CN107338324B - Serum lncRNA marker for diagnosing recurrent abortion caused by unknown reasons, primer set, application and kit - Google Patents
Serum lncRNA marker for diagnosing recurrent abortion caused by unknown reasons, primer set, application and kit Download PDFInfo
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Abstract
The invention belongs to the field of genetic engineering, and relates to a serum lncRNA marker, a primer group, application and a kit for diagnosing recurrent spontaneous abortion of unknown reasons. The serum lncRNA markers include: and SEQ ID NO: 1, a first lncRNA having more than 90% homology with the nucleotide sequence shown in SEQ ID NO: 2, a second lncRNA having more than 90% homology with the nucleotide sequence shown in SEQ ID NO: 3, and a third lncRNA having more than 90% homology with the nucleotide sequence shown in SEQ ID NO: 4, and a fourth lncRNA having more than 90% homology with the nucleotide sequence shown in SEQ ID NO: 5 and a fifth lncRNA having more than 90% homology with the nucleotide sequence shown in SEQ ID NO: 6 has more than 90% homology with the nucleotide sequence shown in the sequence table. Serum lncRNA is a novel biomarker, and can greatly improve the sensitivity and specificity of diagnosis of recurrent abortion.
Description
Technical Field
The invention belongs to the field of genetic engineering, and particularly relates to a serum lncRNA marker for diagnosing unexplained recurrent spontaneous abortion, a primer set, application and a kit.
Background
Recurrent Spontaneous Abortion (RSA) refers to a pregnancy loss of less than 500g before 20W pregnancy or a fetus that is clinically continuous for 2 or more times, and the incidence rate of the pregnancy loss is about 1% -3% of the pregnancy outcome. The causes of RSA are extremely complex, and the currently known causes include chromosomal abnormality, genital anatomical abnormality, endocrine dyscrasia, reproductive system infection, autoimmune and environmental factors, but 50% to 60% of RSA cannot find a known cause clinically, which is called unknown recurrent abortion (URSA). Research has shown that embryonic dysplasia is closely related to URSA. As a pathological pregnancy, URSA has adverse effects on the physiology and the psychology of pregnant women, and the etiology and pathogenesis of URSA are hot spots of researches in the reproductive field at home and abroad.
Long non-coding RNA (lncRNA) generally refers to non-coding RNA transcripts greater than 200 nucleotides in length. In recent years, lncRNA regulates gene expression at multiple levels, including epigenetics, transcription, and posttranscription, and has become a focus of life science research. The results of the human genome research project indicate that only 1.5% of the nucleic acid sequences in the human genome sequence are used for protein coding, while the vast majority of non-protein coding sequences, which occupy 98.5% of the human genome, are transcribed into so-called "long non-coding RNAs". Although lncRNA lacks overall sequence homology, it contains several conserved local segments within the molecule, and these regions may interact with specific protein factors, which all suggest that lncRNA has important biological functions.
Researches show that the expression or function abnormality of lncRNA is closely related to the occurrence of human diseases, and can play a role through mechanisms such as gene imprinting, cell cycle regulation, splicing regulation, translation regulation, chromatin remodeling and mRNA degradation. Embryonic development is a complex process of an organism from its initiation of life to maturity, regulated by a range of environmental factors, genetic factors and epigenetic modifications. Numerous studies have shown that lncRNA, a key transcription element, plays an important role in embryonic development and disease development, is probably one of the earliest molecules that determine temporal and spatial expression of genes in embryonic development, and is probably involved in regulating growth and development related genes. Research reports that lncRNA plays a key role in a complex epigenetic process by regulating the opening or closing of specific genes during the development of germ cells. In the aspect of embryonic stem cells, Samir and the like find that lncRNA and a target gene thereof can regulate embryonic heart development, and prove that lncRNA also plays a certain role in the process of differentiating embryonic stem cells into myocardial cells.
Researches find that hundreds of lncRNA exist in serum, the characteristics are relatively stable, the expression quantity is rich, the specificity is higher, and the quantitative detection is easy. The expression profile of plasma lnRNA has been proved to have a certain prompting function for early diagnosis in lung cancer, colon cancer and other diseases, and the plasma lnRNA can be used as a potential biomarker of the diseases. Therefore, we reasonably believe that serum lncRNA serves as a long-chain non-coding RNA, and the differential expression profile of the long-chain non-coding RNA can also serve as a biomarker for early diagnosis of recurrent abortion.
At present, the application of lncRNA to the auxiliary diagnosis of recurrent abortion in clinic is not reported, if lncRNA related to recurrent abortion can be screened out to be used as a biomarker for early diagnosis, and a corresponding diagnosis kit is developed, the current situation of diagnosis of recurrent abortion is strongly promoted, and a new way is opened for drug screening, drug effect evaluation and targeted therapy.
Disclosure of Invention
The invention aims to provide a serum lncRNA marker, a primer set, application and a kit for diagnosing recurrent abortion of unknown causes. The kit has specificity and sensitivity for determining the expression quantity of the lncRNA marker in serum.
The invention selects lnRNAs in the serum of an abortion control through separating and detecting recurrent abortion cases and non-medical reasons, discovers that lncRNA combinations with specificity and sensitivity for evaluating the occurrence risk of recurrent abortion exist in the serum, provides a serum lncRNA marker combination of a recurrent abortion patient, and application of the serum lncRNA marker or a primer thereof in preparing a recurrent abortion auxiliary diagnostic reagent, and develops a recurrent abortion diagnostic kit convenient for clinical application.
Specifically, the first aspect of the present invention provides a set of serum lncRNA markers for diagnosing recurrent spontaneous abortion of unknown cause, said serum lncRNA markers comprising: and SEQ ID NO: 1, a first lncRNA having more than 90% homology with the nucleotide sequence shown in SEQ ID NO: 2, a second lncRNA having more than 90% homology with the nucleotide sequence shown in SEQ ID NO: 3, and a third lncRNA having more than 90% homology with the nucleotide sequence shown in SEQ ID NO: 4, and a fourth lncRNA having more than 90% homology with the nucleotide sequence shown in SEQ ID NO: 5 and a fifth lncRNA having more than 90% homology with the nucleotide sequence shown in SEQ ID NO: 6 has more than 90% homology with the nucleotide sequence shown in the sequence table.
Preferably, the serum lncRNA markers comprise: and SEQ ID NO: 1, a first lncRNA having a homology of 95% or more with the nucleotide sequence shown in SEQ ID NO: 2, and a second lncRNA having a homology of 95% or more with the nucleotide sequence shown in SEQ ID NO: 3, and a third lncRNA with homology of more than 95% and SEQ ID NO: 4, and a fourth lncRNA with homology of more than 95% with the nucleotide sequence shown in SEQ ID NO: 5 and a fifth lncRNA having more than 95% homology with the nucleotide sequence shown in SEQ ID NO: 6 has more than 95 percent of homology with the nucleotide sequence shown in the sequence table.
Further preferably, the serum lncRNA markers comprise: and SEQ ID NO: 1, a first lncRNA having homology of 96%, 97%, 98% and 99% or more with the nucleotide sequence shown in SEQ ID NO: 2, a second lncRNA having homology of 96%, 97%, 98% and 99% or more with the nucleotide sequence shown in SEQ ID NO: 3, a third lncRNA with homology of 96%, 97%, 98% and 99% or more, a nucleotide sequence shown in SEQ ID NO: 4, a fourth lncRNA with homology of 96%, 97%, 98% and 99% or more with the nucleotide sequence shown in SEQ ID NO: 5 and a fifth lncRNA with the nucleotide sequence shown in SEQ ID NO: 6 has homology of 96%, 97%, 98% and 99%.
Most preferably, the serum lncRNA markers comprise: has the sequence shown in SEQ ID NO: 1 (lnc-CHAC1-1), a first lncRNA having the nucleotide sequence set forth in SEQ ID NO: 2 (lnc-FMN1-1), a second lncRNA having the nucleotide sequence set forth in SEQ ID NO: 3 (lnc-TAX1BP1-4), a third lncRNA having the nucleotide sequence set forth in SEQ ID NO: 4 (lnc-C2CD4A-3), a fourth lncRNA having the nucleotide sequence shown in SEQ ID NO: 5 (lnc-CES1-1) and a fifth lncRNA having the nucleotide sequence shown in SEQ ID NO: 6 (lnc-ATF 3-3).
A second aspect of the present invention provides a primer set for detecting the serum incrna marker, wherein the primer set comprises:
an upstream primer and a downstream primer for detecting the first lncRNA;
an upstream primer and a downstream primer for detecting a second incRNA;
an upstream primer and a downstream primer for detecting a third lncRNA;
an upstream primer and a downstream primer for detecting a fourth lncRNA;
an upstream primer and a downstream primer for detecting a fifth lncRNA;
and the upstream primer and the downstream primer are used for detecting the sixth ncRNA.
The above-mentioned primer can be designed by a person skilled in the art according to a conventional means, and preferably,
the upstream primer for detecting the first lncRNA has the sequence shown in SEQ ID NO: 7, and the downstream primer has a nucleotide sequence shown in SEQ ID NO: 8;
the upstream primer for detecting the second incrna has the sequence shown in SEQ ID NO: 9, and the downstream primer has a nucleotide sequence shown in SEQ ID NO: 10;
the upstream primer for detecting the third lncRNA has the sequence shown in SEQ ID NO: 11, and the downstream primer has a nucleotide sequence shown in SEQ ID NO: 12;
the upstream primer for detecting the fourth lncRNA has the sequence shown in SEQ ID NO: 13, and the downstream primer has a nucleotide sequence shown in SEQ ID NO: 14;
the upstream primer for detecting the fifth lncRNA has the sequence shown in SEQ ID NO: 15, and the downstream primer has a nucleotide sequence shown in SEQ ID NO: 16;
the upstream primer for detecting the sixth lncRNA has the sequence shown in SEQ ID NO: 17, and the downstream primer has a nucleotide sequence shown in SEQ ID NO: 18.
The third aspect of the invention provides the application of the serum IncRNA marker and/or the primer group in the preparation of a reagent for diagnosing or monitoring unexplained recurrent spontaneous abortion.
The fourth aspect of the invention provides a kit for diagnosing or monitoring unexplained recurrent abortion, which comprises the primer set.
The kit may also include internal controls, as well as other reagents commonly used in the relevant PCR technology. Preferably, the kit further comprises:
(1) detecting forward and reverse primers of GAPDH; and/or
(2) DNA polymerase, PCR buffer, MgCl2At least one of dNTPs, water and a nucleic acid dye.
The invention has the advantages that:
(1) the serum lncRNA is a novel biomarker, is stable, minimally invasive and easy to detect, has accurate quantification, and can greatly improve the sensitivity and specificity of diagnosis of recurrent abortion. The successful development of the micromolecule non-coding RNA biomarker subverts the detection method of the traditional biomarker mainly based on protein, creates a brand new situation for preventing and treating recurrent abortion and provides reference for the development of other disease biomarkers.
(2) The serum lncRNA marker provided by the invention can be used as an auxiliary diagnosis marker for recurrent abortion, and early auxiliary diagnosis is carried out on recurrent abortion in a minimally invasive manner, so that a basis is provided for further deep examination of a clinician, and the effect of rapidly and accurately mastering the disease state and the disease severity of a patient is achieved. Meanwhile, support is provided for the next step of timely adopting a more personalized prevention and treatment scheme, and the disease progress is delayed and prevented.
(3) The invention adopts samples which accord with recurrent abortion cases and non-medical reasons and select abortion contrast crowd for verification, and proves that the expression quantities of the several lncRNA markers have significant difference and certain stability, so that the marker has specificity and can be applied as a recurrent abortion biomarker.
(4) The present invention employs a rigorous, multi-stage validation and evaluation system. In the first stage, a plurality of decidua tissue differential lncRNAs are screened by pre-experiment high-throughput sequencing. And in the second stage, qRT-PCR and other methods are applied to verify serum samples of research population and independent population, and a hierarchical scoring system is adopted to normalize diagnosis results. And in the third stage, blind evaluation is carried out on the serum lncRNA marker and the diagnostic kit in another independent group of people, so that the reliability of the serum lncRNA biomarker and the diagnostic kit is ensured.
Additional features and advantages of the invention will be set forth in the detailed description which follows.
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Exemplary embodiments of the present invention will be described in more detail by referring to the accompanying drawings.
FIG. 1 is a schematic diagram of Real-time PCR detection of the expression of lnc-CHAC1-1, lnc-FMN1-1, lnc-TAX1BP1-4, lnc-C2CD4A-3, lnc-CES1-1 and lnc-ATF3-3 in decidua tissue samples from recurrent abortion;
FIG. 2 is a schematic diagram of Real-time PCR detection of the expression of lnc-CHAC1-1, lnc-FMN1-1, lnc-TAX1BP1-4, lnc-C2CD4A-3, lnc-CES1-1 and lnc-ATF3-3 in a serum sample from recurrent abortion;
FIG. 3 is an analysis of the volatility of the serum lncRNA expression level of an individual; the vertical axes are the expression quantity of the lncRNA in serum;
FIG. 4 is a ROC curve between the normal control group and the group of cases of recurrent abortion.
Detailed Description
Preferred embodiments of the present invention will be described in more detail below. While the following describes preferred embodiments of the present invention, it should be understood that the present invention may be embodied in various forms and should not be limited by the embodiments set forth herein.
The test procedures used in the examples below are, unless otherwise specified, conventional in the art and are generally carried out according to the conditions and procedures recommended by the manufacturer.
Example 1: decidua tissue lncRNA screening
1 Material
1.1 study selection and sample Collection
(1) According to the diagnostic criteria: pregnant women with a pregnancy loss of less than 500g before 20 weeks of pregnancy or with a fetal weight of 2 or more are selected as a case group; according to the indexes of age, gestational week number, BMI, etc., selecting pregnant women with normal fertility and non-medical reasons for abortion (without signs and signs of abortion, no history of abortion, and at least one normal child birth) as control group by frequency matching method.
(2) The B-ultrasonic shows no embryo or fetal heart beat, and the abortion caused by monogenic genetic diseases, polygenic genetic diseases, chromosome abnormality, gene mutation and other factors needs to be eliminated.
(3) Study subject grouping:
group A: selecting a control group of abortion pregnant women for non-medical reasons (n is 63, 3 persons are subjected to sequencing screening, 30 persons are subjected to primary verification, and 30 persons are subjected to independent population verification);
group B: pregnant women with recurrent abortion of unknown origin (n is 63, 3 persons for sequencing screening, 30 persons for primary verification, and 30 persons for independent population verification).
(4) With informed consent, the samples were collected and incorporated into the serum and periostracum tissues of pregnant women in this study.
Note: BMI (Body Mass Index, BMI for short) is the ratio of weight kilograms to height meters squared, and is a standard for measuring the Body fat and thin degree and whether the Body is healthy or not.
1.2 reagents
TRIZOlTM Reagent was purchased from Invitrogen, USA. A reverse transcription kit (DRR036A) was purchased from Takara, Japan. SYBR reagents for fluorescent Real-time (Real-time) quantitative PCT were purchased from Biotech, Inc. of Nanjing Novowed. All primers were designed and synthesized by Shanghai Yingjun (Invitrogen) Bio Inc. Transcriptome sequencing and data analysis work was performed by shanghai sky biotechnology limited.
2 method
3 cases of recurrent abortion caused by unknown reasons and 3 cases of non-medical reasons are selected, decidua tissues of abortion control samples are selected, and an Illumina Hiseq 2500 high-throughput sequencing platform is adopted to complete transcript RNA sequencing (bidirectional paired end sequencing). Based on RNA extraction reagent (
Reagent) instructions for extracting decidua tissue total RNA. After the extracted RNA sample is strictly controlled, rRNA is removed, residual mRNA is enriched, and a sequencing library is constructed by a long noncoding RNA method. Performing joint filtration and pollution sequence filtration on the original data (Fastq format) obtained by sequencing, and filtering out read length (read) with low quality and length less than 35 bp. And then performing overall evaluation on the quality of the sequencing data by using FastQC software, wherein the quality comprises the mass value distribution, the read length quality, the GC content, the PCR repeated content and the like of the basic group. After quality control, the read length data is aligned (mapping) to a reference genome (hg19 version) by using TopHat software, and fragments and repeated fragments which cannot be mapped to the genome are removed, so that the FPKM value of each transcript is finally obtained. In order to ensure the accuracy of the analysis result, two methods (Cuffdiff and DESeq) are selected for lncRNA differential expression analysis, and the intersection of the two methods is taken as the final result. The differential lncRNA screening criteria were: fold Change>2 or Fold Change<0.5，FDR<0.05. Performing gene pathway annotation based on KEGG and GO databases on the screened differential genes to obtain all pathway signals in which the genes participate, calculating the significance level of each pathway by adopting Fisher test based on hyper-geometric distribution (hyper-geometric distribution), and screening significant pathway signals (FDR) embodied by the differential genes after multiple comparison and correction<0.05)。
3 results
Screening the different lncRNAs by Cuffdiff and DESeq, and taking the intersection of the two as a final result. Combining bioinformatics analysis, screening 6 candidate lncRNA related to recurrent abortion and carrying out next verification, specifically: lnc-CHAC1-1, lnc-FMN1-1, lnc-TAX1BP1-4, lnc-C2CD4A-3, lnc-CES1-1 and lnc-ATF 3-3.
Example 2: detecting the expression level of lnc-CHAC1-1, lnc-FMN1-1, lnc-TAX1BP1-4, lnc-C2CD4A-3, lnc-CES1-1 and lnc-ATF3-3 in decidua tissues
1 Material
1.1 study selection and sample Collection
Subject selection and sample collection for this example were the same as in example 1.
1.2 reagents
The reagents used for qPCR were the same as in example 1.
2 method
2.1 extraction of Total RNA
Decidua tissue of 63 normal control groups and 63 recurrent abortion cases were used as subjects.
(1) Grinding 50-100mg decidua tissue in liquid nitrogen, adding 1mL TRIzol, homogenizing with a homogenizing ultrasonic instrument, standing at room temperature (15-30 deg.C) for 5min, and completely separating nucleic acid protease. (2) 200. mu.l of chloroform was added to 1mL of TRIzol, and the mixture was vigorously shaken for 30 seconds and allowed to stand on ice for 15 to 30 min. (3) After centrifugation at 12000rpm for 15min at 4 ℃, the sample was divided into 3 layers: the bottom layer is a red organic phase, the upper layer is a colorless aqueous phase and an intermediate layer, and RNA is mainly in the aqueous phase. (4) The aqueous phase was transferred to a fresh RNase-free 1.5mL EP tube, and RNA in the aqueous phase was precipitated with an equal volume of isopropanol to the aqueous phase, and after standing at room temperature for 10min, the RNA was precipitated overnight at-20 ℃. (5) Centrifuge at 12000rpm for 15min at 4 ℃ and discard the supernatant. (6) The RNA pellet was washed with 1mL fresh 75% ethanol, centrifuged at 4 ℃ no more than 7500g for 10min, the supernatant was discarded, and the process was repeated twice. (7) The RNA precipitate was dried at room temperature to be transparent or translucent, 200. mu.l of DEPC water was added thereto, and the mixture was left at 4 ℃ for 2 to 3 hours to dissolve the RNA sufficiently, and the concentration was measured by a Nanodrop 2000. (8) The extracted RNA was stored at-70 ℃.
2.2 Synthesis of cDNA by reverse transcription
Taking pretreated RNA of decidua tissue, and passing through PrimeScriptTMThe cDNA sample was obtained by reverse transcription reaction using RT reagent Kit (Perfect Real Time) (TaKaRa RR 036A). Reverse transcription reaction time setting: the cDNA was obtained by incubation at 37 ℃ for 15 minutes and at 85 ℃ for 5 seconds. The cDNA can be used for the subsequent IncRNAs Real-time PCR detection.
The reverse transcription system was formulated as shown in the following table:
2.3 primer design
All primers were designed and synthesized by Shanghai Yingjun (Invitrogen) Bio Inc. The specific primer sequences are shown in the following table:
2.4 real-time quantitative PCR
The following system was used to determine the expression levels of lncRNAs in the samples:
the qPCR reaction was performed under the following conditions: pre-denaturation (1 cycle): 30s at 95 ℃; cycling reaction (40 cycles): 10s at 95 ℃ and 30s at 60 ℃; dissolution profile (1 cycle): 95 ℃ for 15s +60 ℃ for 1min +95 ℃ for 15 s. Detecting and comparing the change of the expression level of the lncRNA in decidua tissues of the normal control group and the recurrent abortion case group. Analyzing the specificity and the amplification efficiency of the primer, and judging the reaction specificity of the primer according to the dissolution curve. Obtaining ct value according to the amplification curve, analyzing the relative expression of the target gene by using a relative quantity method and an internal reference GAPDH, and using equation 2–△ctRelative lncRNA expression (Δ ct ═ ctlncRNA-ctGAPDH) was calculated.
3 results
According to the analysis of the results, the 6 lncRNAs, lnc-CHAC1-1, lnc-FMN1-1, lnc-TAX1BP1-4, lnc-C2CD4A-3, lnc-CES1-1 and lnc-ATF3-3, have significant differences between the two groups (as shown in FIG. 1). The lncRNAs are suggested to be possibly involved in regulating and controlling the occurrence process of recurrent abortion, and have potential possibility of being applied to auxiliary diagnosis and treatment of recurrent abortion.
Example 3: detecting the expression level of lnc-CHAC1-1, lnc-FMN1-1, lnc-TAX1BP1-4, lnc-C2CD4A-3, lnc-CES1-1 and lnc-ATF3-3 in serum
1 Material
1.1 study selection and sample Collection
Subject selection and sample collection for this example were the same as in example 1.
1.2 reagents
The reagents used for qPCR were the same as in example 1.
2 method
2.1 extraction of Total RNA
Serum samples from 63 normal control groups and 63 recurrent abortion cases were used as subjects.
(1) 5ml of fresh heparin anticoagulation blood is centrifuged for 5min at 3000rpm of a centrifuge, and the supernatant is taken and separately filled into clean 1.5ml of EP tubes, each tube being 100 mul. (2) Add 900. mu.l TRIzol to the EP tube, mix well with shaking, centrifuge at 12000rpm for 15min at 4 ℃, immediately take the supernatant and transfer to a clean 1.5ml EP tube. (3) Adding absolute ethanol with the volume of 1.5 times of the supernatant liquid phase into an EP tube, fully and uniformly mixing, transferring to a centrifugal column, centrifuging at 10000rpm and 4 ℃ for 15 seconds, and discarding the lower-layer waste liquid. (4) Mu.l of RWT buffer (Qiagen miRNeasy Mini Kit, cat # 217004) was added to the column, centrifuged at 10000rpm at 4 ℃ for 15 seconds, and the lower waste liquid was discarded. (5) Mu.l of RPE buffer (Qiagen miRNeasy Mini Kit, cat # 217004) was added to the spin column, and the column was centrifuged at 10000rpm at 4 ℃ for 15 seconds, and the lower waste liquid was discarded. This step is repeated. (6) A new 2ml tube was added to the column and centrifuged at 10000rpm for 1 minute at 4 ℃ in order to remove the RPE buffer. (7) The column was mounted in a new 1.5ml centrifuge tube while 50. mu.l DEPC treated water was added to the column and centrifuged for 1 minute. (8) The treated samples were stored at-70 ℃.
2.2 Synthesis of cDNA by reverse transcription
Taking the pre-treated serum RNA, passing through PrimeScriptTMThe cDNA sample was obtained by reverse transcription reaction using RT reagent Kit (Perfect Real Time) (TaKaRa RR 036A). Reverse transcription reaction time setting: the cDNA was obtained by incubation at 37 ℃ for 15 minutes and at 85 ℃ for 5 seconds.The cDNA can be used for the subsequent IncRNAs Real-time PCR detection.
The reverse transcription system was formulated as shown in the following table:
2.3 primer design
All primers were designed and synthesized by Shanghai Yingjun (Invitrogen) Bio Inc. The specific primer sequences are shown in table 5:
TABLE 5
2.4 real-time quantitative PCR
The following system was used to determine the expression levels of lncRNAs in the samples:
the qPCR reaction was performed under the following conditions: pre-denaturation (1 cycle): 30s at 95 ℃; cycling reaction (40 cycles): 10s at 95 ℃ and 30s at 60 ℃; dissolution profile (1 cycle): 95 ℃ for 15s +60 ℃ for 1min +95 ℃ for 15 s. Detecting and comparing the change of the lncRNA expression level in the serum of the normal control group and the recurrent abortion case group. Analyzing the specificity and the amplification efficiency of the primer, and judging the reaction specificity of the primer according to the dissolution curve. Obtaining ct value according to the amplification curve, analyzing the relative expression of the target gene by using a relative quantity method and an internal reference GAPDH, and using equation 2–△ctRelative lncRNA expression (Δ ct ═ ctlncRNA-ctGAPDH) was calculated.
3 results
According to the result analysis, the 6 lncRNAs of lnc-CHAC1-1, lnc-FMN1-1, lnc-TAX1BP1-4, lnc-C2CD4A-3, lnc-CES1-1 and lnc-ATF3-3 have significant differences between the two groups (as shown in figure 2) and are consistent with the expression trend level in decidua tissues, and the possibility of the lncRNAs applied to the recurrent abortion auxiliary diagnosis and treatment is further verified.
Example 4: stability analysis of serum lncRNA expression level of individual
1 Material
1.1 study selection and sample Collection
Subject selection and sample collection for this example were the same as in example 1. From these 6 adults (3 control groups and 3 recurrent abortion cases) were selected and serum from the subject (interval 1 week, no disease at the interval) was collected three times in succession as a sample.
1.2 reagents
The reagents used for qPCR were the same as in example 1.
2 method
The stability of 6 adult human serum lncRNA levels (3 control groups and 3 recurrent abortion cases) was evaluated using the method of example 3.
3 results
The expression levels of 6 lncRNA in serum, lnc-CHAC1-1, lnc-FMN1-1, lnc-TAX1BP1-4, lnc-C2CD4A-3, lnc-CES1-1 and lnc-ATF3-3 are stable (as shown in FIG. 3). These results suggest that the expression level of the aforementioned individual serum lncRNA is stable and has a characteristic as a diagnostic marker.
Example 5: judgment of recurrent abortion by lncRNA combination
According to the qRT-PCR method, the inventor analyzes the expression levels of lncRNAs of serum samples of a case and a control group, scores lnc-CHAC1-1, lnc-FMN1-1, lnc-TAX1BP1-4, lnc-C2CD4A-3, lnc-CES1-1 and lnc-ATF3-3 by taking a quintuple number of the expression amount of the lncRNAs of a normal control group as a threshold value, further obtains a total score, draws a ROC curve to evaluate the sensitivity and the specificity of prediction, and further evaluates the evaluation capacity of the expression levels of the 6 lncRNAs on recurrent abortion. The results of ROC analysis showed that lnc-CHAC1-1, lnc-FMN1-1, lnc-TAX1BP1-4, lnc-C2CD4A-3, lnc-CES1-1, lnc-ATF3-3 separated the normal control group and the recurrent abortion case group by an AUC (area under ROC curve) of 73.7% (as shown in fig. 4), the sensitivity at the optimal cut-off point was 0.796, and the specificity was 0.621.
The results of the above series of studies demonstrated that the use of lnc-CHAC1-1, lnc-FMN1-1, lnc-TAX1BP1-4, lnc-C2CD4A-3, lnc-CES1-1 and lnc-ATF3-3 separated the recurrent abortion cases from the normal control group well, thus demonstrating that lnc-CHAC1-1, lnc-FMN1-1, lnc-TAX1BP1-4, lnc-C2CD4A-3, lnc-CES1-1 and lnc-ATF3-3 in serum are effective and stable biomarkers for auxiliary diagnosis of recurrent abortion.
Example 6: preparation of lncRNA diagnostic kit for recurrent abortion monitoring and risk assessment
The preparation process and the operation flow of the lncRNA kit provided by the inventor are mainly based on the qRT-PCR technology. Firstly, lncRNA with difference between a control group and a recurrent abortion case group is screened out by an RNA sequencing method, and candidate lncRNA with difference in the two groups is verified by a qRT-PCR method. Combining bioinformatics, the number of the selected corresponding serum lncRNA is controlled to be 6, and the selected serum lncRNA is used as an index for monitoring recurrent abortion and evaluating risks, and the optimization is simplified on the basis of a pre-experiment.
The kit comprises a batch of serum lncRNA primers, wherein the primers of lncRNA mainly comprise forward and reverse primers of lnc-CHAC1-1, lnc-FMN1-1, lnc-TAX1BP1-4, lnc-C2CD4A-3, lnc-CES1-1, lnc-ATF3-3 and GAPDH. Meanwhile, the kit also comprises related reagents commonly used in the PCR technology, such as DEPC water, PCR buffer solution, ROX dye and the like, and corresponding commercial products can be adopted as the reagents. The kit has the value that the change trend of the serum lncRNA biomarker can be detected only by providing a small amount of blood (2ml) once, the possibility of recurrent abortion can be predicted according to the change trend, and dynamic monitoring is easy to carry out.
Having described embodiments of the present invention, the foregoing description is intended to be exemplary, not exhaustive, and not limited to the embodiments disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments.
Claims (1)
1. The application of a primer group for detecting a serum lncRNA marker in preparing a reagent for diagnosing or monitoring recurrent abortion with unknown causes is characterized in that the primer group consists of the following primers:
an upstream primer and a downstream primer for detecting the first lncRNA; the first lncRNA is lnc-CHAC 1-1;
an upstream primer and a downstream primer for detecting a second incRNA; the second lncRNA is lnc-FMN 1-1;
an upstream primer and a downstream primer for detecting a third lncRNA; the third lncRNA is lnc-TAX1BP 1-4;
an upstream primer and a downstream primer for detecting a fourth lncRNA; the fourth lncRNA is lnc-C2CD 4A-3;
an upstream primer and a downstream primer for detecting a fifth lncRNA; the fifth lncRNA is lnc-CES 1-1;
an upstream primer and a downstream primer for detecting the sixth ncRNA; the sixth lncRNA is lnc-ATF 3-3;
the upstream primer for detecting the first lncRNA has the sequence shown in SEQ ID NO: 7, and the downstream primer has a nucleotide sequence shown in SEQ ID NO: 8;
the upstream primer for detecting the second incrna has the sequence shown in SEQ ID NO: 9, and the downstream primer has a nucleotide sequence shown in SEQ ID NO: 10;
the upstream primer for detecting the third lncRNA has the sequence shown in SEQ ID NO: 11, and the downstream primer has a nucleotide sequence shown in SEQ ID NO: 12;
the upstream primer for detecting the fourth lncRNA has the sequence shown in SEQ ID NO: 13, and the downstream primer has a nucleotide sequence shown in SEQ ID NO: 14;
the upstream primer for detecting the fifth lncRNA has the sequence shown in SEQ ID NO: 15, and the downstream primer has a nucleotide sequence shown in SEQ ID NO: 16;
the upstream primer for detecting the sixth lncRNA has the sequence shown in SEQ ID NO: 17, and the downstream primer has a nucleotide sequence shown in SEQ ID NO: 18;
the method for judging recurrent abortion by using lncRNA combination comprises the following steps: and (3) detecting the expression level of the amplified fragments of the primer group by detecting the expression level of the amplified fragments, obtaining a ct value by an amplification curve, and analyzing the relative expression quantity of the target gene by a relative quantity method and internal reference GAPDH so as to detect the expression level of the amplified fragments of the primer group, wherein compared with a normal control group, the expression quantity of lnc-CHAC1-1, lnc-FMN1-1, lnc-TAX1BP1-4, lnc-C2CD4A-3 and lnc-ATF3-3 in the serum of a subject suffering from recurrent abortion is up-regulated, and the expression quantity of lnc-CES1-1 in the serum of the subject suffering from recurrent abortion is down-regulated.
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| CN107937512A (en) * | 2017-11-23 | 2018-04-20 | 南京医科大学 | For diagnosing serum microRNA markers, primer sets and application and the kit of recurrent miscarriage |
| CN108486241B (en) * | 2018-03-13 | 2022-04-12 | 南京医科大学 | Serum messenger ribonucleic acid biomarker for diagnosing recurrent abortion, primer group, application and kit |
| CN108753950B (en) * | 2018-06-12 | 2021-10-08 | 山东省医学科学院基础医学研究所 | Application of LncRNA in serum as URSA diagnosis and pregnancy outcome assessment marker |
| CN108424963B (en) * | 2018-06-12 | 2021-10-22 | 山东中医药大学 | Application of circ _0079591 in serum as URSA diagnosis and pregnancy outcome assessment marker |
| CN108707659B (en) * | 2018-06-12 | 2021-10-12 | 山东中医药大学 | Application of LncRNA in serum as URSA diagnosis and pregnancy outcome assessment marker |
| CN109554490B (en) * | 2018-12-18 | 2020-09-22 | 蚌埠医学院第一附属医院 | Microorganism related to recurrent abortion and application thereof |
| CN111826433B (en) * | 2019-04-23 | 2023-08-18 | 清华大学深圳研究生院 | Application of LncRNA in prognosis evaluation of diabetes and early warning of recurrent abortion |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101169653B1 (en) * | 2011-12-09 | 2012-08-03 | 의료법인 성광의료재단 | Method of providing information for diagnosis of idiopathic recurrent spontaneous abortion using microrna polymorphism and diagnostic composition |
| CN106319031A (en) * | 2015-06-23 | 2017-01-11 | 上海市计划生育科学研究所 | Recurrent spontaneous abortion relevant microRNA and applications thereof |
-
2017
- 2017-09-08 CN CN201710806212.2A patent/CN107338324B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101169653B1 (en) * | 2011-12-09 | 2012-08-03 | 의료법인 성광의료재단 | Method of providing information for diagnosis of idiopathic recurrent spontaneous abortion using microrna polymorphism and diagnostic composition |
| CN106319031A (en) * | 2015-06-23 | 2017-01-11 | 上海市计划生育科学研究所 | Recurrent spontaneous abortion relevant microRNA and applications thereof |
Non-Patent Citations (2)
| Title |
|---|
| Differential expression profile of long noncoding RNAs in human chorionic villi of early recurrent miscarriage;Leilei Wang et al.;《Clinica Chimica Acta》;20170131;第464卷;摘要，第18页左栏第3段-右栏第3段，第20页右栏第2段，表2 * |
| Polymorphisms in lncRNA HOTAIR and susceptibility to breast cancer in a Chinese population;RuiYan et al.;《Cancer Epidemiology》;20151231;第39卷(第6期);第978-985页 * |
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