CN108707656B - Markers at the gene level for preeclampsia - Google Patents
Markers at the gene level for preeclampsia Download PDFInfo
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- CN108707656B CN108707656B CN201810575897.9A CN201810575897A CN108707656B CN 108707656 B CN108707656 B CN 108707656B CN 201810575897 A CN201810575897 A CN 201810575897A CN 108707656 B CN108707656 B CN 108707656B
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Abstract
The invention discloses a PLEK gene as a diagnosis and treatment target in preeclampsia. The invention can judge whether the subject has preeclampsia or diagnose whether the subject has the risk of preeclampsia by detecting the content of the PLEK gene and the expression product thereof in the placenta tissue of the subject. In addition, the invention proves that the PLEK gene is used as a drug target for treating preeclampsia by researching the proliferation index of preeclampsia cells cultured in vitro.
Description
Technical Field
The invention relates to the field of biomedicine, in particular to application of a PLEK gene in preparation of a product for diagnosing and treating preeclampsia.
Background
Preeclampsia is one of the main diseases causing high maternal and child morbidity and mortality in the obstetrical field, has obvious genetic tendency, the pathophysiological changes relate to the pathological changes of multiple systems and multiple organs, the etiology and pathogenesis are not completely clarified so far, and the preeclampsia is always the focus of obstetrical research. The change of gene expression is the core mechanism for regulating the cell life activity process, abnormal metabolic process or pathological change, whether controlled by a single gene or a multi-gene system, and is essentially caused by gene differential expression, so genes capable of regulating blood pressure, body fluid volume, placenta growth, thrombosis, vascular recasting, vascular endothelial function, lipid metabolism, immunity, mitochondrial function and the like can be susceptibility genes in preeclampsia (Wanxipeng, Lin De. study of susceptibility genes for hypertensive syndrome in pregnancy. J. Zhonghua J. obstetrics. 2001, (36) 4: 248-251), the abnormal expression of the genes is the fundamental cause of preeclampsia occurrence and development, in recent years, with the rapid development of basic experimental methods, particularly the complete completion of human genome plan, the center of gravity of human genome research is shifting from 'structure' to 'function', the search for differentially expressed genes has become one of the research hotspots in molecular biology (Gabrielson E, Berg K, Anbazhagan R, equivalent functional genes, gene arrays, and the future of pathology Mod Pathol,2001, (14)12:1294-1299), the isolation and cloning of genes differentially expressed in different tissues or different development stages of the same tissue has important significance for understanding the molecular mechanism of diseases, and the emphasis on the research of the causes and pathogenesis of eclampsia has gradually shifted from the initial summary of clinical data and the observation of histocytology to the exploration of susceptibility genes of diseases and the research of the expression thereof. Although many preeclampsia-related genes, such as the Angiotensinogen (AGT) gene (Ward K, Hata A, Jeunemitre X, et al. A molecular variant of angiotensinogen associated with preimplantation. Nature Genet,1993,4:59-61.), the prothrombin gene [ Kupfermic MJ, Eldor A, Steinman N, et al. incorporated and acquired with genetic of genetic thrombophilia in tissue with compatibility of diagnosis. N Engl J New, 1999,340:9-13 ], tumor necrosis factor a (TNF a) gene [ Rinehart BK, Terra DA, Lagoenayayalan S, et al. Exuk of tissue diagnosis of preeclampsia, et al. expression of tissue diagnosis of preeclampsia, et al. A. the expression of tissue gene of tissue is still requires to be studied for the occurrence of abnormality of the expression of tissue in about the gene, No. 10. A. Adhesis, No. 7. A. expression of tissue diagnosis of tissue of human tissue infection, No. 7. A. expression of tissue diagnosis of human tissue infection, No. 1. A. As. A. As a. related to study of human tissue diagnosis of human tissue infection, No..
The placenta, as a special organ in gestation, plays a role in almost all organs of immature fetus, can secrete various hormones, enzymes, cytokines, growth factors and other substances, coordinates maternal endocrine, immune and metabolic functions, and plays an important role in maintaining normal pregnancy. The placenta ischemia theory is one of the etiology theories of preeclampsia, during normal pregnancy, placenta trophoblasts reversely infiltrate along spiral arterioles to gradually replace vascular endothelium so as to enlarge the vascular cavity and increase blood flow, and the process is called vascular recasting, and the depth can reach 1/3 in an myometrium. The infiltration capacity of placenta trophoblasts is reduced in the preeclampsia, and the placenta trophoblasts are only immersed into a periostracum section, so that the physiological recasting of uterine spiral arterioles is obstructed, and the placenta is ischemic and anoxic; the theory of vascular endothelial injury is the currently accepted central link of preeclampsia onset, vasoconstrictors and cytotoxic factors such as Endothelin (ET), AGT, thromboxane A2, TNF a and the like secreted by the placenta of preeclampsia patients are increased, and vasorelaxors such as prostacyclin, Nitric Oxide (NO) and the like are reduced, so that the injury of vascular endothelial cells of the body can be aggravated; and clinical application proves that the pre-eclampsia disease can be relieved or even disappeared after the fetal placenta is delivered, which indicates that the placenta and the pre-eclampsia are inseparable in occurrence and development. The experiment can know the expression condition of the gene by the difference of the placenta tissue gene expression product (mRNA) by applying the technology, and can directly discover and clone the placenta tissue differential expression gene of the preeclampsia patient and the normal pregnant woman from the gene level. Not only can detect the difference of known genes, but also can discover new genes related to preeclampsia, thereby providing a new idea for the research of genetics and susceptibility genes of preeclampsia.
Disclosure of Invention
One of the purposes of the invention is to provide a method for realizing preeclampsia diagnosis by detecting the expression difference of the PLEK genes.
It is a further object of the present invention to provide a method of treating preeclampsia by inhibiting the expression of the PLEK gene.
The invention also aims to provide a method for screening a therapeutic drug for preeclampsia.
The fourth purpose of the invention is to provide a medicine for treating preeclampsia.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides application of a PLEK detection reagent in preparation of a tool for diagnosing preeclampsia.
Further, the reagent for detecting the PLEK comprises a reagent for detecting the expression quantity of the PLEK gene.
Still further, the agent for detecting PLEK comprises an agent capable of quantifying PLEK gene mRNA, and/or an agent capable of quantifying PLEK protein.
The agent for quantifying the mRNA of the PLEK gene of the present invention can exert its function based on known methods using nucleic acid molecules: such as PCR, e.g., Southern hybridization, Northern hybridization, dot hybridization, Fluorescence In Situ Hybridization (FISH), DNA microarray, ASO methods, high throughput sequencing platforms, etc. The assay can be performed qualitatively, quantitatively, or semi-quantitatively using the reagent.
Further, the PCR method is a known method, for example, ARMS (Amplification Refractorymutation System) method, RT-PCR (reverse transcriptase-PCR) method, nested PCR method, or the like. The amplified nucleic acid can be detected by using a dot blot hybridization method, a surface plasmon resonance method (SPR method), a PCR-RFLP method, an in situ RT-PCR method, a PCR-SSO (sequence specific oligonucleotide) method, a PCR-SSP method, an AMPFLP (amplifiable fragment length polymorphism) method, an MVR-PCR method, and a PCR-SSCP (single strand conformation polymorphism) method.
The reagent capable of quantifying the mRNA of the PLEK gene can be a specific primer of the PLEK gene or a transcript, can also be a specific recognition probe of the PLEK gene or the transcript, or comprises the primer and the probe.
The primers specific to the PLEK gene or transcript described above include those used in real-time quantitative PCR to specifically amplify the PLEK gene. In a specific embodiment of the invention, the primer sequences are shown as SEQ ID NO.1 and SEQ ID NO. 2.
The primer can be prepared by chemical synthesis, appropriately designed by referring to known information using a method known to those skilled in the art, and prepared by chemical synthesis.
The probe may be prepared by chemical synthesis, by appropriately designing with reference to known information using a method known to those skilled in the art, and by chemical synthesis, or may be prepared by preparing a gene containing a desired nucleic acid sequence from a biological material and amplifying it using a primer designed to amplify the desired nucleic acid sequence.
The agent for quantifying the PLEK proteins of the invention may exert its function based on known methods using antibodies: for example, ELISA, radioimmunoassay, immunohistochemistry, Western blotting, etc. may be included.
The reagent for quantifying the PLEK protein comprises an antibody or a fragment thereof which specifically binds to the PLEK protein. An antibody or fragment thereof of any structure, size, immunoglobulin class, origin, etc., may be used so long as it binds to the target protein. The antibodies or fragments thereof included in the assay products of the invention may be monoclonal or polyclonal. An antibody fragment refers to a portion of an antibody (partial fragment) or a peptide containing a portion of an antibody that retains the binding activity of the antibody to an antigen. Antibody fragments may include F (ab')2Fab', Fab, single chain fv (scfv), disulfide-bonded fv (dsfv) or polymers thereof, dimerized V regions (diabodies), or CDR-containing peptides. The reagent for quantifying the PLEK protein of the present invention may comprise an isolated nucleic acid encoding the amino acid sequence of an antibody or encoding a fragment of an antibody, a vector comprising the nucleic acid, and a cell carrying the vector.
Antibodies can be obtained by methods well known to those skilled in the art. For example, mammalian cell expression vectors that retain all or part of the target protein or incorporate polynucleotides encoding them are prepared as antigens. After immunizing an animal with an antigen, immune cells are obtained from the immunized animal and myeloma cells are fused to obtain hybridomas. The antibody is then collected from the hybridoma culture. Finally, monoclonal antibodies against the PLEK proteins can be obtained by performing antigen-specific purification of the obtained antibodies using the PLEK proteins or parts thereof used as antigens. Polyclonal antibodies can be prepared as follows: an animal is immunized with the same antigen as above, a blood sample is collected from the immunized animal, serum is separated from the blood, and then antigen-specific purification is performed on the serum using the above antigen. The antibody fragment can be obtained by treating the obtained antibody with an enzyme or by using sequence information of the obtained antibody.
Binding of the label to the antibody or fragment thereof can be carried out by methods generally known in the art. For example, proteins or peptides may be fluorescently labeled as follows: the protein or peptide is washed with phosphate buffer, a dye prepared with DMSO, a buffer, or the like is added, and the solution is mixed and left at room temperature for 10 minutes. In addition, labeling may be carried out using commercially available labeling kits, such as biotin labeling kit, e.g., biotin labeling kit-NH 2, biotin labeling kit-SH (Dojindo laboratories); alkaline phosphatase labeling kits such as alkaline phosphatase labeling kit-NH 2, alkaline phosphatase labeling kit-sh (dojindo laboratories); peroxidase labeling kits such as peroxidase labeling kit-NH 2, peroxidase labeling kit-NH 2(Dojindo Laboratories); phycobiliprotein labeling kits such as phycobiliprotein labeling kit-NH 2, phycobiliprotein labeling kit-SH, B-phycoerythrin labeling kit-NH 2, B-phycoerythrin labeling kit-SH, R-phycoerythrin labeling kit-NH 2, R-phycoerythrin labeling kit SH (dojindo laboratories); fluorescent labeling kits such as fluorescein labeling kit-NH 2, HiLyte Fluor (TM)555 labeling kit-NH 2, HiLyte Fluor (TM)647 labeling kit-NH 2(Dojindo Laboratories); and DyLight 547 and DyLight647(Techno Chemical Corp.), Zenon (TM), Alexa Fluor (TM) antibody labeling kit, Qdot (TM) antibody labeling kit (Invitrogen Corporation), and EZ-marker protein labeling kit (Funakoshi Corporation). For proper labeling, a suitable instrument can be used to detect the labeled antibody or fragment thereof.
The obtaining of the sample for detecting the PLEK gene expression level detection of the present invention is a routine technique in the art, and preferably can be achieved by a method selected from non-invasive and minimally invasive methods.
The sample may be (but is not limited to): tissue, peripheral blood, bone marrow, lymph nodes, peritoneal cavity wash, urine, sweat. In a specific embodiment of the invention, the sample is from a tissue of a subject.
The invention also provides a tool for diagnosing preeclampsia, and the tool can detect the expression level of the PLEK gene.
Further, the means comprise reagents capable of quantifying the mRNA of the PLEK gene, and/or reagents capable of quantifying the PLEK protein.
Typically, the reagents are present in suitable containers. Each of the primers or probes can be adjusted to at least one desired amount of concentration using a diluent such as deionized water and dispensed into a container.
Further, the reagent capable of quantifying the mRNA of the PLEK gene comprises a primer for specifically amplifying the PLEK gene used in real-time quantitative PCR, and the sequence of the primer is shown as SEQ ID NO.1 and SEQ ID NO. 2.
Further, the means for diagnosing preeclampsia includes, but is not limited to, a chip, a kit, a test strip, or a high throughput sequencing platform; the high-throughput sequencing platform is a special tool, and with the development of high-throughput sequencing technology, the construction of a gene expression profile of a person becomes very convenient work. By comparing the gene expression profiles of patients with diseases and normal people, the abnormality of which gene is related to the disease can be easily analyzed. Therefore, the fact that the abnormality of the PLEK gene is related to the occurrence of preeclampsia in high-throughput sequencing also belongs to the novel application of the PLEK using the invention and is also in the protection scope of the invention.
The kit of the present invention may further comprise a reagent for extracting nucleic acid, a reagent for PCR, a reagent for staining or developing color, and the like. For example, such agents include, but are not limited to: an extraction solution, an amplification solution, a hybridization solution, a color development solution, a washing solution, and the like.
In addition, the kit can also comprise instructions and the like for describing a method for detecting the expression of the PLEK gene.
The kit of the present invention may contain a plurality of different reagents suitable for practical use (e.g., for different detection methods), and is not limited to the reagents listed so far, and is included in the scope of the present invention as long as the reagents are used for diagnosing preeclampsia based on the detection of the PLEK gene or transcript.
The invention also provides a method for diagnosing preeclampsia, which comprises the following steps:
(1) obtaining a sample from a subject;
(2) detecting the expression level of the PLEK gene in a sample of the subject;
(3) correlating the measured PLEK gene expression levels with a disease association in the subject.
(4) The statistically reduced levels of PLEK gene expression, as compared to a normal control, indicates that the subject is judged to have, or is at high risk of having, pre-eclampsia.
The invention also provides a method for treating preeclampsia, which comprises promoting the expression of the PLEK gene or promoting the activity of the expression product of the PLEK gene.
The invention also provides a screening method of candidate drugs for treating preeclampsia, which can measure the effect of the candidate drugs on improving prognosis by measuring the expression level of the PLEK gene or PLEK protein at a certain period after adding test drugs to model cells. More specifically, when the expression level of the PLEK gene or PLEK protein increases or returns to normal levels after addition or administration of a test drug, the drug may be selected as a therapeutic agent for ameliorating preeclampsia.
The invention also provides a medicament for treating preeclampsia, which comprises an agent for promoting PLEK.
The agent for promoting PLEK of the present invention is not limited as long as the agent is capable of promoting the expression or activity of PLEK or a substance involved in a pathway upstream or downstream of PLEK, and is an effective drug for the treatment of preeclampsia.
The invention also provides application of the PLEK gene or the expression product thereof in preparing a medicament for treating preeclampsia.
Further, the medicine comprises naked gene sequences, expression vectors containing the gene sequences, promoting miRNAs, transcription regulating factors or targeted small molecule compounds and the like.
The medicaments of the present invention may be used by formulating pharmaceutical compositions in any manner known in the art. Such compositions comprise the active ingredient in admixture with one or more pharmaceutically acceptable carriers, diluents, fillers, binders and other excipients, depending on the mode of administration and the dosage form envisaged. Therapeutically inert inorganic or organic carriers known to those skilled in the art include, but are not limited to, lactose, corn starch or derivatives thereof, talc, vegetable oils, waxes, fats, polyols such as polyethylene glycol, water, sucrose, ethanol, glycerol and the like, various preservatives, lubricants, dispersants, flavoring agents. Moisturizers, antioxidants, sweeteners, colorants, stabilizers, salts, buffers and the like may also be added as needed to aid in the stability of the formulation or to aid in the enhancement of the activity or its bioavailability or to produce an acceptable mouthfeel or odor upon oral administration, formulations which may be used in such compositions may be in the form of their original compounds as such, or optionally in the form of their pharmaceutically acceptable salts, and the agents of the present invention may be administered alone, in various combinations, and in combination with other therapeutic agents. The compositions so formulated may be administered in any suitable manner known to those skilled in the art, as desired. When using pharmaceutical compositions, a safe and effective amount of the drug of the present invention is administered to a human, and the specific dosage will depend on factors such as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.
The medicine of the present invention may be prepared into various preparation forms. Including, but not limited to, tablets, solutions, granules, patches, ointments, capsules, aerosols or suppositories for transdermal, mucosal, nasal, buccal, sublingual or oral use.
The route of administration of the drug of the present invention is not limited as long as it exerts the desired therapeutic or prophylactic effect, and includes, but is not limited to, intravenous, intraperitoneal, intraocular, intraarterial, intrapulmonary, oral, intravesicular, intramuscular, intratracheal, subcutaneous, transdermal, transpleural, topical, inhalation, transmucosal, dermal, gastrointestinal, intraarticular, intraventricular, rectal, vaginal, intracranial, intraurethral, intrahepatic, intratumoral. In some cases, the administration may be systemic. In some cases topical administration.
The dose of the drug of the present invention is not limited as long as the desired therapeutic effect or prophylactic effect is obtained. The dose of the therapeutic agent or prophylactic agent of the present invention can be determined using, for example, the therapeutic effect or prophylactic effect on a disease as an index.
The sequences of the "PLEK gene" (Chromosome 2, NC-000002.12 (68365190..68397453)) of the present invention can be queried in the NCBI database.
In the context of the present invention, "preeclampsia diagnosis" includes determining whether a subject has suffered from preeclampsia, determining whether a subject is at risk of suffering from preeclampsia, or determining that a preeclampsia patient has relapsed.
As used herein, "treatment" encompasses treatment-related diseases or disease states in a mammal, such as a human, having the associated disease or disorder, and includes:
(1) preventing the occurrence of a disease or condition in a mammal, particularly when the mammal is susceptible to said disease condition but has not been diagnosed as having such a disease condition;
(2) inhibiting a disease or disease state, i.e., preventing its occurrence; or
(3) Alleviating the disease or condition, i.e., causing regression of the disease or condition.
The term "treatment" generally refers to the treatment of a human or animal (e.g., as applied by a veterinarian) wherein some desired therapeutic effect is achieved, e.g., inhibiting the progression of a condition (including slowing the progression, stopping the progression), ameliorating the condition, and curing the condition. Treatment as a prophylactic measure (e.g., prophylaxis) is also included. The use of a patient who has not yet developed a condition but who is at risk of developing the condition is also encompassed by the term "treatment".
The invention has the advantages and beneficial effects that:
the invention discovers and confirms the close correlation between the PLEK gene expression and preeclampsia generation for the first time, and has more verified sample amount and accurate result. The proposal of the correlation provides a new approach for the diagnosis and treatment of preeclampsia.
Drawings
FIG. 1 shows a statistical graph of the differential expression of the PLEK gene in pre-eclamptic patient placental tissue versus normal placental tissue using QPCR;
FIG. 2 shows a statistical plot of the differential expression of the PLEK protein in pre-eclampsia patient placental tissues versus normal placental tissues using a Western blot assay;
FIG. 3 shows a statistical chart of the detection of the overexpression of the PLEK gene by Western blot assay;
FIG. 4 is a graph showing the results of examining the effect of PLEK gene expression on cell proliferation potency by the CCK-8 method.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention. Experimental procedures without specific conditions noted in the examples, generally following conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the laboratory Manual (New York: Cold Spring harbor laboratory Press,1989), or according to the manufacturer's recommendations.
Example 1 screening for genes aberrantly expressed in placental tissues of preeclamptic patients
1. Tissue collection
The placenta tissues of 5 patients in preeclampsia of obstetric and gynecologic delivery in a hospital are collected, 5 placenta tissues of late pregnant women with the same period of normal blood pressure and operation termination of pregnancy due to social factors or pelvic abnormality are selected as normal control groups, multiple pregnancies, infectious diseases, chemical drug dependence, pregnant woman smoking, fetal congenital malformation and other pregnancy complications and complications are eliminated in both groups, the gestational week is strictly verified according to B ultrasonic data in the early pregnancy, the gestation days are calculated, and all included research objects are signed with informed consent before collecting specimens. The diagnosis and scale-up criteria for preeclampsia were made with reference to the seventh edition of the national academy of higher medical sciences gynaecology (Lejie's eds).
The placenta tissue size is about 0.5cm x 0.5cm x 0.5cm after the placenta is delivered in a caesarean section, the placenta tissue size is about 0.5cm x 0.5cm immediately cut at the placenta maternal surface avoiding calcification, blood is removed as much as possible by repeatedly cleaning with sterile normal saline until cleaning fluid is clear, then the normal saline is drained, the tissue is cut into pieces as soon as possible, and the cut tissue is placed in a sterile freezing tube and put in a liquid nitrogen tank for freezing.
2. Tissue RNA extraction
RNA extraction kit (Trans Zol) from Beijing Quanji Biotech Ltd is usedTMUp PlusRNA Kit) tissue sample RNA was extracted. The method comprises the following specific steps:
(1) weighing the ultra-low temperature frozen sample, quickly transferring to a mortar precooled with liquid nitrogen, sufficiently grinding with a pestle until the sample is ground into powder, transferring the powder-ground sample to a centrifuge tube, and adding 1ml of Trans Zol into every 50-100mg of the sampleTMHomogenizing Up with a homogenizer, or repeatedly blowing and sucking with a gun, mixing, and standing at room temperature for 5 min.
(2) 1ml of Trans Zol is usedTMUp, 0.2ml of chloroform was added thereto, followed by vigorous shaking for 30 seconds and incubation at room temperature for 3 minutes.
(3) Centrifugation at 10000 Xg for 15 minutes at 4 ℃. The sample now separated into three layers, a colorless aqueous phase (upper layer), an intermediate layer, and a pink organic phase (lower layer). RNA was in the colorless aqueous phase and 500. mu.l of the colorless aqueous layer was pipetted.
(4) The aspirated 500. mu.l of colorless aqueous phase was transferred to a new centrifuge tube, 500. mu.l of absolute ethanol was added, and the mixture was gently inverted and mixed.
(5) Mu.l of the resulting solution was added to a spin column together with the precipitate, centrifuged at 12000 Xg at room temperature for 30 seconds, and the effluent was discarded.
(6) Add 500. mu.l of CB9, centrifuge at 12000 Xg at RT for 30 seconds and discard the effluent.
(7) Repeating the step (6) once.
(8) Adding 500 μ l WB9 (checking whether absolute ethanol is added before use), centrifuging at 12000 Xg for 30 s at room temperature, and discarding the effluent
(9) Repeating the step (8) once.
(10) Centrifuging at 12000 Xg room temperature for 2min, completely removing residual ethanol, standing at room temperature for several min, and completely air drying the column.
(11) The column was placed in an RNase-free Tube (kit prepared), and 30. mu.l of RNase-free Water was added to the center of the column, and the column was allowed to stand at room temperature for 1 minute.
(12) The RNA was eluted by centrifugation at 12000 Xg for 1 minute at room temperature.
(13) The RNA was stored in a freezer at-80 ℃.
3. Determination of concentration and purity of extracted RNA
RNA concentration and purity were determined using an ND-1000 instrument from Nano Drop, USA.
Using an ultraviolet spectrophotometer Nano Drop 1000 to double click a Nano Drop icon on a computer screen and entering a system menu; after selecting a nucleic acid measurement option, adding 2 mu l of double distilled water into the sample adding hole according to system prompt; clicking to determine to initialize the system; add 2. mu.l of double distilled water to the wells, select RNA items, and click Blank to measure system background. And (4) wiping the double distilled water with a piece of rear lens wiping paper, then dropping the RNA sample to be detected, and clicking Measure. Reading the value and recording the RNA concentration (ng/. mu.l), wherein the care of the A value 260/280 is necessary, and if the value is between 1.8 and 2.0, the RNA sample quality is better. The RNA concentration of each sample was labeled completely and then frozen in a freezer at-70 ℃.
4. Extracted RNA integrity assay
Agarose gel detection of RNA samples the procedure:
1) electrophoresis tank, cleaning of glue making tool: cleaning with detergent (generally soaking overnight), washing with water, and washing with 3% H2O2Filling the electrophoresis tank, standing at room temperature for 10min, washing with 0.1% (V/V) DEPC water, and air drying for use.
2) Preparing glue: 0.5g of agarose powder was weighed into an Erlenmeyer flask containing 45ml of DEPC water, and heated to completely dissolve the agarose. After a little cooling, 5ml of 10 XTAE electrophoresis buffer and a final concentration of 0.5. mu.g/ml ethidium bromide were added. Then pouring gel into the gel groove, inserting a comb, horizontally placing for use after solidification.
3) Sample adding: the sample is mixed with 6 Xelectrophoresis buffer solution and loaded into the gel sample application hole.
4) Electrophoresis: and opening the electrophoresis apparatus, and stabilizing the voltage of 80V electrophoresis.
5) After electrophoresis (about 30 minutes), the gel was stored by UV light and photographed with a gel imaging system.
Integrity determination by agarose gel electrophoresis detection, if can clearly see 28s rRNA, 18s R RNA, 5srRNA three bands, and 28s rRNA brightness should be 18s rRNA two times. The completeness of the extracted total RNA is better, and the RNA quality meets the requirement.
4. High throughput transcriptome sequencing
(1) RNA-seq read mapping
The clean reads were first removed from the low quality reads and then matched to the UCSC h. sapiens reference genome (hg19) using TopHat v1.3.1, a pre-constructed index of h.sapiens UCSC hg19 version was downloaded from the TopHat homepage and used as the reference genome, allowing multiple matching sites per read (default to 20) with up to 2 mismatches when matched to the genome using TopHat. TopHat builds a pool of possible cleavage sites based on the exon regions and GT-AG cleavage signals, from which reads that are not mapped to the genome are mapped to the genome. We use the system default parameters of the TopHat method.
(2) Transcript abundance assessment
The matched read files are processed by Cufflinks v1.0.3, and the Cufflinks v1.0.3 standardizes the number of RNA-seq segments to calculate the relative abundance of the transcript. The FPKM value refers to the number of fragments that match to a region of the exon 1kb long for a particular gene per million sequenced fragments. And calculating a confidence interval of the FPKM estimated value by a Bayesian inference method. The referenced GTF annotation file used by Cufflinks was downloaded from the Ensembl database (Homo _ sapiens. grch37.63. GTF).
(3) Detection of differentially expressed genes
The downloaded Ensembl GTF file and the original file matched by TopHat are transmitted to Cuffdiff, and the Cuffdiff uses the original matched file to re-estimate the expression abundance of the transcripts listed in the GTF file and detect differential expression. Only q values < 0.01 in the Cuffidff output, tests showed that successful comparisons were considered differential expression.
4. Results
The RNA-seq result shows that compared with a normal pregnant woman, 458 genes with high expression and 579 genes with low expression in the placenta tissue of a preeclamptic patient have statistical significance (P is less than 0.05).
Example 2 Large sample validation of expression of differentially expressed genes at the transcriptional level
1. Tissue collection
35 placental tissues of preeclamptic patients and 40 placental tissues of normal pregnant women were collected according to the standard of example 1.
2. Tissue RNA extraction and identification
The procedure is as in example 1.
3. Design and preparation of primers
The primer sequence for detecting PLEK gene expression by qRT-PCR and the primer sequence for amplifying internal reference GAPDH by qRT-PCR are designed and synthesized by biological engineering (Shanghai) corporation, and the sequence information is recorded into NCBI software design primer by searching UCSC database, and is confirmed to be correct in blast in the gene bank.
PLEK gene primers:
an upstream primer: 5'-ATTGACTTAGGTGCCTTAT-3' (SEQ ID NO. 1);
downstream primer 5'-AACAGACTGGTTGGATAC-3' (SEQ ID NO.2),
GAPDH gene primers:
an upstream primer: 5'-GGGAGCCAAAAGGGTCA-3' (SEQ ID NO. 3);
a downstream primer: 5'-GAGTCCTTCCACGATACCAA-3' (SEQ ID NO. 4).
6. Real-time fluorescent quantitative cDNA reverse transcription detection step
Mu.g of total RNA was reverse transcribed with reverse transcription buffer to synthesize cDNA. A25-mu-l reaction system is adopted, 1 mu g of total RNA is taken from each sample as template RNA, and the following components are respectively added into a PCR tube: DEPC water, 5 Xreverse transcription buffer, 10mmol/L dNTP, 0.1mmol/L DTT, 30. mu. mmol/L Oligo dT, 200U/. mu. L M-MLV, template RNA. Incubate at 42 ℃ for 1h, 72 ℃ for 10min, and centrifuge briefly.
7、PCR
(1) A Bio-RAD real-time fluorescence quantitative PCR instrument was used to prepare a reaction system as shown in Table 1.
TABLE 1 PCR reaction System
(2) The following qPCR reaction parameters were used: pre-denaturation at 95 ℃ for 10min, subsequent denaturation at 95 ℃ for 15s, annealing at 52.6 ℃ and extension for 1min, and 45 cycles; then, the fluorescence signal was collected and the product dissolution curve was prepared, and 3 replicates of each sample were averaged. By using 2-△△CtAnd analyzing the expression level of the PLEK by a relative quantification method, wherein Ct is the intensity value of a fluorescence signal detected in a reaction system by a thermal cycler. The calculation method comprises the following steps: delta Ct ═ (Ct target gene-Ct reference gene) preeclampsia patient placental tissue experimental group- (Ct target gene-Ct reference gene) normal pregnant woman placental tissue control group, 2-△△CtThe expression of the target gene in the experimental group is shown as the fold change relative to the control group, and the analysis of the experimental data is performed by the Bio-RAD analysis software.
5. Statistical analysis statistical software SPSS19.0 is used for data analysis, and paired T test is used to judge whether the PLEK expression in the preeclamptic placenta tissue sample and the normal pregnant woman placenta tissue sample is different in statistical significance. The statistical tests are bilateral tests, and the difference is statistically significant when P is less than 0.05.
6. Results
Compared with the placenta tissue of a normal pregnant woman, the PLEK gene expression in the placenta tissue of 35 patients in the pre-epilepsy patients is obviously reduced, and the difference has statistical significance. As shown in FIG. 1, the PLEK gene in the placental tissue of preeclamptic patients was significantly reduced compared to that of normal pregnant women, and the difference was statistically significant (P < 0.05).
Example 2 Large sample validation of expression of differentially expressed genes at the protein level
1. Extraction of Total protein from the tissue of example 2
The protein extraction procedure was performed according to the instructions of the EpiQuik tissue/cell total protein extraction kit.
2. Electrophoresis
β -actin is used as an internal reference, 50 mu g of total protein is subjected to SDS-PAGE separation, then is electrically transferred to a PVDF membrane, is slightly shaken and sealed for 1h at room temperature by 1 xTBST containing 5% skimmed milk powder, PLEK monoclonal antibody and β -actin monoclonal antibody are respectively added, the mixture is subjected to overnight standing at 4 ℃, the membrane is washed for 4 times by 1 xTBST, secondary antibody is added, the mixture is incubated for 1h at room temperature, after the membrane is washed for 4 times by 1 xTBST, the mixture is placed in a Super Signal chemiluminescence reagent for reaction for 2min, and an X-ray film is exposed in a dark room and developed and fixed by a conventional method.
3. Statistical treatment
The grey values of the protein bands were analyzed by using Image J software, with β -actin as an internal reference, and the grey values of the PLEK protein bands were normalized, the data were expressed as mean. + -. standard deviation, and statistically analyzed by using SPSS13.0 statistical software, and the difference between the two was considered to be statistically significant when P <0.05 by using t-test.
4. Results
The levels of PLEK protein were significantly reduced in placental tissue from 35 out of 35 pre-epileptic patients compared to normal pregnant woman placental tissue, with statistical significance for the differences. As shown in FIG. 2, the levels of PLEK proteins in the placental tissues of preeclamptic patients were significantly reduced compared to normal pregnant placental tissues, with statistical significance for the differences (P < 0.05).
Example 3 promotion of PLEK Gene expression
1. Construction of PLEK Gene expression vectors
The amplification primers were designed based on the coding sequence of the PLEK gene (shown in SEQ ID NO. 5). The coding sequence of the full-length PLEK gene was amplified from a cDNA library of adult fetal brain (Clontech, cat # 638831), which was digested simultaneously with restriction enzymes BamHI and XhoI, inserted into the eukaryotic cell expression vector pcDNA3.1 digested simultaneously with restriction enzymes BamHI and XhoI, and the resulting recombinant vector pcDNA3.1-PLEK was ligated for subsequent experiments.
2. Culture of placental trophoblast cells
JEG-3 cells were cultured in DMEM high-pond medium containing double antibody and 10% fetal bovine serum at 37 deg.C and 5% CO2The culture medium is changed for 1 time every 24 hours, and the culture medium is subcultured for 1 time in 48 hours. Cells in logarithmic growth phase were taken for subsequent experiments.
3. Cell transfection
Liposome Lipofectamine2000 was used as the transfection reagent. Experiment was divided into 2 groups: negative control group (pcDNA3.1 transfected); experimental group (pcDNA3.1-PLEK transfected). JEG-3 cells in the logarithmic growth phase were inoculated to 6-well cell culture plates. After 24h, the coverage rate of the cell culture plate is about 70-80%. The transfection protocol was performed according to Lipofectamine2000 instructions.
4. Western blot experiment for detecting promotion efficiency of pcDNA3.1-PLEK
1) Protein sample preparation
The medium is poured off and the bottle is inverted over absorbent paper to allow the absorbent paper to suck the medium dry (or the bottle is placed upright for a while to allow the residual medium to flow to the bottom of the bottle and then removed by pipetting). 3M of 14 ℃ pre-cooled PBS (0.01M pH7.2-7.3) was added to each flask of cells. The cells were washed by gentle shaking for 1min and then the wash solution was discarded. The above operation was repeated twice, and the cells were co-washed three times to wash out the culture solution. After discarding the PBS, the flasks were placed on ice. Mu.1 PMSF (100mM) was added to the lml lysate and shaken well on ice. 400 μ 1 of lysis solution containing PMSF was added to each flask of cells and lysed on ice for 30min, and the flask was shaken back and forth often to fully lyse the cells. After lysis, the cells were scraped to one side of the flask using a clean scraper, and then the cell debris and lysate were transferred to l.5m1 centrifuge tubes using a gun for storage at-20 ℃.
2) Electrophoresis
The procedure of example 2 was followed.
5. Results
As shown in FIG. 3, the expression level of the PLEK protein in the cells of the experimental group (pcDNA3.1-PLEK transfected) was significantly increased compared to that of the empty vector group (pcDNA3.1 transfected), and the difference was statistically significant (P < 0.05).
Example 4 Effect of PLEK Gene expression on placental trophoblast cell proliferation
The cell proliferation is detected by CCK-8 method
(1) Transfection was performed according to the method of example 3;
(2) JEG-3 cells after 24h transfection were aspirated off the medium, cells were digested with 0.25% trypsin, counted with crystal violet stain, resuspended in DMEM/F12 low serum medium containing 0.5% FBS and adjusted to a cell concentration of 2X104/ml；
(3) Add 100. mu.l of cell suspension to each well of a new 96-well plate under sterile conditions, i.e., 2X103Single cell, 37 ℃, 5% CO2Standing for 20 h;
(4) adding 10 mul of CCK solution into each hole respectively, and culturing for 2 h;
(5) the wavelength of 450nm, and the light absorption value of each experimental group is measured by an ultraviolet spectrophotometer.
As a result:
as shown in FIG. 4, the cell proliferation of the experimental group (pcDNA3.1-PLEK transfected) was significantly inhibited compared to the empty vector group (pcDNA3.1 transfected), and the difference was statistically significant (P < 0.05).
The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.
Sequence listing
<110> Beijing, the deep biometric information technology GmbH
<120> marker at the Gene level for preeclampsia
<160>5
<170>SIPOSequenceListing 1.0
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attgacttag gtgccttat 19
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<213> Artificial Sequence (Artificial Sequence)
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aacagactgg ttggatac 18
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<212>DNA
<213> Artificial Sequence (Artificial Sequence)
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gggagccaaa agggtca 17
<210>4
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<213> Artificial Sequence (Artificial Sequence)
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<213> human source (Homo sapiens)
<400>5
atggaaccaa agcggatcag agagggctac cttgtgaaga aggggagcgt gttcaatacg 60
tggaaaccca tgtgggttgt attgttagaa gatggaattg aattctataa gaagaaaagt 120
gacaacagcc ccaaaggaat gatcccgctg aaagggagca ctctgactag cccttgtcaa 180
gactttggca aaaggatgtt tgtgtttaag atcactacga ccaaacagca ggaccacttc 240
ttccaggcag ccttcctgga ggagagagat gcctgggttc gggatatcaa gaaggccatt 300
aaatgcattg aaggaggcca gaaatttgcc aggaaatcta ccaggaggtc cattcgactg 360
ccagaaacca ttgacttagg tgccttatat ttgtccatga aagacactga aaaaggaata 420
aaagaactga atctagagaa ggacaagaag atttttaatc actgcttcac aggtaactgc 480
gtcattgatt ggctggtatc caaccagtct gttaggaatc gccaggaagg cctcatgatt 540
gcttcatcgc tgctcaatga ggggtatctg cagcctgctg gagacatgtc caagagtgca 600
gtggatggaa ctgctgaaaa ccctttcctg gacaaccctg atgccttcta ctactttcca 660
gacagtgggt tcttctgtga agagaattcc agtgatgatg atgtgattct gaaagaagaa 720
ttcagagggg tcattatcaa gcagggatgt ttactgaagc aggggcatag aaggaaaaac 780
tggaaagtga ggaagttcat cttgagagaa gaccctgcct acctgcacta ctatgaccct 840
gctggggcag aagatcccct gggagcaatt cacttgagag gctgtgtggt gacttcagtg 900
gagagcaact caaatggcag gaagagtgag gaagagaacc tttttgagat catcacagca 960
gatgaagtgc actatttctt gcaagcagcc acccccaagg agcgcacaga gtggatcaga 1020
gccatccaga tggcctcccg aactgggaag taa 1053
Claims (4)
1. Application of a reagent for detecting the expression level of PLEK gene mRNA or PLEK protein in preparing a diagnostic tool for preeclampsia.
2. The use according to claim 1, wherein the reagents for detecting the mRNA expression level of the PLEK gene comprise primers for the specific amplification of the PLEK gene used in real-time quantitative PCR; the reagent for detecting the expression level of the PLEK protein comprises an antibody which specifically binds to the PLEK protein.
3. The use according to claim 2, wherein the primer sequences are shown as SEQ ID No.1 and SEQ ID No. 2.
4. The use of any one of claims 1-3, wherein said means comprises a kit, a chip, a strip, a high throughput sequencing platform.
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| CN103031374A (en) * | 2012-11-06 | 2013-04-10 | 中国农业大学 | Gene chip for evaluating transport stress model and application of gene chip for evaluating transport stress model |
| EP3260540A1 (en) * | 2010-11-12 | 2017-12-27 | The General Hospital Corporation | Polycomb-associated non-coding rnas |
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| CN103031374A (en) * | 2012-11-06 | 2013-04-10 | 中国农业大学 | Gene chip for evaluating transport stress model and application of gene chip for evaluating transport stress model |
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