CN109750096A - AGXT gene expression is inhibited to cause the molecular mechanism and method for early warning of oxalic acid calculus - Google Patents
AGXT gene expression is inhibited to cause the molecular mechanism and method for early warning of oxalic acid calculus Download PDFInfo
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Abstract
The invention belongs to gene engineering technology fields, disclose a kind of molecular mechanism and method for early warning for inhibiting AGXT gene expression to cause oxalic acid calculus, the influence of Bioinformatics Prediction, analysis miR-4660/miR-6797 to 3 ' UTR of Gene A/G XT is carried out, determines target site, the detection using cytology horizontal analysis miR-4660 to the expression of Gene A/G XT, miRNAs and gene expression dose: immunohistochemistry detection, RT-PCR experiment detection.The present invention starts with from the post-transcriptional control mechanism of the most important Gene A/G XT of Oxalate metabolism, discovery non-coding tiny RNA (microRNA) hsa-miR-4660 can be by binding directly the transcription and translation of AGXT-3 ' UTR region suppressor, the final incidence for increasing oxalic acid calculus for the first time.
Description
Technical field
The invention belongs to gene engineering technology fields more particularly to a kind of inhibition AGXT gene expression to cause oxalic acid calculus
Molecular mechanism and method for early warning.
Background technique
Currently, the prior art commonly used in the trade is such that
Since primary hyperoxaluria I type is that AGT protein function missing is caused to cause by the homozygous mutation of AGXT gene
, therefore the main method for treating the disease at present is to utilize RNA interference or gene editing technology targeting interference ethyl alcohol acid oxidase
The expression of enzyme (GO).This is mainly due to the upstreams that glycolate oxidase (GO) acts on AGT enzyme, and glycolic is oxidized to acetaldehyde
Acid, the afunction mutation for generating GO enzyme will lead to ethyl alcohol acid content in urine and increase 20 times, and with normal oxalic acid level
With normal renal function.GO enzyme gene deletion is prevented with the co-inheritance characteristic of AGXT gene mutation in mouse experiment
The morbidity of PH1.These mankind and mouse genetics data, which have been strongly suggested that, strikes low GO enzyme and making HAO1mRNA silencing
Expression can block destructive glyoxalic acid the way of production and safely reduce the oxalic acid load of PH1 sufferer.
Kidney stone is the common disease of Chinese population, frequently-occurring disease, and China is also one of big lithangiuria district occurred frequently in the world three, is had
The crowd of 5%-15% can suffer from kidney stone in life.Although minimally invasive blast wave stone-breaking and the extensive of endoscopy make in recent years
With having made great progress the therapeutic effect of the disease, but the recurrence rate of kidney stone is still high, and recurrence rate generally exists
5%-10%, if it is Infective calculus, Relapse rate can exceed that 50%.Lithiasis recurrent exerbation, serious person can be final
Lead to renal failure, rest, social activity and the other daily lifes of extreme influence patient, the economy and psychology for increasing patient are born
Load or even threat to life.The stone composition of most of kidney stone patient is calcium oxalate crystal (calcium oxalate, CaOx).
Therefore, the Crack cause of calcinm oxalate calculus and mechanism are always the key content of kidney stone analysis.
Oxalate metabolism correlation zymetology dysfunction is to cause the major reason of calcinm oxalate calculus:
Ox is mainly formed by organic acid in liver metabolism, mainly by peroxisome alanine Glyoxylate transaminase
Glyoxylate reductase (GR, 260000) or hydroxypyruvate reductase in (AGT1/AGXT, 604285), mitochondria (HPR,
604296) it is metabolized with the catalysis of intracytoplasmic 4-Hydroxy-2-oxoglutarate aldolase -1 (HOGA-l, 613597), these three eggs
If white gene mutates, cause enzymatic function abnormal, internal Ox can be caused to accumulate, clinical manifestation is the kidney knot of recurrent exerbation
Stone, nephrocalcinosis companion's kidney function damage, referred to as primary hyperoxaluria (primary hyperoxaluria, PH),
It is a kind of autosomal recessive hereditary diseases.The mutation of above-mentioned three kinds of enzymes respectively corresponds PH1, PH2 and PH3, and wherein PH1 is most common
(80%), clinical very harmful.However, hyperoxaluria, there are higher heterogeneity, gene mutation can not be explained effectively
The cause of disease of all hyperoxaluria patients.Therefore, the present invention speculates, other than gene mutation, there are also other biology machines
System participates in the formation of hyperoxaluria, but the biological mechanism about other formation for participating in hyperoxaluria has not yet to see report
Road.
MicroRNAs may participate in inhibiting the regulation of Oxalate metabolism enzyme:
MiRNAs is the important molecule regulated and controled after genetic transcription, mainly by directly regulating and controlling with the downstream 3 ' of target gene
Area combines the transcription and translation of (3 ' UTR) suppressor.Analytical proof, miRNAs are participating in development, tumour and genetic disease
Generation.Discovered in recent years, miRNAs participate in the regulation of a variety of renal system complex diseases including kidney stone.Hou
J has found that there are many miRNA abnormal expressions in kidney stone patient using miRNA microarray chip analysis technology analysis.
The analyses such as Gong find that miR-9 and miR-374 participate in the synthesis of claudin-14 albumen and adjust and lead to high Ca in kidney2+, promote
The formation of the high calcium calculus of kidney.However, whether miRNAs participates in the generation of hyperoxaluria, analysis there is no at present.
In conclusion problem of the existing technology is:
(1) in the prior art, whether miRNAs participates in the generation of hyperoxaluria, there is no analysis at present；
But the biological mechanism about other formation for participating in hyperoxaluria has not been reported yet.
(2) whether the prior art such as can be to except reduction oxalic acid calculus to the interference of upstream gene glycolate oxidase (GO)
Other metabolic pathways impact, current and indefinite.
A kind of difficulty of above-mentioned technical problem: small molecule of the MiRNAs wide targeting is solved, it is in general special
It is anisotropic poor.Therefore, it is necessary to find the stronger miRNA of a species specificity, can specificity it is higher targeting and act on AGXT base
Cause, this is the difficult point of technical problem.
Solve the meaning of above-mentioned technical problem: if the higher targeting of specificity can be found and act on AGXT gene
MiRNAs is expected to realize the treatment of AGXT related disease.
Summary of the invention
In view of the problems of the existing technology, the present invention provides a kind of inhibition AGXT gene expressions to cause oxalic acid calculus
Molecular mechanism and method for early warning.
The invention is realized in this way a kind of MiR-4660 inhibition AGXT gene expression causes the molecule machine of oxalic acid calculus
System and early stage method for early warning, the MiR-4660 inhibit AGXT gene expression to cause the molecular mechanism of oxalic acid calculus and early stage pre-
Alarm method includes:
The first step, Bioinformatics Prediction: using DIANA-mT, miRanda, miRDB, miRWalk, RNAhybrid,
The miRNAs and target gene binding site forecasting software pair of PICRAR4, PICTAR5, PITA, RNA22, TargetScan
The miRNAs target sequence of AGXT-3 ' UTR is predicted, and detects binding site conservative by IBIVU server, UCSC,
Analyze combination AGXT 3- ' the UTR characteristic of miR-4660 and miR-6797；
Second step analyzes influence of the miR-4660/miR-697 to 3 ' UTR of Gene A/G XT, determines target site: will
AGXT-3 ' UTR is subcloned to pMIR-Report luciferase reporter gene downstream, and the recombination of expression fusion AGXT-3 ' UTR is glimmering
Light element enzyme；Report carrier is turned with hsa-miR-4660/hsa-miR-6797mimics and random miRNA sequence jointly respectively
Hela cell is contaminated, after culture, analysis fusion recombination luciferase reporter vector fluorescent value variation；
Third step, the expression using cytology horizontal analysis miR-4660 to Gene A/G XT: progress RT-PCR analysis,
Western blot analysis；
The detection of 4th step, miRNAs and gene expression dose: immunohistochemistry detection, RT-PCR experiment detection are carried out.
Further, in second step, after culture 48 hours, whether analysis fusion recombination luciferase reporter vector fluorescent value has
Variation；By the method for continuous site point mutation, the target sequence SEQ ID that miR-4660/miR-697 is specifically combined is obtained
NO:1, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6.
Table 1:The primer information.
Further, the method for RT-PCR analysis includes:
Respectively by hsa-miR-4660mimics or hsa-miR-4660 silencer antagomir and random miRNA sequence
Body of listing transfects respectively to L02 cell and extracts cell total rna after transfection 48 hours, after reverse transcription is at cDNA, utilize design
The expression that AGXT quantifies AGXT in primer pair cDNA is quantitative determined, and detection miRNAs is overexpressed or interference miRNAs
After expression, whether the mRNA level in-site of AGXT is changed, and obtaining hsa-miR-4660 influences information to gene transcription level.
It includes SEQ ID NO:2, SEQ ID NO:3 that AGXT, which quantifies primer,.
Forward primer: 5 '-GGCATCCAGTACGTGTTCCA-3 '；
Reverse primer: 5 '-TGCACTCGGGCTCCTATG-3 '.
Further, Western blot analysis method includes:
Respectively by hsa-miR-4660mimics or hsa-miR-4660 silencer antagomir and random miRNA sequence
Column (negative miRNA mimics) carrier is transfected respectively to L02 cell, after transfection 48 hours, extracts total protein of cell, inspection
After surveying miRNAs overexpression or interference miRNAs expression, AGXT protein level difference obtains hsa-miR-4660 and turns over to gene
Translate horizontal influence information.
Further, immunohistochemical experiment method includes:
To the liver organization for needing to carry out transcutaneous aspiration biopsy of liver lesions, AGXT and hsa-miR-4660 specific probe, stone are customized
It is sliced dyeing after wax investing tissue, analyzes the difference of AGXT and hsa-miR-4660 expression.
Further, RT-PCR experimental method includes:
Oxalic acid calculus group and Normal group periphery blood specimen total serum IgE are extracted, is drawn using miR-4660 specific reverse transcriptase
Object reverse transcription and the expression for detecting miR-4660, analysis oxalic acid calculus group and AGXT and hsa-miR- in Normal group peripheral blood
The difference of 4660 expressions.
In conclusion advantages of the present invention and good effect are as follows:
MiR-4660 is proved to be the higher targeting of a species specificity and significantly inhibits a kind of non-volume of AGXT gene expression
Code RNA utilizes RNA interference or gene editing technology targeting interference ethyl alcohol relative to the main method for treating the disease at present
For the technology of the expression of acid oxidase (GO), glycolate oxidase (GO) interference whether can to except reduce oxalic acid calculus its
He impacts metabolic pathway, current and indefinite；And the present invention is specific regulatory control AGXT gene expression, specificity is higher,
It may cause to obtain influence less to other metabolic pathways.And the present invention is to report that AGXT gene expression may be influenced for the first time
MiRNA has filled up the blank of domestic and international correlative study.
Post-transcriptional control (post- of the present invention from the most important Gene A/G XT of Oxalate metabolism
Transcriptionregulation) mechanism is started with, and finds that non-coding tiny RNA (microRNA) hsa-miR-4660 can for the first time
To eventually lead to the generation of oxalic acid calculus by the transcription and translation for binding directly AGXT-3 ' UTR region suppressor.This hair
The new molecular mechanism of the influence oxalic acid calculus of bright offer, provides foundation for the early diagnosis and targeted therapy of the disease.
Detailed description of the invention
Fig. 1 is the molecule machine that MiR-4660 inhibition AGXT gene expression provided in an embodiment of the present invention causes oxalic acid calculus
System and early stage method for early warning flow chart.
Fig. 2 is in human serum and liver organization provided in an embodiment of the present invention, and miR-4660 expression and the expression of AGXT are aobvious
Write negatively correlated figure.
Fig. 3 is that mRNA and protein expression level provided in an embodiment of the present invention prove that miR-4660 can the significant suppression of specificity
The transcript and expression figure of AGXT gene processed.
Fig. 4 is luciferase reporter gene provided in an embodiment of the present invention experiments have shown that miR-4660 specific can be tied
Close AGXT gene map.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
In the prior art, whether miRNAs participates in the generation of hyperoxaluria, there is no analysis at present；But about other participations
The biological mechanism of the formation of hyperoxaluria has not been reported yet.
In order to solve the above technical problems, below with reference to concrete analysis, the present invention is described in detail.
Whether the relative enzyme gene transcriptional regulatory of Oxalate metabolism is participated in for miRNAs there is not yet clearly reporting.Therefore, originally
It is the main analysis content that invention, which selects the most important Gene A/G XT gene of Oxalate metabolism,.The present invention has carried out miRNAs combination to AGXT
The biology in site is predicted and is tested including Dual-Luciferase, primarily determines that miR-4660 can bind directly AGXT-3 ' UTR shadow
The expression of luciferase is rung, on this basis, the quasi- miR-4660 that furthers elucidate through the following experiment of the present invention regulates and controls AGXT
Molecular mechanism:
Normal Human Liver cell line L02 can be analyzed with specific expressed AGXT using L02, it is possible to reduce external source turns
Contaminate experimental error caused by related gene.Therefore present invention selection L02 cell line is seen by height expression or silencing miR-4660
The mRNA transcription and albumen response situation of AGXT are examined, whether confirmation miR-4660, which has the transcription and translation level of AGXT, inhibits
Effect.
The present invention is compared by homology the kind between AGXT-3 ' UTR, finds source of people AGXT-3 ' UTR and rat
And the homology of mouse agxt-3 ' UTR is lower, collection human peripheral blood sample also liver organization puncture sample, in identifier's body
Influence of the miR-4660 to AGXT.
The present invention is directed to horizontal from cell in vitro and horizontal joint exploration miR-4660 participation Oxalate metabolism disorder in vivo is drawn
Rise kidney stone/kidney failure molecular mechanism, confirmation miRNAs a kind of Novel marker oxalic acid calculus early diagnosis and
Clinical meaning in early warning, also to cause kidney stone/renal failure to Oxalate metabolism disorder by shot design drug of miRNAs from now on
The targeted therapy exhausted provides foundation.
Below with reference to concrete scheme, the invention will be further described.
As shown in Figure 1, MiR-4660 provided in an embodiment of the present invention inhibits AGXT gene expression to cause oxalic acid calculus
Molecular mechanism and early stage method for early warning include:
1) Bioinformatics Prediction:
Bioinformatics Prediction miRNAs binding site, this analysis first with DIANA-mT, miRanda, miRDB,
The miRNAs such as miRWalk, RNAhybrid, PICRAR4, PICTAR5, PITA, RNA22, TargetScan are in conjunction with target gene
Site estimation software predicts the miRNAs target sequence of AGXT-3 ' UTR, and is detected by IBIVU server, UCSC
Binding site conservative, it is thus identified that this 2 miRNAs may combine AGXT 3- ' UTR including miR-4660 and miR-6797.
2) influence of the analysis miR-4660/miR-697 to 3 ' UTR of Gene A/G XT, determines target site:
AGXT-3 ' UTR is subcloned to pMIR-Report luciferase reporter gene downstream, expression fusion is enabled
The recombination luciferase of AGXT-3 ' UTR；By report carrier respectively with hsa-miR-4660/hsa-miR-6797 mimics and
Random miRNA sequence (negative miRNA mimics) transfects Hela cell jointly, after culture 48 hours, observation fusion weight
Whether group luciferase reporter vector fluorescent value has significant change, further by the method for continuous site point mutation, further
Specify the target sequence that miR-4660/miR-697 is specifically combined.It is tested by luciferase reporter gene, the present invention is finally true
The expression of AGXT-3 ' UTR influence luciferase can be bound directly by determining miR-4660.
The target sequence that miR-4660/miR-697 is specifically combined are as follows:
Table 1:The primer information.
3) influence that cytology horizontal analysis miR-4660 expresses Gene A/G XT:
A) RT-PCR is analyzed: Normal Human Liver cell line L02 can be analyzed with specific expressed AGXT using L02, can
To reduce experimental error caused by external source transfection related gene.Therefore, respectively by hsa-miR-4660mimics or hsa-miR-
4660 silencer antagomir and random miRNA sequence (negative miRNA mimics) carrier are transfected respectively to L02
Cell is extracted cell total rna, after reverse transcription is at cDNA, is quantified in primer pair cDNA using the AGXT of design after transfection 48 hours
The expression of AGXT is quantitative determined, after detection miRNAs is overexpressed or interferes miRNAs expression, the mRNA water of AGXT
It is flat whether to have significant change, specify influence of the hsa-miR-4660 to gene transcription level.
AGXT quantifies primer
Forward primer: 5 '-GGCATCCAGTACGTGTTCCA-3 ' SEQ ID NO:2.
Reverse primer: 5 '-TGCACTCGGGCTCCTATG-3 ' SEQ ID NO:3.
B) Western blot is analyzed: respectively by hsa-miR-4660mimics or hsa-miR-4660 silencer
Antagomir and random miRNA sequence (negative miRNA mimics) carrier are transfected respectively to L02 cell, transfection 48
After hour, total protein of cell is extracted, after detection miRNAs is overexpressed or interferes miRNAs expression, AGXT protein level difference,
Specify influence of the hsa-miR-4660 to gene translation level.
4) detection of oxalic acid calculus patient and normal group control miRNAs and gene expression dose:
The present invention is compared by homology the kind between AGXT-3 ' UTR, finds source of people AGXT-3 ' UTR and rat
And the homology of mouse agxt-3 ' UTR is lower, therefore this analysis is not suitable for using rat or mouse as analysis banknote validation
Influence of the miR-4660 to AGXT.Therefore, the quasi- human peripheral blood sample also liver organization of collecting of this analysis punctures sample, identifier
Internal influence of the miR-4660 to AGXT.
I) it immunohistochemical experiment: because AGXT is liver specific expression's gene, therefore could only be detected in human liver organization
It arrives, and transcutaneous aspiration biopsy of liver lesions is not the necessary diagnostic method that oxalic acid calculus patient makes a definite diagnosis at present, and transcutaneous aspiration biopsy of liver lesions is invasive
Operation, therefore this analysis can not collect and collect oxalic acid calculus patient liver organization sample.This analysis is living to needing to carry out liver puncture
The liver organization (making a definite diagnosis remaining tissue after the cause of disease) of the patient of inspection confirmation liver disease (cirrhosis, fatty liver etc.), customization
AGXT and hsa-miR-4660 specific probe is sliced dyeing after paraffin-embedded tissue, by fluorescence microscope AGXT and
The difference of hsa-miR-4660 expression；This analysis passes through Hospital Ethical Committee, Tongji University, Tongji Medical College, Huazhong Science and Technology Univ.
Ratify, the patient for sampling this in analysis signs informed consent form.
Ii) RT-PCR is tested: being collected oxalic acid calculus patient and Normal group periphery blood specimen, is extracted total serum IgE, utilizes
MiR-4660 specific reverse transcriptase primer reverse transcription and the expression for detecting miR-4660 determine oxalic acid calculus patient and normal
The difference of AGXT and hsa-miR-4660 expression in control group peripheral blood.
Below with reference to effect, the invention will be further described.
In the present invention, shown in Fig. 2, in human serum and liver organization, miR-4660 expression and the expression of AGXT are significantly negative
It is related.Illustrate in human body, miR-4660 can significantly inhibit the expression of Gene A/G XT.
Shown in Fig. 3, mRNA and protein expression level prove that miR-4660 specific can significantly inhibit turning for AGXT gene
Record and expression.
Shown in Fig. 4, luciferase reporter gene is experiments have shown that miR-4660 can specifically bind AGXT gene.This grinds
Study carefully prove miR-4660 can by and the combination of AGXT gene 3 ' UTR region specificity inhibit its expression.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Sequence table
<110>HuaZhong Science University, TongJi medical school, TongJi Hospital
<120>AGXT gene expression is inhibited to cause the molecular mechanism and method for early warning of oxalic acid calculus
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Claims (6)
1. a kind of MiR-4660 inhibition AGXT gene expression causes the molecular mechanism and early stage method for early warning of oxalic acid calculus, special
Sign is that the MiR-4660 inhibition AGXT gene expression causes the molecular mechanism and early stage method for early warning packet of oxalic acid calculus
It includes:
The first step, Bioinformatics Prediction: using DIANA-mT, miRanda, miRDB, miRWalk, RNAhybrid,
The miRNAs and target gene binding site forecasting software pair of PICRAR4, PICTAR5, PITA, RNA22, TargetScan
The miRNAs target sequence of AGXT-3 ' UTR is predicted, and detects binding site conservative by IBIVU server, UCSC,
Analyze combination AGXT 3- ' the UTR characteristic of miR-4660 and miR-6797；
Second step analyzes influence of the miR-4660/miR-697 to 3 ' UTR of Gene A/G XT, determines target site: by AGXT-3 '
UTR is subcloned to pMIR-Report luciferase reporter gene downstream, the recombination luciferase of expression fusion AGXT-3 ' UTR；
Report carrier is transfected into Hela with hsa-miR-4660/hsa-miR-6797 mimics and random miRNA sequence respectively jointly
Cell, after culture, analysis fusion recombination luciferase reporter vector fluorescent value variation；
The expression using cytology horizontal analysis miR-4660 to Gene A/G XT: third step carries out RT-PCR analysis, Western
Blot analysis；
The detection of 4th step, miRNAs and gene expression dose: immunohistochemistry detection, RT-PCR experiment detection are carried out.
2. molecular mechanism and early stage that MiR-4660 inhibition AGXT gene expression as described in claim 1 causes oxalic acid calculus
Method for early warning, which is characterized in that in second step, after culture 48 hours, analysis fusion recombination luciferase reporter vector fluorescent value
Whether change；By the method for continuous site point mutation, the target sequence that miR-4660/miR-6797 is specifically combined is obtained
SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6.
3. molecular mechanism and early stage that MiR-4660 inhibition AGXT gene expression as described in claim 1 causes oxalic acid calculus
Method for early warning, which is characterized in that RT-PCR analysis method include:
Hsa-miR-4660mimics or hsa-miR-4660 silencer antagomir and random miRNA sequence are carried respectively
Body transfects respectively to L02 cell and extracts cell total rna after transfection 48 hours, after reverse transcription is at cDNA, utilize the AGXT of design
The expression of AGXT is quantitative determined in quantitative primer pair cDNA, and detection miRNAs is overexpressed or interference miRNAs expression
Afterwards, whether the mRNA level in-site of AGXT changes, and obtaining hsa-miR-4660 influences information to gene transcription level；
It includes SEQ ID NO:2, SEQ ID NO:3 that AGXT, which quantifies primer,.
4. molecular mechanism and early stage that MiR-4660 inhibition AGXT gene expression as described in claim 1 causes oxalic acid calculus
Method for early warning, which is characterized in that Western blot analysis method includes:
Hsa-miR-4660mimics or hsa-miR-4660 silencer antagomir and random miRNA sequence are carried respectively
Body is transfected respectively to L02 cell, after transfection 48 hours, extracts total protein of cell, detection miRNAs is overexpressed or interference
After miRNAs expression, AGXT protein level difference, obtaining hsa-miR-4660 influences information to gene translation level.
5. molecular mechanism and early stage that MiR-4660 inhibition AGXT gene expression as described in claim 1 causes oxalic acid calculus
Method for early warning, which is characterized in that immunohistochemical assay includes:
To the liver organization for needing to carry out transcutaneous aspiration biopsy of liver lesions, AGXT and hsa-miR-4660 specific probe, paraffin packet are customized
It is sliced dyeing after burying tissue, analyzes the difference of AGXT and hsa-miR-4660 expression.
6. molecular mechanism and early stage that MiR-4660 inhibition AGXT gene expression as described in claim 1 causes oxalic acid calculus
Method for early warning, which is characterized in that RT-PCR experimental method includes:
Oxalic acid calculus group and Normal group periphery blood specimen total serum IgE are extracted, it is inverse using miR-4660 specific reverse transcriptase primer
Transcribe and detect the expression of miR-4660, AGXT and hsa-miR-4660 in analysis oxalic acid calculus group and Normal group peripheral blood
The difference of expression.
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| CN101583375A (en) * | 2006-08-02 | 2009-11-18 | 阿尔特斯制药公司 | Crystallized oxalate decarboxylase and methods of use |
| CN104411339A (en) * | 2012-06-21 | 2015-03-11 | 北京命码生科科技有限公司 | Microparticle comprising functional microRNA/siRNA and application thereof |
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