CN107400728A - Applications of the miR 155 as molecular marked compound in preeclampsia is diagnosed - Google Patents
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Abstract
The invention discloses a kind of applications of miR 155 as molecular marked compound in preeclampsia is diagnosed, miR 155 nucleotide sequence is as shown in SEQ.ID.NO.1.The present invention is found through experiments that in PE patients serums that miR 155 expression is increased with normal pregnant than more significant, PE patients serums miRNA and urine miRNA ratio relatively dramatically increases with normal pregnancy group, and make that ROC charts are bright, and miR 155 has medium diagnostic value to preeclampsia.Therefore, by circulating miR 155 measure to patient peripheral, and its clinicopathological characteristics is combined, reliable an index and foundation is provided for the diagnosis, treatment and condition assessment of preeclampsia.
Description
Technical field
The invention belongs to biomedicine technical field, and in particular to a kind of miR-155 is as molecular marked compound in diagnosis
Application in epilepsy early stage.
Background technology
Preeclampsia (Preeclampsia, PE) is the common complication of obstetrics, and the disease is to cause pregnant and lying-in women and perinatal feruses
One of ill and main causes of death.2006, the data that WHO is announced were shown, in developing country, because PE or eclampsia cause
Death account for the 15%-20% of maternal death.According to state of an illness weight, PE points are slight and severe；According to disease time sooner or later
It is divided into Early onset and late hair style.
PE Pathological Physiology can be divided into two stages：The shallow implantation of first stage fine hair causes placental perfusion to reduce；Second
The extensive activation and damage of stage pregnant woman's system vascular endothelial cell, cause multiple organ dysfunction.At present, to the two stages
Pathogenesis be still not clear.There is scholar to think, oxidative stress (oxidative stress, OS) be most likely to be connection this two
The primary factor in individual stage.Because other factors can either promote OS or can be promoted by OS.In serum of women during normal pregnancy
In, level of lipid gradually rises with the increase of pregnant week, and the antioxidation of placenta tissue is also with pregnant week
Increase and gradually strengthen, normal pregnancy remains the balance of oxidative and anti-oxidative.But research finds PE patient but because in vivo
, there is OS in the dysequilibrium of oxidative and anti-oxidative.Due to whole body arteriolar spasm, ischemia-reperfusion disease occurs PE patient for internal organs
Reason changes, so as to produce substantial amounts of ROS, such as oxygen radical, hydrogen peroxide, these materials and lipid, protein, DNA in vivo
With reference to and peroxidization occurs, destroy cell tissue structure, so as to causing cell function decline, apoptosis and necrosis.
Diagnosis of the clinician for PE at present relies primarily on blood pressure and Urine proteins.Twenty-four-hour urine protein quantification is as evaluation
The important evidence of disease severity, but because PE progression of disease is rapid, and twenty-four-hour urine protein quantification generally requires 1-2 days
It can just clarify a diagnosis, so the state of an illness of PE patient can not be reflected in time as diagnosis basis from twenty-four-hour urine protein quantification
Progress.Simultaneously as PE pathogenesis is indefinite, effective predictive index there is no to be applied to clinic at present, pregnant woman occurs high
The terminal stage of a disease is often had evolved to during the clinical symptoms such as blood pressure, albuminuria, so how to be carried out quick and precisely to PE patient
Condition assessment be important topic that we face.
Our research is found, by circulating miR-155 measure to the PE patient peripherals of the different orders of severity, is understood
Expression of the miR-155 in PE peripheral circulations, and its clinicopathological characteristics is combined, it can be commented for PE diagnosis, treatment and the state of an illness
Estimate and reliable an index and foundation are provided.
The content of the invention
It is an object of the invention to provide a kind of applications of miR-155 as molecular marked compound in preeclampsia is diagnosed.
The technical solution adopted in the present invention is applications of the miR-155 as molecular marked compound in preeclampsia is diagnosed,
MiR-155 nucleotide sequence is as shown in SEQ.ID.NO.1.
The features of the present invention also resides in,
MiR-155 is as the molecular marked compound for diagnosing preeclampsia.
MiR-155 is as the molecular marked compound for diagnosing serious preeclampsia.
MiR-155 is as the molecular marked compound assessed for eclampsia illness state.
Applications of the miR-155 as molecular marked compound in eclampsia is treated.
Relative to prior art, beneficial effects of the present invention are：
The invention provides applications of the miR-155 as molecular marked compound in preeclampsia is diagnosed, miR-155 nucleosides
Acid sequence is as shown in SEQ.ID.NO.1.MiR-155 expression and normal pregnancy in PE patients serums is found through experiments that in the present invention
Person increases than more significant, and PE patients serums miRNA and urine miRNA ratio relatively dramatically increases with normal pregnancy group, and
It is bright to make ROC charts, miR-155 has medium diagnostic value to preeclampsia.Therefore, by circulating miR-155 to patient peripheral
Measure, and combine its clinicopathological characteristics, present invention firstly discovers that early diagnosis of the miR-155 to eclampsia plays an important role,
Reliable an index and foundation are provided for the diagnosis, treatment and condition assessment of preeclampsia.
Brief description of the drawings
Fig. 1 is expression of the serum miR-155 in placenta in preeclampsia and normal pregnancy pregnant woman；
Fig. 2 is expression of the urine miR-155 in placenta in preeclampsia and normal pregnancy pregnant woman；
Fig. 3 is the ROC curve figure that serum miR-155 diagnoses preeclampsia；
Fig. 4 is the ROC curve figure that serum miR-155 diagnoses serious preeclampsia；
Fig. 5 is that serum miR-155 and urine miR-155 ratio diagnoses the ROC curve figure of preeclampsia.
Embodiment
The present invention is described in detail with reference to the accompanying drawings and detailed description.
MiR-155 nucleotide sequences are as shown in SEQ.ID.NO.1 in the present invention.Research finds morning of the miR-155 to eclampsia
Phase diagnosis, which has, clearly to be acted on, and is further discovered that miR-155 provides one reliably for PE diagnosis, treatment and condition assessment
Index and foundation.
The nucleotide sequence for the miR-155 that the present invention is studied is as follows：
ACACTCCAGCTGGGTTAATGCTAATCGTGATA (same to SEQ.ID.NO.1)
Below to early diagnosis specific experiments and result of the miR-155 provided by the invention to eclampsia caused by oxidative stress
It is described in detail.
1st, general information is analyzed
The serum and urine of 20 Cases with Preeclampsia patients and 20 normal pregnants are have collected altogether.Wherein serious preeclampsia
Patient 13, mild pre-eclampsia patient 7.PE groups and normal pregnancy group are in pregnant number of days, systolic pressure, diastolic pressure, fibrin ferment
Time, creatinine, significant difference (P be present on birth weight<0.05).Case-data is shown in Table 1.
1 general case-data of table
Note：PT：Prothrombin time, APTT：Activated partial thromboplastin time, TT：Thrombin time
2nd, experimental method
2.1RNA extraction：
1. taking out patients serum/urine that -80 DEG C of ultra low temperature freezers preserve is placed in room-temperature dissolution, fully 4 DEG C after dissolving
12000g centrifuges 10min.
2. drawing 300 μ L serum/urine, 1mLTrizol LS solution is added, rapid concussion shakes up 30s, fully mixes sample
Product.
3. adding 200 μ L chloroforms, 3min is stood after strong concussion 20s；
4. at 4 DEG C, 12000g centrifugation 10min, sample can be divided into three layers：The organic phase of yellow, intermediate layer and colourless water
Phase, RNA is mainly in aqueous phase.
5. upper aqueous layer is carefully moved into another new centrifuge tube；
6. adding 0.5mL isopropanols, fully mix, stand 10min, then at 4 DEG C, 12000g centrifuges 10min；
7. supernatant is abandoned in suction, add and use 75% ethanol 1mL, centrifuge tube 15s is acutely swayed on oscillator, fully mixing, 4
DEG C, 7500g centrifugations 5min；
8. supernatant is abandoned in suction, 5min is dried at room temperature, adds 25 μ LDEPC water dissolving RNA.
2.2 total rna concentrations detect and markization
Absorbance, and ratio calculated are detected under spectrophotometer 260nm and 280nm wavelength, determines RNA purity and dense
Degree, and take appropriate RNA quantitative to 1 μ g, standby subsequent experimental according to testing result.
2.3 reverse transcription
Each sample takes 1 μ g RNA to carry out reverse transcription.MiRNAs reverse transcription has its particularity, first against each
MiRNA, design its specific neck-ring reverse transcription primer.MiRNAs sequence comes from sanger databases, according to Chen et al.
Transcription primers are designed (quoted from Chen C, Ridzon DA, Broomer AJ, et al.Nucleic in the method that 2005 establish
Acids Res.2005,33(20):e179)。
1. primer synthesizes
Using U6RNA as internal reference, it is normalized.
The reverse transcriptase primer sequence of table 2
2. following solution is configured in the PCR pipe for go RNase
The solution composition of table 3
3. above-mentioned solution is mixed, 5min is placed at 85 DEG C, is subsequently placed in cooled on ice.
4. add following reaction system：
The reaction system of table 4
5. place 60min at 42 DEG C after mixing；
6. mixed system is placed into 10min at 85 DEG C.
2.4 real-time fluorescence quantitative PCR
1. design of primers：
The miR-155 of table 5 and internal control primer sequence
MiRNA-R is general anti-sense primer, and each miRNA sense primer is arranged in pairs or groups therewith.
2. reaction system：
The reaction system of table 6
3. reaction condition：
The reaction condition of table 7
2.5Real-time PCR interpretations of result
MiRNA relative expression levels' result uses relative quantitation method (2-ΔCT) represent.The CT values of same sample subtract
Its internal reference U6 CT values, obtain Δ CT values (△ CT sample=CT sample-CT U6sample), and each sample repeats three
It is secondary.
3rd, statistical procedures
Statistical analysis is carried out with SPSS13.0 softwares.Meet the data of normal distribution, continuous data mean ± standard
DifferenceRepresent, compare between two groups using two independent samples t tests；Multigroup data carry out one-way analysis of variance between each group
One-Way ANOVA, compare two-by-two, with Bonferroni methods when meeting homogeneity of variance requirement, when being unsatisfactory for homogeneity of variance requirement
Examined with Tamhane ' s T2.The data of normal distribution are not met, are compared between two groups using rank test；Compare two-by-two in multigroup
Examined using Kruskal-Wallis.Meet the data of normal distribution, linear correlation uses pearson correlation analyses；Do not meet
The data of normal distribution, using spearman Rank correlations, P<0.05 thinks that difference is statistically significant.
Fig. 1 is expression of the serum miR-155 in placenta in preeclampsia and normal pregnancy pregnant woman, and women with pre-eclampsia is clear
Middle miR-155 expression is significantly increased compared with normal pregnant, difference statistically significant (Z=-2.326, P=
0.020)。
Fig. 2 is expression of the urine miR-155 in placenta in preeclampsia and normal pregnancy pregnant woman, placenta in preeclampsia urine
Middle miR-155 expression is compared with normal pregnant, no difference of science of statistics (Z=-0.054, P=0.968).
Fig. 3 is the ROC curve figure that serum miR-155 diagnoses preeclampsia, it can be seen that miR-155 is to eclampsia
There is early stage medium diagnostic value：AUC is 0.715, and area standard is mistaken for 0.081, for diagnosing preeclampsia significance
(P=0.020), 95% credibility interval (CI) of area is (0.556-0.874), not including 0.5, therefore with diagnostic value meaning
Justice.Sensitiveness (sensitivity) and specificity corresponding to diagnostic test effect critical value (cut off value)
(specificity) data, 8 are shown in Table.
Diagnostic values of the serum miR-155 of table 8 to preeclampsia
Fig. 4 is the ROC curve figure that serum miR-155 diagnoses serious preeclampsia, it can be seen that miR-155 pairs
Serious preeclampsia has medium diagnostic value：AUC is 0.703, and area standard is mistaken for 0.084, before diagnosing severe eclampsia
Phase significance (P=0.036), 95% credibility interval (CI) of area is (0.539-0.868), not including 0.5, therefore is had
There is diagnostic value meaning.Diagnostic test effect critical value, corresponding Sensitivity and Specificity data, is shown in Table 9.
Diagnostic values of the serum miR-155 of table 9 to serious preeclampsia
Fig. 5 is that serum miR-155 and urine miR-155 ratio diagnose the ROC curve figure of preeclampsia, can be with from figure
Find out, serum miR-155 and urine miR-155 ratio has medium diagnostic value to preeclampsia：AUC is 0.718, area
Standard is mistaken for 0.08, and for diagnosing sub- preeclampsia significance (P=0.019), 95% credibility interval (CI) of area is
(0.560-0.875), not including 0.5, therefore there is diagnostic value meaning.Diagnostic test effect critical value, corresponding sensitivity
Property and specific data, are shown in Table 10.
Diagnostic values of the serum miRNA/ urines miRNA of table 10 to preeclampsia
With urine miRNA ratio compared with normal pregnant, miR-155 ratios exist the clear miRNA of women with pre-eclampsia
Preeclampsia group dramatically increases compared with normal pregnancy group, and difference is statistically significant (P=0.01), is shown in Table 11.
The serum miRNA of table 11 and urine miRNA ratio
●Compared with normal pregnancy group, P<0.05
Due to serum, urine miR-155 Non-Gaussian Distributions, examined using Spearman rank correlations；Serum, urine miR-
210 normal distributions, using Pearson related checks.Testing result is shown in Table 12.
The Cases with Preeclampsia patient miRNAs of table 12 20 and clinical indices coefficient correlation
Women with pre-eclampsia miR-155 is proportionate with urine miR-155, coefficient correlation 0.573, P=0.000.
To sum up, the present invention is found through experiments that, early diagnosis of the miR-155 to eclampsia caused by oxidative stress has clear and definite
Effect, and it is further discovered that miR-155 provides reliable an index and foundation for PE diagnosis, treatment and condition assessment.
<110>Xi'an Medical University
<120>Applications of the miR-155 as molecular marked compound in preeclampsia is diagnosed
<160>1
<210>1
<211>32
<212> RNA
<213>Artificial sequence
<400> SEQ.ID.NO.1
ACACTCCAGC TGGGTTAATG CTAATCGTGA TA 32
Claims (5)
- Applications of the 1.miR-155 as molecular marked compound in preeclampsia is diagnosed, it is characterised in that miR-155 nucleotides Sequence is as shown in SEQ.ID.NO.1.
- 2. application according to claim 1, it is characterised in that the miR-155 is as point for diagnosing preeclampsia Sub- label.
- 3. application according to claim 1, it is characterised in that the miR-155, which is used as, to be used to diagnose serious preeclampsia Molecular marked compound.
- 4. application according to claim 1, it is characterised in that the miR-155 is as point assessed for eclampsia illness state Sub- label.
- 5. application according to claim 1, it is characterised in that the miR-155 is as molecular marked compound in treatment eclampsia Application in early stage.
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Cited By (4)
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| CN108932976A (en) * | 2018-06-11 | 2018-12-04 | 西安医学院 | A kind of non-alcohol fatty liver Noninvasive diagnosis program |
| CN112007044A (en) * | 2019-09-10 | 2020-12-01 | 四川大学 | Medicine for preventing oxidative stress of retinal ganglion cells and wet macular degeneration |
| CN112391462A (en) * | 2020-12-04 | 2021-02-23 | 天津医科大学第二医院 | Specific miRNAs in peripheral plasma in preeclampsia and application thereof |
| CN113718028A (en) * | 2021-10-15 | 2021-11-30 | 青岛大学附属医院 | micro-RNA based preeclampsia diagnostic application and composition |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108932976A (en) * | 2018-06-11 | 2018-12-04 | 西安医学院 | A kind of non-alcohol fatty liver Noninvasive diagnosis program |
| CN112007044A (en) * | 2019-09-10 | 2020-12-01 | 四川大学 | Medicine for preventing oxidative stress of retinal ganglion cells and wet macular degeneration |
| CN112391462A (en) * | 2020-12-04 | 2021-02-23 | 天津医科大学第二医院 | Specific miRNAs in peripheral plasma in preeclampsia and application thereof |
| CN113718028A (en) * | 2021-10-15 | 2021-11-30 | 青岛大学附属医院 | micro-RNA based preeclampsia diagnostic application and composition |
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