CN105039536B - Purposes of the mo-miR-877 in renal toxicity biomarker is prepared - Google Patents
Purposes of the mo-miR-877 in renal toxicity biomarker is prepared Download PDFInfo
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Abstract
The invention discloses a kind of purposes of mo miR 877 in renal toxicity biomarker is prepared and kits and primer.Mo miR 877 can be used as unique renal toxicity biomarker to be used alone, and can also be used in combination, and play a role in injury of kidney caused by exogenous compounds is detected with conventional renal toxicity biomarker.The kit can high specificity, feasibility highland detection renal toxicity biomarker, injury of kidney caused by so as to detect exogenous compounds.The primer can expand to high specificity mo miR 877, and applied to injury of kidney caused by detection exogenous compounds in the kit.
Description
It it is on July 10th, 2014 this application claims the applying date, application No. is 201410328042.8, entitled " one
The priority of the Chinese patent application of kind renal toxicity biomarker and application thereof ".The application quotes the Chinese patent application
In full.
Technical field
The invention belongs to biotechnologies, and in particular to a kind of mo-miR-877 is in renal toxicity biomarker is prepared
Purposes and kit and primer.
Background technology
Kidney is one of the internal first target organs of most important excretory organs and exogenous poisonous substance in vivo.Very much
Compound (drug) enter Toxicity of Kidney can be caused to damage in vivo, have according to investigations, in acute renal failure 32.4% be due to
Caused by medicine renal toxicity.Therefore it is badly in need of establishing finding and confirming new renal toxicity biomarker, it being capable of early detection kidney damage
Wound, and can reflect kidney injury improvement or exacerbation in addition can reflect damage mechanism.
Current conventional renal toxicity evaluation method, clinically non-clinical Drug safety assessment and common renal damage index
For serum creatinine (CRE) and urea nitrogen (BUN), then carry out histopathologic examination.CRE is easily by age, sex, race, diet
With the influence of organismic internal environment, and unpredictable early lesion, it is impossible to reflect the damage and necrosis of renal tubule；BUN not only by
The influence of renal function is also influenced by kidney other factor such as high-protein diet, hemorrhage of digestive tract etc..And the two needs patient
Acute kidney injury (AKI) just restricted raising after a few days, can be delayed renal toxicity early detection, diagnose and treat.
With going deep into for renal toxicity research, it was found that the Testing index of some new Toxicity of Kidney damages such as urinates total protein
(uTP), biomarker of the serum Cystatin C (CysC) as drug glomerular injury；Clusterin, kidney
Molecular injury (KIM-1), trefoil factor -3 (TFF-3) can as the biomarker of renal damage, but there are sensibility,
The problem of specificity and detection method, it is difficult to traditional renal toxicity be replaced to detect.Therefore, find in time, reliable, feasibility it is strong
The biomarker of renal toxicity is very significant.
MiRNA is a kind of endogenous, non-coding, single-stranded tiny RNA.They can hold non-translational region with target gene mRNA-3 '
(3 '-UTR) is partly or entirely complementary to be combined, Level tune gene expression upon translation, cause target gene occur Translational repression or
MRNA degrades.Ripe about 18 to 25 nucleotide of miRNA sizes.They are transcribed first in nucleus forms primary miRNA
(pri-miRNA), then in the protein complexes and rna plymerase ii formed by RNAase III, Dicer and Drosha
Precursor miRNA (pre-miRNA) is formed under effect.Pre-miRNA enters from nucleus in cytoplasm, is about by Dicer digestions
The double-strand miRNA of 22 nucleotide.Subsequent double-strand is cut off, and forms a ripe single-stranded miRNA.Single-stranded miRNA and RNA are lured
The silencing complex led combines and the reverse complements of selectively acting target gene mRNA, the expression of final regulation and control target gene.
Invention content
Therefore, the technical problem to be solved by the present invention is to be directed to existing renal toxicity biomarker specificity it is low, cannot
The problem of early detection detection, detection method excessively complexity etc., provide a kind of mo-miR-877 and are preparing renal toxicity biological marker
Purposes and kit and primer in object.Mo-miR-877 can be applied to the system of renal toxicity biomarker by the purposes
Standby injury of kidney caused by so as to detect exogenous compounds；The kit can specific detection exogenous compounds cause
Injury of kidney；The primer can specific amplification preparing the mo-miR-877 of renal toxicity biomarker.
The present invention provides purposes of the rno-miR-877 as renal toxicity biomarker.
In the present invention, the rno-miR-877 is a kind of miRNA.In the present invention, the rno-miR-877 of rat has
Base sequence in sequence table described in SEQ ID No.1.
Heretofore described renal toxicity preferably derives from injury of kidney caused by exogenous compounds.The rno-
MiR-877 can be used for injury of kidney caused by detecting exogenous compounds as renal toxicity biomarker.
Exogenous compounds of the present invention are conventional for this field, it is preferred that gentamicin (GM), cis-platinum, N- phenyl
Ortho-aminobenzoic acid or adriamycin, more preferably gentamicin.
In the present invention, preferably, rno-miR-877 is used alone as unique renal toxicity biomarker, Huo Zhehe
Conventional renal toxicity biomarker is used in combination.Preferably, the conventional renal toxicity biomarker is serum creatinine, urine
Plain nitrogen, urine total protein and clusterin.
Rno-miR-877 of the present invention is used for injury of kidney caused by detecting exogenous compounds as renal toxicity biomarker
Method, may comprise steps of, detect nephridial tissue in rno-miR-877 content.
The present invention also provides a kind of kit of injury of kidney caused by detection exogenous compounds, including specific detection
The primer of mo-miR-877, the primer are DNA fragmentation of the nucleotide sequence as shown in SEQ ID No.2 in sequence table.
Preferably, the kit further includes RNA extraction agents, Reverse Transcription and/or qPCR reagents.Described
RNA extraction agent of the RNA extraction agents for this field routine, preferably TRIZOL.The Reverse Transcription is this field
Conventional Reverse Transcription, preferably RNA reverse transcriptases.The qPCR reagents qPCR examination conventional for this field
Agent, preferably archaeal dna polymerase.
The present invention also provides a kind of primers of specific detection mo-miR-877, are in nucleotide sequence such as sequence table
DNA fragmentation shown in SEQ ID No.2.
The inventors discovered that rno-miR-877 is in the rat blood leucocyte of injury of kidney caused by exogenous compounds
Height expression.The blood leucocyte can derive from the blood of each tissue, preferably, rear abdominal aorta.
The method that the present invention finds renal toxicity biomarker, including the analysis composed to miRNA chip expressions, compares male and female
The different time and the express spectra of control group without any processing that rat is handled respectively through exogenous compounds, then according to
The possible renal toxicity biomarker of function prediction known.
On the basis of common knowledge of the art, above-mentioned each optimum condition can be combined arbitrarily to get each preferable reality of the present invention
Example.
The reagents and materials used in the present invention are commercially available.
The positive effect of the present invention is：Draw the inventors found that mo-miR-877 is exogenous compounds
The biomarker of the specificity of the injury of kidney risen, the high table in the blood leucocyte of injury of kidney caused by exogenous compounds
It reaches, the purposes in renal toxicity biomarker is prepared can be played, injury of kidney caused by so as to detect exogenous compounds.
Kit provided by the invention can high specificity, feasibility highland detection renal toxicity biomarker, it is exogenous so as to detect
Injury of kidney caused by compound.Primer provided by the invention can expand to high specificity mo-miR-877, and applied to described
Kit in detection exogenous compounds caused by injury of kidney.
Description of the drawings
Fig. 1 is renal histopathology change (negative control group D2) after the continuous intramuscular injection gentamicin of rat.
Fig. 2 is renal histopathology change (80mg/kg group D4) after the continuous intramuscular injection gentamicin of rat.
Fig. 3 is renal histopathology change (80mg/kg group D8) after the continuous intramuscular injection gentamicin of rat.
Specific embodiment
The present inventor, by miRNA chip of expression spectrum data analyses, has found rno-miR-877 by taking rno-miR-877 as an example
It is the renal toxicity biomarker of injury of kidney caused by exogenous compounds.Detection finds rno-miR-877 in exogenous chemical combination
High expression in the nephridial tissue of injury of kidney caused by object.It is renal toxicity biomarker using rno-miR-877, external source can be detected
Property injury of kidney caused by compound.
It is further illustrated the present invention below by the mode of embodiment, but does not therefore limit the present invention to the reality
It applies among a range.Test method without specific conditions in the following example, according to conventional methods and conditions or according to quotient
Product specification selects.
Embodiment 1 finds renal toxicity biomarker
The foundation of 1.1 renal injury models
1.1.1 experiment material and instrument
Experiment reagent and instrument
Positive drug：Gentamicine sulphate injection (lot number：20110406, purchased from Hubei Pharmaceutical Co., Ltd., specification：
2mL：80000 units；80mg is 80,000 units)；
Vehicle controls product：0.9% sodium chloride injection (physiological saline) (lot number 11010463：Rich crystallization is grown purchased from Shandong
Pharmaceutcal corporation, Ltd)
CRE (R1) (creatinine) assay kit is produced by Japanese Wako Pure Chemical Industries, Ltd. (Wako), BUN (urea
Nitrogen) detection kit produces by German Roche Diagnostics GmbH.
7060 type automatic clinical chemistry analyzers of HITACHI are purchased from Hitachi, Japan Industry Co., Ltd.
Experimental animal
SPF grades of SD rats 200, half male and half female, during first administration weight between 170~230, week old 6~8 weeks it
Between.Animal is purchased from the western Poole-Bi Kai experimental animals Co., Ltd in Shanghai, and credit number is SCXK (Shanghai) 2008-0016.Animal is adopted
It is identified with picric acid and Yihong, 5/cage, has cage board per cage animal.Animal feeding is in national Shanghai new drug safety evaluation
The SPF grade animal houses in research center, animal house temperature are controlled at 22~26 degrees Celsius, and humid control is between 40~70%, often
Minute ventilation number >=15 time, the light and shade periodicity of illumination of 12 hours/12 hours, animal freely ingest, and feed is by Beijing Australia of section
Pull together the 60Coradiation sterilizing big MOUSE REPRODUCTION pellets of SPF of feed corporation,Ltd's offer, animal freely drunk by drinking bottle
With homemade deionized water.
1.1.2 experimental method
1.1.2.1 the preparation of positive drug
Positive drug gentamicine sulphate injection is diluted to required concentration with physiological saline.It is now with the current, room temperature preservation.
Specifically it is shown in Table 1：
1 positive drug gentamicine sulphate injection of table is with tabulation
1.1.2.2 animal experiment dosage is set
The clinical usage of gentamicine sulphate injection is intravenous drip after adult uses intramuscular injection or dilutes, 1 time
80mg (i.e. 80,000 units) or according to 1~1.7mg/kg of weight, 1 time for every eight hours, dilution is using physiological saline or 5%
Glucose injection.This experiment is wished to cause after giving gentamicin sulphate：1. there is different degrees of damage in animal kidneys；
2. occurs different degrees of damage in different time：Slight extremely serious kidney injury；3. there is certain dosage according to lazyness in
Property and time dependence damage.Therefore this experimental setup 5,20 and 80mg/kg groups, while negative control group is set, give physiology
Brine is specifically shown in Table 2：
2 test dose of table designs
1.1.2.3 it is administered and observes and check
The administration route of 3 dosage groups of negative control group and gentamicin sulphate is intramuscular injection, and medicine-feeding part is big
Mouse or so back leg quadriceps muscle of thigh, is administered once daily, and administration capacity is 2mL/kg.The left and right back leg of each every animal is injected
1mL/kg, each administered volume are calculated all in accordance with the weight that the last time measures.
Daily morning and afternoon respectively observes 1 animal dead and dying situation during experiment.2 clinics are observed during administration daily
Symptom, each 1 time of upper and lower noon.
It weighs before administration in the 1st, 3,5,7 and 14 day is administered and convalescence 7,14,21 and 28 days weighs, every time before dissection
It weighs and is only used for calculating organ coefficient.
After first administration after 24 hours, 3 days, 7 days, 14 days and 28 days 14 day convalescences of administration, each time point point
10 animals (female is fifty-fifty) are not taken, from jugular vein blood collection about 0.5mL, with Aspirate supernatant after 1660g centrifugations 15min, are used
Urease methods detection BUN, creatinine enzyme process detection CRE concentration.
1.1.2.4 results of serum biochemical detection
After 1 day (D2) and 3 days (D4) is administered, serum BUN and the CRE mean value and negative control of gentamicin group animal are given
Group is compared and is showed no apparent significant difference.After administration 7 days (D8), serum BUN and CRE mean value and the feminine gender of 5 and 20mg/kg groups
Control group compared to being showed no apparent significant difference, and the serum BUN and CREA of the jenny of 80mg/kg groups obviously higher than
Negative control group (P<0.05), buck change of serum C RE is also apparently higher than negative control group (P<0.05).It is administered 14 days (D15)
Afterwards, serum BUN and the CRE mean value of 5mg/kg groups female animals also has not yet to see notable difference, the serum of 20mg/kg group jennies
CRE is apparently higher than same period negative control group (P<0.05), the serum BUN and CRE and negative control group of 20mg/kg groups buck
Compared to having no notable difference, and the serum BUN and CRE of the female buck of 80mg/kg groups is obviously higher than negative control group (P<
0.05) 3, are the results are shown in Table, table 4.
After 14 day 28 days convalescence (R29) is administered, the serum BUN and CRE and feminine gender of 5 and 20mg/kg group female animals are right
It is compared according to group and is showed no apparent significant difference.The change of serum C RE mean values of the female buck of 80mg/kg groups are right significantly lower than negative
According to group (P<0.05), and serum BUN mean values are showed no obvious abnormalities compared with negative control group.
Serum biochemistry result after 3 jenny intramuscular injection gentamicin of table
*P<0.05,**P<0.01, compared with 0mg/kg groups；BUN units mmol/L；CRE units umol/L；D：Day is administered
Number, R restore number of days.
Serum biochemistry result after 4 buck intramuscular injection gentamicin of table
*P<0.05,**P<0.01, compared with 0mg/kg groups；BUN units mmol/L；CRE units umol/L；D：Day is administered
Number, R restore number of days.
1.1.2.5 histopathological examination result
Fig. 1-3 is shown in the change of histopathology.After administration 1 day, negative control group and each dosage group animal renal pathology
Have no significantly with relevant change (Fig. 1) is administered；After administration 3 days, negative control group and 5mg/kg group animal renal pathologies
Have no that, significantly with relevant change is administered, 20 is visible with 80mg/kg groups part female animals kidney slightly to slight focal kidney
Tubule dilatation (Fig. 2), remaining shows no obvious abnormalities；7 days, the moon similar with female animals renal pathology change after 14 days of administration
Property control group and 5mg/kg group animal kidneys are showed no and relevant change are significantly administered, and portion can be seen in 20mg/kg group female animals
Transfer object is slightly to slight cell infiltration, slightly to the slight changes such as slight focal tubular ectasia, 80mg/kg
The visible apparent focal renal cells degeneration necrosis of all animals of group, focal renal tubule basophilla become, different journeys
The cell infiltration of degree, the abnormal changes such as different degrees of focal tubular ectasia and different degrees of focal cast
(Fig. 3)；Administration restores 28 days groups for 14 days, and negative control group, 5 and 20mg/kg groups have no exception significantly related with administration, but respectively
Lesions, the high dose group female animals such as the accidental spontaneous slight cell infiltration of group, slight basophilla tubule are still visible focal
Property cell infiltration and different degrees of basophilla tubule and renal cast.
1.1.2.6 conclusion
This part experiment gives 5,20 and 80mg/kg of gentamicin and saline control using the injection of SD rat muscles,
Simultaneously set administration 1 day (D2), 3 days (D4), 7 days (D8), 14 days (D15) and administration 14 days recovery 28 days (R29) afterwards five when
Between point blood sampling carry out biochemical index, gross anatomy observation, kidney is weighed and pathological study, to observe gentamicin
The injury of kidney situation of induction.
Biochemical index, after 7 days (D8) is administered, 80mg/kg group animals BUN and CRE are increased.It is administered 14 days (D15)
Afterwards, 80mg/kg groups animal BUN and CRE are increased, and 20mg/kg group animals CRE is also increased.After administration restores 28 days (R29) in 14 days
BUN and CRE ends can restore substantially.
The damage of the main visible renal tubule of histopathologic examination, including necrosis, swelling, comes off.Renal pathology
It checks and finds, after 3 days (D4) is administered, 20 and 80mg/kg group kidneys are shown in focal tubular ectasia；7 days (D8) and 14 days are administered
(D15) after, 20mg/kg group kidneys are shown in inflammatory cell infiltration and focal tubular ectasia, and 80mg/kg group kidneys are shown in that focal kidney is small
Pipe epithelial cell degeneration necrosis, inflammatory cell infiltration, focal cast (slight) etc..After administration restores 28 days (R29) in 14 days, only
See that 80mg/kg group kidneys are shown in basophilla tubule, renal cast.
To sum up, successfully induced rat injury of kidney, and occurring after gentamicin is given in the injection of SD rat muscles
Apparent time dependence and dose-dependent change, therefore gentamicin induction renal injury model is set up, available for kidney poison
Property biomarker is further explored.
The preparation of 1.2miRNA chip of expression spectrum
1.2.1 sampling
Same breed is taken to belong to kind, the uniform consistent normal rat of age close size and the injury of kidney having had built up respectively
There is each three of the rat of injury of kidney in model.Kidney is gone to weigh at once after rat dissection, about 0.5*0.5cm sizes are taken after weighing
Renal tissue is added immediately to the overnight EP pipes of 4 DEG C of precoolings equipped with about 1mL RNAlater liquid, and is cut into small pieces, and places into 4
DEG C refrigerator, makes it fully infiltrate, then continues at -80 DEG C and save backup.
1.2.2 the extraction of total serum IgE
Using mirVanaTMPARISTM (Cat#AM1556, Ambion, Austin, TX, US) and according to production firm
The Standard Operating Procedure of offer carries out the total RNA extractings of sample, and extracting gained total RNA are through Agilent
It is spare after Bioanalyzer 2100 (Agilent technologies, Santa Clara, CA, US) electrophoresis quality inspection qualification.
Operating procedure is as follows：
(1) 70% ethyl alcohol is prepared：35mL absolute ethyl alcohols and 15mL nuclease-free water mixings.
(2) centrifugation should all carry out under the conditions of 4 DEG C, centrifugal speed 13200rpm.
(3) it is homogenized：Per 100mg, tissue adds in 1mL TRIZOL reagents, and is homogenized with homogenizer.
(4) leucocyte for the TRIZOL for having been added to 1mL is taken；
(5) TRIZOL per 1mL adds in the chloroform of about 1/5 volume, and turn upside down abundant mixing 1min or so, and room temperature is quiet
Put 5min.Centrifuge 15min.It is careful to take out supernatant, it avoids touching middle layer, supernatant is transferred to new 1.5mL centrifuge tubes, is added
Enter isometric isopropanol, gently overturn mixing, be stored at room temperature 5min.Centrifugation 15min sucks supernatant, retains precipitation, and to heavy
70% ethyl alcohol of 1mL, centrifugation 10min washing precipitations are added in shallow lake.It sucks supernatant, precipitates after room temperature naturally dry in right amount without RNA enzyme
Water, with Tip pressure-vaccums, abundant dissolving precipitation.
(6) sample is stored in -80 DEG C.
1.2.3RNA Quality Identification
Use NanoDrop ND-1000 spectrophotometer measurement RNA concentration.Use Agilent 2100Bioanalyzer
Verify RNA mass.As a result it shows that all samples are qualified, is analyzed available for RT-PCR and chip hybridization.
1.2.4miRNA label and purifying
Laboratory sample RNA is using the mating kit of Agilent miRNA chips, miRNA Complete Labeling
And Hyb Kit (Cat#5190-0456, Agilent technologies, Santa Clara, CA, US), in sample
MiRNA molecule carries out fluorescent marker.
Operating procedure is as follows：Use preceding dilution Spike-In solution；Dephosphorylation dephosphorylations；By as follows
Shown sequence prepares dephosphorylation mixed liquor：
It takes in above-mentioned 2 μ L to sample tube of mixed liquor, total volume is 4 μ L.Mixing, centrifugation, is placed in 37 DEG C of metal baths and incubates
Educate 30min；Denaturing samples：2.8 μ L 100%DMSO are added in every pipe sample, is placed in 100 DEG C of metal baths and is incubated 7min；Even
It connects：On ice, coupled reaction mixed liquor is prepared as described below：
4.5 μ L reaction mixtures is taken to move in sample tube, total volume is 11.3 μ L, and mixing, centrifugation, 16 DEG C are incubated 2 hours；
Dry sample：(1) 16 DEG C is incubated 2 hours after reaction, need to thoroughly dry sample；It is small that 3 are drained in (2) 45 DEG C of vacuum concentration instrument
When.
1.2.5 chip hybridization and washing
According to the Standard Operating Procedure and matched reagent box of the mating offer of Agilent miRNA chips, miRNA
Complete Labeling and Hyb Kit(Cat#5190-0456,Agilent technologies,Santa Clara,
CA, US) hybridization portion, carry out the hybrid experiment of sample.In hybrid heater is rolled, Hybridization Oven (Cat#
G2545A, Agilent technologies, Santa Clara, CA, US), 55 DEG C, 20rpm, roll hybridization 20 hours.Hybridization
It develops a film, develops a film in cylinder staining dishes (Cat#121, Thermo Shandon, Waltham, MA, US) are washed after the completion
Reagent used is Gene Expression Wash Buffer Kit (Cat#5188-5327, Agilent
technologies,Santa Clara,CA,US)。
Operating procedure is as follows：
Prepare hybridization sample
1st, the sample drained is re-dissolved in 22.5 μ L mixed liquors as follows；
2nd, 22.5 μ L 2 × Hi-RPM Hybridization Buffer are often added in pipe, be slightly vortexed mixing；
3rd, it is incubated 5min in 100 DEG C of metal baths；
4th, after reaction, it is gone to rapidly and 5min is cooled down in ice-water bath；
5th, reaction solution is collected by centrifugation and carries out following step immediately.
Prepare hybrid device
1st, 45 μ L reaction solutions are slowly drawn to fence center；
2nd, chip point sample face (carrying " Agilent " printed words face) is slowly placed on cover plate downward；
3rd, assembled hybridization storehouse is placed on the shelf of hybrid heater, temperature is set in 55 DEG C, rotating speed 20rpm；
4th, hybridize 20 hours.
Chip washs
Specific wash time and temperature are as follows：
1.2.6 chip scanning
Chip results use Agilent Microarray Scanner (Cat#G2565BA, Agilent
Technologies, Santa Clara, CA, US) it is scanned, with Feature Extraction software10.7
(Agilent technologies, Santa Clara, CA, US) reads data, finally using Gene Spring Software
11.0 (Agilent technologies, Santa Clara, CA, US) are normalized, and algorithm used is
Quantile。
Specific setting sweep parameter is as follows：
1.2.7 array experiment Quality Control
The coefficient of variation (CV values) is to judge whether the system is stable by the comparative analysis between two groups of data.This
Chip is with the CV values of repetition probe points (10 repetitions) signal come the stability of computing chip and the stability of technology.As a result it shows
Show, in addition to individual samples, the CV values of most of sample are controlled within 10%, as a result reliably, available for further analyzing.
The analysis of 1.3miRNA chip of expression spectrum is determined with renal toxicity biomarker
1.3.1 the screening of difference miRNA
Using Gene Spring Software 11.0 (Agilent technologies, Santa Clara, CA, US)
Principal component analysis (Principle component analysis, PCA) is carried out between each time point each group, while in group
Animal carries out correlation analysis etc..The selection of group difference miRNA using multiple variation (Fold changes, FC) more than 2 or
Person is less than 0.5 and P<0.05.Cluster analysis is carried out at the same time to selected difference miRNA.
MiRNA can combine the 3 ' UTR in target gene, lower target gene.MiRNA microRNA target predictions utilize TargetScan
Database, miRbase databases, PicTar databases, MirTarget 2.0 and PITA database associations microRNA target
Gene.Since disparate databases have many identical target genes, the target gene method that this experiment is repeated using removal leaves
The target gene result of merging carries out subsequent analysis.
1.3.2GO analysis
The online SAS softwares (http provided by Shanghai Biochip company is used for the target gene of prediction://
Sas.ebioservice.), the online database (http based on GO://www.geneontology.org/) DEGs is carried out
The GO classification analysis of biological process, GO functional annotations and the enrichment analysis of GO functions including DEGs.It is mainly listed in this research
Be biological process classification.
MiRNA-GO Network utilize the functional annotation of target gene and miRNA-mRNA Targeted-control relationships, structure
The network of miRNA function controllings.Network can find the several genes function of miRNA regulation and control, and pass through network analysis, it is desirable to
Obtain the core gene function of core regulation and control miRNA and miRNA regulation and control.
1.3.3Pathway analysis
The target gene of prediction gives birth to differential gene using the online SAS softwares that Shanghai Biochip company provides
Object access (Pathway) is analyzed, and Pathway analyses mainly include KEGG metabolic pathways, BioCarta or the cell of target gene
Signal transduction pathway (Signal Pathway) Pathway enrichment analysis charts etc., but mainly list KEGG herein
(Kyoto Encyclopedia of Genes and Genomes, http://www.genome.adjp/kegg) analysis knot
Fruit.
It is carried out at the same time the work of miRNA-Pathway Network, miR-Pathway Network and miR-GO
Network is similar, using interaction relationship between the Pathway of target gene, with microRNA-mRNA Targeted-control relationships, structure
Build the network of miR-Pathway regulation and control.Network can find many A signal pathways of MicroRNA regulation and control, and pass through network point
Analysis obtains the core signal access of core regulation and control microRNA and microRNA regulation and control.
1.3.4 Microarray results
1.3.4.1 it summarizes
By table 1 as it can be seen that the selection criteria of difference miRNA differs, the miRNA numbers of difference also have difference, but whole become
Gesture is consistent.This experiment is more than 2 or less than 0.5 and P using FC<0.05, different time points animal difference miR-96 gene, administration 1
Difference miRNA numbers of (D2) female animals are respectively 9 and 8 after it, the difference miRNA numbers of (D4) female animals after administration 3 days
It it is 7, the difference miRNA numbers of (D8) female animals are respectively 28 and 9 after administration 7 days, and (D15) male and female are moved after administration 14 days
The difference miRNA numbers of object are respectively 44 and 51, and the difference miRNA numbers point of (R29) female animals after restoring 28 days are administered 14 days
It Wei not be 13 and 11.It can be seen that the miRNA numbers of difference increase also with the increase of administration time, wherein the difference after being administered 14 days
MiRNA quantity is most.And after restoring 28 days, quantity of the difference miRNA quantity close to after being administered 1 day.Further in P<0.05
Under the conditions of, intersection analysis is carried out to the kidney difference miRNA of D2, D4, D8 and D15, the difference miRNA numbers of female animals are sent out respectively
It is 7 and 8 existing.
To rat kidney miRNA difference numbers after 1 gentamicin of table induction injury of kidney
2mean3 represents that FC is more than 2, and average signal value is more than 3.
1.3.4.2GO analysis result
After giving gentamicin, rat kidney is taken, and compared with negative control group in D2, D4, D8, D15 and R29 time point
Obtain difference miRNA.The target gene number of difference miRNA is very more, and GO analyses and KEGG path analysis are carried out to target gene, may
All functions and access are annotated, therefore concentrates and intersection is carried out to D4, D8 and D15 time point, and to the difference miRNA of intersection
The target gene of prediction further makees GO analyses and KEGG path analysis.
To the target gene number of rat kidney difference miRNA predictions after 2 gentamicin of table induction injury of kidney
One of difference miRNA of each time point intersection of jenny is rno-miR-877 (its nucleotide sequence such as sequence tables
Shown in middle SEQ ID No.1), regulation and control target gene core gene function be mainly nitrogen phosphorus compound metabolism, RNA metabolism and
(tables 3) such as transcription, protein kinase adjusting, gene expression regulation, embryo cuttings.
One of difference miRNA of each time point intersection of buck be rno-miR-877, regulation and control target gene core
Gene function is mainly the metabolism of nitrogen phosphorus compound, RNA metabolism and transcription, protein kinase adjusting, embryo cutting, neuron hair
Educate differentiation etc., it is seen that the oligogene function being related to of female animals is similar (table 4).
The target gene GO analyses of each time point intersection of 3 jenny of table
The target gene GO analyses of each time point intersection of 4 buck of table
1.3.4.3Pathway analysis result
By table 5 as it can be seen that the change of the biological pathways of the target gene enrichment of female rats intersection miRNA, relates generally to cancer
Disease signal path, such as prostate cancer, Small Cell Lung Cancer, melanogenesis, cell cycle, prion disease, MARK signal paths etc..
By table 6 as it can be seen that the change of the biological pathways of the target gene enrichment of male rat intersection miRNA, is mainly directed to
Cancer signal path, such as prostate cancer, clear-cell carcinoma, Small Cell Lung Cancer, melanogenesis, the cell cycle, phagocytosis, MARK,
P53, TGF-β and T cell receptor signal path, protein breakdown, leucocyte movement etc..It can be seen that the biology being related to of female animals
It is similar to learn access.
The Pathway analyses of the target gene of each time point intersection of 5 jenny of table
The Pathway analyses of the target gene of each time point intersection of 6 buck of table
1.3.4.4 it summarizes
The miRNA chip results of this experiment nephridial tissue show that each the miRNA expression patterns of group animal are similar, often
The correlation of sample is more slightly worse than full genome detection of expression result in group, it may be possible to since miRNA numbers are less related.But this experiment
The animal miRNA expression patterns of (D15) differ farthest with the expression pattern of negative control group animal after administration 14 days, are secondly administered
The animal miRNA expression patterns of (D8) after 7 days.Difference miRNA numbers increase also with the increase of administration time, and restore 28 days
Afterwards, quantity of the difference miRNA quantity close to after being administered 1 day, it is seen that the change of miRNA is related with giving gentamicin toxicity kidney
Connection.
The target gene number of nephridial tissue each time point difference miRNA is very more, but each time point intersection to jenny
One of difference miRNA be rno-miR-877, the core gene function of the target gene of regulation and control be mainly the metabolism of nitrogen phosphorus compound,
RNA metabolism and transcription, protein kinase adjusting, gene expression regulation, embryo cutting etc..And each time point intersection of buck
One of difference miRNA be rno-miR-877, the core gene function of the target gene of regulation and control is similar with jenny.Further
Path analysis show that female relates generally to cancer signal path, such as prostate cancer, Small Cell Lung Cancer, melanogenesis, cell week
Phase, prion disease, MARK signal paths etc..And male rat is mainly directed to cancer signal path, as prostate cancer, kidney are thin
Born of the same parents' cancer, Small Cell Lung Cancer, melanogenesis, cell cycle, phagocytosis, MARK, p53, TGF-β and T cell receptor signal path,
Protein breakdown, leucocyte movement etc..It can be seen that the biological pathways that female animals are related to are similar.It can be seen that above-mentioned difference miRNA
Can be the neoformation marker of detection renal toxicity, especially female animals are total to discrepant rno-miR-877.
Embodiment 2 detects the expression quantity of mo-miR-877 in blood in injury of kidney rat
In Example 1, the rat three and same breed normal growth of administration (80mg/kg, gentamicin) after 7 days
Rat three.Their total serum IgE is extracted respectively, and abstracting method is same as Example 1.
By the total serum IgE of extraction carry out reverse transcription obtain cDNA (Reverse Transcription and system referring to II RT kit of miScript,
Purchased from German Qiagen companies).Wherein, the 5 ╳ miScript containing 4 μ L in the reaction solution that the system of reverse transcription is every 20 μ L
HiFlex Buffer, 10 ╳ miScriptNucleics Mix of 2 μ L, 2 μ L miScript Reverse
Transcriptase Mix, remainder for RNA templates and remove RNA enzyme water.The program of reverse transcription is：37 DEG C, 1h；95 DEG C, 5min；4
DEG C, heat preservation.
Then by the expression quantity of qPCR detections mo-miR-877, (qPCR reagents and system are referring to KAPAFAST
QPCR Kit, purchased from Kapa Biosystems companies of the U.S.).Wherein, the primer of rno-miR-877 is rno-miR-877f (its
Nucleotide sequence is referring to specification sequence table SEQ ID No.2).7900HT Sequence Detection System (are purchased from
American AB I companies) on qPCR programs be：95 DEG C, 2min；94 DEG C, 15s；60 DEG C, 1min.So as to detect mo- in sample in real time
The expression quantity of miR-877.
The result shows that using the rat after 80mg/kg gentamicins 3 days and 14 days and normal rat are administered in embodiment 1
It compares, the content of mo-miR-877 significantly reduces, and jenny is the content of the mo-miR-877 of intact animal after administration 3 days
0.73 times, buck is 0.86 times of intact animal；But to after being administered 14 days, jenny is the mo- of intact animal
0.46 times of the content of miR-877, and buck is 0.65 times of intact animal.
Meanwhile pathological diagnosis shows that the slight damage of rat kidney of the 80mg/kg gentamicins after 3 days is administered in embodiment 1
Hinder and significantly produce injury of kidney after causing 14 days.It follows that mo-miR-877 can be used as a kind of renal toxicity biomarker
Injury of kidney caused by detecting exogenous compounds.
Embodiment 3 detects the expression quantity of mo-miR-877 in renal tissue in injury of kidney rat
The renal tissue of rat and normal rat is administered in Example 1, extracts RNA respectively, reverse transcription obtains cDNA, logical
The expression quantity for crossing mo-miR-877 in qPCR detection renal tissues (extracts RNA, reverse transcription obtains cDNA, detects by qPCR
Method is same as Example 2).
The results show that embodiment 3 is similar to 2 result of embodiment, illustrate that mo-miR-877 can be used as a kind of renal toxicity to give birth to
Injury of kidney caused by object marker detection exogenous compounds.
It should be understood that the foregoing is merely illustrative of the preferred embodiments of the present invention, the limit to right is not formed
System, other substantially equivalent replacements that those skilled in that art are contemplated that all fall in the scope of protection of the present invention.
Claims (9)
- Purposes of the 1.rno-miR-877 in renal toxicity biomarker is prepared, which is characterized in that the renal toxicity derives from Injury of kidney caused by gentamicin.
- 2. purposes as described in claim 1, which is characterized in that rno-miR-877 is as unique renal toxicity biomarker It is used alone or is used in combination with conventional renal toxicity biomarker.
- 3. purposes as claimed in claim 2, which is characterized in that the conventional renal toxicity biomarker is serum flesh Acid anhydride, urea nitrogen, urine total protein and/or clusterin.
- 4. the kit of injury of kidney caused by a kind of detection gentamicin, which is characterized in that it includes specific detection rno- The primer of miR-877, the primer are DNA fragmentation of the nucleotide sequence as shown in SEQ ID No.2 in sequence table.
- 5. kit as claimed in claim 4, which is characterized in that its further include RNA extraction agents, Reverse Transcription and/or QPCR reagents.
- 6. kit as claimed in claim 5, which is characterized in that the RNA extraction agents include TRIZOL.
- 7. kit as claimed in claim 5, which is characterized in that the Reverse Transcription includes RNA reverse transcriptases.
- 8. kit as claimed in claim 5, which is characterized in that the qPCR reagents include archaeal dna polymerase.
- 9. a kind of primer of specific detection rno-miR-877 is in the preparation of injury of kidney caused by preparation detection gentamicin Purposes, which is characterized in that it is DNA fragmentation of the nucleotide sequence as shown in SEQ ID No.2 in sequence table.
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| Concordant Changes of Plasma and Kidney MicroRNA in the Early Stages of Acute Kidney Injury: Time Course in a Mouse Model of Bilateral Renal Ischemia-Reperfusion;Melissa A. Bellinger et al;《PLOS》;20140430;第9卷(第4期);e93297 * |
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