CN110241199A

CN110241199A - MiR-584-5p is as the application in acute respiratory distress syndrome biomarker

Info

Publication number: CN110241199A
Application number: CN201910484822.4A
Authority: CN
Inventors: 张思泉; 丁先锋; 洪一诺; 周可欣
Original assignee: Hangzhou Xixi Hospital
Current assignee: Hangzhou Xixi Hospital
Priority date: 2019-06-05
Filing date: 2019-06-05
Publication date: 2019-09-17

Abstract

The invention discloses a kind of miR-584-5p as the application in acute respiratory distress syndrome biomarker.It is related to acute respiratory distress syndrome that present invention firstly discloses miR-584-5p, analysis of the present invention is found, the expression in serum when serum of the miR-584-5p in normal serum, ARDS morbidity and ARDS death has significant difference, it is inferred that and demonstrate miR-584-5p can be as the Serology biological marker of ARDS, it can be used alone to carry out quickly non-invasive screening to ARDS patient, assist ARDS pathological identification and clinical diagnosis；It is predicted convenient for judging on serum levels the occurring degree of ARDS patient, to the developing direction of the ARDS state of an illness; to ensure that patient obtains active treatment; being also convenient for medical worker takes suitable treatment measure according to diagnostic result simultaneously, has good potential applicability in clinical practice.

Description

MiR-584-5p is as the application in acute respiratory distress syndrome biomarker

Technical field

The invention belongs to biotechnologys and medical domain, and in particular to miR-584-5p is comprehensive as acute respiratory distress Levy the application in biomarker.

Background technique

Acute respiratory distress syndrome (ARDS) is as caused by intrapulmonary reason and/or extrapulmonary reason, and characteristic feature is just It is intractable hypoxemia, is a kind of clinical syndrome, the death rate of patient is high.The pathogenic factor of ARDS is various, different diseases The pathogenesis because caused by is had nothing in common with each other, and same individual is exposed to the disease incidence and lethality generated under different environmental factors All different, the most complicated is that the disease can change with the migration of time.Clinical manifestation is in Acute onset, breathing more It is poverty-stricken and be difficult to the hypoxemia etc. corrected with conventional oxygen therapy；About the identification of ARDS, " Berlin definition " is mostly used in the world The guidance diagnosed, treated and severe degree divide.Currently, the treatment method for ARDS is mainly controlled including mechanical ventilation Treatment and on-mechanical ventilation therapy two major classes, but its therapeutic effect is extremely limited, needs to develop new treatment method.

MicroRNA (miRNA) is a kind of non-coding single stranded RNA for being about 15-22bp by the length of endogenous gene point Son has specificity on tissue and time, participates in the expression for adjusting its specific target gene, to control the generation of albumen, has There are adjusting development timing, cell Proliferation, differentiation, metabolism, apoptosis and functions stress be waited, participates in adjusting a variety of diseases.Have big Amount the experiment proves that miRNA shows different expressions, Ke Yizuo in the generation and development process in a variety of diseases For the potential index of screening and diagnosis and the selection of therapeutic scheme and prognostic analysis.

In traditional detection method, the detection of miRNA expression generally requires acquisition patient's lesion, and materials are very not Just, and wound can be caused to patient.Recent study personnel discovery in blood plasma and serum there is also independently of cell except And stable miRNA molecule can be obviously kept under harsh environment, and as biological detection sample, serum, which has, to be taken Material convenience, non-invasive and can continuous vitro detection the advantages of so that based on miRNA is qualitative and quantitative measurement technology is found The serum miRNA of cancer specific will be more efficient than traditional protein molecular labeling method as the method for molecular labeling, into And molecular labeling can be overcome to develop encountered bottleneck in Antibody preparation and quantitative analysis.Therefore, developing one kind can assist The serum miRNA of ARDS treatment and diagnosis has extensive scientific research value and potential applicability in clinical practice as biomarker.

Summary of the invention

Present invention purpose is to provide miR-584-5p in as acute respiratory distress syndrome biomarker Application.

For achieving the above object, the technical solution of the application is as follows:

MiR-584-5p is as the application in acute respiratory distress syndrome biomarker.

MiR-584-5p is that well known to a person skilled in the art miRNA, the miR-584-5p to have such as SEQ for one kind Nucleotide sequence shown in ID No.1.It is authorized to Chinese invention patent that notification number is 107034283 B of CN for miR-584- 5p and other several miRNA combinations (miR-153, miR-188 and miR-483) are used as pancreatic cancer drug tolerance screening and mark Object；The miR-584-5p Chinese invention patent application that also number of being applied is CN 201810878450.9 is used as to be assisted with adenocarcinoma of lung Diagnose relevant serum miRNA marker.

Either be used in diagnose which kind of disease, the effect of miRNA is all auxiliary diagnosis, need combine predominantly detect means Testing result judges.Adenocarcinoma of lung occurs mainly in bronchial gland body, wherein it is in the majority with bronchium, it is normal with peripheral lump See, main detection means is chest X-ray or CT；And the Major Clinical of acute respiratory distress syndrome (hereinafter referred to as ARDS) Performance is that the oxygen saturation of lung is low, mainly carries out detection and diagnosis by blood gas analyzer.As it can be seen that ARDS and lung adenopathy Clinical manifestation, pathogenic process are entirely different, and miR-584-5p has not yet been disclosed related to acute respiratory distress syndrome at present.

Analysis of the present invention finds that serum and ARDS patient of the miR-584-5p in normal serum, ARDS morbidity are dead The expression in serum when dying has significant difference, it is inferred that and demonstrate miR-584-5p can be as the serum of ARDS Biomarker object can be used alone to carry out quickly non-invasive screening to ARDS patient, assist ARDS pathological identification and clinic Diagnosis；Developing direction convenient for judging on serum levels the occurring degree of ARDS patient, to the ARDS state of an illness carries out pre- It surveys, to ensure that patient obtains active treatment, while being also convenient for medical worker and suitable treatment measure is taken according to diagnostic result, have There is good potential applicability in clinical practice.

Present invention also provides a kind of for detecting the PCR primer of miR-584-5p expression, this is used to detect miR- The PCR primer of 584-5p expression includes:

MiR-584-5p upstream primer: 5 '-GGTTATGGTTTGCCTGGGA-3 '；

MiR-584-5p downstream primer: 5 '-CAGTGCGTGTCGTGGAGT-3 '.

Present invention also provides a kind of kit for assisting screening or diagnosing ADRS, contain above-mentioned use in the kit In the PCR primer of detection miR-584-5p expression, and the PCR primer for detecting reference gene expression.

Preferably, the reference gene is 5SrRNA, the PCR for detecting reference gene expression draws Object includes:

5SrRNA upstream primer: 5 '-GTCTACGGCCATACCACCCTGAA-3 '；

5SrRNA downstream primer: 5 '-AAGCCTACAGCACCCGGTATTCC-3 '.

Preferably, the kit further includes the Total RNAs extraction system for extracting test serum sample total serum IgE, And the total serum IgE reverse transcription for that will extract is the reverse transcription system of cDNA.

The reverse transcription system includes miR-584-5p reverse transcriptase primer, which has The nucleotide sequence as shown in SEQ ID No.2.

Compared with prior art, the beneficial effects of the present invention are embodied in:

Present invention firstly discovers that miR-584-5p serum and when ARDS death in normal serum, ARDS morbidity Expression in serum has significant difference, it is inferred that and demonstrate miR-584-5p can be as the Serology biological of ARDS Marker can be used alone to carry out quickly non-invasive screening to ARDS patient, assist ARDS pathological identification and clinical diagnosis；Just In judging on serum levels the occurring degree of ARDS patient, predict the developing direction of the ARDS state of an illness, with true It protects patient and obtains active treatment, while being also convenient for medical worker and suitable treatment measure is taken according to diagnostic result, have good Potential applicability in clinical practice.

Detailed description of the invention

Fig. 1 is differential expression of the miR-584-5p in ARDS morbidity in serum sample and normal serum sample point Analyse result；

Fig. 2 is miR-584-5p serum sample and difference when ARDS morbidity in serum sample in ARDS death Anisotropic expression analysis result；

Fig. 3 is differential expression of the miR-584-5p in ARDS death in serum sample and normal serum sample point Analyse result；

Wherein, Disease indicates that serum sample when ARDS morbidity, health indicate normal serum sample, Death table Serum sample when showing ARDS death；Relative expression indicates relative expression quantity.

Specific embodiment

Further details of the technical solution of the present invention with reference to the accompanying drawings and detailed description.

Embodiment 1

ARDS serum sample used in the present embodiment (including serum sample 11 when ARDS morbidity, ARDS patient Serum sample 10 when dead) from December, 2013~2016 year, in Hangzhou, Zhejiang province city, Xi Xi hospital receives treatment between 2 months ARDS patient whole blood, patient in group requires the age to be greater than or equal to 18 years old, and heart function is sound, and non-pregnant woman and nursing period suffer from Person.Normal serum check sample of the present invention comes from healthy volunteer (20).

Reagent used in the present embodiment, such as chloroform, isopropanol, ethyl alcohol, DNA polyacrylamide gel electrophoresis reagent etc. Conventional biochemical reagent is purchased from Shanghai Sangon Biotech Company；PCR amplification reagent (containing 10 × buffer, dNTPs mixture) and reversion Record reaction agents useful for same, as dNTP mixing is employed in RNase inhibitor, M-MLV reverse transcriptase, 5 × M-MLV buffer and reversion Object (each 10mM, RNase-free), is purchased from TaKaRa company；Fluorescent quantificationally PCR detecting kit based on DNA dye method (Master of PCR containing real-time Mix, 50 × ROX reference) is that Products are only praised in Nanjing promise；All primer committees Hold in the palm the synthesis of Shanghai bioengineering Co., Ltd.

1, serum sample is prepared

Whole blood sample comes from ARDS patient and healthy volunteer.

1) centrifuge tube for taking peripheral blood 3ml injection cleaning dry, is immediately placed on 37 DEG C of tilting 1-2h of constant incubator；

2) 4 DEG C of tilting 3-4h, the serum of precipitation move on in new centrifuge tube；

3) sludged blood is centrifuged 10min, is carefully drawn supernatant with 4 DEG C, 4000rpm；

4) merge step 2) and the resulting serum twice of step 3), 4 DEG C, 4000rpm is centrifuged 10min, carefully draws supernatant It goes in 1.5ml centrifuge tube, obtains serum.

Each whole blood sample is stored in -80 DEG C of refrigerators after being all made of above method processing respectively, for use.

2, total serum IgE 1 is extracted) ARDS serum is taken out at -80 DEG C, it is slowly dissolved at 4 DEG C；

2) 200 μ l of serum sample is drawn with pipettor, is added to 1.5ml without in RNase EP pipe, 800 μ l is added Trizol is placed in vortex instrument and is acutely vortexed mixing to abundant cracking, until can't see suspended matter；It is stored at room temperature 15min；

3) add 200 μ l chloroforms；It is vigorously mixed 15s, stratification 5min；See 12000g when having lamination, 4 DEG C of centrifugations 15min takes supernatant fluid (general 500 μ l) to be transferred in new 1.5ml collecting pipe, adds isometric times of supernatant volume with pipettor Isopropanol, mixing of turning upside down, stand 20min；

4) at 4 DEG C, 12000g is centrifuged 10min；Abandon supernatant；

5) plus 1ml75% ethyl alcohol (with dehydrated alcohol and the preparation of DEPC water) washs precipitating, and when washing bounces precipitating；

6) at 4 DEG C, 7500g is centrifuged 5min, abandons supernatant, drying at room temperature；

7) add 60 DEG C of the molten precipitating of DEPC water weight；

8) NanoDrop 2000 detects the content and purity of total serum IgE.

3, Solexa is sequenced

It randomly selects 5 ARDS patients serum samples and 5 normal serum samples carries out solexa sequencing, experiment is by joining river Biotech firm (http://www.lc-bio.com/) completes, and company uses a new generation's illumina/solexa platform, passes through survey Sequence we can obtain in sample known and unknown miRNA and their size and length.Its advantage is exactly to can be found that Unknown miRNA, meanwhile, compared with prior-generation microarray dataset, the reliability of data is greatly improved.

4, design primer

According to test result, designed for the primer of reverse transcription and amplification each miRNA and reference gene 5sRNA, wherein The reverse transcriptase primer and PCR primer of miR-584-5p (its nucleotide sequence is as shown in SEQ ID No.1) and reference gene 5sRNA It is as follows respectively:

MiR-584-5p reverse transcriptase primer (SEQ ID No.2): 5 '-GTCGTATCCAGTGCGTGTCGTGG

AGTCGGCAATTGCACTGGATACGACCTCAGTC-3'；

MiR-584-5p upstream primer (SEQ ID No.3): 5 '-GGTTATGGTTTGCCTGGGA-3 '；

MiR-584-5p downstream primer (SEQ ID No.4): 5 '-CAGTGCGTGTCGTGGAGT-3 '.

5SrRNA upstream primer (SEQ ID No.5): 5 '-GTCTACGGCCATACCACCCTGAA-3 '；

5SrRNA downstream primer (SEQ ID No.6): 5 '-AAGCCTACAGCACCCGGTATTCC-3 '.

5, reverse transcription

Reverse transcription reaction system is as follows:

1 reverse transcription reaction system of table

By each reagent by being added sequentially in the Ep pipe of no RNase shown in table 1, reverse transcription reaction is carried out, reaction condition is set It is set to:

42℃ 60min

70℃ 15min

4℃ ∞

After completion of the reaction, 4 DEG C of refrigerators are stored in.

The present embodiment devises the loop-stem structure reverse transcription primer of specificity for miR-584-5p, is individually as above grasped Make, single tube synthesizes cDNA.

6, quantitative fluorescent PCR

Reaction system such as table 2 for quantitative fluorescent PCR:

2 quantitative fluorescent PCR reaction system of table

By each reagent by be sequentially added sequentially to shown in table 2 in 200 μ l Ep pipes mix after be slightly centrifuged, in ABI 7500 Carry out quantitative fluorescent PCR reaction, response parameter setting are as follows:

7, data processing

It is analyzed according to Solexa sequencing result, obtains miR-584-5p and exist in normal person's sample and ARDS clinical samples Significant difference expression, detects that expression of the miR-584-5p in two samples is above Average expression level, difference table It is higher than twice up to level；Specific data see the table below.

3 Solexa sequencing result of table and fold differences analysis

Note: the valid data sum of miRNA is 7093348 in normal serum, the valid data in ARDS patients serum Sum is 8657112；It shares 20 miRNA in sequencing to lower, 10 up-regulations.

When using relative expression's variable quantity of quantitative fluorescent PCR quantitative detection miRNA, using 5sRNA as reference gene, come Target gene is normalized, to ensure to compare the amount of target gene in the sample of equal amount.Expression quantity multiple Variation calculated with following formula:

RQ=2^-△△CT；

△ △ CT=(C_{T miRNA}-C_{T 5sRNA})_{ARDS r}-(C_{T miRNA}-C_{T 5sRNA})Mean_Normal；

Wherein, RQ represents relative expression quantity (relative quantation), C_{T miRNA}And C_{T 5sRNA}Respectively represent fluorescence The Ct value of target miRNA and reference gene 5sRNA that quantitative detection arrives, ARDS represent ARDS serum, and Health is represented and ARDS The corresponding Normal group of serum, Mean health represent the average value in all Normal groups.

The inspection software that above data, i.e. Ct value can be worn by fluorescent quantitative detector is read out.Weight is set up in quantitative Multiple experiment and negative control experiment, each sample of quantitative experiment are repeated 3 times, and template cDNA is not added in negative control, and with water Instead of being polluted there are PCR and the pollution of higher primer dimer for checking whether.

The statistical analysis of fluorescent quantitation data uses SPSS statistical analysis software.Opposite table of the miRNA in two samples It is examined up to amount analysis using levene ' s Chi-square Test and independent-samples t, when P value≤0.05, it is believed that knot Fruit statistically has significant difference, when P value < 0.01, it is believed that result statistically has extremely significant sex differernce.

The intuitive performance for the difference analysis that miR-584-5p is expressed in the serum of ARDS morbidity and health passes through The histogram (such as Fig. 1) containing error line is drawn to realize；MiR-584-5p in ARDS death serum with respectively and normally MiR-584-5p expression quantity is compared in serum, and the intuitive performance of expression fold difference analysis is realized by drawing histogram, Such as Fig. 2；In ARDS morbidity and death, the intuitive performance of different expression analysis is missed miR-584-5p by drawing to contain in serum The histogram (such as Fig. 3) of poor line.7 software of GraphPad Prism is all made of to draw.

The present invention carries out list sample T inspection with SPSS software and independent sample T is examined, and analyzes target miRNA and is receiving The otherness expressed in the 11 ARDS morbidity serum samples and 20 normal serum samples collected, as seen from Figure 1, miR- Expression of the 584-5p when ARDS falls ill in serum and normal serum has extremely significant otherness (RQ=0.377, P= 0.003)；The result shows that expression of the miR-584-5p in serum can reflect the pathological characteristic of ARDS, and then determine MiR-584-5p can be used as the biomarker of ARDS on serological levels to indicate the clinical pathology situation of the disease.

The miR-584-5p expression correlation of the different stages of development of disease is analyzed, can be used for predicting the hair of ARDS Exhibition direction judges developing stage locating for patient.The present embodiment suffers from 11 ARDS morbidity serum samples and 10 ARDS When person's death in serum sample miR-584-5p differential expression analysis's result such as Fig. 2.From Figure 2 it can be seen that the serum in morbidity Sample and it is dead when serum sample in the expression quantity of miR-584-5p (P=0.017) there were significant differences, miR-584-5p is in patient Expression quantity when just expression quantity when morbidity is than death is low；This has patient's ARDS staging diagnosis important directive significance.

The difference that miR-584-5p is expressed in serum sample and its 20 normal serum samples in 10 ARDS deaths Specific analysis result such as Fig. 3.As seen from Figure 3, expressing in serum of the miR-584-5p in ARDS death significantly reduces, and Extremely significant horizontal (RQ=0.63, P=0.004) is had reached.It therefore can be bright according to expression of the miRNA in ARDS serum Aobvious serum when distinguishing dead and normal serum, it may have preferable reference value: can be with by the analysis result of Fig. 1, Fig. 2 and Fig. 3 Find out, morbidity serum sample and it is dead when serum sample in the expression quantity of miR-584-5p significantly dropped than normal serum sample It is low, and fall ill serum sample than it is dead when serum sample have lower miR-584-5p expression quantity, clinically can basis Comparison result whether there is dead trend after judging morbidity, because, with disease, either controlling after the onset of ARDS More deathward, the expression quantity of miR-584-5p exist rise trend, but cure after miR-584-5p expression quantity It is higher than dead, and there are significant differences for the two.This with judged to send out according to the occurring degree of ARDS in conventional method Disease after being ill have higher accuracy rate and precision, can in time for medical staff adjustment orientation treatment provide according to According to.

Sequence table

<110>Hangzhou Xi Xi hospital

<120>miR-584-5p is as the application in acute respiratory distress syndrome biomarker

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 22

<212> RNA

<213>people (human)

<400> 1

uuaugguuug ccugggacug ag 22

<210> 2

<211> 55

<212> DNA

<213>artificial synthesized sequence (Unknown)

<400> 2

gtcgtatcca gtgcgtgtcg tggagtcggc aattgcactg gatacgacct cagtc 55

<210> 3

<211> 19

<212> DNA

<213>artificial synthesized sequence (Unknown)

<400> 3

ggttatggtt tgcctggga 19

<210> 4

<211> 18

<212> DNA

<213>artificial synthesized sequence (Unknown)

<400> 4

cagtgcgtgt cgtggagt 18

<210> 5

<211> 23

<212> DNA

<213>artificial synthesized sequence (Unknown)

<400> 5

gtctacggcc ataccaccct gaa 23

<210> 6

<211> 23

<212> DNA

<213>artificial synthesized sequence (Unknown)

<400> 6

aagcctacag cacccggtat tcc 23

Claims

1.miR-584-5p as the application in acute respiratory distress syndrome biomarker.

2. miR-584-5p as described in claim 1 is as the application in acute respiratory distress syndrome biomarker, It is characterized in that, the miR-584-5p has the nucleotide sequence as shown in SEQ ID No.1.

3. miR-584-5p as described in claim 1 is as the application in acute respiratory distress syndrome biomarker, It is characterized in that, the acute respiratory distress syndrome biomarker is Serology biological marker.

4. a kind of for detecting the PCR primer of miR-584-5p expression characterized by comprising

MiR-584-5p upstream primer: 5 '-GGTTATGGTTTGCCTGGGA-3 '；

MiR-584-5p downstream primer: 5 '-CAGTGCGTGTCGTGGAGT-3 '.

5. a kind of kit for assisting screening or diagnosing ADRS, which is characterized in that containing being used for as claimed in claim 4 Detect the PCR primer of miR-584-5p expression, and the PCR primer for detecting reference gene expression.

6. as claimed in claim 5 for assisting the kit of screening ADRS, which is characterized in that the reference gene is 5SrRNA, the PCR primer for detecting reference gene expression include:

5SrRNA upstream primer: 5 '-GTCTACGGCCATACCACCCTGAA-3 '；

5SrRNA downstream primer: 5 '-AAGCCTACAGCACCCGGTATTCC-3 '.

7. as claimed in claim 6 for assisting the kit of screening ADRS, which is characterized in that further include to be measured for extracting The Total RNAs extraction system of blood serum sample total serum IgE, and total serum IgE reverse transcription for that will extract are the reverse transcription system of cDNA.

8. as claimed in claim 7 for assisting the kit of screening ADRS, which is characterized in that the reverse transcription system packet MiR-584-5p reverse transcriptase primer and 5SrRNA reverse transcriptase primer are included, which has such as SEQ ID Nucleotide sequence shown in No.2.