CN107058581A - A kind of application of antisense long-chain non-coding RNA in terms of gene therapeutic agents are prepared - Google Patents
A kind of application of antisense long-chain non-coding RNA in terms of gene therapeutic agents are prepared Download PDFInfo
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Abstract
The present invention relates to a kind of application of antisense long-chain non-coding RNA in terms of gene therapeutic agents are prepared, the action target spot of the gene therapeutic agents is MEF2D AS, particular by the expression for improving MEF2D AS, MEF2D expression is lowered, come what is played a role.
Description
Technical field
The invention belongs to biology field, and in particular to a kind of antisense long-chain non-coding RNA is preparing gene therapy
Application in terms of agent.
Background technology
Natural antisense RNA (natural antisense transcripts, NATs) is expressed in multiple species
RNA molecule, can carry out complementary pairing with just RNA and form natural double-stranded RNA, pass through the table of the just gene of different mechanism regulatings
Reach.Natural antisense RNA breeds in cell, important regulating and controlling effect is played during differentiation etc., with the close phase of disease such as tumour, angiocarpy
Close.So far, people are also a glance from a corner to the research of antisense RNA and its function, but have been scientific research person for gene table
The new visual field has been opened up up to the mechanism with tumour.
Myocyte enhancer factor 2 (myocyte enhancer factor 2, MEF2) is a kind of transcription factor, adjusts bone
The transcription of multiple genes in bone myocyte, immunocyte.MEF2D be MEF2 families in a member, it cell survival with point
There is very important effect in terms of change.There are some researches show MEF2D can not only promote the activation of VEGF signal paths, to cell
Multiplication capacity also improves, and the generation to hepatocellular carcinoma (HCC) develops important role.
Highest attention due to people to liver cancer and antisense RNA relation, more and more antisense RNAs related to liver cancer are sent out
Now and further investigate；At the same time, their mechanism of action in liver cancer will gradually be enriched, and be expected to help people to liver cancer
Occur, develop it is further understood, and expect have new breakthrough in the prediction of liver cancer, diagnosis, prognosis and treatment.
The present invention obtains a kind of MEF2D antisense RNAs by studying HepG2 liver cancer cells double-stranded RNAs library, its
There is obvious inhibitory action to the expression of MEF2D genes, be named as MEF2D-AS.
The content of the invention
The present invention provides a kind of antisense long-chain non-coding RNA (being named as MEF2D-AS), it is characterised in that MEF2D-AS's
CDNA sequence has SEQ ID NO：Nucleotide sequence shown in 1.
The present invention provides a kind of kit, it is characterised in that the kit includes the positive and negative justice specificity of MEF2D genes
Primer：5 '-TTACCAGTTGACCAGTGCAG-3 ' and 5 '-TTGCCAGGCAGTGACATT-3 '.
The positive and negative adopted specific primer that another embodiment of the present invention provides above-mentioned MEF2D genes is preparing detection liver
Application in cancer cell HepG2 in MEF2D-AS expression quantity kit.
The present invention provides 2 specific primers：
3’GSP：5′-TGAGAGATACAGGGACCAGGTGGGA-3′
5’GSP：5′-TCCTCCTTACCAGCCTTTAGTTCACCT-3′.
Another technical scheme of the present invention provides the above-mentioned GSP and 5 ' of specific primer 3 ' GSP and is determining MEF2D-AS total lengths
Application in sequence.
The present invention provides a kind of method of measure MEF2D-AS full length sequences, it is characterised in that comprise the following steps：
(1) extraction of total serum IgE；
(2) specific reverse transcriptase is carried out by specific reverse transcriptase primer pair total serum IgE of 3'CDS primer, the first chain is synthesized
After cDNA, enter performing PCR amplification to obtain MEF2D-AS cDNA3' ends, i.e. 3'-RACE with specific primer and kit primer
(3'- ends amplified production)；
(3) specific reverse transcriptase is carried out by specific reverse transcriptase primer pair total serum IgE of 5'CDS primer, is drawn with specificity
Thing and kit primer enter performing PCR amplification to obtain MEF2D-AS cDNA5' ends, i.e. 5'-RACE (5'- ends amplified production)；
(4) 3'-RACE the and 5'-RACE fragments of step (3), (4) all acquisitions are cloned into pGEM-T Easy carriers
In, sequencing is carried out, determines that 3'-RACE fragment lengths are 800bp through sequencing, 5'-RACE fragment lengths are 662bp；
(5) RNA that length is 1265bp is obtained with the splicing of DNAMAN V6 softwares, MEF2D-AS genes is found by BLAST
Transcription initiation matches base in the antisense strand of MEF2D the 8th bit base of introne the 100th, completely aobvious outside with MEF2D the 8th
Sub overlapping (241bp).
Wherein the extraction of total serum IgE can be according to TRIzol in step (1)TMMethod described in reagent specification is extracted,
Or extracted according to the extracting method of the conventional total serum IgE in other this areas；Specific primer described in step (2) is 3 ' GSP
(5 '-TGAGAGATACAGGGACCAGGTGGGA-3 '), kit primer be UPM Long primer (5 '-
CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3 ') and UPM Short primer (5 '-
CTAATACGACTCACTATAGGGC-3′)；Specific primer described in step (3) for 5 ' GSP (5 '-
TCCTCCTTACCAGCCTTTAGTTCACCT-3 ') and SMARTer II AOligonucleotide (5 '-
AAGCAGTGGTATCAACGCAGAGTACATGGG-3 '), kit primer be UPM Long primer (5 '-
CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3 ') and UPM Short primer (5 '-
CTAATACGACTCACTATAGGGC-3′)。
After above-mentioned steps (5), performing PCR amplification, PCR productions can be entered by designing after specific reverse transcriptase primer, synthesis cDNA
Thing identified and determined dna sequence with 1% agarose gel electrophoresis, sequencing result such as SEQ ID NO：Nucleosides shown in 1
Acid sequence.The specific reverse transcriptase primer sequence is as follows：
GSP：5′-GGTGGTTTTTAAAAATAGTGTTGAAC-3′
Reverse primer：5′-CTCTTGGTCGGATGCGGT-3′
Forward primer：5′-CAGAGGGGGTGAGTGACAGAA-3′.
The present invention provides a kind of method for detecting MEF2D-AS expression quantity in hepatocellular carcinoma H22, it is characterised in that including
Following steps：
(1) the positive and negative adopted specific primer of MEF2D genes is designed：5 '-TTACCAGTTGACCAGTGCAG-3 ' and 5 '-
TTGCCAGGCAGTGACATT-3′；
(2) extraction of total serum IgE；
(3) RNA that the specific primer designed with step (1) is obtained to step (2) carries out specific reverse transcriptase, and laggard
Performing PCR, PCR conditions are 94 DEG C of 5min denaturation；94 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 40s (40 circulations)；72 DEG C of 5min extensions；
(4) the PCR results of step (3) using the softwares of GraphPad Prism 5 analyze obtaining hepatocellular carcinoma H22
Middle MEF2D-AS expression quantity.
Wherein the extraction of total serum IgE can be according to TRIzol in step (2)TMMethod described in reagent specification is extracted,
Or extracted according to the extracting method of the conventional total serum IgE in other this areas.
Another embodiment of the present invention provides a kind of gene therapeutic agents, it is characterised in that the gene therapeutic agents pass through increase
The expression of above-mentioned antisense long-chain non-coding RNA (MEF2D-AS) plays a role.Further the gene therapeutic agents are anti-by increase
The expression of adopted RNA (MEF2D-AS), reduces corresponding justice RNA (MEF2D-S) expression to play a role.
Another embodiment of the present invention provides the antisense long-chain non-coding RNA (MEF2D-AS) and prepared with MEF2D-
AS is the purposes in the medicine of target spot.
Another embodiment of the present invention provides the antisense long-chain non-coding RNA (MEF2D-AS) in medicine is prepared
Purposes, it is characterised in that the action target spot of the medicine is MEF2D-AS, particular by the expression for improving MEF2D-AS, is lowered
MEF2D expression, come what is played a role.
The present invention provides a kind of application of DNA methylation enzyme inhibitor in treatment liver-cancer medicine is prepared, it is characterised in that
The DNA methylation enzyme inhibitor is by improving antisense RNA (MEF2D-AS) expression, so as to lower cancer associated gene
MEF2D, and then suppress hepatoma cell proliferation, come what is played a role.
It is related to the design synthesis of primer in the present invention, is according to gene order in Gene Bank or other analysis results
The characteristics of, it is designed by the softwares of Primer Primer 5.0, send company to be synthesized.
Brief description of the drawings
Agarose gel electrophoresis figure (the M.2000bp DNA marker of Fig. 1 PCR primers (MEF2D-AS)；RT. it is specific
Reverse transcription；+ adds reverse transcriptase；- is not added with reverse transcriptase)
Fig. 2 MEF2D-AS RACE agarose gel electrophoresis figure (M.4500bp DNAmarker；5 ' RACE.5 ' expand at end
Increase production thing；3 ' RACE.3 ' hold amplified production)
Fig. 3 UCSC Genome Browser comparison result figures
Fig. 4 MEF2D-AS gene open reading frame prognostic charts
The change figure of the positive antisense rna expression of MEF2D genes after Fig. 5 HepG2 liver cancer cells are handled through 5-Aza-dC
Embodiment
The experiment material that the present invention is used and equipment are as follows：
Experimental cell
Human hepatoma cell line HepG2, from Yangzhou University's epigenetics laboratory cell storehouse.Use DMEM high glucose mediums
In in (containing 10% hyclone, 1% dual anti-) 37 DEG C of incubators of culture.
Cell processing
With DMEM complete mediums (containing 10% hyclone, 1% dual anti-), in 5%CO2, routinely train in 37 DEG C of incubators
Support HepG2.5-Aza-dC dry powder is stored in -70 DEG C of refrigerators, now with the current.Prepare：With dimethyl sulfoxide (DMSO) (Dimethyl
Sulfoxide, DMSO) dissolving 5-Aza-dC dry powder, it is made into the working solution that concentration is 1mM.Take the logarithm growth period HepG2 it is thin
Born of the same parents, culture dish is inoculated in by 20-30% cell concentration, cultivates 24h.Experimental group cell (the HepG2 cells of 5-Aza-dC processing),
10ml culture mediums add 5-Aza-dC to 5 μM of final concentration, cultivate 48h, and 24h changes liquid once.Cellular control unit adds same volume DMSO (to replace
For the 5-Aza-dC in experimental group), 48h is cultivated, same 24h changes liquid once.
Main agents
Capital equipment
The measure of the MEF2D-AS full length sequences of embodiment 1
(1) Total RNAs extraction
The extraction of total serum IgE can be according to TRIzolTMMethod described in reagent specification is extracted, or according to other abilities
The extracting method of the conventional total serum IgE in domain extract cancer cell HepG2 total serum IgE.
(2) the 3'CDS primer (see the table below) provided with 3'- Full Race Core set kits are inverse for specificity
Transcription primers carry out specific reverse transcriptase to total serum IgE, synthesize the first chain cDNA.With the GSP of specific primer 3 ' and kit primer
UPM Long primer and UPM Short primer enter performing PCR amplification to obtain MEF2D-AS cDNA3' ends, i.e. 3'-RACE
(3'- ends amplified production).
(3) it is that specific reverse transcriptase primer pair total serum IgE carries out specific reverse transcriptase with 5'CDS primer (see the table below), with
Specific primer 5 ' GSP and SMARTer II AOligonucleotide and kit primer UPMLong primer and UPM
Short primer enter performing PCR amplification to obtain MEF2D-AS cDNA5' ends, i.e. 5'-RACE (5'- ends amplified production).
(4) 3'-RACE the and 5'-RACE fragments of all acquisitions are cloned into pGEM-T Easy carriers, public by gold only intelligence
Department carries out determined dna sequence, determines that 3'-RACE fragment length is 800bp through sequencing, 5'-RACE length is 662bp.
(5) RNA that length is 1265bp is obtained with the splicing of DNAMAN V6 softwares.MEF2D-AS genes are found by BLAST
Transcription initiation matches base in the antisense strand of MEF2D the 8th bit base of introne the 100th, completely aobvious outside with MEF2D the 8th
Sub overlapping (241bp).
CDNA ends rapid amplifying technology (rapid amplification of cDNAends, RACE) primer sequence
Comprise the following steps that：
(1) 3' ends rapid amplifying technology
The transcription initiation site and termination site for determining antisense RNA are tested by 3 '-RACE.Use SMARTerTMRACE
CDNAAmplification kit (Clontech) carry out the experiment, and experimental procedure is as follows：
1) cell total rna is extracted with Trizol methods, be dissolved in DEPC water, to the RNA of experimental group, with 3'CDS primer A
Specific primer synthesizes cDNA.Primer sequence is seen the above table.Involved RNA operating process in experiment, the vessel used are nothings
RNase's.The solution being related in operating process is configured with DEPC water, and gloves and mouth mask are worn in operating process, to prevent
RNA is contaminated, it is ensured that RNA integralities.
2) RACE-Ready cDNA are prepared
1. following reagent is added into different PCR pipes respectively, mixed and brief centrifugation.Reaction system is shown in Table 3.2
The RACE-Ready cDNA reaction systems of table 3.2 3 '
2. PCR pipe is put into PCR instrument to be denatured, is incubated 72 DEG C of 3min, cool down 42 DEG C of 2min.Centrifugation, 14,000g ×
30sec。
3. prepare 2.5 parts of Buffer Mix, mix following reagent (table 3.3), room temperature is placed stand-by：
The reagent mixed system of table 3.3
4. at ambient temperature, following reagent (table 3.4) is added into 3 ' RACE reaction tubes, is gently mixed, brief centrifugation.
The reagent mixed system of table 3.4
5. in PCR instrument 42 DEG C incubation 90min, 70 DEG C heating 10min.
Plus the ddH after 100 μ L high pressures 6.2O dilution gained cDNA, packing, for subsequent experimental, remaining is put in -20
DEG C refrigerator is preserved.
3) 3 ' RACE PCR react, and reaction system is shown in Table 3.5, and PCR instrument response procedures are shown in 3.6
The RACE PCR reaction systems of table 3.5 3 '
The PCR programs of table 3.6 are set
(2) 5' ends rapid amplifying technology
The transcription initiation site and termination site for determining antisense RNA are tested by 5 '-RACE.Use SMARTerTMRACE
CDNA Amplification kit (Clontech) carry out the experiment, and experimental procedure is as follows：
1) cell total rna is extracted with Trizol methods, be dissolved in DEPC water, to the RNA of experimental group, with 5'CDS primer A
Specific primer synthesizes cDNA.Primer sequence is seen the above table.Involved RNA operating process in experiment, the vessel used are nothings
RNase's.The solution being related in operating process is configured with DEPC water, and gloves and mouth mask are worn in operating process, to prevent
RNA is contaminated, it is ensured that RNA integralities.
2) RACE-Ready cDNA are prepared
1. following reagent is added into different PCR pipes respectively, mixed and brief centrifugation.Reaction system is shown in Table 3.7
The RACE-Ready cDNA reaction systems of table 3.7 5 '
2. PCR pipe is put into PCR instrument to be denatured, is incubated 72 DEG C of 3min, cool down 42 DEG C of 2min.Centrifugation, 14,000g ×
30sec。
3. prepare 2.5 parts of Buffer Mix, mix following reagent (being shown in Table 3.8), room temperature is placed stand-by：
The reagent mixed system of table 3.8
4. mixed to 200 μ L without 1 μ L SMARTer IIAoligo, concussion is added in enzyme centrifuge tube, brief centrifugation.
5. at ambient temperature, gently mixed, instantaneously to 3 ' RACE without following reagent (table 3.9) is added in enzyme centrifuge tube
Centrifugation.
The reagent mixed system of table 3.9
6. biased sample is put into 42 DEG C of incubations 90min, 70 DEG C of heating 10min in PCR instrument.
Plus the ddH after 100 μ L high pressures 7.2O dilution gained cDNA, packing is put in -20 DEG C of refrigerator preservations.
3) 5 ' RACE PCR react, and reaction system is shown in Table 3.10, and PCR instrument response procedures are shown in 3.11
The RACE PCR reaction systems of table 3.10 5 '
The PCR programs of table 3.11 are set
(3) electrophoresis detection is carried out to 3'-RACE and 5'-RACE
1-2% Ago-Gels and electrophoretic buffer are prepared, takes 10 μ L pcr amplification products to run electrophoresis, 4500bp
Ladder Marker are DNA molecular amount standard, 80-120V voltages, electrophoresis 30-60min, when bromophenol blue is moved to gel 2/3
When, stop electrophoresis, taken pictures (Fig. 2) using gel image scanning analyzer.
The MEF2D-AS of embodiment 2 full length sequence is determined
MEF2D-AS full length sequence determines analysis：Known MEF2D be located at X chromosomes (chrX (p11.3)), but due to
During high-flux sequence, sequencing sample is cDNA fragments, so MEF2D-AS transcription initiation site and termination site and indefinite,
Then test to confirm MEF2D-AS transcription initiation site and termination site We conducted 5 ' RACE and 3 ' RACE.Set first
Count specificity to hold for MEF2D-AS 5 ' and the 3 ' amplimers held, the liver cancer cells 5 ' treated with 5-Aza-dC respectively
The RACE RACE of cDNA and 3 ' cDNA are template, and entering performing PCR with MEF2D-AS specific primer expands.DNA electrophoresis results show
Show, 3 ' RACE obtain a 800bp or so band；5 ' RACE obtain 662bp or so.DNA is carried out to obtained band
After sequencing, we obtain the exact sequence of the 5 ' of MEF2D-AS and 3 ' ends, and confirm its transcription initiation site and termination
Site (Fig. 2).By the MEF2D announced in above-mentioned DNA sequencing result and UCSC Genome Browser databases sequence (its
The sequence of 5 ' and 3 ' ends is inaccurate) it is combined, we have obtained the MEF2D-AS of a kind of shear pattern full length sequence (figure
3).To confirm the accuracy for the MEF2D-AS full length sequences that splicing is obtained, we devise specifically according to the full length sequence of acquisition
Property amplimer, enter performing PCR checking, sequencing result is consistent with our splicing result, full length sequence is shown in SEQ ID NO：1.I
(Fig. 4) is predicted to MEF2D-AS ORFs with ORF finder softwares, it is found that this transcripton is not substantially opened
Reading frame.Thus it is concluded that it is probably a long-chain non-coding RNA (long non-coding RNA, lncRNA).
MEF2D-AS expression quantity in the quantitative RT-PCR of embodiment 3 detection hepatocellular carcinoma H22
(1) the positive and negative adopted specific primer of design synthesis MEF2D genes：5 '-TTACCAGTTGACCAGTGCAG-3 ' and
5′-TTGCCAGGCAGTGACATT-3′；
(2) extraction of total serum IgE is carried out to experimental group and control group respectively；
(3) RNA that the specific primer designed with step (1) is obtained to step (2) respectively carries out specific reverse transcriptase, and
Laggard performing PCR, PCR conditions are 94 DEG C of 5min denaturation；94 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 40s (40 circulations)；72 DEG C of 5min prolong
Stretch；Internal reference is used as using β-Actin and GAPDH simultaneously.All primers are listed in the table below：
(4) data analysis is carried out to the PCR results of step (3) using 2- Δs Δ Ct methods, calculates Δ Ct=target gene
Ct- reference gene Ct, Δ Δ Ct=Δs Ct (treatment group) ,-Δ Ct (control group), obtained experimental data GraphPad Prism
5 softwares carry out analysis mapping, calculate P values and carry out data analysis.
5-Aza-dC is detected by quantitative RT-PCR, and MEF2D mRNA and MEF2D-AS RNA are thin in liver cancer before and after the processing
Expression in born of the same parents HepG2.As a result find, handled through DNA methylation enzyme inhibitor (5-Aza-dC), MEF2D-AS expression quantity
There is conspicuousness to improve (p<0.01), and the expression of MEF2D genes in itself then substantially lower (p<0.05) (Fig. 5).
After HepG2 cells are handled through 5-Aza-dC, quantitative fluorescence analysis result is shown, MEF2D-AS expression quantity is significantly carried
Height, and justice MEF2D mRNA expression is inhibited.DNA methylation enzyme inhibitor substantially have activated MEF2D-AS table
Reach, show that MEF2D-AS genes may be regulated and controled by DNA methylation.MEF2D-AS high methylation causes MEF2D-AS silences,
Cause just gene M EF2D overexpression.
The above results show, by increasing MEF2D-AS expression, reduce the expression of MEF2D genes, available for suppression liver
Cancer cell multiplication, the novel targets that MEF2D-AS can be treated as anti-liver cancer and anti-.
The antisense long-chain non-coding RNA that the present invention is provided --- MEF2D-AS can be used for diagnosing cancer of liver and treatment.
The extraction of total serum IgE of the present invention can be according to TRIzolTMMethod described in reagent specification is extracted, or
Extracting method according to the conventional total serum IgE in other this areas is extracted, for example, can be extracted as follows：
1) sample treatment
Culture dish cell is taken, old culture medium is discarded.Plus 1mL Trizol reagents, cell lysis is blown and beaten repeatedly with liquid-transfering gun,
Then lysate is moved on in centrifuge tube；
2) two-phase laminated flow
5min is stored at room temperature, (volume ratio is 5 to addition 0.2mL chloroform in the sample that the Trizol reagents per 1mL are cracked:
1) lid, is covered tightly.Be vortexed mixing 15sec, and 2-3min is incubated under the conditions of room temperature (15-30 DEG C).12,000xg is centrifuged at 4 DEG C
15min.Liquid will be divided into three layers in EP pipes after centrifugation, be nethermost red phenol chloroform phase respectively, white in interbed and
The top colourless aqueous phase.So RNA is dissolved in upper strata aqueous phase.The volume of upper strata aqueous phase is about added Trizol examinations
6/10ths of agent volume.
3) RNA precipitate
Upper strata aqueous phase is drawn into another clean 1.5mL without in enzyme EP pipes.Add 500 μ L different according to every 1mL Trizol reagents
The amount of propyl alcohol adds isopropanol, after being incubated 10min under the conditions of room temperature (15-30 DEG C) after being well mixed, 12 under the conditions of 4 DEG C,
000xg centrifuges 20min.After centrifugation, RNA will appear white precipitation and be located at ttom of pipe or side wall.
4) RNA is cleaned
Remove supernatant, each 1.5ml without added in enzyme EP 500ul-1000uL 75% without enzyme ethanol, (75% ethanol is used
DEPC H2O is prepared), clean RNA precipitate.After mixing, 12,000xg centrifuges 5min under the conditions of 4 DEG C.RNA is cleaned twice.
5) RNA is dried
Abandon after supernatant ethanol solution, 4 DEG C of centrifuge brief centrifugations, bottom of the tube is then left into liquid with small liquid-transfering gun and inhaled
Go out, RNA precipitate is dried 5-10min at ambient temperature.
6) RNA precipitate is dissolved
When dissolving RNA, add the μ L of DEPC water 20 and blown and beaten back and forth several times with liquid-transfering gun, it is fully dissolved, the RNA of acquisition is molten
Liquid be stored in -80 DEG C it is stand-by.
7) RNA quality testings
RNA mass is detected using ultraviolet absorption method.Use DEPC water zeroing spectrophotometer.Then 2 μ L RNA solutions are taken
It is added in test well to be detected, reads the absorption value at OD 260nm and 280nm, the concentration of measure RNA solution and pure
Degree.
Note：Unlisted specific experiment method belongs to experimental implementation conventional in molecular biology in the present invention, is this
The basic skills that art personnel should grasp, reference can be made to the similar opinion that this area textbook or inventor formerly deliver
Text.
SEQUENCE LISTING
<110>Yangzhou University
<120>A kind of application of antisense long-chain non-coding RNA in terms of gene therapeutic agents are prepared
<130> 2017
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1265
<212> cDNA
<213>Human hepatoma cell line HepG2
<400> 1
gtacatggga taaaaacaga gggggtgagt gacagaacaa gtgataggac accagacaga 60
aagtgagaga gaacaagagg atgagaatat gggggatggg gtgttgggga gaggcaattg 120
gatggagagt gatcagaaga gggtcgaatg aagggaagag aagggaaata agaaatatta 180
gttctctttc catcccatgt catcttctgc aacacagggc cccccacctc ccacccccta 240
ccctggggct ggctctcaga aggggttcag ggtgagcctc ttgggtctgt acaagagagc 300
ccaggggtgc cactgttgcc taacagactg tgcagtgcac agcctcatag gatgtccact 360
agaaccctgc agggaaccca gctcccaaga ggtccctcct cttcccgttc aattctccct 420
tcccacacac tcacatgcag tctccccagg actcacatga ggttgctgag agatacaggg 480
accaggtggg actgttgtga ggtggctgtt gcggctgctg aggctgctgt ggctgtggct 540
gctgtggctg cggtggctgc tgctgtggag gctgtggctg ctgcggctgc tggggctgct 600
gtggctgttg ccaggcagtg acattgccta gcgacagccc cccaggtgaa ctaaaggctg 660
gtaaggagga gagctctgca ctggtcaact ggtaatctgc atggaggaaa agtgaggatg 720
aacctggagt tggggagagc acacaggctc ctatgaggga gctgcctcca ggaggactgt 780
ggggatgagg ggacagggag tgtgtaatga cagatgtaag gaattcaaga agaccacaaa 840
aattcagtga agacggtact cagagttaaa gaaacctaag tcataataac catgaggtgc 900
tgtgttttaa caggatgaaa agatgtggtt tttgttttgt tttgtttttt tgagatggag 960
tctcactcta ttgcccaggc tggagtgcag tggtgtgatc ttggctcact acaacctctg 1020
cctcccacgt tcaagcaaat ctcctgcctc ggcctcctaa atggctggga ttacaggcac 1080
atgccaccac gcccagctaa tttttgtatt tttagtagag atggggtttt gccatgctgg 1140
caaggctggt ctcaaactcc tgacctcaag tgattcgccc acctcggcct cccaaagtgc 1200
tgggattaca ggcatgagcc accgcatccg accaagaggt ggtttttaaa aatagtgttg 1260
aactt 1265
Claims (6)
1. a kind of kit, it is characterised in that the kit includes the positive and negative adopted specific primer of MEF2D genes：5′-
TTACCAGTTGACCAGTGCAG-3 ' and 5 '-TTGCCAGGCAGTGACATT-3 '.
2. the positive and negative adopted specific primer of the MEF2D genes described in claim 1 is in detection hepatocellular carcinoma H22 is prepared
Application in MEF2D-AS expression quantity kits.
3. a kind of method for detecting MEF2D-AS expression quantity in hepatocellular carcinoma H22, it is characterised in that comprise the following steps：
(1) the positive and negative adopted specific primer of design synthesis MEF2D genes：5 '-TTACCAGTTGACCAGTGCAG-3 ' and 5 '-
TTGCCAGGCAGTGACATT-3′；
(2) extraction of total serum IgE；
(3) RNA that the specific primer designed with step (1) is obtained to step (2) carries out specific reverse transcriptase, then carries out
PCR, PCR condition are 94 DEG C of 5min denaturation；94 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 40s (40 circulations)；72 DEG C of 5min extensions；
(4) the PCR results of step (3) using the softwares of GraphPad Prism 5 analyze obtaining in hepatocellular carcinoma H22
MEF2D-AS expression quantity.
Purposes of the 4.MEF2D-AS in preparing using MEF2D-AS as the medicine of target spot.
Purposes of the 5.MEF2D-AS in medicine is prepared, it is characterised in that the action target spot of the medicine is MEF2D-AS, specifically
It is the expression by improving MEF2D-AS, MEF2D expression is lowered, come what is played a role.
6. a kind of application of DNA methylation enzyme inhibitor in treatment liver-cancer medicine is prepared, it is characterised in that the DNA methylation
Enzyme inhibitor is, by improving MEF2D-AS expression, so as to lower cancer associated gene MEF2D, and then to suppress liver cancer cells increasing
Grow, come what is played a role.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107447040A (en) * | 2017-09-26 | 2017-12-08 | 扬州大学 | Long-chain non-coding RNA lnc ALVE1 AS1 FISH detection probes and kit |
| CN107447039A (en) * | 2017-09-26 | 2017-12-08 | 扬州大学 | Long-chain non-coding RNA lnc ALVE1 AS1 expression Northern blot detection primers, kit |
| CN107488750A (en) * | 2017-09-26 | 2017-12-19 | 扬州大学 | Long-chain non-coding RNA lnc ALVE1 AS1 detection of expression primers and kit |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107447040A (en) * | 2017-09-26 | 2017-12-08 | 扬州大学 | Long-chain non-coding RNA lnc ALVE1 AS1 FISH detection probes and kit |
| CN107447039A (en) * | 2017-09-26 | 2017-12-08 | 扬州大学 | Long-chain non-coding RNA lnc ALVE1 AS1 expression Northern blot detection primers, kit |
| CN107488750A (en) * | 2017-09-26 | 2017-12-19 | 扬州大学 | Long-chain non-coding RNA lnc ALVE1 AS1 detection of expression primers and kit |
| CN107488750B (en) * | 2017-09-26 | 2019-11-05 | 扬州大学 | Long-chain non-coding RNA lnc-ALVE1-AS1 detection of expression primer and kit |
| CN107447039B (en) * | 2017-09-26 | 2019-11-26 | 扬州大学 | Long-chain non-coding RNA lnc-ALVE1-AS1 expresses Northern blot detection primer, kit |
| CN107447040B (en) * | 2017-09-26 | 2020-07-31 | 扬州大学 | FISH detection probe and kit for long-chain non-coding RNA lnc-A L VE1-AS1 |
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Application publication date: 20170818 |