CN108624678B - Biomarker for diagnosis and treatment of preeclampsia - Google Patents
Biomarker for diagnosis and treatment of preeclampsia Download PDFInfo
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- CN108624678B CN108624678B CN201810669608.1A CN201810669608A CN108624678B CN 108624678 B CN108624678 B CN 108624678B CN 201810669608 A CN201810669608 A CN 201810669608A CN 108624678 B CN108624678 B CN 108624678B
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/08—Antiepileptics; Anticonvulsants
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/28—Neurological disorders
- G01N2800/2857—Seizure disorders; Epilepsy
Abstract
The invention discloses a biomarker for preeclampsia diagnosis and treatment, wherein the biomarker KIAA1949 is expressed and reduced in placenta tissues and blood of a pregnant woman in preeclampsia, and the fact that the KIAA1949 can be used as a detection target for early diagnosis of preeclampsia is suggested; overexpression of the KIAA1949 gene can promote proliferation and invasion of trophoblasts, so that the KIAA1949 gene can be used as a molecular target for treating preeclampsia.
Description
Technical Field
The invention belongs to the field of biological medicines, and relates to a biomarker for diagnosis and treatment of preeclampsia, in particular to a biomarker which is KIAA1949 obviously reduced in preeclampsia.
Background
Preeclampsia (PE), eclampsia (eclampsia) and gestational hypertension are specific diseases in pregnancy, belong to The category of pregnancy hypertension diseases (hyperkinetic disorders in pregnancy), and have morbidity of about 10% in China, wherein The preeclampsia (Redman CW, Sargent I L. L. atest acquired in unsubstantial standing pre-eclampsia [ J ] Science,2005, 308:1592 1594) is mainly expressed by hypertension and proteinuria which appear after 20 weeks of pregnancy and can cause systemic multiple organ functional impairment and functional failure, can cause maternal mortality of up to 15% and is The main cause of maternal and perinatal death, and is The key point of pathological obstetrical research until now, and The pathogenesis is unknown (Duley L. The prognosis of pregnancy clinical study of The maternal death of at least a part of pregnancy and peripartum death, and The pregnancy of pregnancy is considered to be The important point, and The pregnancy of The study of pregnancy is that The pregnancy of pregnancy is only The pregnancy induced by The clinical pregnancy of pregnancy induced by The clinical change of pregnancy of The pregnancy induced by.
In addition to environmental factors such as early birth, nutritional deficiency, multiple pregnancy and prior Hypertension, nephritis, diabetes history, the risk of preeclampsia is genetically influenced by the interaction of multiple genes or multiple polymorphic sites, and has obvious genetic tendency (Serrano N C, case JP, Diaz L A, oral No. synthetic gene and isk of a clinical trial: a multicentropic-control study [ J ] Hypertension,2004,44(5):702 and 707.) since 19 th century, familial onset of preeclampsia has been confirmed, and the genetic factors involved in the onset of preeclampsia has been shown to be effective in the clinical research on the pathogenesis of preeclampsia of women and clinical diagnosis of preeclampsia, and the early eclampsia is considered to be important for the clinical research on the early pregnancy and early pregnancy, and early pregnancy research on the clinical diagnosis of preeclampsia, early pregnancy diagnosis of pregnancy, early pregnancy.
Disclosure of Invention
In order to make up for the defects of the prior art, the invention aims to provide a biomarker for preeclampsia, and sensitively and specifically realizes the diagnosis and treatment of preeclampsia.
The invention also aims to provide application of the biomarker in screening the biomarker for treating preeclampsia.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides application of a detection reagent of KIAA1949 in preparing a product for diagnosing preeclampsia.
Further, the product comprises: a chip, a preparation, a kit or a nucleic acid membrane strip.
The invention provides a product for diagnosing preeclampsia, which comprises a detection reagent of KIAA 1949.
Further, the reagent comprises:
a probe that specifically recognizes the KIAA1949 gene; or
A primer for specifically amplifying the KIAA1949 gene; or
An antibody or ligand that specifically binds to a KIAA1949 protein.
The products of the invention can be used to detect the expression levels of multiple genes including the KIAA1949 gene and encoded proteins (e.g., multiple genes and encoded proteins associated with preeclampsia).
The invention provides an application of KIAA1949 in preparing a pharmaceutical composition for treating preeclampsia.
Further, the pharmaceutical composition comprises an accelerant of the KIAA1949 functional expression, wherein the accelerant comprises a substance which improves the stability of the KIAA1949 gene or an expression product thereof, up-regulates the expression level of the KIAA1949 gene or the expression product thereof, and increases the effective action time of the KIAA1949 gene or the expression product thereof.
Further, the promoter is a carrier containing KIAA 1949.
The invention also provides a pharmaceutical composition, which comprises the promoter for the functional expression of the KIAA1949 and a pharmaceutically acceptable carrier.
Wherein the promoter comprises a substance which improves the stability of the KIAA1949 gene or an expression product thereof, up-regulates the expression level of the KIAA1949 gene or an expression product thereof, and increases the effective action time of the KIAA1949 gene or an expression product thereof.
Further, the promoter is a carrier containing KIAA 1949.
The invention provides application of KIAA1949 in screening medicines for treating preeclampsia.
Further, the step of screening for a drug for the treatment of preeclampsia comprises:
treating a system expressing or containing the KIAA1949 gene or a protein encoded by it with a substance to be screened; and
detecting the expression or activity of the KIAA1949 gene or the protein encoded by the gene in the system.
Wherein, if the substance to be screened can promote the expression or activity of KIAA1949, the substance to be screened is a medicament for treating preeclampsia.
In the present invention, the system includes (but is not limited to): a cell system, a subcellular system, a solution system, a tissue system, an organ system, or an animal system.
In the present invention, the steps further include: the obtained candidate drugs are subjected to further cell experiments and/or animal experiments to further select drugs from the candidate drugs that can treat preeclampsia.
The invention has the advantages and beneficial effects that:
the invention discovers a biomarker KIAA1949 related to the occurrence and development of preeclampsia for the first time, realizes early diagnosis of preeclampsia by detecting the level of KIAA1949 of a subject, and indicates that a patient may suffer from preeclampsia when the KIAA1949 shows differential low expression.
The invention provides a molecular target KIAA1949 for treating preeclampsia, and the molecular target KIAA1949 has sensitivity and specificity for treating diseases by targeting a molecular marker and changing the level of the marker.
The invention provides a new means for the research and development of the medicament for treating preeclampsia.
Drawings
FIG. 1 is a graph showing the detection of KIAA1949 gene expression in preeclamptic patients using QPCR; wherein A is the expression level in placental tissue and Panel B is the expression level in blood;
FIG. 2 is a graph of the expression level of the KIAA1949 gene;
FIG. 3 is a graph of the expression level of KIAA1949 protein;
FIG. 4 is a graph showing the effect of the MTT method to detect KIAA1949 on cell proliferation activity;
FIG. 5 shows a graph of the effect of the KIAA1949 gene on cell invasion detected using a Transwell chamber.
Detailed Description
Through extensive and intensive research, the invention detects the gene expression levels in the blood of preeclamptic patients and normal pregnant women through high-throughput sequencing technology and bioinformatics analysis, finds genes with obvious differences, and discusses the relationship between the genes and the occurrence of preeclampsia, thereby finding better ways and methods for early detection and targeted treatment of preeclampsia. The invention discovers that KIAA1949 is related to the occurrence and the development of preeclampsia for the first time through screening, and verifies that the KIAA1949 is low expressed in the preeclampsia. KIAA1949 can be used as an independent predictor of preeclampsia, and can also be combined with other gene markers for application.
The term "biomarker" in the present invention is any gene or protein whose expression level in a tissue or cell is altered compared to the expression level in a normal or healthy cell or tissue.
The KIAA1949 gene is located on zone 1 of chromosome 6 short arm 2, and the KIAA1949 gene in the invention comprises wild type, mutant or fragment thereof. In a specific embodiment of the invention, KIAA1949 has a sequence as shown by the KIAA1949 gene (NM — 001134870.1) in GeneBank, the current international public nucleic acid database.
Biomarkers described herein include genes and proteins. Such biomarkers include DNA comprising the complete or partial sequence of the nucleic acid sequence encoding the biomarker or the complement of this sequence. Biomarker nucleic acids also include RNA that contains the entire or partial sequence of any nucleic acid sequence of interest. Biomarker proteins are proteins encoded by or corresponding to the DNA biomarkers of the invention. The biomarker protein comprises the complete or partial amino acid sequence of any biomarker protein or polypeptide. Fragments and variants of biomarker genes and proteins are also included within the scope of the invention.
The present invention may utilize any method known in the art for determining gene expression. It will be appreciated by those skilled in the art that the means by which gene expression is determined is not an important aspect of the present invention. The expression level of the biomarker can be detected at the transcriptional or translational (i.e., protein) level.
The genes and proteins of the invention can be detected using a variety of nucleic acid techniques known to those of ordinary skill in the art, including but not limited to: nucleic acid sequencing, nucleic acid hybridization and nucleic acid amplification technology, and protein immunodetection technology.
Illustrative, non-limiting examples of nucleic acid sequencing techniques include, but are not limited to, chain terminator (Sanger) sequencing and dye terminator sequencing. One of ordinary skill in the art will recognize that RNA is typically reverse transcribed into DNA prior to sequencing because it is less stable in cells and more susceptible to nuclease attack in experiments.
Illustrative, non-limiting examples of nucleic acid amplification techniques include, but are not limited to, Polymerase Chain Reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), transcription-mediated amplification (TMA), ligase chain reaction (L CR), Strand Displacement Amplification (SDA), and nucleic acid sequence-based amplification (NASBA). one of ordinary skill in the art will recognize that some amplification techniques (e.g., PCR) require reverse transcription of RNA to DNA (e.g., RT-PCR) prior to amplification, while others amplify RNA directly (e.g., TMA and NASBA).
The polymerase chain reaction, commonly known as PCR, uses multiple cycles of denaturation, annealing of primer pairs to opposite strands, and primer extension to exponentially increase the copy number of the target nucleic acid sequence, transcription-mediated amplification of TMA (autocatalytically synthesizing multiple copies of the target nucleic acid sequence under conditions of substantially constant temperature, ionic strength, and pH, where multiple RNA copies of the target sequence autocatalytically generate additional copies; the ligase chain reaction of L CR uses two sets of complementary DNA oligonucleotides that hybridize to adjacent regions of the target nucleic acid; other amplification methods include, for example, nucleic acid sequence-based amplification, commonly known as NASBA, amplification of the probe molecule itself using RNA replicase (commonly known as Q β replicase), transcription-based amplification methods, and self-sustained sequence amplification.
Non-amplified or amplified nucleic acids of the invention can be detected by any conventional means.
Protein immunoassays include, but are not limited to, sandwich immunoassays, such as sandwich E L ISA, where detection of a biomarker is performed using two antibodies that recognize different epitopes on the biomarker, Radioimmunoassays (RIA), direct, indirect or contrast enzyme-linked immunosorbent assays (E L ISA), Enzyme Immunoassays (EIA), Fluorescence Immunoassays (FIA), western blots, immunoprecipitations, and any particle-based immunoassays (e.g., using gold, silver or latex particles, magnetic particles, or quantum dots).
Any direct (e.g., using a sensor chip) or indirect method may be used in the detection of the biomarkers of the invention.
Chip, preparation, nucleic acid membrane strip and kit
The invention provides products, including (but not limited to) chips, formulations, nucleic acid membrane strips or kits, for detecting the expression level of the KIAA1949 gene. Wherein the chip includes: a solid support; and oligonucleotide probes orderly fixed on the solid phase carrier, wherein the oligonucleotide probes specifically correspond to a part or all of a sequence shown by KIAA 1949.
The solid phase carrier comprises an inorganic carrier and an organic carrier, wherein the inorganic carrier comprises but is not limited to a silicon carrier, a glass carrier, a ceramic carrier and the like; the organic vehicle includes a polypropylene film, a nylon film, and the like.
The term "probe" refers to a molecule that binds to a specific sequence or subsequence or other portion of another molecule. Unless otherwise indicated, the term "probe" generally refers to a polynucleotide probe that is capable of binding to another polynucleotide (often referred to as a "target polynucleotide") by complementary base pairing. Depending on the stringency of the hybridization conditions, a probe can bind to a target polynucleotide that lacks complete sequence complementarity to the probe. The probe may be directly or indirectly labeled, and includes within its scope a primer. Hybridization modalities, including, but not limited to: solution phase, solid phase, mixed phase or in situ hybridization assays.
Exemplary probes in the present invention include PCR primers as well as gene-specific DNA oligonucleotide probes, such as microarray probes immobilized on a microarray substrate, quantitative nuclease protection test probes, probes attached to molecular barcodes, and probes immobilized on beads.
These probes may be DNA or RNA, or may be polynucleotides obtained by replacing some or all of the nucleotides with an artificial Nucleic Acid such as PNA (peptide Nucleic Acid), L NA (registered trademark), locked Nucleic Acid, ENA (registered trademark, 2 '-O, 4' -C-Ethylene-Bridged Nucleic Acid), GNA (Glycerol Nucleic Acid), TNA (Threose Nucleic Acid ), or the like.
In the present invention, the term "antibody" includes, but is not limited to, monoclonal antibodies, polyclonal antibodies. Antibodies specific for the KIAA1949 protein include intact antibody molecules, any fragment or modification of an antibody (e.g., chimeric antibodies, scFv, Fab, F (ab') 2, Fv, etc., so long as the fragment retains the ability to bind to the KIAA1949 protein.
In the present invention, a nucleic acid membrane strip comprises a substrate and oligonucleotide probes immobilized on the substrate; the substrate may be any substrate suitable for immobilizing oligonucleotide probes, such as a nylon membrane, a nitrocellulose membrane, a polypropylene membrane, a glass plate, a silica gel wafer, a micro magnetic bead, or the like.
Exemplary probes in the present invention include PCR primers as well as gene-specific DNA oligonucleotide probes, such as microarray probes immobilized on a microarray substrate, quantitative nuclease protection test probes, probes attached to molecular barcodes, and probes immobilized on beads.
The invention provides a kit which can be used for detecting the expression of KIAA 1949. The kit comprises a specific primer pair for amplifying KIAA 1949; a standard DNA template; and (3) PCR reaction liquid.
In a preferred embodiment, the specific primer pair comprises an upstream primer and a downstream primer, and the sequences are shown as SEQ ID NO. 3-4.
As a more preferable embodiment, the kit is a fluorescent quantitative PCR detection kit, and the primer is suitable for detection of SYBR Green, TaqMan probes, molecular beacons, double-hybrid probes and composite probes.
In a more preferred embodiment, the PCR reaction solution in the kit is a fluorescent quantitative PCR reaction solution, and further comprises a fluorescent dye.
In a more preferred embodiment, the fluorescent quantitative PCR reaction solution comprises dNTP and Mg2+The fluorescent dye is SYBR Green II, and the Taq enzyme is hot start enzyme.
Accelerator and pharmaceutical composition
The pharmaceutical compositions of the invention include an inhibitor of the functional expression of KIAA1949, and a pharmaceutically acceptable carrier.
First, at the DNA level, for example, by deletion or disruption OF the gene, or no transcription occurs (in both cases preventing synthesis OF the relevant gene product). a deletion OF transcription may, for example, result from epigenetic changes (e.g., DNA methylation) or from loss OF function mutations, "loss OF function" or "L OF mutations as used herein is mutations that prevent, reduce or eliminate the function OF the gene product relative to gain OF function mutations that confer enhanced or new activity.
Second, at the RNA level, for example by lack of efficient translation, for example because of instability of the mRNA (e.g. by UTR variants), can lead to degradation of the mRNA prior to translation of the transcript. Or by lack of efficient transcription, e.g. because mutations induce new splice variants.
As an alternative embodiment of the present invention, the promoter comprises a substance that improves the stability of the KIAA1949 gene or its expression product, up-regulates the expression level of the KIAA1949 gene or its expression product, increases the effective duration of action of the KIAA1949 gene or its expression product.
Typically, these enhancers will be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, typically having a pH of about 5 to about 8, preferably a pH of about 6 to about 8, although the pH will vary depending on the nature of the material being formulated and the condition being treated. The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to: intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, or topical administration.
As a preferable mode of the invention, the promoter of the KIAA1949 is an expression vector of the KIAA 1949. The expression vector usually further contains a promoter, an origin of replication, and/or a marker gene.
Methods well known to those skilled in the art can be used to construct the expression vectors required by the present invention. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The expression vector preferably comprises one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, such as kanamycin, gentamicin, hygromycin, ampicillin resistance.
In the present invention, there are various vectors known in the art, such as commercially available vectors, including plasmids, cosmids, phages, viruses, and the like. The expression vector can be introduced into the host cell by a known method such as electroporation, calcium phosphate method, liposome method, DEAE dextran method, microinjection, viral infection, lipofection, or binding to a cell membrane-permeable peptide.
In the present invention, a "host cell" cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: coli, bacterial cells of the genus streptomyces; fungal cells such as yeast; a plant cell; insect cells of Drosophila S2 or Sf 9; CHO, COS, or 293 cell.
Transformation of a host cell with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is prokaryotic, e.g., E.coli, competent cells capable of DNA uptake can be harvested after exponential growth phase using CaCl2Methods, the steps used are well known in the art. Another method is to use MgCl2. If desired, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, etc.
The invention also provides a pharmaceutical composition, which contains an effective amount of the promoter of the KIAA1949 and a pharmaceutically acceptable carrier. The compositions are useful for treating preeclamptic patients. Any of the foregoing promoters for KIAA1949 may be used in the preparation of the composition. The pharmaceutically acceptable carrier and/or adjuvant includes (but is not limited to) diluent, binder, surfactant, humectant, adsorption carrier, lubricant, filler, and disintegrant.
Wherein the diluent is lactose, sodium chloride, glucose, urea, starch, water, etc.; binders such as starch, pregelatinized starch, dextrin, maltodextrin, sucrose, acacia, gelatin, methyl cellulose, carboxymethyl cellulose, ethyl cellulose, polyvinyl alcohol, polyethylene glycol, polyvinyl pyrrolidone, alginic acid and alginates, xanthan gum, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, and the like; surfactants such as polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, glyceryl monostearate, cetyl alcohol, etc.; humectants such as glycerin, starch, etc.; adsorption carriers such as starch, lactose, bentonite, silica gel, kaolin, and bentonite, etc.; lubricants such as zinc stearate, glyceryl monostearate, polyethylene glycol, talc, calcium stearate and magnesium stearate, polyethylene glycol, boric acid powder, hydrogenated vegetable oil, sodium stearyl fumarate, polyoxyethylene monostearate, monolaurocyanate, sodium lauryl sulfate, magnesium lauryl sulfate, etc.; fillers such as mannitol (granular or powder), xylitol, sorbitol, maltose, erythrose, microcrystalline cellulose, polymeric sugar, coupling sugar, glucose, lactose, sucrose, dextrin, starch, sodium alginate, laminarin powder, agar powder, calcium carbonate, sodium bicarbonate, etc.; disintegrating agent such as crosslinked vinylpyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl methyl, crosslinked sodium carboxymethyl cellulose, soybean polysaccharide, etc.
The pharmaceutical composition of the present invention may further comprise additives such as stabilizers, bactericides, buffers, isotonizing agents, chelating agents, pH control agents, and surfactants.
The term "treatment" in the present invention refers to the medical management of a patient for the purpose of curing, ameliorating, stabilizing or preventing a disease, pathological condition or disorder. The term includes active therapy, i.e., treatment aimed specifically at ameliorating a disease, pathological condition, or disorder, and also includes causal treatment, i.e., treatment aimed at removing the cause of the associated disease, pathological condition, or disorder. Moreover, the term also includes palliative treatments, i.e., treatments designed to alleviate symptoms rather than cure a disease, pathological condition, or disorder; prophylactic treatment, i.e., treatment aimed at minimizing or partially or completely inhibiting the development of the associated disease, pathological state, or disorder; and supportive treatment, i.e. treatment for supplementing another specific therapy with the purpose of improving the associated disease, pathological state or condition.
Statistical analysis
In the specific embodiment of the present invention, the experiments were performed by repeating at least 3 times, the data of the results are expressed as mean ± standard deviation, and the statistical analysis is performed by using SPSS18.0 statistical software, and the difference between the two is considered to have statistical significance by using t test when P is less than 0.05.
The following examples are intended to illustrate the invention in further detail with reference to the accompanying drawings and examples, and are not intended to limit the scope of the invention the experimental procedures, for which specific conditions are not indicated in the examples, are generally performed according to conventional conditions, such as those described in Sambrook et al, molecular cloning, A laboratory Manual (New York: Cold Spring Harbor L aboratoryPress,1989), or according to the manufacturer's recommendations.
Example 1 screening of Gene markers associated with preeclampsia
1. Sample collection
1) Collection of serum specimens
Collecting blood of 45 normal pregnant women and untreated preeclamptic patients, standing with EDTA anticoagulant tube for 10min, centrifuging to separate serum, and storing at-20 deg.C.
2) Collection of placenta specimen
Placenta tissues of 45 cases of preeclampsia and normal pregnant women are collected, rinsed for 2 times with normal saline, dehydrated and then subpackaged in a freezing storage tube, and stored at-80 ℃ for later use.
Both groups excluded multiple pregnancy, infectious disease, chemical drug dependence, pregnant woman smoking, fetal congenital malformation and other pregnancy complications and complications, and all subjects enrolled in the study signed an informed consent before collecting specimens. All the specimens were obtained with the consent of the tissue ethics committee. 5 samples of each group were taken for gene expression profile detection and analysis, differential expression gene screening was performed, and a validation experiment was performed on all 45 samples of each group.
2. Preparation of RNA samples
The total RNA in the placenta tissue is extracted by using a tissue RNA extraction kit of QIAGEN, and the specific steps refer to the instruction.
3. Mass analysis of RNA samples
And (2) carrying out agarose gel electrophoresis on the extracted RNA, detecting the concentration and purity of the extracted RNA by using Nanodrop2000, detecting the RNA integrity by using the agarose gel electrophoresis, and determining an RIN value by using Agilent 2100. the total amount of the RNA is required to be 5 mu g by single library building, and the concentration is more than or equal to 200 ng/mu L260/280 and is between 1.8 and 2.2.
4. Removal of rRNA
Ribosomal RNA was removed from total RNA using Ribo-Zero kit.
5. Construction of cDNA library
The construction of cDNA library was carried out using Illumina Truseq RNA sample Prep Kit, the specific procedures were as described in the specification.
6. Sequencing on machine
And (3) sequencing the cDNA library by using an Illumina X-Ten sequencing platform, wherein the specific operation is carried out according to the instruction.
7. High throughput transcriptome sequencing data analysis
And (3) performing bioinformatics analysis on a sequencing result, performing RNA-seq reading positioning by using TopHat v1.3.1, standardizing the number of RNA-seq fragments by using Cufflinks v1.0.3 to calculate the relative abundance of the transcript, detecting differential expression by using cuffdiff, and considering that the gene is significantly differentially expressed when the p value is less than 0.05.
8. Results
The RNA-seq result shows that the expression level of the KIAA1949 gene in preeclampsia tissues is remarkably reduced compared with the placenta tissues of normal pregnant women.
Example 2 QPCR sequencing validation of differential expression of the KIAA1949 Gene
1. Large sample QPCR validation was performed on differential KIAA1949 gene expression.
2. RNA extraction
Extracting total RNA in placenta tissue by using a tissue RNA extraction kit of QIAGEN, extracting RNA in serum by using a blood RNA extraction kit, and referring to the instruction.
3. Reverse transcription:
1) mu.l dNTP mix 1. mu.l, 1. mu.l Oligo dT primer, 2. mu.g total RNA, RNase FreedH2O to make the total volume to 10 μ l, carrying out denaturation and annealing reaction on a PCR instrument at 65 ℃ for 5min, and placing at 4 ℃ after the reaction is finished.
2) A20. mu.l reaction system was constructed, and 5 × Primer Script Buffer 4. mu.l, RNaseINHIBITor 0.5. mu.l, Prime Script RTase 0.5. mu.l, RNase Free ddH were added2O5.0. mu.l, and carrying out reverse transcription reaction on a PCR instrument according to the following conditions: and (3) 15-30 min at 42 ℃ and 5min at 95 ℃, and placing on ice after the reaction is finished.
3) Heating in water bath at 42 deg.C for 15min, heating at 95 deg.C for 3min, and storing at-20 deg.C for use.
4. QPCR detection of the expression level of KIAA1949
1) Primer design
QPCR amplification primers were designed based on the coding sequences of the KIAA1949 gene and GAPDH gene from Genebank and were synthesized by Bomader Biotech. The specific primer sequences are as follows:
the primer sequence of housekeeping gene GAPDH is as follows:
a forward primer: 5'-CTCTGGTAAAGTGGATATTGT-3' (SEQ ID NO.1)
Reverse primer: 5'-GGTGGAATCATATTGGAACA-3' (SEQ ID NO.2)
KIAA1949 gene:
the forward primer is 5'-CAGGAACAGAGTTTGGTA-3' (SEQ ID NO. 3);
the reverse primer was 5'-TCCGAGTAGTCTTGTCTT-3' (SEQ ID NO. 4).
2) And (3) PCR reaction system: 1. mu.l each of forward and reverse primers, 10. mu.l of SYBR Green PCR master mix, 1. mu.l of cDNA, ddH2O 7μl。
3) The PCR reaction conditions are that × 40 cycles are carried out at 95 ℃ for 10min, (95 ℃ for 10s, 60 ℃ for 30s and 72 ℃ for 15s), the PCR reaction is carried out at 65-95 ℃ and the temperature rising speed is 0.5 ℃/5s on a Bio-Rad iQ5 fluorescence quantitative PCR instrument, a target band is determined through melting curve analysis and electrophoresis, and the relative quantification is carried out by a delta CT method.
5. Statistical method
And calculating the experimental results of fluorescent quantitative RT-PCR of the preeclampsia tissue and the normal placenta tissue and KIAA1949 in the blood of the preeclampsia patient and the normal pregnant woman by taking GAPDH as an internal reference, performing statistical analysis by adopting SPSS18.0 statistical software, and performing t test on the difference between the two, wherein P <0.05 has statistical difference.
6. Results
As shown in fig. 1, compared with normal pregnant women, the expression of the KIAA1949 gene in preeclamptic pregnant women is significantly reduced, the difference has statistical significance (P <0.05), the positive detection rate in placental tissues is × 100%: 43/45 × 100%: 95.56% and the positive detection rate in blood is 42/45 × 100%: 93.33%, which suggests that the detection of the expression level of KIAA1949 in blood or placental tissues can be used for the auxiliary diagnosis of preeclampsia.
Example 3 overexpression of the KIAA1949 Gene
1. Cell culture
Human early pregnancy chorionic trophoblast line (HTR-8/SVneo) was cultured in RPIM-1640 medium containing 10% fetal bovine serum at 37 ℃ in 5% CO2Cultured in an incubator. The solution was changed 1 time 2-3 days and passaged by conventional digestion with 0.25% EDTA-containing trypsin.
2. Transfection
1) Treatment of cells prior to transfection
Trophoblast HTR-8/SVneo at log phase at 1 × 105Respectively planted in six-hole plates at 37 ℃ with 5% CO2Cultured in an incubator.
2) Construction of Gene overexpression vectors
Specific PCR amplification primers were synthesized based on the sequence of KIAA1949 in GeneBank, and the sequences of the primers were as follows:
a forward primer: 5'-CCGGTTTAAACGCCACCATGGCCACCATCCCAG-3' (SEQ ID NO.5)
Reverse primer: 5'-CGGGCGGCCGCCCGCCGGCAGGACTCATCCA-3' (SEQ ID NO.6)
Two restriction enzyme sites PmeI and NotI are added to the 5 'end primer and the 3' end primer respectively. cDNA extracted and reverse transcribed from preeclamptic patients is used as an amplification template, the cDNA sequence is inserted into a eukaryotic cell expression vector pcDNA3.1 which is subjected to double enzyme digestion by restriction enzymes PmeI and NotI, and the obtained recombinant vector pcDNA3.1-1 is connected for subsequent experiments.
3) Transfection
The nerve cells were divided into 3 groups, namely, a control group (HTR-8/SVneo), a blank control group (transfected pcDNA3.1-NC) and an experimental group (transfected pcDNA3.1-1). Transfection of the vector was performed using liposome 3000, and the specific transfection method was performed as indicated in the specification. The transfection concentrations of the pcDNA3.1 empty vector and pcDNA3.1-1 were 0.5. mu.g/ml.
3. QPCR detection of the transcript level of the KIAA1949 gene
3.1 extraction of Total RNA from cells
Extracting total RNA of cells by using QIAGEN cell RNA extraction kit, wherein the detailed steps are described in the kit specification
3.2 reverse transcription procedure as in example 2.
3.3 QPCR amplification step as in example 2.
4. Statistical method
The experiments were performed in 3 replicates, the data were expressed as mean ± sd, statistically analyzed using SPSS18.0 statistical software, and the differences between the over-expressed KIAA1949 gene expression group and the control group were determined to be statistically significant when P <0.05 using a t-test.
5. Results
As shown in FIG. 2, the expression level of the KIAA1949 gene was significantly increased in the experimental group compared to the control group HTR-8/SVneo and the transfected no-load pcDNA3.1-NC.
Example 4E L ISA detection of protein expression of KIAA1949 in HTR-8/SVneo cells
The protein level of KIAA1949 in the supernatant of HTR-8/SVneo cells is determined by using a double-antibody sandwich Enzyme-labeled immunoassay (Enzyme-L keyed immunological Assay, E L ISA) analysis method, the supernatants of three groups of HTR-8/SVneo cells are collected 48h after transfection, and the concentration of KIAA1949 in the supernatant of tumor cells is quantitatively detected according to the operation flow of an E L ISA kit.
1. A standard substance with a concentration of 70000pg/ml is prepared, and after 10-fold dilution, 2-fold dilution is carried out, and 7 dilutions are carried out in total.
2. Sample adding: respectively setting a blank hole, a standard hole and a sample hole to be measured. And adding 50 mul of sample diluent into the blank hole, and respectively adding 50 mul of standard sample and sample to be detected with different concentration gradients into the rest holes. Gently shaking and mixing, covering the enzyme label plate, and reacting for 2h at 37 ℃.
3. The liquid was discarded and dried. Add 200. mu.l of VEGF-C conjugate per well. After 120min at 37 ℃, the liquid in the wells was discarded, dried and the plate was washed 3 times with PBS.
4. Add substrate solution 200. mu.l to each well in sequence, and develop color for 30min at 37 ℃ in the dark.
5. The reaction was stopped by adding 50. mu.l of stop solution to each well in sequence.
6. The optical density (OD value) of each well was measured sequentially at a wavelength of 450nm using an enzyme-linked analyzer. And (4) subtracting the OD value of the zero hole from the OD values of all the standard samples and the samples to be detected to obtain a corrected value.
7. The actual concentration of the sample is calculated.
8. As shown in FIG. 3 below, the KIAA1949 gene of HTR-8/SVneo cells was overexpressed, and the protein content of KIAA1949 was also increased, and the difference was statistically significant.
Example 5 MTT assay for HTR-8/SVneo cell proliferation Activity
1. 24h after cell transfection, 0.25% trypsin digestion, counting after medium heavy suspension, diluting cell suspension, adjusting concentration to 10 per well4/ml；
2. Inoculating 150 μ l of cells to a 96-well plate, and repeating 5 parallel wells;
3. after 1-6 days of transfection, the medium in each well was discarded, and 100. mu.l (0.5mg/ml) of MTT medium was added to continue culturing for 5 hours. Discarding MTT culture solution, adding 150 μ l DMSO into each well to dissolve MTT reducing substance formazan, shaking on a shaking table for 10min to dissolve the crystal completely, and detecting absorbance value at 490nm with enzyme-linked immunoassay instrument;
4. the cellular absorbance values were counted every day and the resulting values were plotted in a graph.
5. Results
As shown in FIG. 4, the proliferation of cells in the pcDNA3.1-1-transfected group was significantly increased compared to the control, suggesting that altering the expression level of KIAA1949 may alter the proliferative capacity of trophoblasts.
Example 7 Transwell cell in vitro invasion assay
Collecting HTR-8/SVneo cells of different groups 48h after cell transfection, and re-suspending in culture solution to make the final concentration of the cells be 106Per ml, aspirate 100. mu.l of cell suspension into a Transwell chamber. The effect of KIAA1949 gene overexpression on HTR-8/SVneo cell invasiveness was observed using the Transwell chamber method.
1. The Matrigel was thawed at 4 ℃ and an ice box (ice bath environment) was prepared. The Matrigel was diluted with RPIM-1640 and used at a final concentration of 1 mg/ml.
2. And taking out the precooled Transwell chambers, putting the precooled Transwell chambers into a 24-well plate, uniformly adding 50 mu 1 of diluted Matrigel glue to each Transwell chamber membrane, and standing the cell culture box for 3-4 hours to coagulate the glue at 37 ℃.
3. Cells were collected in logarithmic growth phase and resuspended in culture medium to a final concentration of 106Perml, gently add 100. mu.1 cell suspension into the chamber.
4. Adding 600 μ 1 culture medium containing 20% serum into 24-well plate, and culturing at 37 deg.C and 5 ℃％CO2Incubating in an incubator for 36 h.
5. Gently wiping the Matrigel glue and cells in the Transwell hole with a cotton swab, fixing the cells at the bottom of the chamber with formaldehyde, standing at room temperature for 25min, taking out the chamber and drying in the air.
6. 0.4% crystal violet is dyed for 10min, washed three times by normal saline, observed under a microscope after being dried, eight different visual field photographs are randomly selected and counted, and the results are counted and analyzed.
7. Results
As shown in FIG. 5, the cell numbers of the lower cell membranes of the experimental groups pcDNA3.1-1 increased after the HTR-8/SVneo, pcDNA3.1-NC and pcDNA3.1-1 were cultured in the transwell chamber, indicating that increasing the expression level of KIAA1949 increased the infiltration capacity of trophoblasts, suggesting that KIAA1949 may be useful in the treatment of preeclampsia.
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.
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Claims (8)
- Use of a detection reagent for KIAA1949 in the preparation of a product for diagnosing preeclampsia, wherein the reagent is a reagent for detecting the mRNA or protein expression level of KIAA 1949.
- 2. Use according to claim 1, characterized in that the product comprises: a chip, a preparation, a kit or a nucleic acid membrane strip.
- 3. The use according to claim 1, wherein the agent comprises:a probe that specifically recognizes the KIAA1949 gene; orA primer for specifically amplifying the KIAA1949 gene; orAn antibody or ligand that specifically binds to a KIAA1949 protein.
- 4. The use of claim 3, wherein the primer sequence for the specific amplification of the KIAA1949 gene is shown as SEQ ID No.3 and SEQ ID No. 4.
- Use of KIAA1949 in the manufacture of a pharmaceutical composition for the treatment of pre-eclampsia.
- 6. The use of claim 5, wherein said pharmaceutical composition comprises an enhancer of functional expression of KIAA 1949.
- 7. The use of claim 6, wherein said promoter is a carrier comprising KIAA 1949.
- Use of KIAA1949 in the screening of a pharmaceutical composition for the treatment of preeclampsia.
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