CN104878117A - Method for screening tiny RNA modified through oxidation - Google Patents
Method for screening tiny RNA modified through oxidation Download PDFInfo
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- CN104878117A CN104878117A CN201510350933.8A CN201510350933A CN104878117A CN 104878117 A CN104878117 A CN 104878117A CN 201510350933 A CN201510350933 A CN 201510350933A CN 104878117 A CN104878117 A CN 104878117A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
- C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
Abstract
The invention relates to a method for screening tiny RNA modified through oxidation. The method comprises the following steps that a cardiac muscle cell of a mammal is selected, 8-oxoguanine is generated through oxidation guanine 8-bit carbon under the oxidative stress state, and oxidative damage processing is carried out on the tiny RNA; the cell tiny RNA on which oxidative damage processing is carried out is extracted through a flash PAGETM distiller system, and the oxidized tiny RNA is purified through the immunity deposition method; a tiny RNA expression profile chip is used for carrying out detection, and the tiny RNA subtype variety with oxidative damage is screened; the tiny RNA sequence is shown in an SEQ IDNO:1-11 in a sequence table. The method is simple in technology process, reliable in damage principle, good in screening effect, wide in application range and user-friendly in using environment.
Description
Technical field:
The present invention relates to a kind of Microrna screening method of oxidative modification, belong to biologic applications technical field.
Background technology:
At present, under oxidative stress (oxidative stress) state, apoptotic molecular mechanism is the key problem of free radical and oxidative damage theory; Noncoding little nucleic acid molecule---Microrna (microRNA, miRNA) be that a newfound class was about the non-coding RNA that 22nt (19-25nt) belongs to conservative in evolution in recent years, this microRNA acts on mRNA by complementary pairing principle, affect protein translation process thus and cause gene silencing, its effect is presented as to regulate and control genetic expression at post-transcriptional level.In the prior art, increasing evidence shows the keying action of miRNA in various biological process, and miRNA has become the important tool of life science and the new tool of various diseases treatment.
But this area is still long-term and continue to need to differentiate the change of miRNA in oxidative stress process and biological action thereof, but have not yet to see the open report of this respect.
Summary of the invention:
The object of the invention is to the deficiency overcoming prior art existence, seek Microrna screening method and application thereof that a kind of oxidative modification is provided, identify H
2o
2the miRNA subtype category of oxidative damage is there is in inducing cardiomyocytes apoptotic process; Examination goes out the difference regulatory gene of miRNA and oxidation miRNA in myocardial cell, and promotes its application.
In order to realize foregoing invention object, the screening method that the present invention relates to comprises the steps:
(1) choose and make mammiferous myocardial cell produce 8-oxygen guanines by oxidation guanine 8 carbon under oxidative stress status to make the process of Microrna generation oxidative damage;
(2) flashPAGE is adopted
tMthe Microrna of the cell after fractionator systems extraction step (1) oxidative damage process, is purified into the Microrna of oxidation by immuno-precipitation;
(3) adopt microrna expression spectrum chip to detect, examination goes out the Microrna subtype category of oxidative damage;
Wherein, described Microrna be selected from rno-miR-184, rno-miR-21*, rno-miR-135a*, rno-miR-139-3p, rno-miR-30c-2*, rno-miR-290, rno-miR-29a, rno-miR-23a, rno-miR-21, rno-miR-204* and rno-miR-106b one or more, its sequence is as shown in SEQ IDNO:1-11 in sequence table:
rno-miR-184(5’-uggacggagaacugauaagggu-3’(SEQ ID NO:1))
rno-miR-21*(5’-caacagcagucgaugggcuguc-3’(SEQ ID NO:2))
rno-miR-135a*(5’-uguagggauggaagccaugaaa-3’(SEQ ID NO:3))
rno-miR-139-3p(5’-uggagacgcggcccuguuggag-3’(SEQ ID NO:4))
rno-miR-30c-2*(5’-cugggagaaggcuguuuacucu-3’(SEQ ID NO:5))
rno-miR-290(5’-cucaaacuaugggggcacuuuuu-3’(SEQ ID NO:6))
rno-miR-29a(5’-uagcaccaucugaaaucgguua-3’(SEQ ID NO:7))
rno-miR-23a(5’-aucacauugccagggauuucc-3’(SEQ ID NO:8))
rno-miR-21(5’-uagcuuaucagacugauguuga-3’(SEQ ID NO:9))
rno-miR-204*(5’-gcugggaaggcaaagggacguu-3’(SEQ ID NO:10))
rno-miR-106b(5’-uaaagugcugacagugcagau-3’(SEQ ID NO:11))
When making Microrna generation oxidative damage in described step (1), with Fenton reagent oxidation system oxidative damage Microrna, or use H
2o
2oxidative damage Microrna in the process of process cell death inducing; Wherein, Fenton reagent oxidation system is by Cu
+or Fe
2+with H
2o
2composition; Cu
+/ Fe
2+concentration be respectively 0.5mM, H
2o
2concentration be 0.1-2mM; Or Fenton reagent oxidation system is by Fe
3+or Cu
2+respectively with xitix (Asc) and H
2o
2be mixed with in phosphate buffered saline buffer; Use H
2o
2in the process of process inducing cardiomyocytes apoptosis, H
2o
2concentration be 0.4-1mM; Use H
2o
2the time of process inducing cardiomyocytes apoptosis is 1-6 hour.
The method of the difference regulatory gene of Microrna and its oxidation Microrna in the screening myocardial cell that the present invention relates to, comprises the following steps:
(1) make cell produce 8-oxygen guanine by oxidation guanine 8 carbon under oxidative stress status, make Microrna generation oxidative damage with Fenton reagent oxidation system; And the Microrna of oxidation is purified into by immuno-precipitation;
(2) adopt the Microrna of method process LAN Microrna and its oxidation in H9c2 myocardial cell of liposome transient transfection cell, extract cell total rna; Adopt H
2o
2process H9c2 myocardial cell;
(3) adopt full-length genome chip of expression spectrum to detect, examination goes out the difference regulatory gene of Microrna and its oxidation Microrna in myocardial cell; Wherein, the Microrna of oxidative damage is selected from the arbitrary sequence as shown in SEQ IDNO:1-11; Microrna be selected from rno-miR-184, rno-miR-21*, rno-miR-135a*, rno-miR-139-3p, rno-miR-30c-2*, rno-miR-290, rno-miR-29a, rno-miR-23a, rno-miR-21, rno-miR-204* and rno-miR-106b one or more.
The H of cell levels in the present invention's Fenton reagent oxidation system in vitro
2o
2inducing cardiomyocytes apoptotic process, and in iron doping and ischemia-reperfusion induction myocardial damage animal model, there is oxidative damage in miRNA, generation 8-oxygen guanine; There is following characteristics:
(1) miRNA is at Cu
+/ Fe
2+with H
2o
2form in Fenton reagent oxidation system and oxidative damage occurs, i.e. guanine 8 carbon oxidized generation 8-oxygen guanines, and separately with Fe
3+/ Asc or H
2o
2process, can not cause miRNA oxidative damage, and the OH free radical that Fenton reagent oxidation system produces is that the reaction of miRNA oxidative damage is necessary;
(2) at H
2o
2in induction H9c2 apoptosis of cardiac muscle process there is oxidative damage in miRNA, and at 0-1mM H
2o
2within the scope of concentration for the treatment of, the miRNA oxidative damage degree of H9c2 cell and H
2o
2process is in dose-dependent relationship; With 0.4mM H
2o
2process H9c2 cell, miRNA oxidative damage degree and H in cell
2o
2process is in time-dependent sexual intercourse;
(3) iron doping and ischemia-reperfusion induction myocardial damage animal model is adopted, find consistent with active oxygen and apoptosis detected result, in iron doping group mouse each organ miRNA sample, 8-oxygen guanine content more obviously raises with placebo, and in the miRNA sample of ischemia-reperfusion group mouse heart hazardous area, 8-oxygen guanine content more obviously raises with sham operated rats.
The present invention determines H
2o
2the miRNA subtype category of oxidative damage is there is in inducing cardiomyocytes apoptotic process, by generating in H9c2 myocardial cell oxidative damage miRNA and degraded, locating features, act synergistically with other biological macromole and the research of oxidative damage miRNA regulate gene expression function difference, contribute to fullying understand the molecular mechanism of the pathological processes such as apoptosis and necrosis under oxidative stress status, it can be used as the strong research means of oxidative stress model of cell apoptosis; Adopt miRNA chip of expression spectrum to analyze and qRT-PCR method validation, the miRNA subtype category of 11 oxidative damages of confirmation is:
rno-miR-184(5’-uggacggagaacugauaagggu-3’(SEQ ID NO:1))
rno-miR-21*(5’-caacagcagucgaugggcuguc-3’(SEQ ID NO:2))
rno-miR-135a*(5’-uguagggauggaagccaugaaa-3’(SEQ ID NO:3))
rno-miR-139-3p(5’-uggagacgcggcccuguuggag-3’(SEQ ID NO:4))
rno-miR-30c-2*(5’-cugggagaaggcuguuuacucu-3’(SEQ ID NO:5))
rno-miR-290(5’-cucaaacuaugggggcacuuuuu-3’(SEQ ID NO:6))
rno-miR-29a(5’-uagcaccaucugaaaucgguua-3’(SEQ ID NO:7))
rno-miR-23a(5’-aucacauugccagggauuucc-3’(SEQ ID NO:8))
rno-miR-21(5’-uagcuuaucagacugauguuga-3’(SEQ ID NO:9))
rno-miR-204*(5’-gcugggaaggcaaagggacguu-3’(SEQ ID NO:10))
rno-miR-106b(5’-uaaagugcugacagugcagau-3’(SEQ ID NO:11))。
In the miRNA of the oxidative damage that the present invention relates to, miR-184 and oxi-miR-184 difference regulatory gene; Adopt rat full-length genome chip of expression spectrum to analyze, final confirmation process LAN miR-184 can make 33 genes in mRNA level in-site generation noticeable change, and process LAN oxi-miR-184 can make 66 genes occur significantly to become in mRNA level in-site; Difference regulatory gene comprises BCL2L1 and BCL2L2; BCL2L1 and BCL2L2 belongs to apoptosis suppressive gene Bcl-2 family together, coded protein product is respectively Bcl-xL and Bcl-w, Bcl-2 family member plays a role with dimeric form usually, Bcl-xL and Bcl-w has the apoptotic function of antagonism, under oxidative stress status, oxidative damage is there is with miRNA, it regulates the function of gene expression dose in cell also to change, and occurs difference regulatory gene.
Compared with prior art, its technological process is simple in the present invention, and damage principle is reliable, and screening effect is good, applied range, and environment for use is friendly.
Accompanying drawing illustrates:
Fig. 1 is the 8-oxygen guanine that HPLC-UV/MS detects in miRNA sample, and wherein a is 8-oxygen guanine standard substance; B is that double-strand miRNA has-let-7a-1 contrasts; C is iron (Fe
3+) and xitix (Asc) treatment group; D is H
2o
2treatment group; E is Fe
3+/ Asc+H
2o
2treatment group; F is Cu
2+/ Asc+H
2o
2treatment group.
Fig. 2 is H
2o
2induce miRNA oxidative damage in myocardium H9c2 apoptosis process, wherein a is the different concns H with 0-200 μM
2o
2process H9c2 cell, Northwestern Blot analyzes 8-oxygen guanine content result; B detects reactive oxygen species result of variations for adopting DCF fluorescent probe; C is with the H of 0.4mM concentration
2o
2process H9c2 cell 0-6h, gets the result that different time points detects 8-oxygen guanine content in miRNA; D is reactive oxygen species result of variations.
Fig. 3 is the oxidative damage of miRNA in iron doping induction myocardial injury models, and wherein a is the foundation of iron doping induction myocardial injury models; B is mouse heart left ventricle cytoactive oxygen level; C is mouse heart left ventricle Level of Apoptosis; D is the result that Northwestern Blot analyzes 8-oxygen guanine content in mouse core, liver, spleen, lung, kidney and each organ miRNA of brain.
Fig. 4 is the oxidative damage of miRNA in Myocardial Ischemia Reperfusion Injury model, and wherein a is the foundation of Myocardial Ischemia Reperfusion Injury model; B is mouse heart frontier district cytoactive oxygen level; C is mouse heart frontier district Level of Apoptosis; D is the result that Northwestern Blot analyzes 8-oxygen guanine content in mouse heart hazardous area and non-hazardous area miRNA.
Fig. 5 is chip detection oxidation miRNA, and wherein a is the result that Northwestern Blot identifies the oxidation miRNA that immuno-precipitation purifying obtains; B is the result of chip detection oxidation miRNA; C is the result utilizing qRT-PCR method validation chip.
Fig. 6 is the difference regulatory gene that the screening of rat full-length genome chip of expression spectrum obtains process LAN miR-184 and process LAN oxi-miR-184.
Embodiment:
Also by reference to the accompanying drawings the inventive method is further elaborated below by specific embodiment.
Embodiment 1: the concrete steps of the present embodiment
(1) Fenton reagent oxidation system causes miRNA oxidative damage, produces 8-oxygen guanine
Cu
+/ Fe
2+with H
2o
2forming Fenton reagent oxidation system and have extremely strong oxidation capacity, is the principal reaction that organism produces OH (hydroxyl radical free radical); Apply the double-strand miRNA mimics sample (hsa-let-7a-1:5 '-UGAGGUAGUAGGUUGUAUAGUU-3 ' SEQ ID NO:12) of this reaction system external oxidation chemistry synthesis, i.e. miRNA (80 μ g) and 0.5mM ironic citrate (Fe
3+) or copper sulfate (Cu
2+), 5mM xitix (Asc) and 2mM H
2o
2, at 10mMNaH
2pO
4/ Na
2hPO
4in damping fluid (pH 7.4) system, hatch 1h for 37 DEG C.After hatching end, add 10 μ l 100mM deferoxamine mesylate (iron ion scavenging agent) or bisoxalydihydrazone (cupric ion scavenging agent) with termination reaction; The 3M NaAc of 1/10 volume is added, 1 μ g/ml glycogen of 1/10 volume and the ethanol of 2.5 times of volumes ,-20 DEG C of precipitation miRNA in above-mentioned solution; Add 10 μ l nuclease P 1s in the miRNA sample that above-mentioned precipitation obtains and (300mM NaAc and 0.2mM ZnCl can be stored in purchased from Promega, 0.4U/ μ l
2, pH 5.3 ,-20 DEG C are frozen), hatch 2h for 37 DEG C, then add 5 μ l alkaline phosphatases (can purchased from Promega, 1U/ μ l), hatch 60min for 50 DEG C; The centrifugal 10s of 2000g; 100 μ l enzymolysis products are transferred to Micropure-EZ filter (can purchased from Millipore), and 0 DEG C, the centrifugal 2min of 14000g is to remove albumen; The centrifugal clear liquid obtained, as described in carry out HPLC-UV/MS analysis;
Result confirms, at Cu
+/ Fe
2+with H
2o
2form in Fenton reagent oxidation system, there is oxidative damage in miRNA, i.e. guanine 8 carbon oxidized generation 8-oxygen guanine (Fig. 1 e and Fig. 1 f), and Fe
3+/ Asc group and H
2o
2group, because not producing OH, therefore does not cause miRNA oxidative damage (Fig. 1 c and Fig. 1 d);
(2) the purify oxidized miRNA of immuno-precipitation and chip detection oxidation miRNA;
(3) adopt microrna expression spectrum chip to detect, examination goes out the Microrna subtype category of oxidative damage.
Supplemental content
Embodiment 2: the concrete steps of the present embodiment
(1) H
2o
2induce miRNA oxidative damage in myocardium H9c2 apoptosis process, produce 8-oxygen guanine
The present embodiment is for detecting H
2o
2miRNA oxidative damage degree in induction H9c2 myocardial cell's (it can derive from American Type CultureCollection) apoptotic process;
Respectively with the different concns H of 0-200 μM
2o
2process H9c2 cell, after 3h, collecting cell carries out active oxygen (Reactiveoxygen species, ROS) detection (it can derive from Invitrogen), and adopts flashPAGE
tMfractionator systems (Ambion, Inc) extracts miRNA, as described in carry out Northwestern Blot analysis, the results are shown in Figure 2a and Fig. 2 b;
Result shows, with 0mM H
2o
2treatment group is contrast, 25 μMs, 50 μMs, 100 μMs and 200 μMs of H
2o
2the intracellular ROS level for the treatment of group raises multiple and is respectively 2.25 ± 0.19,3.43 ± 0.31,5.54 ± 0.46,6.72 ± 0.57 (p<0.05); Correspondingly, 25 μMs, 50 μMs, 100 μMs and 200 μMs of H
2o
2in the cell for the treatment of group, 8-oxygen guanine content raises multiple and is respectively 1.41 ± 0.11,2.01 ± 0.19,3.35 ± 0.32,3.69 ± 0.29 (p<0.05).Confirm thus, at 0-1mM H
2o
2within the scope of concentration for the treatment of, the miRNA oxidative damage degree of H9C2 cell and H
2o
2concentration is proportionate; In addition, with the H of 0.4mM concentration
2o
2process H9c2 cell, the time point collecting cell respectively after 0h, 0.5h, 1h, 2h, 4h and 6h carries out ROS and detects and Northwestern Blot analysis (Fig. 2 c and Fig. 2 d);
Result shows, and with 0h treatment group for contrast, the intracellular ROS level of 1h, 2h, 4h and 6h treatment group raises multiple and is respectively 3.29 ± 0.21,4.71 ± 0.35,5.35 ± 0.49,6.79 ± 0.62,5.99 ± 0.51 (p<0.05); Correspondingly, in the cell of 1h, 2h, 4h and 6h treatment group, 8-oxygen guanine content raises multiple and is respectively 1.69 ± 0.55,2.14 ± 0.18,3.01 ± 0.26,3.72 ± 0.31,4.06 ± 0.39 (p<0.05); Confirm thus, with 0.4mM H
2o
2process H9c2 cell, miRNA oxidative damage degree and H in cell
2o
2treatment time is proportionate;
(2) the purify oxidized miRNA of immuno-precipitation and chip detection oxidation miRNA;
For qualification H
2o
2cell death inducing cause the miRNA subtype category of oxidative damage, the present embodiment is with 0.4mM H
2o
2process H9c2 myocardial cell, collecting cell after 3h, extracts control group (0mM H respectively
2o
2treatment group) and H
2o
2(0.4mM H
2o
2treatment group) cell miRNA, immuno-precipitation purifying is adopted to obtain being oxidized miRNA, then miRNA chip detection is carried out, details are as follows for concrete grammar: 2.5 μ g miRNA and 2.5 μ g anti-8-oxoG antibody (can derive from QEDBioscience) are at room temperature hatched 1h, and negative control group omits antibody incubation or antibody hatches 1h in advance with 24ng/ μ l 8-oxoG; Add Protein L gel beads (can Pierce be derived from, 20 μ l) and hatch 15h under 4 DEG C of conditions; After hatching end, wash 3 times by the PBS solution containing 0.04% (v/v) NP-40; Subsequently, add successively in ProteinL gel beads by under such as order: 300 μ l containing the PBS solution of 0.04% (v/v) NP-40,10% (w/v) SDS of 30 μ l and the PCI (phenol: chloroform: primary isoamyl alcohol is 25:24:1) of 300 μ l; This mixed solution is hatched under 37 DEG C of conditions 15min (every 5min vibration once), the centrifugal 5min of 14,000rpm, in order to water phase separated and organic phase; Collect aqueous phase, and with 40 μ l 3M sodium-acetate (pH5.2), 2 μ l 5mg/ml glycogens and 1ml 95% (v/v) ethanol mixing; By frozen for this sample in-80 DEG C, more than 1h, subsequently again with the miRNA that the centrifugal 20min of 14,000rpm is precipitated; To go forward side by side line space air dry by 75% washing with alcohol, by miRNA sample dissolution in 10 μ l DEPC process water, obtain being oxidized miRNA; The oxidation miRNA that adopts Northwestern Blot to identify immuno-precipitation purifying to obtain (Fig. 5 a);
(3) adopt microrna expression spectrum chip to detect, examination goes out the Microrna subtype category of oxidative damage;
MiRNA chip of expression spectrum (miRCURY
tMlNA Array (v.10.0), becomes biological company limited purchased from health) screening, utilize miRCURY
tMchip mark test kit and RNEasy Mini test kit (can derive from Exiqon) carry out miRNA mark and concentrated mark sample, adopt miRCURY
tMchip microarray test kit carries out miRNA chip hybridization, and at 635nm place fluorescence excitation, utilizes Genepix 4000B to carry out image scanning and use Genepix Pro 6.0 to carry out data analysis (Fig. 5 b); By to the analysis of oxidation miRNA group (oxi-miR) standard value, oxi-miR group finds that there is 11 miRNA altogether, is respectively rno-miR-184, rno-miR-21*, rno-miR-135a*, rno-miR-139-3p, rno-miR-30c-2*, rno-miR-290, rno-miR-29a, rno-miR-23a, rno-miR-21, rno-miR-204* and rno-miR-106b (table 1); As described in adopt qRT-PCR method to verify oxi-miR group 11 miRNA, its result consistent with chip detection result (Fig. 5 c);
The screening of table 1 gene chip and qRT-PCR verify the microRNA of oxidative damage
Embodiment 3: miRNA oxidative damage in iron doping induction myocardial injury models, produces 8-oxygen guanine
The present embodiment can cause ROS level in born of the same parents significantly to raise for iron doping (iron overload) in myocardial cell, and causes the impaired and apoptosis of cardiac muscle degree of myocardial function to increase; Adopt and regularly quantitatively sets up iron doping model fast to male C57BL/6 mouse (it can derive from Institute of Zoology, Academia Sinica's Experimental Animal Center) abdominal injection Iron Dextran, dosage is 12mg/ days, one Friday sky, put to death after injection surrounding; Heart tissue paraffin section finds after Prussian blue NiHCF thin films, iron doping (Fe) organizes visible blue iron particle deposition in tenuigenin, and placebo (Plecebo) is organized cell and had no iron particle deposition, confirm the successful foundation of mouse iron doping model (Fig. 3 a); Meanwhile, Fe group cell ROS and level of apoptosis and the obvious rising of Plecebo group (Fig. 3 b and Fig. 3 c); In order to evaluate miRNA oxidative damage degree in iron doping model further, extract each organ miRNA of mouse core, liver, spleen, lung, kidney and brain respectively and carry out Northwestern Blot analysis, in Fe group mouse each organ miRNA sample, 8-oxygen guanine content more obviously raises (Fig. 3 d) with Plecebo group.
Embodiment 4: miRNA oxidative damage in Myocardial Ischemia Reperfusion Injury model, produces 8-oxygen guanine
Each group of mouse is opened following coronary artery occlusion left anterior descending branch after chest by the present embodiment, records the electrocardiogram(ECG after ECG electrocardiogram(ECG and ligation 30min after Reperfu-sion before ligation respectively; Engender after animal coronary artery ligation that ST section is raised, illustrate that mouse left room antetheca has obvious ischemic; After perfusion 2h terminates, the two dye method of azovan coerulen-TTC is adopted to measure myocardial infarct size; Hazardous area (areaat risk, AAR) is red and white indicating area; Infarcted region (infarct area, INF) is white indicating area, and the successful foundation of confirmed myocardial ischemia reperfusion injury model (Fig. 4 a); Meanwhile, ischemia-reperfusion (I/R) is organized cell ROS and level of apoptosis and sham-operation (Sham) and is organized obvious rising (Fig. 4 b and Fig. 4 c); In order to evaluate miRNA oxidative damage degree in myocardial ischemia-reperfusion model further, extract mouse heart hazardous area (AAR) and non-hazardous area (areanot at risk respectively, ANAR) miRNA carries out Northwestern Blot analysis, 8-oxygen guanine content and the obvious rising of Sham group (Fig. 4 d) in I/R group mouse heart hazardous area miRNA.
Embodiment 5: the examination that process LAN miR-184 and oxidation miR-184 (oxi-miR-184) difference regulatory gene are expressed
The present embodiment is that evaluation miR-184 and oxi-miR-184 is on the impact of H9c2 cellular gene expression level, adopt method process LAN miR-184 and oxi-miR-184 of liposome transient transfection H9c2 cell, extract cell total rna, carry out rat full-length genome expression pattern analysis, details are as follows for concrete grammar:
Adopt Fe described in embodiment 1
2+with H
2o
2form Fenton reagent oxidation system, the miR-184mimics of chemosynthesis is carried out external oxidation, and then adopt immunoprecipitation method purifying described in embodiment 2 to obtain oxi-miR-184; Adopt liposome method transient transfection H9c2 cell 5nM miR-184 and oxi-miR-184 respectively, collecting cell after 24h, extract process LAN respectively with Trizol reagent (can Invitrogen be derived from) and contrast 1 group (miR-NC-1 group) and process LAN miR-184 (miR-184 group), process LAN contrasts 2 groups (miR-NC-2 groups) and process LAN oxi-miR-184 cell total rna, and uses further
rNA clean-up test kit carried out column purification to total serum IgE, quantitative with spectrophotometer, the quality inspection of denaturing formaldehyde gel electrophoresis;
The screening of rat full-length genome chip of expression spectrum (27K Rat Genome Array, purchased from CapitalBio company).Adopt
cRNA amplification label test kit carries out fluorescent mark to sample RNA; Chip LuxScan 10KA twin-channel laser scanner (CapitalBio company) scans, LuxScan 3.0 image analysis software (CapitalBio company) is adopted to analyze chip image, picture signal is converted into numerary signal, observes the gene (Fig. 6) of differential expression; By analyzing each group of standard value, (miR-184/miR-NC≤0.5 is for significantly lowering in mRNA level in-site generation noticeable change to find to make 33 genes by process LAN miR-184, miR-184/miR-NC>=2 are significantly raise), process LAN oxi-miR-184 can make 66 genes, and in mRNA level in-site generation noticeable change, (oxi-miR-184/miR-NC≤0.5 is for significantly lowering, oxi-miR-184/miR-NC>=2 are significantly raise), difference regulatory gene comprises PRKCB1, BCL2L1 and BCL2L2 etc., the results are shown in Table 2;
The difference regulatory gene of table 2 gene chip screening process LAN miR-184 and oxi-miR-184
The present embodiment achieves the screening of the Microrna of oxidative modification, and reaches application purpose.
Sequence table
SEQ ID NO:1
rno-miR-184 (5’-uggacggagaacugauaagggu-3’)
SEQ ID NO:2
rno-miR-21* (5’-caacagcagucgaugggcuguc-3’)
SEQ ID NO:3
rno-miR-135a* (5’-uguagggauggaagccaugaaa-3’)
SEQ ID NO:4
rno-miR-139-3p (5’-uggagacgcggcccuguuggag-3’)
SEQ ID NO:5
rno-miR-30c-2* (5’-cugggagaaggcuguuuacucu-3’)
SEQ ID NO:6
rno-miR-290 (5’-cucaaacuaugggggcacuuuuu-3’)
SEQ ID NO:7
rno-miR-29a (5’-uagcaccaucugaaaucgguua-3’)
SEQ ID NO:8
rno-miR-23a (5’-aucacauugccagggauuucc-3’)
SEQ ID NO:9
rno-miR-21 (5’-uagcuuaucagacugauguuga-3’)
SEQ ID NO:10
rno-miR-204* (5’-gcugggaaggcaaagggacguu-3’)
SEQ ID NO:11
rno-miR-106b (5’-uaaagugcugacagugcagau-3’)
SEQ ID NO:12
5’-UGAGGUAGUAGGUUGUAUAGUU-3’
Claims (3)
1. a Microrna screening method for oxidative modification, is characterized in that comprising the steps: that (1) is chosen mammiferous myocardial cell and make the process of Microrna generation oxidative damage by oxidation guanine 8 carbon generation 8-oxygen guanines under oxidative stress status; (2) flashPAGE is adopted
tMthe Microrna of the cell after fractionator systems extraction step (1) oxidative damage process, is purified into the Microrna of oxidation by immuno-precipitation; (3) adopt microrna expression spectrum chip to detect, examination goes out the Microrna subtype category of oxidative damage; Wherein, described Microrna be selected from rno-miR-184, rno-miR-21*, rno-miR-135a*, rno-miR-139-3p, rno-miR-30c-2*, rno-miR-290, rno-miR-29a, rno-miR-23a, rno-miR-21, rno-miR-204* and rno-miR-106b one or more, its sequence is as shown in SEQ IDNO:1-11 in sequence table.
2., according to the Microrna screening method of a kind of oxidative modification shown in claim 1, when it is characterized in that making Microrna generation oxidative damage in described step (1), with Fenton reagent oxidation system oxidative damage Microrna, or use H
2o
2oxidative damage Microrna in the process of process cell death inducing; Wherein, Fenton reagent oxidation system is by Cu
+or Fe
2+with H
2o
2composition; Cu
+/ Fe
2+concentration be respectively 0.5mM, H
2o
2concentration be 0.1-2mM; Or Fenton reagent oxidation system is by Fe
3+or Cu
2+respectively with xitix and H
2o
2be mixed with in phosphate buffered saline buffer; Use H
2o
2in the process of process inducing cardiomyocytes apoptosis, H
2o
2concentration be 0.4-1mM; Use H
2o
2the time of process inducing cardiomyocytes apoptosis is 1-6 hour.
3. one kind is screened the method for the difference regulatory gene of Microrna and its oxidation Microrna in myocardial cell, comprise the following steps: (1) makes cell produce 8-oxygen guanine by oxidation guanine 8 carbon under oxidative stress status, makes Microrna generation oxidative damage with Fenton reagent oxidation system; And the Microrna of oxidation is purified into by immuno-precipitation; (2) adopt the Microrna of method process LAN Microrna and its oxidation in H9c2 myocardial cell of liposome transient transfection cell, extract cell total rna; Adopt H
2o
2process H9c2 myocardial cell; (3) adopt full-length genome chip of expression spectrum to detect, examination goes out the difference regulatory gene of Microrna and its oxidation Microrna in myocardial cell; Wherein, the Microrna of oxidative damage is selected from the arbitrary sequence as shown in SEQ IDNO:1-11; Microrna be selected from rno-miR-184, rno-miR-21*, rno-miR-135a*, rno-miR-139-3p, rno-miR-30c-2*, rno-miR-290, rno-miR-29a, rno-miR-23a, rno-miR-21, rno-miR-204* and rno-miR-106b one or more.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115052590A (en) * | 2019-11-28 | 2022-09-13 | 高丽大学校产学协力团 | RNA interference-inducing nucleic acids comprising 8-oxoguanine, modified nucleic acids binding to microRNAs comprising 8-oxoguanine, and uses thereof |
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2015
- 2015-06-24 CN CN201510350933.8A patent/CN104878117A/en active Pending
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| JIAN-XUN WANG等: "Oxidative Modification of miR-184 Enables It to Target Bcl-xL and Bcl-w", 《MOLECULAR CELL》 * |
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| CN115052590A (en) * | 2019-11-28 | 2022-09-13 | 高丽大学校产学协力团 | RNA interference-inducing nucleic acids comprising 8-oxoguanine, modified nucleic acids binding to microRNAs comprising 8-oxoguanine, and uses thereof |
| EP4144349A4 (en) * | 2019-11-28 | 2024-04-24 | Univ Korea Res & Bus Found | Rna interference-inducing nucleic acid comprising 8-oxoguanine, modified nucleic acid binding to microrna comprising 8-oxoguanine, and uses thereof |
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