CN108753950B - Application of LncRNA in serum as URSA diagnosis and pregnancy outcome assessment marker - Google Patents
Application of LncRNA in serum as URSA diagnosis and pregnancy outcome assessment marker Download PDFInfo
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Abstract
The invention discloses application of LncRNA NONHSAT167899.1 in serum as a URSA diagnosis and pregnancy outcome assessment marker, and LncRNA NONHSAT167899.1 as the URSA diagnosis and pregnancy outcome assessment marker, and develops a corresponding detection kit.
Description
Technical Field
The invention belongs to the field of biomedicine, and particularly relates to application of LncRNA NONHSAT167899.1 in serum as a marker for diagnosis of Unexplained Recurrent Spontaneous Abortion (URSA) and pregnancy outcome assessment.
Background
Spontaneous abortion occurs 2 or more times continuously and is called recurrent abortion (RSA), the incidence rate is about 1% -5% of women of childbearing age, the etiology is complex, except for few chromosome, endocrine, anatomical, infection and other factors, about 80% of etiology is unknown, and the abortion is called unexplained recurrent abortion (URSA). URSA seriously harms reproductive health of women of childbearing age, but biological indexes with strong specificity, high sensitivity and good stability are not available at present, and no clear and uniform standard exists in clinical intervention treatment, so that the pregnancy outcome cannot be effectively predicted.
With the development of deep sequencing technologies and the completion of multiple genome projects, large amounts of non-coding RNA have been identified, and more research has found that what was once referred to as "dark material" or "junk RNA" in the genome because it was unable to encode proteins plays an important role in many life activities. Epigenetic study of non-coding RNA is one of the hottest biological subjects in recent years, and the knowledge of non-coding RNA is continuously refreshed by the results of infinite studies. The LncRNA is an RNA having a length of more than 200 nucleotides, does not have an open reading frame for encoding a protein, is encoded by RNA polymerase II, is spliced, and is polyadenylated in many cases. LncRNA can be used for carrying out mechanisms such as posttranscriptional regulation or posttranslational modification on target gene mRNAs, plays a role in epigenetics regulation, has important function in cell growth and development, and participates in the occurrence and development of diseases. In 2014, Wang et al detected LncRNA in abortive tissue genome by using a human long-chain non-coding RNA (LncRNA) array, and found that infection and inflammation pathways regulated by LncRNA are main potential causes of spontaneous abortion. At present, LncRNAs are reported to be applied to the auxiliary diagnosis and treatment of recurrent abortion, but the diagnosis efficiency is low. For example, Luchneicheng, fang 36191, et al, studied a panel of serum LncRNA markers for diagnosis of recurrent abortion of unknown origin, showed that 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 from the recurrent abortion cases by 73.3% AUC.
Therefore, there is a need to further search for more effective long-chain non-coding RNA as a marker for clinical diagnosis, treatment and prognosis detection of URSA, which provides a basis for diagnosing and treating URSA and developing related drugs.
Disclosure of Invention
In view of the above prior art, the inventors have conducted extensive technical studies and long-term clinical practice to provide a product related to diagnosis of URSA and/or evaluation of pregnancy outcome of patients with URSA, and to provide the use of LncRNA NONHSAT167899.1 as a marker for diagnosis of URSA and evaluation of pregnancy outcome.
In a first aspect of the invention, there is provided the use of LncRNA non hsat167899.1 as a marker for diagnosing URSA and/or assessing pregnancy outcome of a patient with URSA in the manufacture of a product for diagnosing URSA and/or for assessing pregnancy outcome of a patient with URSA.
Wherein the nucleic acid sequence of the LncRNA NONHSAT167899.1 is as follows:
AAAATATTATCACTTTTAGTCCTTATGAAGTATCTTCTAGTGGATGTATTATTTTTTTCTTACTCAGTTAAGTTAAAAAATAATTTTCAGAGAATTTAATGAGAACCATCTCTTCAATTGCTGTCTTAAAATATTATTCAGCCTTGAGGCAGTTGGTGGTGTGGAGAGAAGGTTGAATGATGTGGTTTTGTTTGTTGTCTGCCTATGTGATGCCAACACCAGATATGGCCATTTTTTCACCACATTCTCAATTATAGGTATAGGCAGGTTACTAACTCATTGTATAAAACAAGATCTCTTTCAACTTATTTAGCTTATTTCGATATGTCAGTTTTTCTGAATACCTCAGGTGGTATTTGAGCACCAGGGATTAATAGGCTGCTCCAAGATCTCATTGGCTTGCAGGAAACTTACTGAGTGTCTAAAGCACGTAACACACTATCCACTGTGTATTTCCATTTAAACATTTTACACATTTTATCTCCCTGGAATTCTTTTCACCTGTAATGTGGCTCAAATCTCCTCTACACTCTCAAAACTCCTAAACTAATGGGTGAATTGCTTAAATTTCAACCCACTCAGGGATCTACCAATATTGAAGTCTTTATATTTCTCCTTATGATTTTCTAGCTCCATATACATAGAGAAGAATAAGCAGATTTGTAAAGTTTAGGTTGCATTGTATCTATCAGATAAATATGTCTTGTGTTTCCCTCAACCTTGCTATCCCTCCAAAAAAAAAAAAAAAAAAAAAAAAGAAAAGGCATAGTTTTTACTTCTTAAATGTCTATTGTCTGAGGAAGGTCAGTCTTTATAATCTAGCCATCTGAATGCAGTAACTATCAAGAAACAGAAAAATCACATAATCTTTTAAAATATCTTCATACTAGACTTTACGTTACACTGAATCTTTCAATTTGACTTCTGC
further, the LncRNA NONHSAT167899.1 is LncRNAONHSAT167899.1 in serum samples.
In a second aspect of the present invention, there is provided the use of a kit or gene chip for detecting LncRNA non hsat167899.1 in the preparation of a product for diagnosing URSA and/or for assessing pregnancy outcome of a patient with URSA.
Further, the kit comprises at least a forward primer 5'-GCACGTAACACACTATCCACTG-3' and a reverse primer 5'-TGGTAGATCCCTGAGTGGGTT-3' against LncRNA nonahsat 167899.1.
Further, the gene chip comprises at least a probe hybridizing with the nucleic acid sequence of LncRNA NONHSAT 167899.1.
Further, the product can diagnose whether the patient has URSA by detecting the expression level of LncRNA NONHSAT167899.1 in serum, and the low expression of LncRNA NONHSAT167899.1 is related to the occurrence, development and pregnancy outcome of URSA.
Further, the product for detecting the expression level of LncRNA non hsat167899.1 comprises: detecting the expression level of LncRNA NONHSAT167899.1 by RT-PCR, real-time quantitative PCR, in situ hybridization, gene chip or gene sequencing to diagnose URSA and/or to evaluate products of pregnancy outcome of URSA patients.
In a third aspect of the invention, there is provided a product for diagnosing URSA and/or assessing pregnancy outcome in a patient with URSA, the product being characterised by: the product can diagnose URSA and/or evaluate pregnancy outcome of URSA patients by detecting the expression level of LncRNA NONHSAT167899.1 in serum, and high-throughput detection results show that LncRNAONHSAT167899.1 has obviously reduced expression in the URSA group compared with normal pregnant women.
Further, the product is a chip or a detection kit; wherein the chip comprises at least a probe hybridizing with the nucleic acid sequence of LncRNAnHSAT167899.1.
Further, the detection kit comprises a reagent for preparing a reverse transcription reaction system and a reagent for preparing a qPCR reaction system.
In a fourth aspect of the invention, there is provided the use of LncRNA non hsat167899.1 in the manufacture of a medicament for the treatment of URSA.
Further, the drug is an agonist of LncRNA non sat 167899.1.
Compared with the prior art, the technical scheme of the invention has the beneficial effects that:
(1) the LncRNAnHSAT167899.1 obtained by screening through high-throughput chip and large clinical sample expression verification can be used as a marker for diagnosing URSA, and the diagnosis efficiency is up to 93.30%.
(2) The LncRNA NONHSAT167899.1 obtained by screening through a high-throughput chip can be used as a marker for pregnancy outcome assessment of URSA patients, and the diagnosis efficiency is up to 91.30%.
(3) The invention provides a basis for developing a medicament for improving the expression level of LncRNA NONHSAT167899.1 in the future.
(4) The invention takes LncRNA NONHSAT167899.1 as a marker for diagnosing URSA and pregnancy outcome assessment, develops a corresponding detection kit, has high detection sensitivity, high specificity and convenient detection, meets the detection requirement of diagnosing URSA patients, and has high diagnosis accuracy through clinical verification.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
FIG. 1: and (3) screening the difference LncRNA between the peripheral blood of the URSA patient and that of a normal pregnant woman.
FIG. 2: the expression of the peripheral blood LncRNA NONHSAT167899.1 of the URSA patients and normal pregnant women is verified.
FIG. 3: the expression of the LncRNA NONHSAT167899.1 in the peripheral blood of the pregnancy failure group and the pregnancy success group in the URSA patients is verified.
FIG. 4: ROC Curve graph of URSA patient and normal pregnant woman, A is LncRNA NONHSAT167899.1ROC graph, B is progestational hormone ROC graph.
FIG. 5: ROC Curve graphs of a pregnancy failure group and a pregnancy success group in URSA patients, wherein A is an LncRNA NONHSAT167899.1ROC graph, and B is a progestogen ROC graph.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of the stated features, steps, operations, and/or combinations thereof, unless the context clearly indicates otherwise.
Interpretation of terms:
LncRNA non hsat 167899.1: the long-chain non-coding ribonucleic acid LncRNA NONHSAT167899.1 has a nucleic acid sequence shown as SEQ ID NO. 1.
As introduced by the background technology, in the prior art, the diagnosis of URSA and the pregnancy outcome assessment by using progestogen and the like have certain defects, and in order to solve the technical problems, LncRNAONHSAT167899.1 obtained by screening through a high-throughput chip can be used as a URSA diagnosis and pregnancy outcome assessment marker.
In another embodiment of the present invention, the LncRNA NONHSAT167899.1 is LncRNA NONHSAT167899.1 in a serum sample.
In a specific embodiment of the present invention, the related specific technical solution includes:
(1) venous blood from URSA patients and normal pregnant women (6 per group) was collected, mononuclear cells were isolated, and differential LncRNA was screened using a high throughput chip.
(2) Sample size was expanded (60 each per group) and fluorescent real-time quantitative pcr (qpcr) was used to verify screening for differential LncRNA expression.
(3) Analysis of clinical data (age, sex).
(4) Logistic regression model analysis.
(5) ROC curve analysis examines the efficacy and optimal cutoff value.
In an exemplary embodiment of the present invention, there is provided a kit or a gene chip for detecting LncRNA non hssat 167899.1 for use in the preparation of a product for diagnosing URSA and/or evaluating pregnancy outcome of a patient with URSA.
In one embodiment of the invention, the kit comprises at least a forward primer 5'-GCACGTAACACACTATCCACTG-3' and a reverse primer 5'-TGGTAGATCCCTGAGTGGGTT-3' directed against LncRNA non sat 167899.1.
In one embodiment of the present invention, the gene chip comprises at least a probe hybridizing to a nucleic acid sequence of LncRNA non sat 167899.1.
In one embodiment of the invention, the product can diagnose whether the patient has URSA by detecting the expression level of LncRNA NONHSAT167899.1 in serum, and the low expression of LncRNA NONHSAT167899.1 is related to the occurrence, development and pregnancy outcome of URSA.
In another embodiment of the present invention, the product for measuring the expression level of LncRNA non hsat167899.1 in serum comprises: detecting the expression level of LncRNA NONHSAT167899.1 by RT-PCR, real-time quantitative PCR, in situ hybridization, gene chip or gene sequencing to diagnose URSA and/or to evaluate products of pregnancy outcome of URSA patients.
Wherein the gene sequencing is capable of detecting a relative change in the level of gene expression, such as Illumina sequencing. The Illumina platform is a Sequencing method based on Sequencing-By-Synthesis (SBS) technology. The reversible blocking technology can realize that only one base is synthesized each time, the fluorescent group is marked, the corresponding laser is used for exciting the fluorescent group, and the exciting light is captured, so that the base information is read. The original image Data file obtained by high-throughput sequencing is analyzed and converted into an original sequencing sequence through Base recognition (Base Calling), the sequence is called Raw Data or Raw Reads, and the result is stored in a FASTQ (fq for short) file format. Sequencing the clear Reads with the designated reference genome using hisat2 to obtain the position information on the reference genome or genes and the sequence characteristic information specific to the sequenced sample. The expression level of a gene is directly reflected by the abundance of the transcript, and the higher the abundance degree of the transcript, the higher the expression level of the gene. In transcriptome sequencing analysis, the expression level of a gene can be estimated by counting the number of sequencing sequences (reads) that map to exon regions of the transcript. The transcript expression was calculated using FPKM (Fragments Per kb Per Million Reads) as the number of Fragments Per kilobase Per Million Fragments from a particular transcript. FPKM considers the influence of sequencing depth and transcript length on fragments counting, and is the most common method for estimating the expression level of the transcripts at present. The FPKM calculation formula is as follows:
in an exemplary embodiment of the invention, there is provided a product for diagnosing URSA and/or assessing pregnancy outcome in a patient with URSA, the product being characterized by: the product can diagnose URSA and/or evaluate pregnancy outcome of URSA patients by detecting the expression level of LncRNA NONHSAT167899.1 in serum, and high-throughput detection results show that the expression of LncRNA NONHSAT167899.1 in the URSA group is obviously reduced compared with that of normal pregnant women.
In one embodiment of the invention, the product is a chip or a test kit; wherein the chip comprises at least a probe hybridizing to the nucleic acid sequence of LncRNA NONHSAT 167899.1.
In one embodiment of the present invention, for the detection kit, the detection system comprises a reverse transcription reaction system and a qPCR reaction system, and the detection kit comprises reagents for preparing the reverse transcription reaction system and reagents for preparing the qPCR reaction system.
In one embodiment of the present invention, the reagents used for preparing the reverse transcription reaction system include at least reverse transcription buffer (MLV-5 XBuffer), dNTP mix, RNAse protein inhibitor (RNAsin), reverse transcriptase mix (M-MLV) and polythymidine (OligodT).
In one embodiment of the invention, the reagents used for preparing the qPCR reaction system comprise at least a forward primer solution and a reverse primer solution against LncRNA nonahsat 167899.1, a SYBR Green mixed solution, and nuclease-free pure water.
In an exemplary embodiment of the invention, there is provided the use of LncRNA non hsat167899.1 in the manufacture of a medicament for the treatment of URSA.
In one embodiment of the invention, the drug is an agonist of LncRNA non sat167899.1, and the agonist of LncRNA non sat167899.1 is a product capable of increasing the expression level of LncRNA non sat167899.1, and the product comprises: an LncRNA NONHSAT167899.1 overexpression vector, an LncRNA NONHSAT167899.1 transcription activation type Cas9-VP64-sgRNA co-expression vector, a compound, a composition or a reagent for improving the expression level of LncRNA NONHSAT167899.1 and the like. In which, Lnc RNA overexpression vectors (e.g., lentiviral expression vectors, adenoviral expression vectors) and transcription-activated Cas9-VP64-sgRNA co-expression vectors have been commercialized, and can also be prepared by conventional technical means.
In order to make the technical solutions of the present invention more clearly understood by those skilled in the art, the technical solutions of the present invention will be described in detail below with reference to specific embodiments.
In the following examples, the reagents used were all analytical grade and were commercially available unless otherwise indicated. Experimental procedures not specifically identified herein are generally carried out under conventional conditions such as those described in the molecular cloning guidelines published by scientific Press, J. SammBruk et al, or under conditions recommended by the manufacturer. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention.
1. Subject inclusion and exclusion criteria:
(one) source of cases
All cases are from 60 cases of stay in the gynecologic hospital and outpatient service of the Shandong Chinese medicine university affiliated hospital in 2016 (01) to 2017 (06). The blood samples of the normal control group are all from normal pregnancy physical examination persons in subsidiary hospitals of Shandong Chinese medicine university, and are diseases without heart, brain, lung, liver and kidney and thrombotic diseases, and diseases without known influence on research indexes, and the total number of the blood samples is 60.
(II) inclusion standard:
(1) URSA patient inclusion criteria:
the patient has 2 or more than 2 times of spontaneous abortion history and no history of live birth;
the chromosomes of both couples and (or) the embryo are normal, and the family genetic disease and the close marriage history are not available;
the gynecological examination of leucorrhea, ultrasonic examination and/or hysterosalpingography and the like eliminates organic lesions, anatomical malformation of reproductive organs and infectious factors of patients;
fourthly, the menstrual cycle is normal, the basal body temperature is two-phase, and the normal ovulation is monitored by ultrasonic;
analyzing the sperm of the male;
sixthly, endocrine tests such as sex hormone, thyroid function, blood sugar, insulin and the like are normal;
seventhly, the examination of autoantibodies such as antinuclear antibody, anticardiolipin antibody, antithyroid antibody and anti-2-glycoprotein I antibody is negative;
examination related to the state before thrombus, including that D-dimer, fibrin (protogen) degradation products, blood coagulation series and whole blood analysis are all normal;
ninthly, negative IgM series;
active immunotherapy has not been performed in r.
(2) Normal controls were included as standards:
the normal pregnancy physical examination women within 12 weeks of gestation have no history of spontaneous abortion, stillbirth and stillbirth, heredity, anatomy and endocrine abnormality, and history of infection and autoimmune diseases; during the pregnancy, there are no symptoms and signs of threatened abortion such as vaginal bleeding and abdominal pain; the embryo development is normal and the heart tube beats as proved by ultrasound.
2. And (3) high-flux chip detection:
peripheral blood from URSA and normal pregnant women was collected, total RNA quantified using NanoDrop ND-2000(Thermo Scientific) and RNA integrity tested by Agilent Bioanalyzer 2100(Agilent Technologies). After the RNA quality is qualified, the total RNA is reversely transcribed into double-strand cDNA, and cRNA labeled by Cyanine-3-CTP (Cy3) is further synthesized. The labeled cRNA was hybridized to an Agilent Human lncRNA Microrray V6 (4X 180K, Design ID:084410) chip, which was eluted and scanned with Agilent Scanner G2505C (Agilent Technologies) to obtain the original image.
Raw data were extracted by processing raw images using Feature Extraction software (version10.7.1.1, Agilent Technologies). Subsequently, the quantile standardization and subsequent processing were carried out using Genespring software (version 13.1, Agilent Technologies). The normalized data is filtered and at least one 100% of the probes labeled "P" from each set of samples used for comparison are left for subsequent analysis. And performing difference circRNA and LncRNA by using the P value and the fold change value of the T test, wherein the screening standard is that the fold change value is more than or equal to 2.0 under the up-regulation or down-regulation and the P value is less than or equal to 0.05.
qPCR confirmed differential LncRNA expression:
(1) cell extraction of RNA:
take 5X 106～1×107Adding 1ml of Trizon into each cell, fully and uniformly mixing, and standing at room temperature for 5-10 min; adding 200 mul/1 ml Trizon chloroform, covering the EP tube tightly and shaking vigorously for 15 s; centrifugation at 4 ℃: 12000rpm is multiplied by 10min, and the upper aqueous phase is taken in a new EP tube; adding isopropanol with the same volume, gently inverting and mixing, and standing at room temperature for 10 min; centrifugation at 4 ℃: 12000rpm is multiplied by 10 min; discarding the supernatant, adding 1ml of 75 (v/v)% ethanol (anhydrous ethanol: DNase/RDase-free water: 3:1), and gently mixing; centrifugation at 4 ℃: 12000rpm is multiplied by 5 min; discarding the supernatant, and adding 1ml of absolute ethyl alcohol; centrifuging at 4 ℃ and at 12000rpm for 5 min; discarding the supernatant (removing residual liquid as much as possible), and drying at room temperature or in vacuum for 10-20 min; adding appropriate amount of DNase/RDase-free water (usually 30-50 μ l) according to the amount of RNA precipitation to dissolve RNA; after mixing, the concentration was measured and recorded for reverse transcription.
(2) RT assay
Taking 0.2ml of EP tube to mark the name and date of the sample, and adding RNA, DNase/RDase-free water and OligodT into the marked EP tube according to requirements; running RT-1 program at 70 deg.C for 5min (Table 1); MIX of MLV-5 XBuffer, dNTP, RNAsin and M-MLV prepared according to the system is added into the EP tube, an RT-2 program is operated, the temperature is 42 ℃, 1h is carried out, cDNA is obtained, and a PCR experiment is carried out (table 2).
TABLE 1 RNA and OligodT
| Volume of the system | 20μl |
| RNA | 11μl |
| DNase/RDase-free water | 11-V1 |
| OligodT | 1μl |
| Volume of RNA used V1 | 2000ng/C1 |
TABLE 2 reverse transcription reaction System
| Volume of the system | 20μl |
| MLV-5×buffer | 4μl |
| dNTP | 2μl |
| RNAsin | 1μl |
| M-MLV | 1μl |
| RNA+OligodT | 11μl |
(3) qPCR experiments: dissolving SYBR, the primers and the cDNA, performing instantaneous centrifugation, mixing uniformly, and placing on ice; DNase/RDase-free water.
TABLE 3 PCR reaction system (20. mu.l)
| Reaction system | 20μl |
| Primer F (10 μm) | 1.2μl |
| Primer R (10 μm) | 1.2μl |
| SYBR | 10μl |
| cDNA | 2μl |
| DNase/RDase-free water | 5.6μl |
Instantaneous centrifugation; the 7500 program was run and conditions were set as in the table below.
TABLE 4 PCR procedure
TABLE 5 LncRNA NONHSAT167899.1 primer sequences
4. Statistical analysis:
SPSS22.0 software (SPSS inc., USA) was used. The continuous variable adopts Median (Median) and mean value + -standard deviation
Represents; the measurement data is tested by t, and the count data is tested by χ2And (6) checking. Diagnostic ability was judged by plotting Receiver Operating Characteristic (ROC) curves and calculating the corresponding area under the curve (AUC). The optimal cutoff value is selected as the value corresponding to the maximum sum of sensitivity and specificity. The area under the curve AUC variability was compared using the medcale10.4.7.0 software. P<0.05 (double-sided) is statistically different. And (4) analyzing the testing efficiency and the sample size by adopting R software, wherein the testing efficiency is that R is more than or equal to 0.8.
Results
1. High throughput assay results
The high throughput results showed that circ _0079591, circ _0082660, LncRNA non sat167899.1, LncRNA ENST00000514235 expressed significantly different in the URSA group compared to the normal pregnancy control group, wherein, as shown in fig. 1, LncRNA non sat167899.1 expressed significantly lower in the URSA group compared to the normal pregnancy control group, P < 0.05. The applicant has already protected as other patent applications with respect to the specific solutions of circ _0082660, circ _0079591 and LncRNA ENST 00000514235.
qPCR verification of LncRNA NONHSAT167899.1 expression
As shown in fig. 2, expression of LncRNA nonahsat 167899.1 was significantly reduced in the URSA group compared to the normal control group (P < 0.05); as shown in fig. 3, LncRNA non hsat167899.1 showed a significant decrease in expression in the pregnancy failure group compared to the pregnancy success group with P <0.05 in the URSA patients.
3. Analysis of clinical data between groups
(1) Age: the age comparison between groups had no statistical significance (P >0.05) and were comparable (see Table 6, Table 7).
TABLE 6 age comparison between Normal and URSA groups
Note: the age between groups was tested for t, P > 0.05.
TABLE 7 comparison of age between pregnancy success and failure groups in URSA
Note: the age between groups was tested for t, P > 0.05.
Logistic regression analysis: the Logistic regression analysis of the normal pregnancy group and the URSA group is shown in Table 8, and the Logistic regression analysis of the pregnancy success group and the pregnancy failure group in URSA is shown in Table 9.
TABLE 8 Logistic regression analysis between normal pregnancy group and URSA group
the results of the single-factor Logistic regression model constructed by taking LncRNA NONHSAT167899.1 as an independent variable and the multi-factor Logistic regression model constructed after age is corrected at the same time show that the statistical results obtained by correction and non-correction are consistent, so that the LncRNA NONHSAT167899.1 is determined to have the potential of URSA diagnosis.
TABLE 9 Logistic regression analysis of pregnancy success and failure groups in URSA
the results of the single-factor Logistic regression model constructed by taking LncRNA NONHSAT167899.1 as an independent variable and the multi-factor Logistic regression model constructed after age correction show that the statistical results obtained by correction and non-correction are consistent, so that the LncRNA NONHSAT167899.1 is determined to have the potential of predicting URSA pregnancy outcome.
ROC curve analysis test efficacy and optimal cutoff value:
the results are shown in fig. 4, where the area under the LncRNA NONHSAT167899.1ROC curve is 0.933, higher than that of the progestin (0.703), p < 0.05. The relative expression level of LncRNA NONHSAT167899.1 is less than 1.85, and URSA is diagnosed; relative expression higher than 1.85, not diagnosed as URSA; the diagnostic efficiency was 93.30%.
The results are shown in fig. 5, where the area under the LncRNA NONHSAT167899.1ROC curve is 0.913, higher than the progestin (0.838) with p < 0.05. The relative expression level of LncRNA NONHSAT167899.1 is lower than 1.863, and the prognosis of URSA patients is evaluated as pregnancy failure; relative expression is higher than 1.863, and the prognosis of URSA patient is evaluated as success of pregnancy; the prognostic evaluation efficiency was 91.30%.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
SEQUENCE LISTING
<110> institute of basic medicine of Shandong province academy of medical sciences
<120> application of LncRNA in serum as URSA diagnosis and pregnancy outcome assessment marker
<130> 2018
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 928
<212> DNA
<213> LncRNA NONHSAT167899.1 sequence
<400> 1
aaaatattat cacttttagt ccttatgaag tatcttctag tggatgtatt atttttttct 60
tactcagtta agttaaaaaa taattttcag agaatttaat gagaaccatc tcttcaattg 120
ctgtcttaaa atattattca gccttgaggc agttggtggt gtggagagaa ggttgaatga 180
tgtggttttg tttgttgtct gcctatgtga tgccaacacc agatatggcc attttttcac 240
cacattctca attataggta taggcaggtt actaactcat tgtataaaac aagatctctt 300
tcaacttatt tagcttattt cgatatgtca gtttttctga atacctcagg tggtatttga 360
gcaccaggga ttaataggct gctccaagat ctcattggct tgcaggaaac ttactgagtg 420
tctaaagcac gtaacacact atccactgtg tatttccatt taaacatttt acacatttta 480
tctccctgga attcttttca cctgtaatgt ggctcaaatc tcctctacac tctcaaaact 540
cctaaactaa tgggtgaatt gcttaaattt caacccactc agggatctac caatattgaa 600
gtctttatat ttctccttat gattttctag ctccatatac atagagaaga ataagcagat 660
ttgtaaagtt taggttgcat tgtatctatc agataaatat gtcttgtgtt tccctcaacc 720
ttgctatccc tccaaaaaaa aaaaaaaaaa aaaaaaagaa aaggcatagt ttttacttct 780
taaatgtcta ttgtctgagg aaggtcagtc tttataatct agccatctga atgcagtaac 840
tatcaagaaa cagaaaaatc acataatctt ttaaaatatc ttcatactag actttacgtt 900
acactgaatc tttcaatttg acttctgc 928
<210> 2
<211> 22
<212> DNA
<213> Artificial sequence
<400> 2
gcacgtaaca cactatccac tg 22
<210> 3
<211> 21
<212> DNA
<213> Artificial sequence
<400> 3
tggtagatcc ctgagtgggt t 21
Claims (2)
1. Use of a primer for detecting expression level of LncRNA non hsat167899.1 in the preparation of a product for diagnosing URSA and/or evaluating pregnancy outcome of a patient with URSA;
the nucleic acid sequence of the LncRNA NONHSAT167899.1 is shown as SEQ ID NO. 1.
2. Use according to claim 1, characterized in that: the primer for detecting the expression level of LncRNA NONHSAT167899.1 consists of a forward primer 5'-GCACGTAACACACTATCCACTG-3' and a reverse primer 5'-TGGTAGATCCCTGAGTGGGTT-3'.
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Citations (1)
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| CN107338324A (en) * | 2017-09-08 | 2017-11-10 | 南京医科大学 | For the serum lncRNA marks of acatalepsia reason recurrent miscarriage, primer sets and application and kit |
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| CN107338324A (en) * | 2017-09-08 | 2017-11-10 | 南京医科大学 | For the serum lncRNA marks of acatalepsia reason recurrent miscarriage, primer sets and application and kit |
Non-Patent Citations (2)
| Title |
|---|
| NONHSAT167899.1;anonymity;《NONCODE数据库》;20140813;第1页 * |
| 长链非编码RNA在妊娠相关疾病中的研究进展;贾文通等;《中华围产医学杂志》;20171031;第20卷(第10期);第700-704页 * |
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