30e-5p sequence (5'-TGTAAACATCCTTGACTGGAAG-3'), a stable overexpression lentivirus of miR-30e-5p and a lentivirus inhibiting (silencing) miR-30e-5p were constructed, and corresponding negative control lentiviruses (overexpression negative control OE-NC and inhibition negative control Inh-NC) were also constructed.

[0015] Construction of overexpression vectors Using the GV369 vector backbone (element: Ubi-MCS-SV40-EGFP-IRES-puromycin, restriction sites AgeI / NheI), the target fragment containing the mature hsa-miR-30e-5p sequence was amplified by PCR using specific primers. After restriction enzyme digestion, the linearized vector was ligated with the target fragment to construct a recombinant overexpression plasmid. Positive clones showed perfect sequence matching during sequencing. The control vector was numbered CON238. After vector construction, HEK293T cells were co-transfected using the Gekkai three-plasmid packaging system to prepare overexpressing lentivirus LV-MIR155 (miR-30e-5p), with a titer of 1×10⁻⁶. 9 TU / mL.

[0016] Inhibition vector construction The GV691 vector backbone (element: hU6-MCS-CBh-gcGFP-IRES-puromycin) was selected, and the reverse complementary sequence 5'-CTTCCAGTCAAGGATGTTTACA-3' of the mature strand of hsa-miR-30e-5p was used as the interference suppressor fragment. The corresponding double-stranded oligo was synthesized, digested with AgeI and EcoRI, and ligated into the GV691 linear vector. To address the issue of sequencing interruption due to the hairpin structure of shRNA, positive clones with completely correct front-end sequences were selected as recombinant suppressor plasmids. The control vector was designated CON313. The recombinant plasmid was co-transfected into HEK293T cells to prepare the lentiviral inhibitor LV-hsa-miR-30e-5p, with a titer of 7 × 10⁻⁶. 8 TU / mL.

[0017] After all recombinant lentiviruses were packaged, cell supernatants were collected, purified by removing impurities, filtering, and ultracentrifugation. Viral titers were detected using a combination of fluorescence assay, drug screening, and absolute quantitative qPCR. All finished viral products had titers ≥1×10⁻⁶. 8 The standard TU / mL was aliquoted and stored at -80 ℃ in the dark, while simultaneously performing a full set of quality control tests for mycoplasma, bacteria, fungi, chlamydia, and endotoxins.

[0018] 1.2 Isolation and culture of primary TED orbital fibroblasts (TED-OFs) Ocular connective tissue specimens obtained during TED (tumor retardation) surgery and HC (hyperplasia) oculoplastic surgery were transported back to the laboratory on ice. The tissues were rinsed with PBS in a sterile operating room to remove as much blood and tissue fluid as possible. Adipose tissue was minced into pieces approximately 1×1 mm. 3Transfer the small pieces of cells to centrifuge tubes, add an appropriate amount of 10% type I collagenase, and mix thoroughly. Place the centrifuge tubes on a shaker at 37°C and 300 rpm overnight. Remove the centrifuge tubes, centrifuge at 1200 rpm for 15 min, discard the supernatant, wash once with PBS, and centrifuge at 1200 rpm for 5 min. Resuspend the bottom pellet in an appropriate amount of DMEM medium containing 10% serum and 1% antibiotics. Seed the cell suspension into 6-well plates, add medium to 4 mL, and change the medium every 3-4 days thereafter. Once the cells have reached confluence, passage them to 3-5 generations for subsequent transfection experiments.

[0019] 1.3 Lentiviral transfection of TED-OFs TED-OFs were seeded in 6-well plates. When the cell density reached 60% confluence, miR30e-5p mimic lentivirus, miR30e-5p inhibitor lentivirus, OE-NC, and Inh-NC negative control lentivirus were added, respectively. A final concentration of 4% p infection solution was added to enhance transfection efficiency. After 16 h of infection, the culture medium was replaced with complete medium. After 72 h, 1 μg / mL puromycin was added to select stable transfected cell lines. Fluorescence microscopy showed that the fluorescence positivity rate was >90%. qPCR was used to verify the expression level of miR30e-5p: the expression level of miR30e-5p in the OE group was upregulated by about 4 times compared with OE-NC; the expression level of miR30e-5p in the Inh group was downregulated by about 50% compared with Inh-NC.

[0020] 1.4 Detection of cell proliferation, inflammation, adipogenesis, and fibrosis-related indicators (1) CCK8 cell proliferation experiment Stable transfected cells from each group were seeded into 96-well plates, and CCK8 reagent was added at 24h, 48h, and 72h, respectively. The absorbance (OD value) at 450nm was measured using an ELISA reader.

[0021] Results: The OD value of the Inh group was significantly lower than that of Inh-NC, and cell proliferation was significantly inhibited; the OD value of the OE group was significantly higher than that of OE-NC, and miR30e-5p promoted the proliferation of TED-OFs.

[0022] (2) ELISA detection of hyaluronic acid (HA), inflammatory factors IL-6 and IL-8 Cell culture supernatants from each group were collected, and the levels of HA, IL-6, and IL-8 were detected using a commercially available ELISA kit.

[0023] Results: The secretion of HA, IL-6 and IL-8 in the supernatant of the Inh group was significantly lower than that of Inh-NC; the secretion of the above factors in the OE group was significantly higher than that of OE-NC.

[0024] (3) RT-qPCR detection of adipogenesis, fibrosis and inflammation-related mRNAs Total RNA was extracted from cells in each group, and cDNA was obtained by reverse transcription. The relative expression levels of CEBPβ, PPARγ, ADPN (adipogenesis), COL1A1, COL1A2, α-SMA (fibrosis), HAS2, HAS3 (hyaluronic acid synthesis), IL6, and ICAM1 (inflammation) mRNA were detected by qPCR.

[0025] Results: Inhibition of miR30e-5p significantly downregulated the mRNA expression of all detected genes; overexpression of miR30e-5p significantly upregulated the mRNA expression of all genes.

[0026] (4) Western Blot detection of protein expression Total protein was extracted from cells, transferred to a membrane by electrophoresis, and then incubated with primary antibodies: COL1A1, ICAM1, α-SMA, ADPN, CEBPβ, and β-actin. The relative expression levels of the proteins were then measured by imaging.

[0027] Results: In the Inh group, the expression of lipid, fibrosis, and inflammatory marker proteins was significantly decreased; in the OE group, the expression of these proteins was significantly increased.

[0028] (5) Oil Red O staining for adipogenic differentiation Cells in each group were induced with adipogenic induction solution for 27 consecutive days, fixed with 4% paraformaldehyde, stained with Oil Red O staining solution, photographed under a microscope, and the relative area of ​​lipid droplets was quantified.

[0029] Results: The number and area of ​​lipid droplets in the Inh group were significantly reduced, and adipogenic differentiation was inhibited; lipid droplets accumulated in large quantities in the OE group, and adipogenic differentiation was enhanced.

[0030] Example 2: Verification of the mechanism by which miR30e-5p regulates the pAKT / pmTOR signaling pathway 2.1 Target gene prediction and enrichment analysis The target mRNAs of hsamiR30e-5p were predicted using three databases: Starbase, Targetscan, and miRDB. The intersection of these databases yielded 655 common target genes. GO biological function enrichment and KEGG pathway enrichment analyses were performed on the target genes. The results showed that the target genes were significantly enriched in the mTOR, MAPK, FoxO, and insulin signaling pathways, identifying the AKT / mTOR pathway as the core regulatory pathway.

[0031] 2.2 Western blot detection of phosphorylated proteins in the AKT / mTOR pathway Total protein was extracted from each group of stably transfected TED-OFs in Example 1. Total AKT, mTOR, and phosphorylated proteins pAKT (Ser473) and pmTOR (Ser2448) were detected. The pAKT / AKT and pmTOR / mTOR ratios were calculated using GAPDH / β-actin as internal controls.

[0032] Results: In the Inh group, the phosphorylation levels of pAKT and pmTOR were significantly lower than those in Inh-NC, indicating that the pathway activity was inhibited; in the OE group, the phosphorylation levels of pAKT and pmTOR were significantly higher than those in OE-NC, indicating that the pathway was continuously activated; this demonstrates that miR30e-5p positively regulates the activity of the AKT / mTOR signaling pathway.

[0033] Example 3: Construction of AAV9-miR30e-5p inhibitory vector and in vivo therapeutic experiment in TED animal model 3.1 Construction of AAV9-miR30e-5p-inh viral vector The GV481 adeno-associated virus vector backbone was selected, with AAV2 inverted terminal repeats (ITRs) at both ends. The element arrangement was U6-MCS-CAG-mCherry, and the multiple cloning site was BsmBI. Using the mouse mmu-miR-30e-5p mature strand inverted complementary sequence 5'-CTTCCAGTCAAGGATGTTTACA-3' as an interfering insert, two complementary oligos were synthesized: P25G2306a (tctaaaaaaTGTAAACATCCTTGACTGGAAG) and P25G2306b (accgCTTCCAGTCAAGGATGTTTACAttttt). The oligo powder was annealed to form double-stranded DNA, and the GV481 vector was linearized using BsmBI restriction endonuclease. Recombination of the target fragment with the vector was then completed via ligation.

[0034] Single clones were selected, expanded, and sequenced for verification. Positive recombinant plasmids with perfectly matched insert sequences were screened. AAV-293 packaging cells were co-transfected using the Jikai AAV Helper-Free triple plasmid packaging system (recombinant GV481 vector, pAAV-RC, pHelper) to prepare AAV9 serotype recombinant adeno-associated virus. Cell pellet and culture supernatant were collected simultaneously 72 h after transfection. Viral particles were released through repeated freeze-thaw cycles, followed by PEG concentration, CsCl density gradient ultracentrifugation, and ultrafiltration desalting purification. The purified virus was then aliquoted after sterilization using a 0.22 μm filter. The viral genome physical titer was detected using ddPCR, with the final titer meeting the requirement of ≥1×10⁻⁶. 12 The standard (vg / mL) was stored at -80 ℃ in the dark. An AAV9-EGFP empty vector negative control virus (INHC) was constructed simultaneously as an experimental control. All viral products underwent a full set of quality control tests for appearance clarity, sterility, and endotoxin, and all indicators met the qualified standards.

[0035] 3.2 Construction of TSHR-immunized TED mouse model Female BALB / c mice aged 6-8 weeks, weighing 16-20 g, were selected. Animals were randomly divided into experimental, control, and blank control groups. A recombinant adenovirus intramuscular injection method was used to establish the model. Six-week-old female BALB / c mice were divided into three groups: one group was injected with recombinant adenovirus expressing the human TSHR A subunit (Ad-TSHR289), hereinafter referred to as the TED group; the other group was injected with an equal dose of adenovirus carrying the control plasmid (Ad-EGFP), hereinafter referred to as the EGFP group. The modeling process followed an extended immunization protocol, including three injections in the initial phase, each three weeks apart, and six injections in the maintenance phase, each four weeks apart. Each injection consisted of 25 μL of adenovirus suspension (titer 10) injected into the left and right quadriceps femoris muscles. 8 After completing 9 injections (PFU), the model was evaluated to determine if the model was successful.

[0036] Model validation metrics: Appearance: The TED group exhibited eyelid redness and swelling, widened palpebral fissures, protruding eyeballs, and weight loss; Serological findings: Serum TT4 and TSAb levels were significantly elevated in the TED group, consistent with hyperthyroidism. Thyroid pathology HE staining: Irregular proliferation of thyroid follicles and extensive infiltration of inflammatory cells; Serum exosome identification: Western blot analysis was performed to detect exosome marker proteins CD63, CD81, and TSG101. qPCR confirmed that the expression of miR30e-5p in serum exosomes of TED mice was significantly upregulated.

[0037] 3.3 Intraorbital injection intervention in mice Mice that successfully developed the TED model and exhibited mild to severe TED phenotypes were randomly assigned to three groups based on disease severity: the TED model control group (CON group, without viral intervention), the AAV9 empty vector control group (INHC group), and the AAV9-mediated miR-30e-5p inhibition group (INH group). All mice received the corresponding intervention via retroorbital injection: the CON group received 100 μL of saline, and the INHC group received 100 μL of AAV9 solution carrying the empty vector (viral titer of 2.5 × 10⁻⁶). 11 ( / animal), while the INH group was injected with an equal volume and titer of AAV9-mmu-miR-30e-5p-inhibition recombinant virus solution.

[0038] Two weeks after the intervention, all mice in all groups were given a second injection of adenovirus to maintain the TED model status. The experiment ended at week 44, and all mice were euthanized. Relevant orbital tissue and serum samples were collected for subsequent histological, molecular biological, and biochemical assays.

[0039] 3.4 Detection of therapeutic effects in animals (1) Ocular appearance and CAS clinical activity score Before administration and 6 weeks after administration, mice's eyes were photographed to record eyelid congestion and swelling, and scores were calculated according to the TED Clinical CAS scoring criteria.

[0040] Mouse eye appearance assessment The score includes two observable features: eyelid edema and eyelid swelling.

[0041] (1) Eyelid edema (0-3): 0 = none; 1 = mild eyelid thickening; 2 = moderate eyelid swelling, with partial narrowing of the palpebral fissure; 3 = severe swelling, almost closing the eyes; (2) Eyelid congestion (0-3): 0 = none; 1 = localized mild redness and swelling; 2 = diffuse moderate redness and swelling; 3 = deep red ("cherry red"); Each parameter is scored from 0 to 3, with higher scores indicating more severe inflammation. 1-2 indicates mild TED, 3-4 indicates moderate TED, and 5-6 indicates severe TED.

[0042] Result: As Figure 2 As shown, after 6 weeks of administration, the eyelid redness and congestion in the INH group significantly subsided, the palpebral fissure retracted, and the CAS score was significantly lower than that in the INHC and TED model groups.

[0043] (2) Small animal MRI orbital tissue imaging The orbits of mice were scanned using a Bruker 9.4T small animal magnetic resonance imaging system to quantify the degree of retro-orbital inflammation in each group of mice.

[0044] Results: Comparison of ocular imaging characteristics among different groups of mice showed that the orbital imaging of the INHC group was basically consistent with that of the TED model group, with obvious extraocular muscle hypertrophy and inflammatory edema. However, the ocular pathological imaging phenotype of the INH group mice was significantly improved, the degree of extraocular muscle hypertrophy was significantly alleviated, and the muscle fiber contour was closer to the normal state. This suggests that inhibiting miR-30e-5p can effectively reduce periorbital inflammatory infiltration and tissue edema in TED mice and significantly alleviate ocular inflammatory pathological damage.

[0045] (3) Orbital histopathological staining After sacrifice, the retroorbital tissue of the mice was separated, fixed, embedded in paraffin, and sectioned. HE staining: such as Figure 4 As shown, the relative area of ​​retroorbital fat (OAT / ON) was quantified, and the fat percentage was significantly reduced in the INH group, indicating a decrease in inflammatory cell infiltration. Masson collagen staining: collagen deposition was significantly reduced in the INH group, and the degree of orbital fibrosis was alleviated.

[0046] (4) Western blot detection of orbital tissue proteins Total protein was extracted from the retroorbital tissue of mice in each group, and the expression of adipogenic (ADPN, CEBPβ), fibrosis (COL1A1, α-SMA), and inflammation (ICAM1) marker proteins was detected.

[0047] Results: The expression levels of the above proteins in the INH group were significantly downregulated compared with those in the INHC group. In vivo inhibition of miR30e-5p could simultaneously inhibit orbital inflammation, fat hyperplasia, and tissue fibrosis.

[0048] Although the invention has been described with reference to preferred embodiments, various modifications can be made thereto and components can be replaced with equivalents without departing from the scope of the invention. In particular, the technical features mentioned in the various embodiments can be combined in any manner, provided there is no structural conflict. The invention is not limited to the specific embodiments disclosed herein, but includes all technical solutions falling within the scope of the claims.

[0049] The technical solution of the present invention has been described above with reference to the preferred embodiments shown in the accompanying drawings. However, it will be readily understood by those skilled in the art that the scope of protection of the present invention is obviously not limited to these specific embodiments. Without departing from the principles of the present invention, those skilled in the art can make equivalent changes or substitutions to the relevant technical features, and the technical solutions after such changes or substitutions will all fall within the scope of protection of the present invention.

Claims

1. The application of a miR-30e-5p inhibitor as a drug for treating thyroid eye disease, characterized in that, The miR-30e-5p inhibitor is used to prepare a pharmaceutical composition for treating thyroid eye diseases.

2. The application of the miR-30e-5p inhibitor according to claim 1 as a drug for treating thyroid eye disease, characterized in that, The miR-30e-5p is hsa-miR-30e-5p.

3. The application of miR-30e-5p as a drug for treating thyroid eye disease according to claim 2, characterized in that, The pharmaceutical composition comprises an effective amount of an hsa-miR-30e-5p inhibitor and a pharmaceutically acceptable carrier.

4. The application of the miR-30e-5p inhibitor according to claim 3 as a drug for treating thyroid eye disease, characterized in that, The pharmaceutical composition is an ophthalmic preparation, including eye drops, ophthalmic gels, subconjunctival injections, periorbital and intravenous injections, etc.

5. The application of the miR-30e-5p inhibitor according to claim 2 as a drug for treating thyroid eye disease, characterized in that, The nucleic acid sequence of the miR-30e-5p inhibitor is an inhibitory sequence based on hsa-miR-30e-5p.

6. The use of the miR-30e-5p inhibitor according to claim 6 as a drug for treating thyroid eye disease, characterized in that, The nucleic acid sequence of the miR-30e-5p inhibitor is as described in SEQ ID NO:1.

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