CN112553316A - Diagnostic markers for cilia-related diseases - Google Patents
Diagnostic markers for cilia-related diseases Download PDFInfo
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- CN112553316A CN112553316A CN201910910288.9A CN201910910288A CN112553316A CN 112553316 A CN112553316 A CN 112553316A CN 201910910288 A CN201910910288 A CN 201910910288A CN 112553316 A CN112553316 A CN 112553316A
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Abstract
The invention discloses a diagnostic marker for cilia-related diseases. The invention provides application of lysophosphatidic acid as a marker in preparing a product for diagnosing or screening cilia defect related diseases, and application of an LPA signal pathway related inhibitor in preparing a product for treating cilia defect related diseases. The present study found that lysophosphatidic acid (LPA) is a major factor in serum that induces ciliary assembly, and LPAR1 is critical for the normal development of cilia on mouse RG cells, VZ/SVZ production, and neocortical formation. The present study indicates that LPA may be a prenatal diagnostic marker for cilia-deficient diseases, and that inhibitors related to the LPA signaling pathway may be potential drugs for the treatment of cilia-related diseases.
Description
Technical Field
The present invention relates to the field of biomedicine, and in particular to diagnostic markers for cilia-related diseases.
Background
The primary cilia are an antenna-like organelle present on the surface of most mammalian cells. It is capable of sensing a number of extracellular physical and chemical signals and plays a key role in a variety of biological processes such as embryonic development and tissue homeostasis. Abnormalities in ciliary function are associated with tumorigenesis and a number of human developmental disorders collectively referred to as ciliary-related disorders. While cilia-related diseases may lead to the production of many aborted fetuses, there is no good prenatal diagnostic marker to determine whether a fetus is afflicted with cilia-related diseases, and it is imperative to find an effective prenatal diagnostic marker for identifying whether a fetus is afflicted.
The process of cilia generation is dynamically regulated by cilia assembly and disassembly. Under in vitro culture conditions, we typically use serum starvation and serum re-stimulation to induce cilia to assemble and disassemble. Upon serum starvation, cells enter the resting phase (G0/G1) and cilia assemble; when the serum is restimulated, the cells re-enter the cell cycle and the cilia must be disassembled. However, it is not clear which component in serum causes the cilia to be disassembled.
Disclosure of Invention
The object of the present invention is to provide a diagnostic marker for cilia-related diseases.
In a first aspect, the invention claims the use of lysophosphatidic acid (LPA) as a marker for the preparation of a product for diagnosing or screening diseases associated with ciliary defects.
In a second aspect, the invention claims the use of a substance for the detection of lysophosphatidic acid (LPA) for the preparation of a product for the diagnosis or screening of diseases associated with ciliary deficiency.
In the first and second aspects, the diagnosing or screening for a cilia deficiency-associated disease may be prenatal diagnosis or screening of the fetus.
In a third aspect, the invention claims the use of lysophosphatidic acid (LPA) for inducing ciliary disassembly or for the preparation of a product for inducing ciliary disassembly.
Further, the use is of lysophosphatidic acid (LPA) and LPAR1 protein (i.e. lysophosphatidic acid receptor 1(LPAR1), the same below) for inducing ciliary disassembly or for preparing a product for inducing ciliary disassembly.
In a fourth aspect, the invention claims the use of an inhibitor associated with the LPA signaling pathway for the manufacture of a product for the treatment of a disease associated with ciliary deficiency.
In a fifth aspect, the invention claims the use of a substance capable of reducing the lysophosphatidic acid (LPA) content in any one of:
(A1) in the preparation of a product for the treatment of a condition associated with ciliary deficiency;
(A2) blocking lysophosphatidic acid (LPA) induces ciliary disassembly, or preparing a product for blocking lysophosphatidic acid induced ciliary disassembly.
In a sixth aspect, the invention claims the use of a substance capable of inhibiting expression of LPAR1 protein in any one of:
(A1) in the preparation of a product for the treatment of a condition associated with ciliary deficiency;
(A2) blocking lysophosphatidic acid (LPA) induces ciliary disassembly, or preparing a product for blocking lysophosphatidic acid induced ciliary disassembly.
Further, the substance capable of inhibiting LPAR1 protein expression may be an siRNA capable of interfering with expression of a gene encoding LPAR1 protein.
In a specific embodiment of the invention, the siRNA capable of interfering with expression of the gene encoding LPAR1 protein has the sequence of SEQ ID No.1 or SEQ ID No. 2.
Further, the substance capable of inhibiting expression of LPAR1 protein may be an inhibitor of LPAR1 protein.
In a specific embodiment of the invention, the inhibitor of LPAR1 protein was Ki 16425.
In each of the above aspects, the cilia deficiency-related disease is a disease caused by a lysophosphatidic acid (LPA) content significantly higher than physiological values causing ciliary disassembly.
In each of the above aspects, the product may be a pharmaceutical product.
In each of the above aspects, the cilia are primary cilia.
The invention is applicable to mammals, such as humans or mice in particular.
The present study found that lysophosphatidic acid (LPA) is the major factor in serum that induces ciliary assembly, and LPAR1 is critical for the normal development of cilia on mouse RG cells, VZ/SVZ production, and neocortical formation. The research result of the invention can link the cilia defect disease and LPA signal transduction, excessive LPA can cause cilia to be directly disassembled to further cause cilia defect, namely abnormal increase of LPA content can cause cilia defect disease. Thus, the present study indicates that LPA may be a prenatal diagnostic marker for cilia-deficient diseases, and that inhibitors related to the LPA signaling pathway may be potential drugs for the treatment of cilia-related diseases.
Drawings
Figure 1 shows that LPA is the major factor in serum that induces ciliary disassembly. a is a flow of inducing primary cilia to be disassembled at a cellular level; b is the minimum concentration of serum induced cilia de-assembly explored in a concentration gradient experiment; c is to explore the influence of serum under different treatment conditions on cilia de-assembly; d is the minimum concentration for exploring the cilia de-assembly induced by LPA in a concentration gradient experiment; e is a representative diagram of d.
Figure 2 shows LPA-dependent 1 induction of ciliary disassembly. a is expression of LPAR1-6 in RPE-1 cells; b abolished serum and LPA de-assembly effect on cilia after siRNA knockdown of LPAR 1; c is the knock-down effect of panel b; inhibitor Ki16425 with d LPAR1 and LPAR3 inhibited the de-assembly effect of serum and LPA on cilia.
In the quantification results for each graph, p < 0.05; denotes p < 0.01; denotes p < 0.001.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
1. Human retinal pigment epithelial cells (RPE-1): is given to the good teacher of the Zhu academy of sciences in Chinese academy. Described in "Tu HQ, Qin XH, Liu ZB, et al, Microtuble asters anchors by FSD1 control axoneme assembly and ciliogenesis. Nat Commun.2018Dec 11; 5277 "the public is available from the applicant, and is used for repeated experiments, but not for other purposes.
2. Reagent:
ki 16425: selleck corporation, cat # S1315. LPA: sigma, cat # L7260; Ac-Tubulin antibody: sigma, cat # T6793; gamma-Tubulin antibody: sigma, cat # T6557; LPAR1 antibody: santa corporation, cat # sc-515665; alpha-Tubulin antibody, Sigma, cat # T5168.
Example 1 related study of LPA as diagnostic marker for cilia-related diseases
First, experiment method
1. Cilium de-assembly experimental system
(1) The seed cell: digesting the RPE-1 cells, seeding the RPE-1 cells at 1/10 density in a 24-well plate with a glass slide, shaking, placing at 37 deg.C and 5% CO2Culturing in an incubator.
(2) After 12h, the culture medium is changed to Opti-MEM for starvation, and after 48h, serum or LPA is added for treatment for 24h according to experimental purposes, and then cells are harvested.
(3) Immunofluorescence:
1) placing the 24-well plate on ice for 10min, then removing the culture medium, and washing twice with PBS;
2) fixing: adding 250 μ l of 4% PFA preheated at 37 deg.C into each well, fixing at 37 deg.C for ten minutes, discarding the fixing solution, and washing twice with PBS;
3) punching: adding 250 μ l of 0.3% PBST into each well, perforating for 10min on ice, and removing the supernatant;
4) and (3) sealing: adding 250 μ l of 3% NGS (normal coat serum) into each well, and sealing at room temperature for 1 h;
5) a first antibody: the slide is pulled out and placed on a glass plate with a sealed film, 30 mu l of Ac-Tubulin primary antibody (1:500 is prepared in a sealing solution) is added into each slide, the incubation is carried out for 1h at room temperature, then the slide is placed back into a pore plate and washed for three times by PBS for 5min each time;
6) secondary antibody: the glass slide is pulled out and placed on a glass plate covered by a sealing film, 30 mu l of Alex-488 secondary antibody (1:400 is prepared in a sealing solution) is added into each glass slide, the incubation is carried out for 1h at room temperature, then the glass slide is placed back into a pore plate, and the washing is carried out for three times by PBS (phosphate buffer solution), and each time is 5 min;
7) dyeing the core: preparing Hoechst into PBS according to the ratio of 1:500, adding 250 mul of dye solution into each hole, keeping the temperature at room temperature for 10min, and then washing twice with PBS;
8) sealing: the slides were marked, 7. mu.l of the blocking tablet was added dropwise, the slides were placed upside down on the slides, dried at room temperature and then placed in a refrigerator at 4 ℃.
9) The cilia development was observed under a fluorescence microscope and counted.
2. siRNA interference:
(1) the seed cell: digesting the RPE-1 cells, seeding the RPE-1 cells at 1/10 density in a 24-well plate and a 6-well plate with a glass slide in advance, shaking, placing at 37 deg.C and 5% CO2Culturing in an incubator.
(2) Interference: after 12h, medium was changed with half the volume of fresh complete medium in normal culture, and after 1h, siRNA interference was performed.
LPAR1 siRNA(1#,sigma)：5′-GAAAUGAGCGCCACCUUUAdTdT-3′(SEQ ID No.1)；
LPAR1 siRNA(2#,Thermo Fisher Scientific)：5′-CAUCUGCUGGACUCCUGGAUUGGUU-3′(SEQ ID No.2)。
Transfecting by using siRAM 50nM concentration, diluting by 400 times, adding iMax, uniformly mixing siRNA and iMax, standing for 15min, and slowly dripping the mixed solution into cells.
(3) After 6h, the medium was changed to Opti-MEM medium, and after 48h, serum or LPA was added for 24h, after which the cells were harvested.
(4) Immunofluorescence: immunofluorescent staining was performed according to the method in step 1.
3. Real-time quantitative PCR:
normally cultured RPE-1 cells were harvested and total RNA was extracted from the cells by Trizol method. RNA was reverse transcribed and then subjected to Real-time quantitative PCR (Real-time PCR), specific primers for LPAR1-6 were designed and expression of these genes was examined, with GAPDH as internal reference and LPAR1 normalized. The detection primer sequences are as follows:
LPAR1(Gene ID 1902) detection primer:
LPAR1-F：5′-GTGTGGGCTGGAACTGTAT CTG-3′；
LPAR1-R：5′-TAGTCCTCTGGCGAACATAG-3′。
LPAR2(Gene ID 9170) detection primers:
LPAR2-F：5′-GGCCAGTGCTACTACAACGAGACC-3′；
LPAR2-R：5′-TGGAGGCGATGGCTGCTATG AC-3′。
LPAR3(Gene ID 23566) detection primers:
LPAR3-F：5′-CCTGGTGGTTCTGCTCCTCGAC-3′；
LPAR3-R：5′-GTGCCATACAT GTCCTCGTCCTTG-3′。
LPAR4(Gene ID 2846) detection primer:
LPAR4-F：5′-ATTGAAGTTGTTGGGTTTATCAT-3′；
LPAR4-R：5′-GCACAAGGTGATTGGGTACAT-3′。
LPAR5(Gene ID 57121) detection primer:
LPAR5-F：5′-CCTGGCGGCGGTGGT CTACTCGTC-3′；
LPAR5-R：5′-GACCGCCAGCGTGCTGTTGTAGGG-3′。
LPAR6(Gene ID 10161) detection primer:
LPAR6-F：5′-TTGTATGGGTGCATGTTCAGC-3′；
LPAR6-R：5′-GCCAATTCCGTGTTGTGAAGT-3′。
GAPDH (Gene ID 2597) detection primer:
GAPDH-F:5′-GGAGCGAGATCCCTC CAAAAT-3′；
GAPDH-R:5′-GGCTGTTGTCATACTTCTCATGG-3′。
4、Western Bolt：
(1) electrophoresis: the collected cell samples were spotted in SDS Page gel and electrophoresed at 80V until after Marker separation, the voltage was adjusted to 120V and then until bromophenol blue bottoms.
(2) Transfer printing: the transfer was performed with PVDF film, 400mA for 3 h.
(3) And (3) sealing: sealing with 5% milk at room temperature for 1 h;
(4) a first antibody: incubation with LPAR1, alpha-tubulin primary antibody, respectively, overnight at 4 ℃, followed by washing the membrane 3 times with TBST for 5min each time;
(5) secondary antibody: incubating with secondary antibody corresponding to the primary antibody, incubating at room temperature for 1h, and washing the membrane with TBST for 5min three times;
(6) and (3) developing: after washing the membrane, dripping to dry, mixing A, B liquid 1:1, putting in a dark box, and developing in a dark room.
5. Ki16425 inhibition assay
(1) The seed cell: digesting the RPE-1 cells, seeding the RPE-1 cells at 1/10 density in a 24-well plate with a glass slide, shaking, placing at 37 deg.C and 5% CO2Culturing in an incubator.
(2) After 12h, the culture medium is changed to Opti-MEM to starve for 48 h.
(3) Cells were pretreated with 40 μ M Ki16425 in Opti-MEM for half an hour before 40 μ M Ki16425 in serum or LPA containing medium and stimulated for 24 h.
(3) Immunofluorescence: the same experimental method as in step 1.
Second, results and analysis
1. LPA is the major factor in serum that induces ciliary disassembly
In order to identify factors inducing cilia de-assembly in serum, the invention establishes a cilia de-assembly experimental system by using human retinal pigment epithelial cells (RPE-1). RPE-1 cells were first serum starved for 48 hours, allowing most cells to induce ciliation. Cells were then re-stimulated with serum for 24 hours to allow cilia to de-assemble (a in figure 1). The effect of serum on ciliary disassembly was dose-dependent, with a minimum concentration of serum effective to induce ciliary breakdown of approximately 1% (b in figure 1).
To determine which components of serum are capable of inducing ciliary disassembly, the present invention next disrupts the protein or nucleic acid components of serum by boiling or treatment with pronase or cyclodextrin, respectively, and then uses the treated serum to stimulate cells. It was found that these treated sera were still able to induce cilia to undergo de-assembly (c in figure 1). These results indicate that the ciliary disorganizing activity of serum may not be caused by proteins or nucleic acids. The invention in turn removes lipids from the serum and treats the starved cells with this serum. It was found that serum after lipid removal did not induce cilia to undergo disassembly (c in figure 1). The results indicate that factors in serum that induce ciliary disassembly are likely lipid molecules.
Subsequently, the present inventors performed a series of screening for lipids, and finally found that lysophosphatidic acid (LPA) was able to induce cilia to undergo disassembly. The present invention uses Ac-Tubulin to indicate primary cilia and gamma-Tubulin to indicate centrosomes, both 2. mu.M LPA and 10% FBS were found to be effective in inducing primary cilia de-assembly. LPA is a phospholipid abundant in serum, with concentrations in the μ M range in human serum. At this concentration LPA alone can induce de-assembly of a large number of cilia (d and e in fig. 1). Thus, these results indicate that high concentrations of LPA in serum are sufficient to induce ciliary disassembly.
2. LPA-induced ciliary disassembly was dependent on LPAR1
LPA function depends on a series of G-protein coupled receptors LPAR1-6, and the present inventors found that only LPAR1 is highly expressed in RPE-1 cells (FIG. 2, a). Furthermore, the present invention knockdown LPAR1 in RPE-1 cells, and found that knockdown LPAR1 blocked serum or LPA-induced ciliary disassembly (b and c in fig. 2). Also, Ki16425(LPAR1 and LPAR3 antagonists) was used in the present invention to treat serum starved RPE-1 cells and was found to significantly inhibit serum and LPA-induced ciliary disassembly (fig. 2 d). These data indicate that the LPA-LPAR1 signaling pathway is critical for serum or LPA-induced ciliary disassembly.
Third, summarize
Cilia deficits lead to cilia-related diseases, and studies of the present invention indicate that LPA overdose leads to direct cilia de-assembly and thus to cilia deficits, i.e. abnormally elevated LPA levels may lead to cilia-deficient diseases. Thus, the present study indicates that LPA may be able to serve as a prenatal diagnostic marker for cilia-deficient diseases, and that inhibitors related to the LPA pathway may be potential drugs for the treatment of cilia-related diseases.
<110> biomedical analysis center of military medical research institute of military science institute
<120> diagnostic marker for cilia-related diseases
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Claims (10)
1. Use of lysophosphatidic acid as a marker in the manufacture of a product for diagnosing or screening for a cilium defect-related disease.
2. Use of a substance for detecting lysophosphatidic acid in the manufacture of a product for diagnosing or screening a cilium defect-related disease.
3. Use of a lysophosphatidic acid to induce ciliary disassembly or to prepare a product for inducing ciliary disassembly.
4. Use according to claim 3, characterized in that: the use is of lysophosphatidic acid and LPAR1 protein in inducing ciliary disassembly or in the preparation of a product for inducing ciliary disassembly.
Use of an inhibitor associated with the LPA signaling pathway for the manufacture of a product for the treatment of a disease associated with ciliary deficiency.
6. Use of a substance capable of reducing the lysophosphatidic acid content in any one of:
(A1) in the preparation of a product for the treatment of a condition associated with ciliary deficiency;
(A2) blocking lysophosphatidic acid from inducing ciliary disassembly, or preparing a product for blocking lysophosphatidic acid from inducing ciliary disassembly.
7. Use of a substance capable of inhibiting LPAR1 protein expression in any of:
(A1) in the preparation of a product for the treatment of a condition associated with ciliary deficiency;
(A2) blocking lysophosphatidic acid from inducing ciliary disassembly, or preparing a product for blocking lysophosphatidic acid from inducing ciliary disassembly.
8. Use according to claim 7, characterized in that: the substance capable of inhibiting expression of LPAR1 protein was an siRNA capable of interfering with expression of a gene encoding LPAR1 protein;
further, the sequence of the siRNA capable of interfering with expression of the gene encoding LPAR1 protein is SEQ ID No.1 or SEQ ID No. 2.
9. Use according to claim 7, characterized in that: the agent capable of inhibiting expression of LPAR1 protein was an inhibitor of LPAR1 protein;
further, the inhibitor of LPAR1 protein was Ki 16425.
10. Use according to any one of claims 1 to 9, characterized in that: the diseases associated with ciliary deficiency are diseases caused by the disorganization of cilia due to abnormally increased lysophosphatidic acid content.
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| CN113913511A (en) * | 2021-11-19 | 2022-01-11 | 中国人民解放军军事科学院军事医学研究院 | Application of OTULIN in preparation of reagents for diagnosing and treating cilia-related diseases |
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| CN101979090A (en) * | 2010-09-25 | 2011-02-23 | 北京师范大学 | Medicament for treating tumors |
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| CN113913511A (en) * | 2021-11-19 | 2022-01-11 | 中国人民解放军军事科学院军事医学研究院 | Application of OTULIN in preparation of reagents for diagnosing and treating cilia-related diseases |
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