Use of kif15 modulators in the preparation of medicaments for the regulation of cholesterol metabolism

Abstract

The application discloses application of a KIF15 modulator in preparation of a drug for regulating cholesterol metabolism. The application first proposes and verifies that intervention of a microtubule-related motor protein KIF15 can realize regulation of cholesterol metabolism of peripheral macrophages and central microglial cells, including cholesterol synthesis and efflux, and provides a new direction for treatment of diseases related to abnormal cholesterol metabolism.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to the application of a KIF15 regulator in the preparation of cholesterol metabolism regulating drugs. Background Technology

[0002] KIF15 (Kinesin Family Member 15) is a member of the kinesin superfamily. As a microtubule-dependent molecular motor, it plays a crucial role in the assembly and maintenance of the mitotic spindle. It can crosslink and slide antiparallel microtubules, driving centrosome separation and ensuring proper chromosome separation. Notably, its function is redundant with another kinesin, Eg5: when Eg5 function is inhibited, KIF15 expression is upregulated, thus becoming an important drug resistance mechanism for tumor cells to evade Eg5 inhibitor therapy. In addition to cell division, KIF15 is also involved in the migration and phagocytosis of immune cells. Recent studies have found that KIF15 is highly expressed in various malignant tumors (such as gallbladder cancer, liver cancer, and prostate cancer), and promotes tumor cell proliferation, migration, and invasion by activating multiple signaling pathways. Its high expression is often closely associated with poor patient prognosis, and therefore it is widely regarded as a potential anti-tumor target.

[0003] Statins are a class of widely used lipid-lowering drugs in clinical practice. Their core mechanism of action lies in inhibiting HMG-CoA reductase, a key rate-limiting enzyme in cholesterol synthesis. By blocking the activity of this enzyme, statins can effectively reduce the production of endogenous cholesterol in liver cells. The decrease in intracellular cholesterol levels will feedback activate sterol regulatory element-binding protein, leading to upregulation and enhanced activity of low-density lipoprotein receptors on the surface of liver cells. This change significantly accelerates the clearance of low-density lipoprotein from the blood, thereby exerting a potent lipid-lowering effect and stabilizing atherosclerotic plaques, serving as a cornerstone of treatment for cardiovascular events such as myocardial infarction and stroke.

[0004] However, there are currently few methods for regulating cholesterol metabolism that can simultaneously increase cholesterol synthesis and excretion. Summary of the Invention

[0005] Purpose of the invention: The purpose of this invention is to provide KIF15 regulators, namely KIF15 inhibitors and KIF15 enhancers, for their application in the regulation of cholesterol metabolism, especially the regulation of cholesterol synthesis and efflux, and in the preparation of related drugs based on this.

[0006] Technical solution: The application of the KIF15 regulator described in this invention in the preparation of cholesterol metabolism regulating drugs.

[0007] Preferably, the application is the use of KIF15 inhibitors in the preparation of cholesterol metabolism regulating drugs; more preferably, the application is the use of KIF15 inhibitors in the preparation of drugs for treating diseases caused by abnormal cholesterol synthesis and efflux; more preferably, the diseases caused by abnormal cholesterol synthesis and efflux include: atherosclerosis, cell dysfunction caused by microglial lipid accumulation after nerve injury, Alzheimer's disease, Tangier disease, sitosterolemia, Niemann-Pick type C disease, Smith-Lemli-Opitz syndrome, etc.

[0008] Preferably, the KIF15 inhibitor is any one of siRNA, shRNA, sgRNA, miRNA, antisense oligonucleotide, or KIF15 nucleic acid aptamer or KIF15 protein inhibitor used to inhibit KIF15 expression.

[0009] Preferably, the application is the use of KIF15 enhancer in the preparation of cholesterol metabolism regulating drugs; more preferably, the application is the use of KIF15 enhancer in the preparation of drugs for treating diseases caused by abnormal cholesterol synthesis and efflux; more preferably, the diseases caused by abnormal cholesterol synthesis and efflux include: hypercholesterolemia, non-alcoholic fatty liver disease, Huntington's disease, and cancers with high levels of cholesterol synthesis.

[0010] Preferably, the KIF15 enhancer is an overexpression vector containing a KIF15 expression cassette or a recombinant KIF15 protein.

[0011] Preferably, the drug contains a KIF15 regulator as an active ingredient and also contains pharmaceutically acceptable excipients; more preferably, the pharmaceutically acceptable excipients include any one or more of excipients, diluents, lubricants, flow aids, wetting agents, emulsifiers, pH buffers, solubilizers, cosolvents, or solvents.

[0012] Preferably, the dosage form of the drug includes tablets, capsules, granules, powders, chewable tablets, effervescent tablets, sustained-release tablets, microcapsules, injections, infusions, suspensions, patches, suppositories, transdermal patches, microemulsions, liposomes, and nanoparticles.

[0013] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The present invention is the first to propose and verify that intervention in the microtubule-associated motor protein KIF15 can regulate cholesterol metabolism in peripheral macrophages and central microglia, including cholesterol synthesis and efflux, providing a new direction for the treatment of cholesterol metabolism-related diseases. Attached Figure Description

[0014] Figure 1 Interference in mouse primary macrophages Kif15The fluorescence intensity bar graph of Bodipy-cholesterol cells discharged into the culture medium was then examined. Figure 2 Interference in mouse primary macrophages Kif15 Post-quantitative PCR detection Hmgcs1 , Fdft1 , Abca1 and Abcg1 A bar chart representing levels; Figure 3 Interference in mouse primary macrophages Kif15 Microscopic images of free cholesterol in cell membranes were then detected using Filipin III staining. Figure 4 Knockdown in zebrafish kif15 Post-transcriptome sequencing and gene ontology pathway enrichment analysis diagram; Figure 5 Knockdown in zebrafish kif15 A bar chart of cholesterol metabolism-related genes detected by qPCR. Figure 6 A schematic diagram of the protocol for constructing Kif15 knockout mice; Figure 7 Quantitative real-time PCR detection of microglia from Kif15 systemic knockout mice Hmgcs1 , Fdft1 , Abca1 and Abcg1 A bar chart representing levels; Figure 8 Microscopic images of microglia from Kif15 systemic knockout mice, obtained by detecting free cholesterol in the cell membrane using Filipin III staining. Detailed Implementation

[0015] The technical solution of the present invention will be further described below.

[0016] Example 1: Application of KIF15 (Kinesin Family Member 15) regulator in the regulation of cholesterol metabolism in macrophages 1. Isolation of primary mouse macrophages Macrophages were obtained from 10-week-old, 25-g, male C57BL / 6 mice using an intraperitoneal inflammation induction method. Specifically, 1 mL of 5% starch broth was injected into the peritoneal cavity of the mice once a day for 3 consecutive days. After the last injection, the peritoneal cavity of the mice was irrigated with serum-free DMEM medium, and 5 mL of the irrigated fluid was collected and repeated twice.

[0017] The recovered irrigation fluids were combined, centrifuged at 1000 rpm, and then cultured in an adherent culture to obtain mouse primary macrophages.

[0018] 2. Treatment of primary mouse macrophages The aforementioned primary mouse macrophages were used at a concentration of 1.5 × 10⁻⁶. 5 Macrophages were seeded at a density of cells / well in 24-well plates. When the cell confluence reached 65±5%, 200 pmol of KIF15-targeting siRNA (Kif15 kd, sequence: 5'-cagcuauaauugcaaaugudTdT-3', 5'-acauuugcaauuauagcugdTdT-3', synthesized by Jiangsu Saisofe Biotechnology Co., Ltd.) was transfected into macrophages using Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific, L3000001). The control group was transfected with control siRNA (Ctrl, sequence: 5'-uucuccgaacgugucacgudTdT-3', 5'-acgugacacguucggagaadTdT-3', synthesized by Jiangsu Saisofe Biotechnology Co., Ltd.) under the same conditions.

[0019] 3. Analysis of cholesterol metabolism in primary mouse macrophages 3.1 Cholesterol Tracing After 48 h of transfection with siRNA or KIF15-IN-1, the original culture medium was replaced with DMEM-F12 complete medium containing a final concentration of 1 μg / mL Bodipy-cholesterol (MedChemExpress, HY-DY1029). After 12 h of culture, the medium was replaced with regular DMEM-F12 complete medium, the culture supernatant was collected, and the amount of Bodipy-cholesterol excreted from the cells in the culture medium was detected using a microplate reader (green fluorescent channel).

[0020] The results are as follows Figure 1 As shown, interference with KIF15 increases the rate at which macrophages excrete cholesterol.

[0021] 3.2 Analysis of transcriptional levels of cholesterol metabolism-related genes Cells were collected 48 h after transfection with siRNA or KIF15-IN-1. Total RNA was extracted using the TRIzol kit (Thermo Fisher Scientific, 15596018CN) and reverse transcribed into cDNA using the HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Novizan, R312-02) primers shown below: Hmgcs1 Upstream primer: 5'-gcccagtggcagaaagag-3'; Hmgcs1Downstream primer: 5'-cccaaaggcttccagtcc-3'; Fdft1 Upstream primer: 5'-ctcaatgggtctgttcct-3'; Fdft1 :5'-atgtgctgtagggtgttg-3'; Abca1 Upstream primer: 5'-ttccgttatccaactccc-3'; Abca1 Downstream primer: 5'-atgtttcattgtccacca-3'; Abcg1 Upstream primer: 5'-ctgctgcctcacctcact-3'; Abcg1 Downstream primer: 5'-ccatctctcgtctgccttcatc-3'; Internal Reference 18S rRNA upstream primer: 5'-cgcggttctattttgttggt-3'; Internal Reference 18S rRNA downstream primer: 5'-cctccgactttcgttcttga-3'; qRT-PCR was performed using the Taq Pro Universal SYBR qPCR Master Mix kit (Novizan, Q712-02), with reaction conditions of 95℃ pre-denaturation for 30 s; 95℃ denaturation for 5 s, 60℃ annealing extension for 30 s, for 40 cycles. Fluorescence values ​​were collected during the extension phase of each cycle. 18S rRNA was used as an internal control, and Ct values ​​were calculated using the ΔΔCt method. Hmgcs1, Fdft1, Abca1 and Abcg1 The relative expression level of mRNA.

[0022] The results are as follows Figure 2 As shown, after KIF15 interference, cholesterol synthesis genes... Hmgcs1 and Fdft1 cholesterol transport genes Abca1 and Abcg1 Significantly upward adjustment.

[0023] 3.3 Filipin III staining After 48 h of transfection with siRNA or KIF15-IN-1, cells were fixed with 4% paraformaldehyde and then stained with 0.05 mg / mL Filipin III (MedChemExpress, HY-N6718) solution. Filipin III was observed using a Zeiss Imager M2 fluorescence microscope.

[0024] The results are as follows Figure 3 As shown, after KIF15 interference, Filipin III staining revealed a decrease in the content of free cholesterol in the cell membrane.

[0025] Example 2: Application of KIF15 regulator in the regulation of cholesterol metabolism in zebrafish 1. Processing of zebrafish Gene Tools, LLC was commissioned to synthesize a target with the sequence ATGTATTAAAAACCTCACCTGGCTG. kif15 KIF15 knockdown was achieved by injecting morpholine oligonucleotide (kif15 MO) and a control morpholine oligonucleotide (Ctrl MO) with the sequence CCTCTTACCTCAGTTACAATTTATA. A 0.7 mM solution of morpholine oligonucleotide was prepared and injected into 1-cell stage AB strain zebrafish fertilized eggs to obtain zebrafish with KIF15 gene knockdown.

[0026] 2. Analysis of cholesterol metabolism in zebrafish 24 h after the above injection, KIF15 gene knockdown zebrafish were collected, and total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific, 15596018CN). Transcriptome sequencing was performed by Shanghai Bio-Tech Co., Ltd., followed by gene functional cluster analysis.

[0027] The results are as follows Figure 4 As shown, knockdown in zebrafish embryos kif15 Subsequently, pathways such as cholesterol reverse transport and high-density lipoprotein assembly are upregulated, resulting in significant enrichment.

[0028] Simultaneously, the aforementioned RNA was reverse transcribed into cDNA using the HiScript III 1st Strand cDNA Synthesis Kit (+gDNAwiper) (Novizan, R312-02), using the primers shown below: abca1a Upstream primer: 5'-gtggagcgaaccaataag-3'; abca1a Downstream primer: 5'-cacatagcagggatacgg-3'; abca1b Upstream primer: 5'-ttcaatgagaccgaccag-3'; abca1b Downstream primer: 5'-atacaatcccagcccaga-3'; abca1g Upstream primer: 5'-cttgacggctcttggcttat-3'; abca1g Downstream primer: 5'-cgctgggctggtgtattg-3'; apoa1a Upstream primer: 5'-ctggacggaaccgactatga-3'; apoa1aDownstream primer: 5'-ggaggtggtctgggcata-3'; apoa1b Upstream primer: 5'-acctccattcccacttcc-3'; apoa1b Downstream primer: 5'-tcagctcatccctgtactcat-3'; apoea Upstream primer: 5'-gctgaggaaatgagggac-3'; apoea Downstream primer: 5'-gttgaacttctgggctct-3'; apoeb Upstream primer: 5'-gcctctgatgctgctggtc-3'; apoeb Downstream primer: 5'-ctgagtgctgcgttcctt-3'; nr1h3 Upstream primer: 5'-gtgcaggatcacgagtta-3'; nr1h3 Downstream primer: 5'-cagaaagtagaggtggaagt-3'; scap Upstream primer: 5'-atccagacctcctgaagaca-3'; scap Downstream primer: 5'-gggacgccaaagagtaaat-3'; srebf1 Upstream primer: 5'-catggagtttgggctgtt-3'; srebf1 Downstream primer: 5'-gctgagaatggaggaggc-3'; srebf2 Upstream primer: 5'-aagccatcgactacatcaaa-3'; srebf2 Downstream primer: 5'-gacacctcagccttcagc-3'; Internal Reference 18S rRNA upstream primer: 5'-tcgctagttggcatcgtttatg-3'; Internal Reference 18S rRNA downstream primer: 5'-cggaggttcgaagacgatca-3'; qRT-PCR was performed using the Taq Pro Universal SYBR qPCR Master Mix kit (Novizan, Q712-02). The reaction conditions were: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 5 s; 60℃ annealing and extension for 30 s, for 40 cycles. Fluorescence values ​​were collected during the extension phase of each cycle. 18S rRNA was used as an internal control, and Ct values ​​were calculated using the ΔΔCt method. abca1a, abca1b, abca1g, apoa1a, apoa1b, apoea, apoeb, nr1h3, scap, srebf1, and srebf2 The relative expression level of mRNA.

[0029] The results are as follows Figure 5 As shown, knockdown in zebrafish embryos kif15 back, abca1a, abca1b, abca1g When cholesterol efflux genes are upregulated.

[0030] Example 3: Application of KIF15 regulator in the regulation of cholesterol metabolism in mice 1. Treatment of mice Jiangsu Jicui Yaokang Biotechnology Co., Ltd. was commissioned to construct KIF15 systemic knockout mice (C57BL / 6J background, with deletions in exons 2 and 3 of the KIF15 gene) systemically. Figure 6 As shown, the construction method is referenced in the literature (DOI: 10.1007 / s11011-023-01238-y).

[0031] A spinal cord contusion model was established in 10-week-old, 25g male KIF15 systemic knockout mice (Kif15_KO) and wild-type C57BL / 6J mice (Kif15_WT). Mice were anesthetized with isoflurane, their back hair was removed, and the area was disinfected with povidone-iodine. A 1.5 cm incision was made at the T10 segment of the spine to expose the spinal cord. A 10 g weight was then dropped from a height of 8 mm from a cannula. The contusion model was an indirect impact model; the weight struck the 7.5 cm cannula from a height of 8 mm, resulting in an actual drop height of approximately 8.3 cm, impacting the spinal cord through the cannula. Exudate was removed with cotton swabs, and the fascia, muscles, and skin of the mice were sutured layer by layer. The skin of the mice was disinfected again with povidone-iodine.

[0032] 2. Analysis of cholesterol metabolism in mice Three days after injury, spinal cord tissue was collected from the injury site of mice. After being ground and lysed in liquid nitrogen, total RNA was extracted using the TRIzol kit and reverse transcribed into cDNA using the HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper). The cDNA was then processed as described in Example 1. Hmgcs1, Fdft1, Abca1 and Abcg1 Primers were used for qRT-PCR detection using the TaqPro Universal SYBR qPCR Master Mix kit. The reaction conditions were: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 5 s; 60℃ annealing extension for 30 s, for 40 cycles. Fluorescence values ​​were collected during the extension phase of each cycle. 18S rRNA was used as an internal control, and Ct values ​​were calculated using the ΔΔCt method. Hmgcs1, Fdft1, Abca1 and Abcg1 The relative expression level of mRNA.

[0033] The results are as follows Figure 7 As shown, after Kif15 knockout, cholesterol synthesis-related genes... Hmgcs1、Fdft1 cholesterol transport genes Abca1 and Abcg1 The expression is adjusted upwards.

[0034] Three days after injury, mice were euthanized, and blood was removed by intracardiac perfusion with ice-cold PBS. The entire spinal cord was rapidly removed and placed in pre-chilled culture medium on ice. The spinal cord was minced and digested at 37°C for 30 min with a mixed solution containing 0.25% trypsin (Dalian Meilun Biotechnology Co., Ltd., MA0233) and 20 µg / mL DNase I (Dalian Meilun Biotechnology Co., Ltd., MB3069-1). After terminating digestion with serum-containing culture medium, the cells were sequentially passed through 70 μm and 40 μm cell sieves. The filtrate was then collected, and the cells were collected by centrifugation. The cells were resuspended in 37% Percoll (Beijing Solarbio Science & Technology Co., Ltd., P8370) and subjected to density gradient centrifugation at 800×g for 20 min (the centrifugation system consisted of 2 mL PBS, 4 mL 30% Percoll, 4 mL 37% Percoll, and 4 mL 70% Percoll from top to bottom). Cells between the 37% and 70% Percoll were collected and resuspended in PBS. Magnetic beads conjugated with anti-CD11b antibody (Miltenyi Biotec, 130-049-601) were added, and the cells were incubated at 4℃ for 15 min. The cells were then loaded onto a MACS sorting column (Miltenyi Biotec, 130-042-401). After discarding the percolation solution, the positive fraction was eluted from the sorting column to obtain high-purity microglia. Cells were fixed with 4% paraformaldehyde and blocked with blocking solution containing 5% BSA (Soluble Biotech, SW3015, Beijing) at 37°C. They were then stained with 0.05 mg / mL Filipin III solution in the dark for 30 min. After staining, the cells were rinsed with PBS, mounted, and observed and images were acquired using a fluorescence microscope.

[0035] The results are as follows Figure 8 As shown, Kif15 After knockout, the free cholesterol load on the microglial cell membrane decreased.

Claims

1. The application of a KIF15 regulator in the preparation of cholesterol metabolism regulating drugs.

2. The application according to claim 1, characterized in that, The application described is the use of KIF15 inhibitors in the preparation of cholesterol metabolism regulating drugs.

3. The application according to claim 2, characterized in that, The application is the use of KIF15 inhibitors in the preparation of drugs for the treatment of diseases caused by abnormal cholesterol synthesis and efflux.

4. The application according to claim 2 or 3, characterized in that, The KIF15 inhibitor is any one of siRNA, shRNA, sgRNA, miRNA, antisense oligonucleotide, or KIF15 nucleic acid aptamer or KIF15 protein inhibitor used to inhibit KIF15 expression.

5. The application according to claim 1, characterized in that, The application described is the use of KIF15 enhancers in the preparation of cholesterol metabolism regulating drugs.

6. The application according to claim 5, characterized in that, The application described is the use of KIF15 enhancers in the preparation of drugs for treating diseases caused by abnormal cholesterol synthesis and efflux.

7. The application according to claim 5 or 6, characterized in that, The KIF15 enhancer is an overexpression vector containing a KIF15 expression cassette or a recombinant KIF15 protein.

8. The application according to claim 1, characterized in that, The drug contains a KIF15 regulator as its active ingredient, as well as pharmaceutically acceptable excipients.

9. The application according to claim 8, characterized in that, The pharmaceutically acceptable excipients include any one or more of the following: excipients, diluents, lubricants, glidants, wetting agents, emulsifiers, pH buffers, solubilizers, cosolvents, or solvents.

10. The application according to claim 1, characterized in that, The dosage forms of the drugs include tablets, capsules, granules, powders, chewable tablets, effervescent tablets, sustained-release tablets, microcapsules, injections, infusions, suspensions, patches, suppositories, transdermal patches, microemulsions, liposomes, and nanoparticles.

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