A new use of human miR-335-3p and a method for regulating expression of target gene IL-16

Abstract

The present application relates to the technical field of biological medicine, disclose a kind of new use of human-derived miR-335-3p and the method for regulating target gene IL-16 expression, its new use is to inhibit yellow ligament cell fibrosis, and hsa-miR-335-3p can be targeted to bind IL-16 mRNA 3'UTR, the 8mer binding site of human-derived miR-335-3p and IL-16 mRNA 3'UTR exists, located at 775-782 of 3'UTR, to inhibit the expression of IL-16 gene, and interfere with the function of IL-16 gene to promote yellow ligament hypertrophy, it has wide application prospect on the drug related to lumbar yellow ligament hypertrophy, based on the present application can be made to treat or prevent the disease related to lumbar yellow ligament hypertrophy drug or detection kit.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a novel use of human miR-335-3p and a method for regulating the expression of the target gene IL-16. Background Technology

[0002] Lumbar spinal stenosis (LSS) is one of the most common spinal diseases endangering the health of the elderly, and its incidence is increasing year by year with the acceleration of my country's aging process. Hypertrophy of the ligamentum flavum is the main pathological factor causing lumbar spinal stenosis, with radiographic evidence of ligamentum flavum hypertrophy present in approximately 25%-50% of LSS patients. Hypertrophy of the ligamentum flavum manifests as fibrosis and thickening of the ligamentum flavum, which is originally rich in elastin, directly compressing the lumbar spinal canal. The pathological mechanism of ligamentum flavum hypertrophy involves multiple links, including mechanical stress, chronic inflammation, and fibrotic remodeling. At the molecular level, pro-fibrotic factors such as transforming growth factor-β (TGF-β) activate downstream signaling pathways, driving the transformation of ligamentum flavum fibroblasts into myofibroblasts, leading to excessive deposition of collagen (such as Collagen I and Collagen III), ultimately resulting in fibrotic hypertrophy of the ligamentum flavum.

[0003] In recent years, microRNAs (miRNAs), as important epigenetic regulators, have been shown to play a key regulatory role in the degeneration of the ligamentum flavum. Existing technologies have already used methods such as miRNA sequencing analysis to screen for differentially expressed miRNAs associated with ligamentum flavum fibrosis. Specifically: Previous studies have reported that miR-21, miR-155, and other miRNAs participate in regulating the fibrosis process in ligamentum flavum hypertrophy. These miRNAs have been found to target systemic fibrosis signaling pathways such as TGF-β / Smad and MAPK, and are involved in the fibrosis process of multiple organs.

[0004] However, current technologies for screening miRNAs related to ligamentum flavum fibrosis employ a general, indiscriminate screening strategy, failing to target the specific inflammation-fibrosis nodes of ligamentum flavum hypertrophy. Existing miRNAs (such as miR-146a and miR-21) primarily target broad-spectrum fibrosis pathways throughout the body, including TGF-β / Smad and MAPK. This results in several technical drawbacks: these miRNAs lack disease specificity for ligamentum flavum hypertrophy; if developed into systemically administered gene therapy drugs, they could easily interfere with the normal fibrosis repair processes of other organs such as the skin, lungs, and kidneys, posing a high risk of potential systemic side effects. Furthermore, current technologies have never established a link between human (hsa)-miR-335-3p and lumbar ligamentum flavum hypertrophy, overlooking the regulatory potential of this miRNA in ligamentum flavum fibrosis, leading to a significant gap in the specificity of miRNA drug candidates in this field.

[0005] Meanwhile, current technologies rely on imaging examinations such as CT and MRI to diagnose lumbar ligamentum flavum hypertrophy, and a diagnosis can only be made after obvious morphological thickening and nerve compression are observed. Treatment primarily involves surgical decompression in the end-stage, lacking non-surgical treatment options for early-stage disease and slowing fibrosis progression. This results in several objective technical drawbacks: patients are often diagnosed at middle or late stages, missing the optimal intervention window; surgical treatment is highly invasive, expensive, and poorly tolerated by elderly patients, failing to meet the clinical needs of early diagnosis and treatment in the aging population; furthermore, current technologies do not provide bifunctional molecular targets that can be used for both diagnosis and treatment, leading to a disconnect between the development of diagnostic biomarkers and therapeutic drugs, resulting in low overall translational efficiency. Summary of the Invention

[0006] The purpose of this invention is to provide a novel use of human miR-335-3p and a method for regulating the expression of the target gene IL-16, in order to solve the problems mentioned in the background art.

[0007] To achieve the above objectives, the present invention provides the following technical solution: a human miR-335-3p, the novel use of which is to inhibit ligamentum flavum cell fibrosis.

[0008] As a further improvement of the present invention, the gene sequence information of hsa-miR-335-3p is as follows: UUUUUCAUUAUUGCUCCUGACC.

[0009] As a further improvement of the present invention, the primer sequence information of the human miR-335-3p is as follows: Human-derived miR-35-3p-F: AATTCGTTTTTCATTATTGCTCCTGACCTTCAAGAGAGGTCAGGAAATAATGAAAAATTTTTTG, Human miR-35-3p-R: GATCCAAAAAATTTTTCATTATTTCCTGACCTCTCTTGAAGGTCAGGAGCAATAATGAAAAACG.

[0010] A method for regulating the expression of the target gene IL-16 using human miR-335-3p, wherein the human miR-335-3p targets and binds to the 3'UTR region of IL-16 mRNA, and the human miR-335-3p has an 8-mer binding site with the IL-16 mRNA 3'UTR, located at positions 775-782 of the 3'UTR.

[0011] As a further improvement of the present invention, overexpression of the human miR-335-3p can antagonize the upregulation of IL-16 induced by excessive stress and inhibit the expression of fibrosis genes driven by excessive stress.

[0012] A kit for detecting lumbar ligamentum flavum hypertrophy, comprising reagents for detecting the expression level of the human miR-335-3p, wherein the reagents are specific primers or probes.

[0013] A medicament for treating or preventing lumbar ligamentum flavum hypertrophy, comprising the human miR-335-3p mimic or a derivative thereof as the active ingredient, and comprising a pharmaceutically acceptable carrier.

[0014] Compared with the prior art, the beneficial effects of the present invention are: I. This invention reveals for the first time a novel mechanism by which hsa-miR-335-3p inhibits IL-16 gene expression by targeting and binding to the 3'UTR (8mer binding site, located at positions 775-782 of the 3'UTR) of IL-16 mRNA. Dual-luciferase reporter gene assays demonstrated that transfection with hsa-miR-335-3p significantly downregulated luciferase expression in the IL16-3'UTR-WT group (P<0.001), directly confirming the targeted regulatory relationship between the two at the molecular level. IL-16, as a pro-inflammatory cytokine, is upregulated under excessive stress conditions and drives the expression of fibrosis genes (COL1A2, COL3A1). This invention, by targeting and inhibiting IL-16 with hsa-miR-335-3p, achieves a dual intervention in ligamentum flavum fibrosis from the inflammation-fibrosis coupling link, demonstrating greater mechanistic depth and interventional efficacy compared to existing technologies that focus only on the single regulatory strategy of the fibrosis pathway.

[0015] II. Compared with existing miRNAs such as miR-21 and miR-155 that target broad-spectrum fibrosis pathways like TGF-β / Smad and MAPK, the mechanism of action of hsa-miR-335-3p targeting IL-16 in this invention is more focused on the inflammatory-immune regulatory links in the ligamentum flavum fibrosis process. hsa-miR-335-3p is significantly downregulated in hypertrophic ligamentum flavum tissue, exhibiting better tissue specificity. Therefore, gene therapy drugs developed based on hsa-miR-335-3p, when administered systemically, cause less interference with the normal fibrosis repair process in other organs such as the skin, lungs, and kidneys, and have a lower risk of potential systemic side effects.

[0016] Third, this invention not only provides the use of hsa-miR-335-3p as a therapeutic target, but also provides a diagnostic kit for lumbar ligamentum flavum hypertrophy based on the detection of hsa-miR-335-3p expression levels. hsa-miR-335-3p is significantly downregulated in ligamentum flavum hypertrophy tissue and can serve as an early molecular diagnostic biomarker, identifying at-risk individuals before significant morphological changes appear on imaging, enabling early diagnosis and treatment. Simultaneously, supplementing with hsa-miR-335-3p mimics enables non-invasive gene therapy, overcoming the limitation of existing technologies where diagnostic biomarkers and therapeutic drug development are often separate, significantly improving clinical translation efficiency.

[0017] IV. Current treatments for lumbar ligamentum flavum hypertrophy primarily rely on surgical decompression in the end-stage, with extremely limited non-surgical options. The hsa-miR-335-3p mimic provided by this invention can inhibit the progression of ligamentum flavum fibrosis in the early stages of the disease through local or systemic administration, delaying or even eliminating the need for surgical intervention. It is particularly suitable for elderly patients and offers significant advantages such as minimal invasiveness, repeatable administration, and high patient compliance. Attached Figure Description

[0018] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is a diagram showing the experimental results of the dual-luciferase reporter gene assay in this invention, in which hsa-miR-335-3p targets and binds to the 3'UTR of IL-16 mRNA. Figure 2 The figure shows the effect of overexpression / inhibition of hsa-miR-335-3p on the expression of IL-16, Collagen I, and Collagen III proteins in ligamentum flavum cells in this invention. Figure 3 The figure shows the experimental results of antagonizing excessive stress after overexpression of hsa-miR-335-3p in this invention. Detailed Implementation

[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] A novel application of human miR-335-3p is to inhibit fibrosis of ligamentum flavum cells.

[0022] The gene sequence information of hsa-miR-335-3p is as follows: hsa-miR-335-3pMIMAT0004703UUUUUCAUUAUUGCUCCUGACC.

[0023] The primer sequence information for hsa-miR-335-3p is as follows: hsa-miR-35-3p-F: AATTCGTTTTTCATTATTGCTCCTGACCTTCAAGAGAGGTCAGGAAATAATGAAAAATTTTTTG, hsa-miR-35-3p-R: GATCCAAAAAATTTTTCATTATTTCCTGACCTCTCTTGAAGGTCAGGAGCAATAATGAAAAACG.

[0024] A method for regulating the expression of the target gene IL-16 using human miR-335-3p, wherein the human miR-335-3p targets and binds to the 3'UTR region of IL-16 mRNA, and the human miR-335-3p has an 8-mer binding site with the IL-16 mRNA 3'UTR, located at positions 775-782 of the 3'UTR.

[0025] This invention verifies the target binding relationship. The verification process involves co-transfecting cells with an IL-16 mRNA 3'UTR wild-type (WT) or mutant (MUT) reporter plasmid and an hsa-miR-335-3p mimic or control, followed by detection of luciferase activity. The final results are as follows: Figure 1 As shown, dual-luciferase gene reporter assays demonstrated that the luciferase activity of IL16-3'UTR-WT in the hsa-miR-335-3p transfection group was significantly lower than that in the control group. P The value was <0.001), while no significant change was observed in the IL16-3'UTR-MUT group, demonstrating that hsa-miR-335-3p specifically binds to the 775-782 site of the IL-16 mRNA 3'UTR. This is the core innovation of this invention, forming the molecular basis for hsa-miR-335-3p as a therapeutic target for ligamentum flavum fibrosis. This invention opens up a completely new regulatory axis: hsa-miR-335-3p / IL-16.

[0026] This invention verifies the function of human miR-335-3p in inhibiting ligamentum flavum cell fibrosis, and the specific process is as follows: A. Preparation of working solutions: Take 20 nmol of each miR-335-3p product (mimic is a miRNA mimic, inhibitor is a miRNA inhibitor, mimic-NC is a miRNA mimic negative control, and inhibitor-NC is an inhibitor negative control), briefly centrifuge, and dissolve in 1 ml of RNase-free H2O (i.e., ribonuclease-free water) to prepare 20 μM working solutions. Aliquot and label, and store in a -20°C freezer, avoiding repeated freeze-thaw cycles.

[0027] B. Cell inoculation: Take P3 generation cells of ligamentum flavum, digest them, and inoculate them into six-well culture plates. Transfection begins when the cell density reaches 80%.

[0028] C. Experimental grouping: Divided into 6 groups. The top row of 3 wells on the 6-well plate: blank control group, mimic, mimic-NC; the bottom row of 3 wells: blank control group, inhibitor, inhibitor-NC.

[0029] D. Preparation of Transfection Solutions: Prepare each transfection solution according to the following ratio: 20μM working solution (mimic, inhibitor, mimic-NC, inhibitor-NC): Ribo FECT Buffer: Ribo FECT Reagent: Complete Culture Medium (20% FBS, antibiotic-free) = 10μl: 120μl: 12μl: 1858μl. Incubate at room temperature for 15 min. Add 2ml of the solution to each well of a six-well plate, resulting in a final transfection solution concentration of 100nM. Incubate the plate in a 5% CO2, 37°C, saturated humidity incubator.

[0030] E. Total protein was extracted from each group of cells 48 hours after transfection for Western blotting detection.

[0031] Overexpression of hsa-miR-335-3p significantly reduced IL-16 protein expression in ligamentum flavum cells, while inhibition of hsa-miR-335-3p significantly increased IL-16 protein expression in ligamentum flavum cells. One novel application of hsa-miR-335-3p described above is that overexpression of hsa-miR-335-3p significantly inhibited the fibrotic phenotypes (Collagen I and Collagen III) in ligamentum flavum cells, while inhibition of hsa-miR-335-3p significantly increased these phenotypes. Figure 2 As shown, the left side reveals that after overexpression of hsa-miR-335-3p (miR-335-3p mimic), the protein expression levels of IL-16, Collagen I, and Collagen III in ligamentum flavum cells were significantly lower than those in the control group (NC). The right side shows that after inhibition of hsa-miR-335-3p (miR-335-3p inhibitor), the expression levels of the above three proteins were significantly higher than those in the control group. These results indicate that hsa-miR-335-3p negatively regulates IL-16 expression and inhibits ligamentum flavum cell fibrosis.

[0032] In some embodiments of the present invention, the process is specifically carried out using an overstressed cell model: P3 generation cells from the ligamentum flavum were digested and seeded onto BioFlex cell stretching plates. Cells were randomly divided into experimental and control groups, with a cell density of 1×10⁶ cells per well. 5 When the cell confluence reached 90%, the cells were starved with 2% serum medium for 6 hours to maintain the cells in the same growth state across all groups. Experimental group: Cells were cyclically stretched using a Flexcell FX-5000 instrument under the following conditions: 20% stretching intensity, 10 seconds of stretching, and 10 seconds of rest. Control group: No stretching stimulation was applied. Cell lysates were collected at 6 hours and 12 hours for qRT-PCR detection of gene expression.

[0033] Excessive stress can promote IL-16 expression in ligamentum flavum fibroblasts, thereby driving the expression of fibrosis genes (COL1A2, COL3A1). Overexpression of hsa-miR-335-3p can antagonize the stress-induced upregulation of IL-16 and inhibit the expression of stress-driven fibrosis genes (COL1A2, COL3A1); conversely, inhibition of hsa-miR-335-3p can enhance the expression of stress-driven fibrosis genes (COL1A2, COL3A1). Figure 3As shown, the expression changes of IL-16, COL1A2, and COL3A1 in ligamentum flavum fibroblasts under excessive stress conditions are illustrated. The top left panel shows a significant increase in IL-16 expression in the excessive stress treatment group, while the expression of fibrosis genes COL1A2 and COL3A1 is upregulated. The bottom two panels show that overexpression of hsa-miR-335-3p significantly inhibits the stress-induced upregulation of IL-16, and the expression of COL1A2 and COL3A1 is correspondingly reduced. The top right panel shows that inhibition of hsa-miR-335-3p further enhances the upregulation effect of excessive stress on IL-16 and fibrosis genes. These results indicate that hsa-miR-335-3p negatively regulates the excessive stress-driven ligamentum flavum fibrosis process by targeting and inhibiting IL-16. This invention reveals for the first time that hsa-miR-335-3p can antagonize the upregulation of IL-16 and the expression of fibrosis genes (COL1A2, COL3A1) induced by excessive stress, elucidating the negative regulatory role of hsa-miR-335-3p in the relationship between mechanical stimulation and fibrosis, and providing a new perspective for understanding the mechanical-biological coupling mechanism of ligamentum flavum hypertrophy.

[0034] In some examples of the present invention, a kit for detecting lumbar ligamentum flavum hypertrophy includes reagents for detecting the expression level of the human miR-335-3p, wherein the reagents are specific primers or probes, enabling early molecular diagnosis of lumbar spinal stenosis and overcoming the limitation of traditional imaging examinations that can only detect mid-to-late stage lesions.

[0035] In some embodiments of the present invention, a medicament for treating or preventing lumbar ligamentum flavum hypertrophy comprises the aforementioned human miR-335-3p mimic or a derivative thereof as an active ingredient, and comprises a pharmaceutically acceptable carrier, preferably LNP (lipid nanoparticles). This medicament for treating or preventing lumbar ligamentum flavum hypertrophy provides a novel option for non-surgical interventions that have long been lacking in clinical practice.

[0036] In some examples of this invention, in addition to using specific primers for RT-qPCR detection of hsa-miR-335-3p, the following methods can also be used: (1) Northern blot hybridization detection; (2) in situ hybridization detection; (3) high-throughput detection based on microarray chips; and (4) detection based on next-generation sequencing (NGS). All of the above methods can achieve quantitative or qualitative detection of hsa-miR-335-3p expression levels, which can be used for the auxiliary diagnosis of lumbar ligamentum flavum hypertrophy.

[0037] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A human-derived miR-335-3p, characterized in that, Its new application lies in inhibiting fibrosis of ligamentum flavum cells.

2. The human-derived miR-335-3p according to claim 1, characterized in that, The gene sequence information of hsa-miR-335-3p is as follows: UUUUUCAUUAUUGCUCCUGACC.

3. The human-derived miR-335-3p according to claim 2, characterized in that, The primer sequence information for the human miR-335-3p is as follows: Human-derived miR-35-3p-F: AATTCGTTTTTCATTATTGCTCCTGACCTTCAAGAGAGGTCAGGAAATAATGAAAAATTTTTTG, Human miR-35-3p-R: GATCCAAAAAATTTTTCATTATTTCCTGACCTCTCTTGAAGGTCAGGAGCAATAATGAAAAACG.

4. A method for regulating the expression of the target gene IL-16 using human miR-335-3p as described in claim 3, characterized in that: The human miR-335-3p targets and binds to the 3'UTR region of IL-16 mRNA. The human miR-335-3p has an 8-mer binding site with the IL-16 mRNA 3'UTR, located at positions 775-782 of the 3'UTR.

5. The method for regulating the expression of the target gene IL-16 using human miR-335-3p according to claim 4, characterized in that, Overexpression of the human miR-335-3p can antagonize the upregulation of IL-16 induced by excessive stress and inhibit the expression of excessive stress-driven fibrosis genes.

6. A kit for detecting lumbar ligamentum flavum hypertrophy, comprising reagents for detecting the expression level of human miR-335-3p as described in claim 3, said reagents being specific primers or probes.

7. A medicament for treating or preventing lumbar ligamentum flavum hypertrophy, comprising a human miR-335-3p mimic or a derivative thereof as the active ingredient, as claimed in claim 3, and comprising a pharmaceutically acceptable carrier.

Images