CN110511933A - A kind of rat long-chain non-coding lncRNA-lncMSTRG10078 and its application for resisting cellular damage - Google Patents
A kind of rat long-chain non-coding lncRNA-lncMSTRG10078 and its application for resisting cellular damage Download PDFInfo
- Publication number
- CN110511933A CN110511933A CN201910663663.4A CN201910663663A CN110511933A CN 110511933 A CN110511933 A CN 110511933A CN 201910663663 A CN201910663663 A CN 201910663663A CN 110511933 A CN110511933 A CN 110511933A
- Authority
- CN
- China
- Prior art keywords
- lncrna
- sequence
- cell
- cellular damage
- chain non
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
Abstract
The invention discloses a kind of rat long-chain non-coding lncRNA-lncMSTRG10078 and its resist cellular damage application, sequence as shown in SEQ ID NO.1 or have and the above sequence homology of SEQ ID NO.190%；The present invention confirms the objective reality of the sequence by PCR；Clone the sequence transcript, construct pcDNA3.0-lncMSTRG10078 over-express vector, transfect GH3 cell, the results showed that mitochondrial respiratory chain subunit, inflammation and apoptosis generation and metabolism change can significantly be regulated and controled by being overexpressed lncMSTRG10078, to resist cellular damage effect；Can be seen that by streaming result be overexpressed lncMSTRG10078 can the significant generation of inhibitory activity oxygen (ROS) and the generation of Apoptosis, further confirm that the effect of cell resistance cellular damage can be enhanced in the sequence.
Description
Technical field
The present invention relates to technical field of molecular biology, more particularly to a kind of rat long-chain non-coding lncRNA-
LncMSTRG10078 and its application for resisting cellular damage.
Background technique
LncRNA is the RNA molecule that a kind of transcript length is more than 200nt, lacks special complete open reading frame,
Ability without coding protein.Just lncRNA, antisense are classified as according to relative position of the lncRNA in genome
LncRNA between lncRNA, two-way lncRNA, gene, 5 class of lncRNA in gene, this positional relationship is for speculating lncRNA's
Biological function tool is very helpful.LncRNA is divided for signaling molecule, bait molecule, guidance molecule and skeleton according to function
The 4 class molecule such as molecule.These lncRNA were once considered as " determined garbage sequence " accumulated in evolutionary process, were II turn of RNA polymerase
The by-product of record does not have biological function, thus is not given to enough attention.With the continuous development of sequencing technologies, people
Gradually unlocked the Mysterious Veil of lncRNA, more and more evidences show that lncRNA is one and turns with multiple functions
Record is originally.Recent studies indicate that lncRNA wide participation is to development and cell differentiation, chromosome silencing, genomic imprinting, dyeing
A variety of important biological processes such as matter modification, transcriptional activation, transcription interference.
Cellular damage is mainly reflected in inflammation, apoptosis, metabolism change and mitochondrial function damage etc..ROS is thin
The product of born of the same parents' metabolism, acts on mitochondria, can cause the damage of mitochondria；Since cell cannot be metabolized processing ROS in time, cause
ROS is largely gathered in the cell, is caused the dysfunction of cell, is caused cellular damage, eventually leads to Apoptosis and CYP450
The change of metabolic enzyme.However compared with the mRNA of coding protein, the gene expression abundance of lncRNA is very low, still there is a large amount of lncRNA
It is not proved and finds.Therefore, research finds and identifies novel lncRNA, and studies its grinding in terms of resisting cellular damage
Study carefully and has great importance.
Summary of the invention
For the above-mentioned prior art, the object of the present invention is to provide a rat long-chain non-coding RNAs-
LncMSTRG10078 and its mechanism of action in resistance cellular damage, and then application long-chain non-coding RNA resistance cell damage
Wound.
To achieve the above object, the present invention provides following schemes:
As the first aspect of the present invention:
The present invention provides a kind of rat long-chain non-coding lncRNA, and the lncRNA is lncMSTRG10078, has such as
Sequence shown in SEQ ID NO.1.
Further, the rat long-chain non-coding lncRNA sequence further includes in the sequence shown in SEQ ID NO.1
Addition, substitution, insertion or deletion one or more nucleotide and mutant, allele or the derivative generated.
As a second aspect of the invention:
The present invention provides a kind of eukaryotic over-expression vector, contains above-mentioned rat long-chain non-coding lncRNA.
As the third aspect of the present invention:
The present invention provides above-mentioned rat long-chain non-coding lncRNA and is resisting the application in cellular damage.
And above-mentioned eukaryotic over-expression vector is resisting the application in cellular damage.
As the fourth aspect of the present invention:
The present invention provides a kind of method for resisting cellular damage, comprising the following steps:
(1) eukaryotic over-expression vector of above-mentioned rat long-chain non-coding RNA is constructed；
(2) eukaryotic over-expression vector is transfected into cell, increases the expression quantity of rat long-chain non-coding RNA.
The invention discloses following technical effects:
The present invention for the first time from rat isolated one to resist the relevant rat long-chain non-coding RNA of cellular damage
lnc-MSTRG10078.It is raw in agricultural at nucleic acid drug that lnc-MSTRG10078 of the invention can be used as genetic resources or exploitation
Production, livestock and poultry cultivation, human disease use in treating, and for resisting exogenous drugs or poisonous substance to the damaging action of body, have
Wide application value.
Detailed description of the invention
It in order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, below will be to institute in embodiment
Attached drawing to be used is needed to be briefly described, it should be apparent that, the accompanying drawings in the following description is only some implementations of the invention
Example, for those of ordinary skill in the art, without creative efforts, can also obtain according to these attached drawings
Obtain other attached drawings.
Fig. 1 is lncRNA-lncMSTRG10078 in rat gene group position and structure chart；
Fig. 2 is the analysis result figure of lncRNA-lncMSTRG10078 code capacity, and A is NCBI ORF-Finder prediction
As a result, B is the website CPC prediction result；
Fig. 3 is the amplification figure for cloning lncRNA-lncMSTRG10078, and M indicates DNA marker, DNA molecular amount mark
Standard is DL5000, and 1-4 swimming lane indicates the amplified band of lncMSTRG10078, size 1964bp；
Fig. 4 is that Quickcut HindIII and Quickcut XhoI double digestion identify over-express vector pcDNA3.0-
LncMSTRG10078, the plasmid size of over-express vector are 7334bp, and DNA marker used is DL15000bp；
Fig. 5 is that pcDNA3.0-EGFP transfection conditions grope figure；
Fig. 6 is that lncMSTRG10078 is overexpressed verification result figure, is as a result expressed as Mean ± SD (n=3), * expression p <
0.05, the significant difference compared in group；* indicates p < 0.01, and difference is extremely significant compared in group；
Fig. 7 is that lncMSTRG10078 regulates and controls mitochondria related gene expression figure, is as a result expressed as Mean ± SD (n=3), *
Indicate p < 0.05, the significant difference compared in group；* indicates p < 0.01, and difference is extremely significant compared in group；
Fig. 8 is that lncMSTRG10078 regulates and controls expressions of inflammation-related genes figure, is as a result expressed as Mean ± SD (n=3), * table
Show p < 0.05, the significant difference compared in group；* indicates p < 0.01, and difference is extremely significant compared in group；
Fig. 9 is lncMSTRG10078 modulating apoptosis related gene expression figure, is as a result expressed as Mean ± SD (n=3), * table
Show p < 0.05, the significant difference compared in group；* indicates p < 0.01, and difference is extremely significant compared in group；
Figure 10 is that lncMSTRG10078 regulates and controls Metabolism-Related Genes Expression figure, is as a result expressed as Mean ± SD (n=3), *
Indicate p < 0.05, the significant difference compared in group；* indicates p < 0.01, and difference is extremely significant compared in group；
Figure 11 is lncMSTRG10078 to ROS regulating and controlling effect figure；
Figure 12 is regulating and controlling effect figure of the lncMSTRG10078 to Apoptosis.
Specific embodiment
It is noted that described further below be all exemplary, it is intended to provide further instruction to the application, make herein
All technical and scientific terms, which have, identical to be contained in the application person of an ordinary skill in the technical field is normally understood
Justice.
As background parts are introduced, lncRNA participates in the multiple biological function of body, but about cellular damage phase
The lncRNA of pass is even insufficient compared to the research of mRNA.Therefore, the purpose of the present invention is to provide a kind of rat cells to damage phase
The long-chain non-coding RNA of pass, and it is applied to the damage for resisting cell.
The present invention obtains long-chain non-coding RNA lncRNA- using the end cDNA rapid amplifying technology (RACE)
lncMSTRG10078.Display lncRNA-lncMSTRG10078 is compared by UCSC and NCBI and is positioned at rat base
Because organizing No. 1 chromosome, length 1964bp, for nucleotide sequence as shown in SEQ ID NO.1, structure is as shown in Figure 1.
Due to the particularity of nucleotide sequence, the variant of polynucleotides shown in any SEQ ID NO.1, as long as itself and the core
Thuja acid has 90% or more homology, and function having the same, belongs within the scope of protection of the invention.Polynucleotides
Variant refers to a kind of polynucleotide sequence changed with one or more nucleotide, this nucleotide variants includes replacing variation
Body, Deletion variants and insertion variant.
Term " homologous " be primarily referred to as it is homologous in sequence, that is, be used to illustrate two or more RNA or DNA sequence dna
Ancestors having the same.Homologous sequence generally has similar function.The homology of RNA and DNA is often through their sequences
Similitude determines that similitude is used to describe identical DNA alkali between detection sequence and target sequence during referring to sequence alignment
The height of base or amino acid residue sequence proportion.In general, when similarity degree is higher than 50%, often speculate detection sequence
It may be homologous sequence with target sequence；When degree of similarity is lower than 20%, just it is difficult to determine if with homology.
The code capacity of web analytics lncMSTRG10078 is predicted by code capacity, display lncMSTRG10078 does not have
There is coding albumen ability.
The present invention also applies methods of homologous recombination to clone lncMSTRG10078 sequence, constructs over-express vector, passes through
HindIII and XhoI digestion over-express vector pcDNA3.0, the over-express vector pcDNA3.0 and purpose piece of glue recycling linearisation
Section is attached, and sequencing identification constructs carrier for expression of eukaryon.With lipofectamine, empty carrier pcDNA3.0 is transfected respectively
In purpose carrier pcDNA3.0-lncMSTRG10078 to GH3 cell, quantitative fluorescent PCR analyzes result lncMSTRG10078
The expression quantity of related target gene, the results show that transfecting purpose carrier pcDNA3.0- compared to transfection empty carrier group
LncMSTRG10078 can significantly resist cellular damage effect.
With lipofectamine, empty carrier pcDNA3.0 and purpose carrier pcDNA3.0- is transfected respectively
With the generation of flow cytomery ROS and Apoptosis in lncMSTRG10078 to GH3 cell, as the result is shown compared to
Empty carrier group is transfected, transfection purpose carrier pcDNA3.0-lncMSTRG10078 can be significantly reduced ROS and Apoptosis
Generation, and then resist cellular damage effect.
In conclusion using eukaryotic over-expression vector, increase rat lncRNA-lncMSTRG10078 by expression quantity, it can be with
The significant damaging action for resisting cell.
In order to enable those skilled in the art can clearly understand the technical solution of the application, below with reference to tool
The application is described in detail in the embodiment of body.
Test material used in the embodiment of the present invention is the test material of this field routine, can pass through commercial channel
It is commercially available.The experimental method that detailed conditions are not specified is said according to conventional methods or according to operation proposed by supplier
What bright book carried out.
The amplification of embodiment 1lncRNA-lncMSTRG10078 full length sequence
1. material and reagent
1.1 material
Cell line used in this test is rat pituitary oncocyte (GH3 cell).
1.2 reagent
Phanta Max Super-Fidelity DNA Polymerase, article No. P515, only praise biology purchased from Nanjing promise
Science and Technology Ltd.；HiScript II 1st Strand cDNA Synthesis Kit (+gDNA wiper), article No. R212-
01, it is purchased from Nanjing Vazyme Biotechnology Co., Ltd.；FastPure Gel DNA Extraction Mini Kit, article No.
DC301 is purchased from Nanjing Vazyme Biotechnology Co., Ltd.；Mighty TA-cloning Reagent Set forArticle No. 6019, purchased from precious day doctor biotechnology (Beijing) Co., Ltd (takara China)；Primer is by south
The synthesis of Jing Jinsirui Biotechnology Co., Ltd.
2. experimental method
The extraction of 2.1 total serum IgEs
In strict accordance with the pillar total RNA from animal tissues extracting and purifying reagent of Sangon Biotech (Shanghai) Co., Ltd.
Box (B518651) separates total serum IgE from each GH3 cell sample.Mentioned total serum IgE sample is measured with Quawell Q3000
OD260 and OD280 calculates RNA concentration.The mentioned total serum IgE sample of 1 μ g is taken to be detected with 1.0% agarose gel electrophoresis method for detecting total
RNA integrity degree.
2.2 design of primers
According to the nucleotide sequence of known part lncRNA-lncMSTRG10078, with reference to Nanjing Jin Sirui Co., Ltd
Primer calculating instrument design primer, primer sequence is as follows:
2.3 reverse transcription PCR
The reverse transcription primer that this test uses carries out primer for QT and GSP-RT, according to the order of primer synthesis dilute
It releases, every 20 μ L reverse transcription system uses 1 μ L, carries out the synthesis of the cDNA of 3 ' RACE and 5 ' RACE, only praises fully according to promise
HiScript II 1st Strand cDNA Synthesis Kit synthesizes 80 μ L cDNA.
RNaseH processing: RNA is removed in DNA-RNA heterozygosis chain, to the every 20 μ L for having synthesized first chain on ice bath
2 μ L, DEPC water of Reaction Buffer (10X) 17.8 μ L, RNase H (5U/ μ L) 0.2 μ L is sequentially added in reaction system, gently
It is light to mix and (be blown and beaten with pipettor and mix or mixed gently with Vortex in minimum speed), subsequent centrifugation liquid.37
DEG C be incubated for 1h, be added 2.5 μ L 0.5M EDTA (pH8.0) mix, with terminate reaction, it is subsequent using ethanol precipitation purifying synthesis
Double-strand cDNA.
Illustrate: the condition that RNA is removed in other situations DNA-RNA heterozygosis chain can be carried out with reference to above-mentioned condition.RNase H
The pH range of reaction is about in 7.5-8.3.
2.4 3 ' RACE of RACE and 5 '
3 ' RACE and 5 ' RACE are carried out with reference to the classical RACE method in round pcr experiment guide (original work second edition).
The analysis of 2.5 lncRNA-lncMSTRG10078 structures and code capacity
The analysis of structure is carried out to the full length sequence of lncRNA-lncMSTRG10078 using the website UCSC and NCBI, and is answered
LncRNA-lncMSTRG10078 code capacity is analyzed with the website NCBI ORF-Finder and CPC.
3. experimental result
The overall length of lncRNA-lncMSTRG10078 is identified using 5 ' and 3 ' RACE technologies, finally obtains its overall length
Sequence is 1964bp, as shown in SEQ ID NO.1.The comparison of sequence is carried out using UCSC and NCBI, as the result is shown
LncRNA-lncMSTRG10078 is predominantly located at No. 1 chromosome (antisense strand, from 153859895-153861846) (figure of rat
1).Using NCBI ORF-Finder and CPC, it can be concluded that, lncRNA-lncMSTRG10078 does not have encoding histone in Fig. 2
Ability, it is that an overall length is 1964bp and does not have encoding histone ability that the above results, which demonstrate lncRNA-lncMSTRG10078,
LncRNA.The clone of embodiment 2lncRNA-lncMSTRG10078 sequence and analysis
1. reagent and carrier
Phanta Max Super-Fidelity DNA Polymerase, article No. P505, only praise biology purchased from Nanjing promise
Science and Technology Ltd.；HiScript II 1st Strand cDNA Synthesis Kit (+gDNA wiper), article No. R212-
01, it is purchased from Nanjing Vazyme Biotechnology Co., Ltd.；FastPure Gel DNA Extraction Mini Kit, article No.
DC301 is purchased from Nanjing Vazyme Biotechnology Co., Ltd.；ClonExpress Ultra One Step Cloning Kit,
Article No. C115 is purchased from Nanjing Vazyme Biotechnology Co., Ltd.；Mighty TA-cloning Reagent Set forArticle No. 6019, purchased from precious day doctor biotechnology (Beijing) Co., Ltd (takara China)；Quickcut
HindIII and Quickcut XhoI, purchased from precious day doctor biotechnology (Beijing) Co., Ltd (takara China)；Glue recycling
(D2500-02) kit and endotoxin plasmid extraction kit (D6950-01) is gone to be purchased from OMEGA company；Primer by
Nanjing Genscript Biotechnology Co., Ltd.'s synthesis.
2. method
The extraction of 2.1 total serum IgEs
Experimental method is the same as 2.1 Total RNAs extractions in embodiment 1.
2.2 design of primers
According to the sequence of lncRNA-lncMSTRG10078 nucleotide, as shown in SEQ ID NO.1, using homologous recombination enzyme
Design principle, design lncRNA-lncMSTRG10078 amplimer, it is specific as follows:
lncMST F actatagggagacccaagcttTTCCCAATGGATTCAAGGAGC；lncMST R ccctcta
gatgcatgctcgagTTTTTTTTTTTTTGACTGTTTCAATCTTTTAAACAAGATT
2.3 the first chain cDNA synthetic reaction
Reverse transcription reaction is divided into three steps, with the total serum IgE (< 5 μ g) of extraction for template, carries out RNA template denaturation, In first
Reaction solution Oligo (dT) is prepared in RNase-free centrifuge tube23VN (50 μM) adds RNA and RNase-free ddH2After O extremely
12 μ L of volume, by above-mentioned reaction solution, 65 DEG C of heating 5min are immediately placed in and are quenched on ice, and stand 2min on ice.It will be above-mentioned anti-
After answering liquid to be denaturalized, continues 4 μ L of addition 4 × gDNA wiper Mix, mixing is gently blown and beaten with pipettor.42 DEG C of 2min remove base
Because of a group DNA.Continue in the mixed liquor of previous step, continues 10 × RT of addition Mix, 2 μ L, HiScript II Enzyme Mix
2 μ L, after gently blowing and beating mixing with pipettor, 50 DEG C of 45min, 85 DEG C of 2min carry out reverse transcription reaction and obtain the first chain cDNA.
2.4 lncRNA-lncMSTRG10078 transcript PCR amplifications
It is enterprising in PCR instrument using high fidelity enzyme Phanta Max Super-Fidelity DNA Polymerase kit
Row PCR reaction, reaction system are as follows: 2 × Phanta Max Buffer 25 μ L, dNTP Mix (10mM each) 1 μ L,
Each 2 μ L of lncMSTRG10078 specificity upstream and downstream primer, 2 μ L, Phanta Max Super-Fidelity DNA of template DNA
Polymerase 1 μ L, ddH2O to 50 μ L.Response procedures are 95 DEG C of 3min of initial denaturation；95 DEG C of 15sec are denaturalized, are annealed 55 DEG C
15sec extends 72 DEG C of 120sec, recycles 35 times；Then 72 DEG C of extension 5min.The PCR product of acquisition carries out agarose electrophoresis inspection
It surveys, is taken pictures using gel imager, analyze resulting clip size (Fig. 3) and cut correct single goal band.
The building of 2.5 lncRNA-lncMSTRG10078 over-express vectors
Over-express vector pcDNA3.0 is subjected to double digestion, agar with Quickcut Hind III and Quickcut XhoI
Correct single band is cut after sugared gel electrophoresis.By in 2.4 target gene band and the item of over-express vector double digestion bring into
The recycling of row glue, glue recycling step are carried out according to the plastic recovery kit of OMEGA.The DNA solution of glue recycling with 2 ×
CloneExpress Mix is attached, and linked system is as follows: 3 μ L of linearized vector, 2 μ L of lncMSTRG10078 target fragment,
2 × CloneExpress Mix, 5 μ L gently blows and beats mixing using pipettor, and of short duration centrifugation collects reaction solution to official bottom, will
Mixed liquor is placed in 50 DEG C of 15 min in PCR instrument, is down to 4 DEG C or is immediately placed on cooled on ice.Connection product is converted to Trans-
T1 phage Resistant Competent cell takes the competent cell melted on 50 μ L ice baths, and 5 μ L connections are added and produce
Object mixes gently, and places 30 minutes in ice bath, 42 DEG C water-bath heat shock 30 seconds, pipe is quickly then transferred in ice bath 2 points
Clock, the process not shake centrifuge tube.The sterile not antibiotic LB culture medium of 500 μ L is added into each centrifuge tube, mixes
Even to be placed on 37 DEG C, 200rpm is cultivated 1 hour, makes bacteria resuscitation.After 5000rpm is centrifuged 1min, discard supernatant to 100 μ of residue
L is resuspended cell precipitation and is added on the LB agar medium containing ammonia benzyl antibiotic, and cell is uniformly spreadable.By plate as
37 DEG C are absorbed to liquid, are inverted plate, and 37 DEG C are incubated overnight, and picking monoclonal shakes bacterium 12h, utilize after extracting plasmid
Quickcut HindIII and Quickcut XhoI carry out double digestion identification (Fig. 4), will identify that correct monoclonal carrier is sent to
Sangon Biotech's sequencing identification.
3. experimental result
Through agarose electrophoresis detection and sequencing result analysis, the fragment length of resulting positive colony is 1964bp, and
Its sequence is correct, it was confirmed that the necessary being of lncRNA-lncMSTRG10078.
Embodiment 3:lncRNA-lncMSTRG10078 functional verification
1.GH3 cell culture
GH3 cell recovery in contain the 5mL DMEM complete medium (dual anti-+ 1mL paddy ammonia of 10%FBS+90%DMEM+1mL
Amide) in be placed in 95% relative humidity, 37 DEG C of temperature, and cultivated in the incubator containing 5%CO2.Work as cell growth state
It when reaching 70%~90%, using trypsase-EDTA digestion method, is passed in 1:2 ratio, passage in every 1-2 days is primary.Cell
It freezes and is frozen using cells frozen storing liquid (90%FBS+10%DMSO).
2. GH3 Cell Transfection Conditions are groped
The day before transfection, trypsin digestion cell and count (make its transfected after about 24 hours before density reach 90% a left side
The right side is advisable), plating cells are contained into serum in 1mL, in the culture medium of not antibiotic normal growth；With 50 μ L serum-frees
Opti-MEM culture medium dilution 1:1,1:1.5,1:2, the ExFect Transfection Reagent reagent of 1:2.5,1:3,
Stand 5 min of activation；For every hole cell, 1 μ g plasmid is diluted using the Opti-MEM culture medium of 50 μ L serum-frees, is mixed gently
Afterwards in being placed at room temperature for 5min；The ExFect Transfection Reagent reagent and DNA that have diluted are mixed, it is soft to mix,
It is placed at room temperature for 20min, to form DNA/ExFect Transfection Reagent reagent mixture；Standing 20min's
Period handles cell, and 900 μ L opti-MEM are then added；ExFect Transfection Reagent reagent mixture is mixed
It closes object to be added in each hole, softly rocks tissue culture plate back and forth, mark；Nonreactive complete medium is replaced with after 6h,
And 12 hours after transfection, 24 hours, 36 hours and 48 hour record fluorescence, best transfection concentrations and time (Fig. 5) are determined.
3. lncRNA-lncMSTRG10078 is transfected
Determine optimal transfection concentrations and time according to 2 (1:2.5, transfection is for 24 hours).The day before transfection, pancreatin digestion are thin
Born of the same parents simultaneously count (density, which reaches 90% or so, before transfecting it after about 24 hours is advisable), and plating cells are contained serum in 1mL,
In the culture medium of not antibiotic normal growth；According to DNA: transfection reagent=1:2.5 ratio, with 50 μ L serum-frees
The diluted 2.5 μ L ExFect Transfection Reagent reagent of Opti-MEM culture medium stands activation 5min；For
Every hole cell dilutes 1 μ g plasmid using the Opti-MEM culture medium of 50 μ L serum-frees, in being placed at room temperature for 5min after mixing gently；
The ExFect Transfection Reagent reagent and DNA that have diluted are mixed, it is soft to mix, it is placed at room temperature for 20min, with
Just DNA/ExFect Transfection Reagent reagent mixture is formed；Cell is handled during standing 20min, so
After 900 μ L opti-MEM are added；ExFect Transfection Reagent reagent mixture is added in each hole,
Tissue culture plate is softly rocked back and forth, is marked, and is replaced with nonreactive complete medium after 6h, and laggard for 24 hours in transfecting
Row receives sample.The fluorescence quantitative PCR detection of 4.lncRNA-lncMSTRG10078 correlation function gene
4.1 Total RNAs extraction
Experimental method is the same as 2.1 Total RNAs extractions in embodiment 1.
4.2 design of primers
According to the nucleotide sequence of related target gene, qRT-PCR primer, product are designed by NCBI primer Blast
Length is between 70-300bp, and the β-actin that uses expression quantity stable is as reference gene.Primer is by Nanjing Jin Siruisheng
The synthesis of object Science and Technology Ltd..
Primer sequence is as follows:
4.3 reverse transcription reaction
Reverse transcription reaction is divided into two steps, using the 1 μ g of total serum IgE of extraction as template, progress genomic DNA removal first, and In
Reaction solution Oligo (dT) is prepared in RNase-free centrifuge tube23VN (50 μM) 1 μ L, Random hexamers (50ng/ μ L) 1 μ
4 μ L of L, 4 × gDNA wiper Mix adds RNA and RNase-free ddH2To 16 μ L of volume after O, gently blown with pipettor
Beat and mix, 42 DEG C of heating 2min are immediately placed in and are quenched on ice, continue in above-mentioned reaction solution 10 × RT of addition Mix, 2 μ L,
2 μ L of HiScript II Enzyme Mix, after gently blowing and beating mixing with pipettor, 50 DEG C of 15min, 85 DEG C of 2min are inverted
Record reaction obtains the first chain cDNA.
4.4 Quantitative real-time(RT)PCR
Using ChamQ Universal SYBR qPCR Master Mix kit in CFX96 Real-Time PCR
PCR amplification is carried out on Detection System real-time fluorescence quantitative PCR instrument, it is as follows to prepare qRT-PCR reaction system on ice:
It is fixed in CFX96 Real-Time PCR Detection System real-time fluorescence after reaction mixture gentle centrifugation
PCR amplification, response procedures are carried out in amount PCR instrument are as follows:
Step 1: 95 DEG C of 30s at；
Step 2: 95 DEG C of 10s at；The corresponding Tm value of 30s at, totally 40 recycle；
Step 3: 95 DEG C of 10s at；65 DEG C 95 DEG C of to, it is incremented by 0.5 DEG C.Amplification curve and the dissolution for analyzing each gene are bent
Line.By Ct comparison method, and using β-actin as reference gene, i.e., 2-ΔΔCtTo calculate transcriptional level difference.Experiment repeats 3
It is secondary.
5. experimental result
It is overexpressed the expression of lncMSTRG10078 in the GH3 cell of pcDNA3.0 and pcDNA3.0-lncMSTRG10078
Situation is shown in Fig. 6, it can be seen that lncMSTRG10078 is overexpressed successfully.It can be seen that from Fig. 7, Fig. 8, Fig. 9, Figure 10
LncMSTRG10078 can significantly inhibit regulation mitochondrial respiratory chain correlation subunit to change, significant inhibition inflammation occurs,
The significant generation for inhibiting apoptosis, the relevant CYP450 enzymic change of significant regulation mitochondrial metabolism.The experimental results showed that
LncMSTRG10078 has important work in the GH3 cell for being overexpressed pcDNA3.0 and pcDNA3.0-lncMSTRG10078
With, can regulate and control mitochondrial respiratory chain subunit, inflammation and apoptosis generation and metabolism change angle, further support
Anti-cell damaging action.
Regulation of the embodiment 4lncRNA-lncMSTRG10078 to ROS and Apoptosis
1. GH3 cell culture
GH3 cell culture condition is the same as 1 in embodiment 3.
2. lncRNA-lncMSTRG10078 is transfected
LncRNA-lncMSTRG10078 is transfected with 3 in embodiment 3.
3. ROS is measured
After GH3 cell culture, 12 orifice plates are inoculated in, transfect pcDNA3.0 and pcDNA3.0-lncMSTRG10078 respectively
After for 24 hours, the culture medium containing 10 μM of DCFH-DA is added.37 DEG C of incubated cell 1h.With 0.25% trypsin digestion cell 30s, add
Enter complete medium and terminate digestion, cell suspension is made, 1000g is centrifuged 5 minutes, is washed 1~2 time with PBS, cell is collected by centrifugation
Sediment is resuspended cell with 500 μ LPBS, carries out ROS measurement with Beckman FC500.
4. Apoptosis measures
After GH3 cell culture, 12 orifice plates are inoculated in, transfect pcDNA3.0 and pcDNA3.0-lncMSTRG10078 respectively
After for 24 hours, with the 0.25% trypsin digestion cell 30s for being free of EDTA, complete medium is added and terminates digestion, cell suspension is made,
2000rpm is centrifuged 5 minutes at room temperature, and primary with pre-cooling PBS resuspension cell, 2000rpm is centrifuged 5 minutes, and it is thin to abandon supernatant washing
1 × Binding Buffer suspension cell of 300 μ L is added in born of the same parents, after adding the Annexin V-FITC mixing of 5 μ L, is protected from light,
Incubation at room temperature 15 minutes；The PI dyeing of 5 μ L is added within 5 minutes before upper machine, 1 × Binding of 200 μ L is added before upper machine
Buffer carries out the measurement of Apoptosis with Beckman FC500.
5. experimental result
It can be concluded that, pcDNA3.0 and pcDNA3.0- is overexpressed in GH3 cell from streaming result figure 11 and Figure 12
LncMSTRG10078, lncMSTRG10078 can significantly inhibit the generation of ROS and the apoptosis of significant inhibition cell
Rate, further explanation lncMSTRG10078 is played an important role in the damage for resisting cell and function.
Embodiment described above is only that preferred embodiment of the invention is described, and is not carried out to the scope of the present invention
It limits, without departing from the spirit of the design of the present invention, those of ordinary skill in the art make technical solution of the present invention
Various changes and improvements, should all fall into claims of the present invention determine protection scope in.
Sequence table
<110>Hua Zhong Agriculture University
<120>application of a kind of rat long-chain non-coding lncRNA-lncMSTRG10078 and its resistance cellular damage
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1964
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
ttcccaatgg attcaaggag ctagcccccg cccctcgtcc gaggccacct gctccgtgct 60
tggttgcgat gttcaccccc gcagccaaca aagcgaaagc aagcccagaa agcagatccc 120
gtgagggtga cagcctgtgc tggcctccat acagagaata ctctccgagg ggtccctgca 180
ccctcagtac ctgaggttgg ccttgtcttc tccacttacc tgcctaactg gagaagctca 240
agtctgtgag aagctcaagt ctgtgcagcc ccactgcagg caggtaggga aagacgctac 300
tgtcccctgt cacactctgg ccaaaggcag caccgcacca ctcaggcagt acgagggact 360
gctgtgttca gtaaaatcct tgccttgcag gaccagactt cccttgagga tacgcgtggg 420
ggctcttcgt atcaagatgt aacaagatga agctcacttt cctcccacat gagggctttt 480
gtgcttttgt accttggcac tctctatcca gaggacttga ggatcatcat gctccctgcc 540
tcttgagcaa ggctctgagc aaggatgctg tcctgtgaag taaccattag ccacatgtag 600
ctgtaatact ctaaataaaa tacaatttaa aatccatttc cccagtccca gcaagcagtt 660
ctttttaaag cagttgggag catgtgtgag tgcatatagg tacagaacac ttccaacttt 720
gcagaacgtt ctcttgtctt gatctgagtc tcccacttcc tagggaggtt tctaagaacc 780
ccatgaagcc agtcatagat gttagggtca caccttaatc cctgaattta aatgtatact 840
tcctctcagt attcaagcga ttccttctga aggaaataca tctcactgtg tatgggataa 900
cacccaagtc gtcaggaggc cttggttaac cccagcctgg ttcctgacaa ggtctaatgc 960
ttgtcctact tcacactgtg ctgtccttga ggtctcccct cacttgcttc tccccatttc 1020
tccaccttac ttccctgcct ccacctcggg gcttttgtat ctgctaatgc ttttgcatgg 1080
atcctcacac tccaatcttc caaacaactg ccacatctca tctctgaggt ggcccctaat 1140
atgtcaactt gggggccttt attgtaggta ttcccttcag tctgaccatc actaggggga 1200
acataggaaa aaggtatccc ctatgtgtaa acgaacaagt cagttaccag ctcacagctc 1260
ttgtgcttta ttctctgcct cccccttaca ctacaggctt cactgggctt gggtctttac 1320
ctgcaaatcc atttgtcctc agggcttgac gtgtctttgt acaataaaaa catgtttggt 1380
gagggaaggt gagtggcttc cctgttgatc aggactatga gctctcagaa tggctgccag 1440
acagatggtg tggttctctc agactcactt tcaaaccgca tcccattctc tggtcccaac 1500
tgcaaggcag gcataatggt actgtcaggg tcctcctgcc tggcctcagg attccatctc 1560
ctcggaagtg atgggaatta gtgccttccc ctgaagcctc tgtcctttct gctccaattt 1620
ctagttctca gctagaatga tctatattca attgtactgg ataaaagaat gtcgattctc 1680
tttagatgat atttggtata taggtcacac ttcagagcag gccccatgcc ctggactaat 1740
tagctatctg ggcaacacag actgaattcc atggggtttg tttgtttgtt tgttcatttg 1800
ttttgagtca gagggcaaac ataaagttgg atgggtagga aggaggggag gacctgggaa 1860
cagctgggag aacaagatga acatgatcga ttatgtttta tgaaacctcc aatgaataaa 1920
taaaaatctt gtttaaaaga ttgaaacagt caaaaaaaaa aaaa 1964
Claims (6)
1. a kind of rat long-chain non-coding lncRNA, the lncRNA are lncMSTRG10078, have such as SEQ ID NO.1 institute
The sequence shown.
2. rat long-chain non-coding lncRNA according to claim 1, which is characterized in that the rat long-chain non-coding
LncRNA sequence further includes addition, substitution, insertion or deletion one or more nucleotide in the sequence shown in SEQ ID NO.1
And mutant, allele or the derivative generated.
3. a kind of eukaryotic over-expression vector, which is characterized in that contain rat long-chain non-coding of any of claims 1 or 2
lncRNA。
4. rat long-chain non-coding lncRNA as claimed in claim 1 or 2 is resisting the application in cellular damage.
5. eukaryotic over-expression vector as claimed in claim 3 is resisting the application in cellular damage.
6. a kind of method for resisting cellular damage, which comprises the following steps:
(1) eukaryotic over-expression vector of rat long-chain non-coding RNA as claimed in claim 3 is constructed；
(2) eukaryotic over-expression vector is transfected into cell, increases the expression quantity of rat long-chain non-coding RNA.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910663663.4A CN110511933B (en) | 2019-07-23 | 2019-07-23 | Rat long-chain non-coding lncRNA-lncMSTRG10078 and application thereof in resisting cell injury |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910663663.4A CN110511933B (en) | 2019-07-23 | 2019-07-23 | Rat long-chain non-coding lncRNA-lncMSTRG10078 and application thereof in resisting cell injury |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110511933A true CN110511933A (en) | 2019-11-29 |
| CN110511933B CN110511933B (en) | 2021-07-09 |
Family
ID=68623103
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910663663.4A Active CN110511933B (en) | 2019-07-23 | 2019-07-23 | Rat long-chain non-coding lncRNA-lncMSTRG10078 and application thereof in resisting cell injury |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110511933B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112458089A (en) * | 2020-11-25 | 2021-03-09 | 中国人民解放军军事科学院军事医学研究院 | Long-chain non-coding gene and related biological material and application thereof |
-
2019
- 2019-07-23 CN CN201910663663.4A patent/CN110511933B/en active Active
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112458089A (en) * | 2020-11-25 | 2021-03-09 | 中国人民解放军军事科学院军事医学研究院 | Long-chain non-coding gene and related biological material and application thereof |
| CN112458089B (en) * | 2020-11-25 | 2022-05-10 | 中国人民解放军军事科学院军事医学研究院 | Long-chain non-coding gene and related biological material and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110511933B (en) | 2021-07-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106754948B (en) | Nilaparvata lugens NlMLP gene, encoding protein and application thereof | |
| CN111387105B (en) | Method for producing seeds of all-male macrobrachium rosenbergii | |
| CN107245498B (en) | Construction method and application of pig pCRTC3-sgRNA expression vector | |
| CN109734798A (en) | Migratory locusts serpin 7 and its encoding gene and application | |
| CN108610427A (en) | Migratory locusts diapause hormone gene and its application in regulating and controlling Insect Diapause | |
| CN106609267A (en) | Application of cucumber CsTTG1 gene to regulating of development of plant epidermal hair, fruit thorns and thorn galls | |
| CN105296519A (en) | Construction and application of mouse CRABP2 gene eukaryotic expression vector | |
| Yin et al. | Identification and expression of the target gene emx2 of miR-26a and miR-26b in Paralichthys olivaceus | |
| CN110511933A (en) | A kind of rat long-chain non-coding lncRNA-lncMSTRG10078 and its application for resisting cellular damage | |
| CN112226425B (en) | Asian locusta extracellular protease ERK and coding gene and application thereof | |
| CN101979547A (en) | Identification of isolation cloning and core region of promoters suitable for gene expression of skeletal muscles in pigs | |
| Elsahookie et al. | An overview on mechanism of gene expression regulation | |
| CN109734789A (en) | Migratory locusts FK506 binding protein 46 and its encoding gene and application | |
| CN108060147A (en) | Migratory locusts protein tyrosine kinase PTK and its encoding gene and application | |
| Douglas et al. | Partial transcriptome of the developing pituitary gland | |
| CN108374003A (en) | Migratory locusts Protein-tyrosine-phosphatase PTP1B and its encoding gene and application | |
| CN110564726B (en) | Strawberry long-chain non-coding RNA-FRILAIR and application thereof in fruit ripening | |
| CN112458080A (en) | siRNA fishing method for obtaining lncRNA LOC157273 | |
| CN110066799A (en) | Target dsRNA and the application of Tetranychus cinnabarinus molting hormone acceptor gene EcR | |
| CN116769810B (en) | Asiatic dolly locust TRE gene and application thereof in locust control | |
| CN108300724A (en) | A kind of spotted maigre superoxide dismutase ecCu/ZnSOD genes and its application | |
| CN108300763A (en) | A method of screening miR-101-3P target genes | |
| CN112029772B (en) | Gustavus gene and application thereof, dsRNA synthesized by Gustavus gene and preparation method and application of dsRNA | |
| CN107365776A (en) | Application of the EMP genes in rice callus differentiation and development | |
| CN101200715A (en) | Human CD40L expression system and method for making same |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |