CN106978417A - A kind of recognizable RNA fragments of HuR albumen and the application in HuR Activity determinations - Google Patents
A kind of recognizable RNA fragments of HuR albumen and the application in HuR Activity determinations Download PDFInfo
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Abstract
The present invention relates to the RNA fragments that a kind of HuR albumen can recognize that, it is characterised in that by SEQ ID NO:DNA sequence dna transcription shown in 1 is obtained；Further relate to the application of the RNA fragments；Further relate to a kind of method for being used to detect intracellular HuR post-transcriptional controls activity.By using the DNA fragmentation and method of the present invention, intracellular HuR post-transcriptional control activity can be detected directly against property, rather than the content of its transcript or protein is only detected, it is enable to more accurately analyze effects of the HuR as mRNA binding factors played in some Developmental Biology and pathological development.
Description
Technical field
The present invention relates to biology field, more specifically it relates to a kind of recognizable RNA fragments of HuR albumen and its
Application in intracellular HuR post-transcriptional controls activity is detected.
Background technology
Human antigen R (human antigen R, HuR) belongs to embryonic death abnormal vision in rna binding protein
(embryonic lethal abnormalvision, ELAV) family, ELAV families include 4 members：HuB, HuC, HuD and
HuR, preceding 3 members are main to express in nerve fiber and reproductive organs, and relevant with neurodevelopment.Recent study shows,
HuR played stably in target gene post-transcriptional control mRNA and promote translation effect.Multinomial experiment is confirmed：The target that HuR is combined
The factor is mostly oncogene, tumor suppressor gene and tumorigenic regulatory factor, thus the generation of HuR and kinds of tumors, development and pre-
It is closely related afterwards.The generation of HuR and human breast carcinoma, colorectal cancer, cervical carcinoma, oophoroma and stomach cancer, attack and shift relevant, can
Can influence the key factor of tumorigenesis and prognosis.
HuR is a kind of mRNA associated proteins, and its activity form can be combined with mRNA 3 '-UTR, and be come by this way
Regulate and control mRNA stability and translation efficiency.Therefore, the research for HuR needs to detect HuR activity form.
However, endogenous HuR still relies on is Western Blot technologies for detection at present, although sensitivity is high, but inspection
The difference of the simply HuR protein contents measured, can not directly embody the change of its activation level.
Accordingly, it would be desirable to design a kind of new method for being used to detect intracellular HuR post-transcriptional controls activity.
The content of the invention
Inventor has found there are 3 RNA identification blocks (RRMs) on HuR albumen by studying, can be with AREs certain bits
Point is combined.RRM1 and RRM2 is combined with rich in U/A copy sequences.RRM3 is closed with poly (A) caudal knot, and help maintains RNA albumen to answer
The stabilization of compound.By analysis, inventor has found, by SEQ ID NO:The RNA sequence of sequential coding shown in 1 is connected on mRNA
During 3 ' end, HuR albumen can effectively combine the 3rd ' area of the mRNA, activate the stability that the mRNA is translated and improved mRNA.
Studied based on more than, the invention provides the RNA fragments that a kind of HuR albumen can recognize that, it is by SEQ ID NO:1 institute
The DNA sequence dna transcription shown is obtained.
Present invention also offers application of the above-mentioned RNA fragments in intracellular HuR post-transcriptional controls activity is detected.
Present invention also offers a kind of method for being used to detect intracellular HuR post-transcriptional controls activity, it includes following step
Suddenly：
S1：Reporter System comprising reporter gene expression frame is imported into the cell, the reporter gene expression frame
3 '-UTR areas include SEQ ID NO:DNA fragmentation shown in 1；
S2：The intracellular HuR post-transcriptional controls activity is calculated by detecting the expression of the reporter gene.It will can report
The expression intensity of gene is accused as the index for indicating HuR post-transcriptional controls activity.
Preferably, the Reporter System be dual-luciferase reporter system, including luciferase I expression cassettes and
Luciferase II expression cassettes, the DNA fragmentation is connected to 3 '-UTR areas of luciferase I expression cassettes, and the luciferase I is excited
The wavelength of fluorescence of generation is different from luciferase II.HuR post-transcriptional control activity is expressed as luciferase I and excites the glimmering of generation
Luminous intensity excites the ratio of the fluorescence intensity of generation with luciferase II.
Preferably, luciferase I expression cassettes and luciferase the II expression cassette is on same carrier.
Preferably, the dual-luciferase reporter system by the DNA fragmentation by being inserted into plasmid psiCHECK
3 '-UTR areas of middle renilla luciferase gene are obtained.
Preferably, S1 is specifically included：
S11：By the cell culture to adherent, and recover form；
S12：The Reporter System is transfected into cell.
Preferably, S2 is specifically included：
S21：Cell is continued into culture 24-36 hours after transfection, cell is washed；
S22：Luciferase I respectively in the cell after detection transfection and luciferase II activity；
S23：Luciferase II activity normalization luciferase I activity is obtained into value as HuR post-transcriptional controls are weighed to live
The index of property.
By using the RNA fragments and method of the present invention, intracellular HuR post-transcriptional control can be detected directly against property
Activity, rather than only detect the content of its transcript or protein, is enable to more accurately to analyze HuR and is tied as mRNA
Close effect of the factor played in some Developmental Biology and pathological development.
Brief description of the drawings
Fig. 1 is that Xho I and Not I carry out the agarose gel photograph after double digestion to recombinant plasmid；
Fig. 2 has psiCHECK2 and psiCHECK2-HuR-RLu human prostate cancer cell line PC-3, people for transfection respectively
Relative HuR activity in cervical cancer tumer line HeLa and human embryonic kidney cell line's HEK-293T cells (that is, live by firefly luciferase
Property with renilla luciferase activity ratio) statistical chart；
Fig. 3 has pcDNA-TAP (that is, empty carrier) and pcDNA-TAP-HuR human prostate cancer cell line for transfection respectively
(that is, firefly is glimmering for relative HuR activity in PC-3, Human cervical cancer cell lines HeLa and human embryonic kidney cell line's HEK-293T cells
The ratio of light element enzymatic activity and renilla luciferase activity) statistical chart.
Embodiment
The principle and feature of the present invention are described below in conjunction with example, the given examples are served only to explain the present invention, and
It is non-to be used to limit the scope of the present invention.
1. build the DNA encoding sequence of the combinative RNA fragments of HuR albumen
Inventor researchs and analyses to HuR, finds the mRNA core sequences of HuR combinations, goes out to compile with reference to the sequences Design
The DNA encoding sequence of the code combinative RNA fragments of HuR albumen, its sequence such as SEQ ID NO:Shown in 1.It is connected to
psiCHECKTM- 2Vector renilla luciferases gene (hRluc) downstream site, i.e. hRluc 3'-UTR areas, constitute hRluc-
HuR target RNA motif-hLuc luciferase reporter gene plasmids.
To ensure the success of vector construction, the cohesive end of corresponding restriction enzyme digestion sites is added in end,
This example adds the cohesive end (CTCGAG) of Xho I restriction enzyme sites at 5 ' ends, and the viscosity end of Not I restriction enzyme sites is added at 3 ' ends
Hold (GCGGCCGC), the design of the cohesive end on sequence both sides contributes to the connection of fragment and carrier.
By the method for de novo formation, two oligonucleotide chains of the fragment are respectively synthesized, its sequence is respectively such as SEQ ID
NO:Shown in 2 and 3.5 ' ends are formed after two oligonucleotide chain complementary pairings and include Xho I restriction enzyme sites cohesive ends and 3 ' end bags
The double-stranded DNA of the restriction enzyme site cohesive ends of I containing Not.100 μ l annealing systems are as follows：Annealing Buffer for DNA
The μ l of Oligos (5X) 20, oligonucleotide chain (50 μM) each 20 μ l, remainder are ddH2O。
After fully mixing, PCR instrument program is set to carry out annealing reaction, specific procedure is：95 DEG C are annealed that (purpose is 2 minutes
DNAoligo is allowed fully to be denatured)；Decline 1 DEG C within every 90 seconds, be down to after 25 DEG C and terminate reaction.DNA annealed products are with 1.5% common fine jade
Sepharose electroresis appraisal, surveys concentration rearmounted standby on ice.
2. above-mentioned DNA fragmentation is inserted into Luciferase Expression Vectors
The luciferase expression reporter plasmid that the embodiment of the present invention is selected is psiCHECKTM2 plasmids.With restricted interior
Enzyme cutting Xho I and Not I (are purchased from TAKARA companies, Xho I article No.s are that 1635, Not I article No.s are 1623) to above-mentioned
psiCHECKTM2 plasmids carry out double digestion, and 20 μ l digestion systems are as follows：10x QuickCut Green buffer 2 μ l, Xho I
1 μ l, Not I 1 μ l, psiCHECKTMThe μ g of 2 empty carrier 1, remainder is ddH2O。
After fully mixing, 37 DEG C of endonuclease reactions 90 minutes identify plasmid double digestion with the sugared gel electrophoresis of 1.5% plain agar
Situation afterwards, then gel extraction (DNA glue reclaims kit is purchased from Tiangeng biochemical technology Co., Ltd, and article No. is DP209), returns
Receipts survey sample concentration after terminating, and put standby on ice.
The double-stranded DNA that annealing is obtained is connected to psiCHECKTMOn 2 plasmid encoding luciferases element enzyme reporter plasmid.10 μ l connect
Junctor system is as follows：DNA ligase (be purchased from TAKARA companies, article No. is 6022) 5 μ l, double chain DNA fragment with through double digestion
psiCHECKTM2 carriers totally 5 μ l.To promote enzyme to be linked to be power, the mol ratio of DNA fragmentation and carrier is controlled 8:1 or so.Fill
Divide after mixing, enzyme disjunctor system is placed in PCR instrument, 16 DEG C are reacted 1 hour, and coupled reaction obtains recombinant plasmid after terminating, and is placed in
It is standby on ice.
By recombinant plasmid (containing coding HuR albumen can binding site DNA sequence dna psiCHECKTM2 reporter gene matter
Grain) convert to Escherichia coli.Specific method is as follows：The competence bacillus coli DH 5 alpha after thawing is taken, above-mentioned connect is added
Recombinant plasmid, soft piping and druming is incubated 20 minutes on ice, 42 DEG C of heat shocks 60 seconds, stands 3 minutes on ice, then adds 450 μ l not
LB fluid nutrient mediums containing antibiotic, are positioned over 1 hour (37 DEG C, 200rpm) of recovery in constant-temperature table.Take the bacterium solution after recovery
To being covered with solid medium (containing ampicillin) culture dish, bacterium solution is equably paved with to whole plate with glass paving bacterium device,
After placing 10 minutes, it is inverted in after 37 DEG C of incubators stay overnight, observes bacterial growth situation.Picking colony is to 5ml containing ampicillin
LB fluid nutrient mediums, shake after bacterium (37 DEG C, 200rpm) breeds 12 hours and extract plasmid, the sugar gel electrophoresis of 1.5% plain agar
Identification.
Double digestion identification, double digestion of the embodiment of the present invention are carried out to the plasmid of said extracted with restriction endonuclease Xho I and Not I
Identification is as shown in Figure 1 (swimming lane 1 is marker, and swimming lane 1-3 is shown as positive colony).Deliver to Wuhan for positive group and hold up section's biotechnology
Co., Ltd is sequenced, and confirms that DNA fragmentation has been integrated into psiCHECKTMOn -2Vector reporter plasmids.So far, containing coding
HuR albumen can binding site DNA fragmentation psiCHECKTM2 reporter plasmids (are represented as psiCHECK2-HuR- below
RLuc) successfully construct.
3.psiCHECK2-HuR-RLuc transfectional cells
The embodiment of the present invention uses human prostate cancer cell line PC-3, Human cervical cancer cell lines HeLa, human embryonic kidney cell line
HEK-293T carries out Dual-Luciferase experiment.The cell that logarithmic phase grows uniformly is planted in 24 orifice plates respectively, per hole about 10
Ten thousand cells, use 10% hyclone, and DMEM in high glucose medium culture is stayed overnight.After cell attachment and after recovering form, it will report
Genic system related plasmids are transfected into cell (cell density is about that 60-80% is advisable during transfection).The transfection reagent used is
Neofect (purchased from zero objective Powerise bio tech ltd, article No. is TF20121201).50 μ l rotaring redyeing systems are as follows：Plasmid
The μ l of 0.5 μ g, Neofect transfection reagent 0.5, remainder is serum free medium.
The plasmid used in this experiment is respectively：psiCHECKTM- 2Vector reporter plasmids, psiCHECK2-HuR-
RLuc (containing coding HuR albumen can binding site DNA fragmentation psiCHECKTM- 2Vector reporter plasmids),
PcDNA-TAP (empty plasmid), pcDNA-TAP-HuR (HuR is overexpressed plasmid).
4. calculate HuR activity
It is standardized using firefly luciferase activity as internal reference, calculates HuR activity.Specific experiment method is as follows：
Cell after transfection continues to cultivate 24-36 hours, then abandons culture medium supernatant, cell is cleaned with 1xPBS 3 times, 5 minutes every time, clearly
Notice that operation is soft when washing cell, it is to avoid front piping and druming cell.Using dual luciferase reporter gene detection kit (purchased from lid
Peaceful biotechnology has new company, and article No. is GN201-01) detected.
Whether the embodiment of the present invention is active for checking this report genic system, by psiCHECKTM- 2 plasmids and
PsiCHECK2-HuR-RLuc plasmid transfections into cell, the activity of examining report genic system, measurement result as shown in Fig. 2
The relative luciferase activity for the treatment of group (transfection psiCHECK2-HuR-RLuc) (transfects psiCHECK apparently higher than control groupTM-
2 empty carriers).
Whether the embodiment of the present invention can be with specific detection HuR activity, by pcDNA-TAP for checking this report genic system
Plasmid and pcDNA-TAP-HuR plasmids are transfected to cell, as a result as shown in figure 3, external source is imported after HuR, positive treatment group respectively
(transfection pcDNA-TAP-HuR) fluorescence activity is apparently higher than control group (transfection pcDNA-TAP empty carriers).
The foregoing is only presently preferred embodiments of the present invention, be not intended to limit the invention, it is all the present invention spirit and
Within principle, any modification, equivalent substitution and improvements made etc. should be included in the scope of the protection.
Sequence table
<110>Wuhan Union Hospital
<120>A kind of recognizable RNA fragments of HuR albumen and the application in HuR Activity determinations
<130> 1
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 66
<212> DNA
<213>Artificial sequence
<400> 1
tggataccgt tcgttgttaa ccgttgatag ccttggtttt taagccgtat atggctgtaa 60
ataatt 66
<210> 2
<211> 73
<212> DNA
<213>Artificial sequence
<400> 2
tcgagtggat accgttcgtt gttaaccgtt gatagccttg gtttttaagc cgtatatggc 60
tgtaaataat tgc 73
<210> 3
<211> 73
<212> DNA
<213>Artificial sequence
<400> 3
ggccgcaatt atttacagcc atatacggct taaaaaccaa ggctatcaac ggttaacaac 60
gaacggtatc cac 73
Claims (8)
1. the recognizable RNA fragments of a kind of HuR albumen, it is characterised in that by SEQ ID NO:DNA sequence dna shown in 1 is transcribed
Arrive.
2. application of the RNA fragments in intracellular HuR post-transcriptional controls activity is detected described in claim 1.
3. a kind of method for being used to detect intracellular HuR post-transcriptional controls activity, it is characterised in that comprise the following steps：
S1：Reporter System comprising reporter gene expression frame is imported into the cell, the reporter gene expression frame 3 '-
UTR areas include SEQ ID NO:DNA fragmentation shown in 1；
S2：The intracellular HuR post-transcriptional controls activity is calculated by detecting the expression of the reporter gene.
4. method according to claim 3, it is characterised in that the Reporter System is luciferase reporter gene
System, including luciferase I expression cassettes and luciferase II expression cassettes, the DNA fragmentation are connected to luciferase I expression cassettes
3 '-UTR areas, the luciferase I excites the wavelength of fluorescence of generation different from luciferase II.
5. method according to claim 4, it is characterised in that luciferase I expression cassettes and luciferase the II expression
Frame is on same carrier.
6. method according to claim 5, it is characterised in that the dual-luciferase reporter system passes through by described in
The 3 '-UTR areas that DNA fragmentation is inserted into renilla luciferase gene in plasmid psiCHECK are obtained.
7. the method according to any one of claim 4-6, it is characterised in that S1 is specifically included：
S11：By the cell culture to adherent, and recover form；
S12：The Reporter System is transfected into cell.
8. method according to claim 7, it is characterised in that S2 is specifically included：
S21：Cell is continued into culture 24-36 hours after transfection, cell is washed；
S22：Luciferase I respectively in the cell after detection transfection and luciferase II activity；
S23：The value that luciferase II activity normalization luciferase I activity is obtained is active as HuR post-transcriptional controls are weighed
Index.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710198729.8A CN106978417B (en) | 2017-03-29 | 2017-03-29 | RNA fragment capable of being identified by HuR protein and application of RNA fragment in HuR activity detection |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710198729.8A CN106978417B (en) | 2017-03-29 | 2017-03-29 | RNA fragment capable of being identified by HuR protein and application of RNA fragment in HuR activity detection |
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| Publication Number | Publication Date |
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| CN106978417A true CN106978417A (en) | 2017-07-25 |
| CN106978417B CN106978417B (en) | 2020-02-21 |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106399461A (en) * | 2016-09-14 | 2017-02-15 | 妙顺（上海）生物科技有限公司 | Method of detection of transcription factor expression activity by luciferase reporter gene system |
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2017
- 2017-03-29 CN CN201710198729.8A patent/CN106978417B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106399461A (en) * | 2016-09-14 | 2017-02-15 | 妙顺（上海）生物科技有限公司 | Method of detection of transcription factor expression activity by luciferase reporter gene system |
Non-Patent Citations (2)
| Title |
|---|
| WANG HONG等: "《The structure of the ARE-binding domains of Hu antigen R (HuR) undergoes conformational changes during RNA binding》", 《ACTA CRYSTALLOGRAPHICA SECTION D-STRUCTURAL BIOLOGY》 * |
| 明洁: "《"HuR依赖性mRNA稳定性对p27Kip1的转录后表达调控》", 《中国博士学位论文全文数据库 医药卫生科技辑》 * |
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