CN108384805A - Promote application of the reagent of circular rna circRNF13 expression on preparing treatment Dendritic cell drug - Google Patents
Promote application of the reagent of circular rna circRNF13 expression on preparing treatment Dendritic cell drug Download PDFInfo
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Abstract
The invention discloses promote application of the reagent of circular rna circRNF13 expression on preparing treatment Dendritic cell drug.It confirms to be overexpressed circRNF13 in Tca8113 cells by studying, the proliferation of Tca8113 cells can be inhibited.Therefore, circRNF13 is overexpressed preparation for treating Dendritic cell, there is far-reaching clinical meaning and important popularizing application prospect.
Description
Technical field
The invention belongs to oncomolecularbiology fields, and in particular to promote the reagent of circular rna circRNF13 expression
Application on preparing treatment Dendritic cell drug.
Background technology
The Human Genome Project and its subsequent DNA element encyclopedia plan (The Encyclopedia of DNA
Elements Project, ENCODE) achievement in research shows that protein coding gene sequence only accounts for the 1- of human genomic sequence
3%, and the transcribed sequence of the overwhelming majority is non-coding RNA (non-coding RNA, ncRNA) in human genome.Non- volume
Code RNA is although non-coding protein, due to wide participation genes within cells expression regulation, in field of biomedical research one
Directly it is concerned.Study more is linear ncRNA molecules, from RNA sequence length can be divided into Microrna (microRNA,
MiRNA, 19-23nt) and long-chain non-coding RNA (long non-coding RNA, lncRNAs,>200nt), they including
Important function is played in the occurrence and development of a variety of mankind's common diseases including malignant tumour.Recently, a kind of completely new
NcRNA molecules, circular rna (circular RNA, circRNA) is due to its unique configuration and the important biomolecule increasingly found
Function is learned, the new forward position of biomedical sector and hot spot are had become.
CircRNA had once once been taken as the wrong product during genetic transcription post-processing.With the lines such as mRNA and lncRNA
Property RNA process it is much like, the formation of circRNA is also to be given birth to when carrying out montage processing to RNA precursors by splicing complex
At, only in this course, the loop-forming sequences of flank combine cyclization, subsequent splicing complex to carry out montage first, most
Flanking sequence cuts off to form circRNA at last.Recently as new-generation sequencings technologies such as sequencing technologies especially RNA-seq
Development, more and more circRNA by research by it has been found that simultaneously disclose part circRNA in numerous life
It all plays an important role in activity.Additionally due to its unique structure causes circRNA insensitive to nuclease, in tumour
CircRNA also has a clear superiority in terms of the development and application of marker, therefore very likely as a kind of important molecular marker
The target spot of object and emerging drug development.But since this field has just emerged, most of circRNA is not yet found or there is no
Research.
Squamous cell carcinoma of tongue (tongue squamous cell carcinoma, TSCC) is that the highest oral cavity of incidence is disliked
Property tumour, Dendritic cell majority is squamous carcinoma, mostly occurs in lingual margin, is often ulcer type or leaching secondly for the tip of the tongue, back and root of the tongue etc.
Profit type.General grade malignancy is higher, and growth is fast, and wellability is stronger, and ordinary wave and lingualis cause tongue limitation of movement, make to speak, feed
And it swallows and difficulty occurs.The chemotherapy that operative treatment is aided with based on suitable platinum medicine is Dendritic cell clinically main treatment side
Case.However, finding that the therapeutic effect of Dendritic cell patient is often not satisfactory in clinic, survival rate is only 55-65% within 5 years, studies carefully it
Reason essentially consists in the remaining cancer cell of operation, and (for example to have invaded the circulating tumor being transferred in blood vessel or lymphatic vessel thin
Born of the same parents) generally existing drug resistance, lead oncogenic recurrence and transfer, cause treatment failure.Therefore, Tca8113 cells are found out to chemotherapy
The resistance mechanisms of drug screen new drug target, final to improve survival and life quality, are to be worth clinical section
The important topic that the workers of grinding study.
In order to study circRNA Tca8113 cells in cis-platinum class Drug-resistant effect and mechanism, we use
The circRNA genetic chip Arraystar Human Circular RNA Microarray V2.0 couple two of Agilent companies
The expression of circRNAs in Tca8113 cells strain (Tca8113 and Cal27) before and after cisplatin treated is analyzed.I
Find circRNF13 expression variation it is the most notable, prompt circRNF13 may be related to the cisplatin resistance of Dendritic cell, and
Up to the present, the biological function of circRNF13 and mechanism of action have no any report.
Since genetic chip can only be based on circRNA ring-type splicing site upstream and downstream sequence design probes, pass through these spies
Needle specificity captures circRNA, to detect the gene expression abundance of circRNA in cell or tissue;Pass through particularly for us
CircRNA chips find circRNF13 for, by genetic chip provide technical data we can find its corresponding spy
The total 60bp of needle, the last period is the sequence of 8 exon, 3 ' the end 30bp of RNF13 genes, and latter section is then RNF13 genes 2
5 ' end 30bp of exon, it is to be joined end to end to add by No. 2 of the precursor RNA of RNF13 genetic transcriptions and 8 exons to prompt it
Work forms, but specifically RNF13 which exon processing generates (whether 2~8 exons are included entirely withinWhether
Also include intron sequencesBecause some circRNA include just intron sequences) it does not know.Then, our design primers are simultaneously
Pass through the PCR successful clones nested twice full length sequence of circRNF13, it was confirmed that circRNF13 is strictly by being located at dye
The 2-8 extras of RNF13 genes (ring finger protein 13, NM_183381.2) on the regions colour solid 3q25.1 are aobvious
Subring is spliced to form, overall length 716bp.
In order to further study the biological function of circRNF13, it is dry that we devise the small molecule for circRNF13
The expression that RNA (siRNA) sequence-specific has struck circRNF13 low is disturbed, while successfully constructing circRNF13 over-express vectors
CircRNF13 is overexpressed in Tca8113 cells.MTT and flow cytometry detection confirm that up-regulation circRNF13 can be bright
Aobvious to inhibit Tca8113 cells proliferation, mechanism is arresting cell cycle and inducing cell apoptosis, and strikes low circRNF13 and then obtain
Opposite result.Further, we successfully obtain Dendritic cell chemotherapy resistance cell strain, in real time by Fiber differentiation
The expression of circRNF13 in quantitative PCR detection cell, find expression of the circRNF13 in drug-resistant cell strain with it is former
Beginning cell strain is lowered compared to significantly;And we are overexpressed circRNF13 in mdr cell, can obviously reverse Dendritic cell thin
The drug-resistant phenotype of born of the same parents, and the expression of low circRNF13 is struck in initial cell strain, then Tca8113 cells can be significantly improved to suitable
The tolerance of platinum.
Finally, we are had detected in Dendritic cell clinical sample by real-time fluorescence quantitative PCR and the method for in situ hybridization
The expression of circRNF13 finds that circRNF13 expresses notable downward in Dendritic cell, and circRNF13 expresses low trouble
Its life span of person is shorter than circRNF13 and expresses high patient, therefore can be used for tongue for the detection preparation of the lncRNA
The auxiliary diagnosis and Index for diagnosis of squamous carcinoma.
Invention content
Treatment Dendritic cell drug is being prepared the object of the present invention is to provide the reagent of circular rna circRNF13 expression is promoted
On application, the sequence such as SEQ NO of the circular rna circRNF13:Shown in 1.
The reagent of circular rna circRNF13 expression that promotes is circRNF13 over-express vectors.
The building process of the circRNF13 over-express vectors is as follows:
(1) it according to the fundamentum of the Forming Mechanism of circRNA and eucaryote RNA montages, designs and is suitable for
The loop-forming sequences of circRNA expression;According to the sequence information of commercial carrier pcDNA3.1, suitable multiple cloning sites are designed,
To obtain completely realizing that the DNA sequence dna that circRNA is overexpressed is as follows:
GTGCTGGGATTACAGGTGTGAGCTACCACCCCCGGCCCACTTTTTCTTAAGCTTGGTACCGAGCTCGGATCCACATC GATTGGTGGAATTCTGCAGATATCCACCGCGGTGGCGGCCGCTCGAGTCTAGAGAAAAGAATTAGGCTCGGCACGGT
AGCTCACACCTGTAATCCCAGCA, intermediate underscore part are multiple cloning sites, and both ends are respectively upstream and downstream loop-forming sequences;
(2) above-mentioned synthesis is realized that NheI restriction enzyme site sequences are added in DNA sequence dna one end that circRNA is overexpressed, separately
One end addition ApaI restriction enzyme site sequences obtain pcCirc by being built into pcDNA3.1 carriers after NheI and ApaI digestions
Empty plasmid, sequencing confirm that plasmid is correct, sequence such as SEQ NO:Shown in 13;
(3) selection Cla I and Sac II restriction enzyme sites are used for digestion pcCirc empty plasmids, and by circRNF13 sequences
It is inserted between the upstream and downstream loop-forming sequences of carrier.
The detailed process of step (3) is as follows:
1) using Tca8113 cells Tca8113cDNA as template, TaKaRa LA are utilizedEnzyme carries out PCR amplification overall length
CircRNF13 sequences;CircRNF13 full length sequence amplimers are as follows:
Sense primer:5’-GTGATTTTACAACGAGAT-3’;
Downstream primer:5’-CTTTCTTGAATTTATGTA-3’;
In upstream and downstream, 5 ' ends of primer add restriction enzyme Cla I and Sac II recognition sites and protection alkali respectively
After base, primer sequence is as follows:
Sense primer:5’-AGGAATCGAT GTGATTTTACAACGAGAT-3 ', underscore part are that Cla I identify position
Point;
Downstream primer:5’-ATGCCCGCGG CTTTCTTGAATTTATGTA-3 ', underscore part are that Sac II identify position
Point;
2) PCR amplification, circRNF13 full length sequences, PCR reaction conditions are as follows:
PCR reaction steps
5 return to the 2nd step, carry out 39 secondary response cycles altogether;
3) by through Cla I and Sac II double digestion rear electrophoresis, glue returns again after PCR product electrophoresis, glue recycling target fragment
It receives;
4) pcCirc empty plasmids recycle target fragment through Cla I and Sac II double digestion rear electrophoresis glue;
5) with the connection of T4 DNA ligases 3) and 4) step glue recovery product to get to the carrier matter for being overexpressed circRNF13
Grain;
6) by the eukaryon expression plasmid transformed competence colibacillus large intestine bar comprising circRNF13 full length sequences that 5) step obtains
Bacterium, to expand plasmid.
After we transfect circRNF13 carrier for expression of eukaryon, had detected in cell by real-time fluorescence quantitative PCR
The expression of circRNF13 finds that circRNF13 expressions are significantly raised;And transfect the siRNA of targeting circRNF13
(siRNA) after, the expression of intracellular circRNF13 is obviously lowered;Compared with the cell of transfection empty carrier, it is overexpressed
The speed of growth of the Tca8113 cells Tca8113 and Cal27 of circRNF13 significantly slow down, and are struck using siRNA interference sequences low
After the expression of circRNF13, vitro growth rates are accelerated.Flow cytometry analysis shows to transfect in Tca8113 cells
After circRNF13, G2/M phase cell proportions dramatically increase, and S phase cell proportions are reduced, and show that circRNF13 can be by Dendritic cell
Cell block makes its cell division slow speed in the G2/M phases.Meanwhile apoptosis after circRNF13 is transfected in Tca8113 cells
Cell proportion obviously increases, this is also one of the reason of circRNF13 inhibits Tca8113 cells growth.And strike low circRNF13
Opposite result is then obtained.Therefore, circRNF13 is overexpressed preparation for treating Dendritic cell, there is far-reaching clinical meaning
Adopted and important popularizing application prospect.
Description of the drawings
Fig. 1:CircRNF13 is demonstrated by qRT-PCR, and significantly up-regulation (left side), sequencing result card are expressed after cisplatin treated
That real qRT-PCR is detected is strictly circRNF13 (right side).Arrow represents splicing site, is 8 extras of RNF13 on the left of arrow
Aobvious son 3 ' is held, and is 5 ' terminal sequences of 2 exon of RNF13 genes on the right side of arrow.
Fig. 2:It is sequenced by nesting PCR- twice, for the first time the successful clone full length sequence of circRNF13 molecules, it was demonstrated that
CircRNA is by the 2-8 exon rings of the RNF13 gene transcripts NM_183381.2 on the regions chromosome 3q25.1
Change is spliced to form, overall length 716bp.
Fig. 3:Design simultaneously successfully build circRNF13 over-express vectors, using pcDNA3.1 carriers as basic framework, by
Two sections of loop-forming sequences and restriction enzyme site is added in its CMV promoter downstream, and the 2-8 exons of RNF13 are passed through
PCR, digestion are connected in carrier (left side), are then transfected into Tca8113 cells, are successfully overexpressed in Tca8113 cells
CircRNF13 (right side).
Fig. 4:It is devised for circRNF13 ring-types splicing site (exon 8 and exon 2 junction) selectively targeted
The siRNA sequence (left side) of the circular rna, is then transfected into Tca8113 cells, is successfully struck in Tca8113 cells low
The expression (right side) of circRNF13.
Fig. 5:MTT proliferation experiments show compared with control group (NC), and being overexpressed circRNF13 (OE-circRNF13) can be with
Inhibit the proliferation of Tca8113 cells, and the expression (si-circRNF13) for striking low circRNF13 can promote Tca8113 cells
Proliferation.
Fig. 6:Flow cytometry analysis is found compared with control group (NC), is overexpressed circRNF13 (OE-circRNF13)
Tca8113 cells G2/M phase distribution proportions obviously increase afterwards, show cell-cycle arrest in the G2/M phases, and strike low circRNF13's
After expressing (si-circRNF13), the S phases, which are distributed, significantly increases, and shows that cell cycle progression accelerates.
Fig. 7:Flow cytometry analysis Apoptosis situation is found compared with control group (NC), is overexpressed circRNF13
(OE-circRNF13) Tca8113 cells apoptosis ratio obviously increases afterwards, in the case of normal culture, apoptosis of tumor cells ratio compared with
Low, so striking the expression of low circRNF13, apoptotic cell ratio is in a slight decrease.
Fig. 8:CircRNF13 expression significantly reduces in chemotherapy resistance cell (CR) compared with control group (NC).
Fig. 9:The expression (si-circRNF13) of low circRNF13 is struck in original Tca8113 cells (NC) or in drug resistance
After being overexpressed circRNF13 (OE-circRNF13) in cell (CR), with the cisplatin treated cell of same concentrations, then streaming is thin
Born of the same parents' instrument detects Apoptosis situation, it is found that the expression for striking low circRNF13 can increase tolerance of the cell to cis-platinum (apoptosis subtracts
It is few), and the drug resistance of mdr cell can be significantly reduced by being overexpressed circRNF13 then.
Figure 10:It is had detected by qRT-PCR in control tissue (N) by 28 pairs of fresh Dendritic cell biopsies (T) and cancer
The expression of circRNF13 finds that circRNF13 expresses notable downward in Dendritic cell biopsy.
Figure 11:It is had detected in control tissue (left side) by the Dendritic cell (right side) and cancer of archive by hybridization in situ technique
The expression of circRNF13 further demonstrates circRNF13 and expresses relatively low or do not express in Dendritic cell biopsy.
Figure 12:We have collected 88 Dendritic cell tissue specimens for having Clinical Follow-up data, and in situ hybridization has detected
The expression of circRNF13, wherein 21 expression (Negative) that can't detect circRNF13, the existence of these patients
Time is shorter compared with other patients's (although circRNF13 expressions are not high, can detect to obtain, Positive), and prognosis is more
Difference.
Specific implementation mode
It further illustrates the present invention, is not intended to limit the present invention below in conjunction with specific implementation mode.
The full length sequence that circRNF13 in Tca8113 cells is determined is sequenced in embodiment 1, PCR
1. materials and methods
1.1 cell line
Tca8113 cells Tca8113 and Cal27 are purchased from Central South University's cell centre, and normal condition is cultivated.
1.2 reagents and kit
TRIZOLTMReagent(Invitrogen);Plastic recovery kit (OMEGA);Reverse Transcriptase kit
(Promega);Proteinase K, DNase I, RNAsin, RNase A (GBICOL companies);LAEnzyme (Takara).
1.3 fluorescence quantitative PCR detection circRNF13 is expressed in Tca8113 cells
Extracted total RNA, 1 μ g RNA carry out real-time fluorescence quantitative PCR after reverse transcription is at cDNA.CircRNF13 forward directions are drawn
Object is 5-GTCCAGGATAGACATAGAGC-3, such as SEQ NO:Shown in 2 and reverse primer 5-GTGTAGACTTGTGTGGCTGA-
3.Such as SEQ NO:Shown in 3.
GAPDH forward primers for control are 5 '-ACCACAGTCCATGCCATCAC-3 ' such as SEQ NO:Shown in 4, and
Reverse primer 5 '-TCCACCACCCTGTTGCTGTA-3 ', such as SEQ NO:Shown in 5.
Real-time fluorescence quantitative PCR reaction system
Real-time fluorescence quantitative PCR reaction step
5 Plate read
6 82℃ 30sec
7 Plate read
8 Go to step 2for more 39times
9 55.0 DEG C of from of Perform melting curve 95.0 DEG C of to, 0.2 DEG C of read every, hold
for 1sec.
The amplification curve and melting curve of real-time fluorescence quantitative PCR, the expression intensity root of each gene are confirmed after reaction
After CT values (threshold cycle values), reference gene (GAPDH) markization, is examined and calculated using group t-test
P values.
Finally, real-time fluorescence quantitative PCR is amplified the segment come and is sequenced by us, and sequence is carried out to the segment expanded
Row compare, confirm we amplification be exactly circRNF13, and obtain circRNF13 cyclisation splice specific site.
1.4 reverse transcription PCRs obtain circRNF13 overall lengths
1) it using Tca8113 cells Tca8113 cDNA as template, by designing two pairs of primers, is overlapped mutually twice embedding
The PCR amplification overall length circRNF13 sequences of set (sequence obtained by design of primers and PCR amplification is as shown in Figure 2).CircRNF13 is complete
Long sequence amplification primer is as follows:
It expands for the first time, sense primer:5-GCTAGAAGAAACAGACTTCGTAAA-3;Such as SEQ NO:Shown in 6;
Downstream primer:5-CTAATGAGGTCATCAGAATCAACA-3, such as SEQ NO:, shown in 7;
Second of amplification, sense primer:5-AATGTTGATTCTGATGACCT-3, such as SEQ NO:Shown in 8,
Downstream primer:5-AGATTGTGTAGACTTGTGTGG-3, such as SEQ NO:Shown in 9.
2) PCR amplification, circRNF13 full length sequences, PCR reaction conditions are as follows:
PCR reaction steps
5 return to the 2nd step, carry out 39 secondary response cycles altogether;
3) sequencing analysis is carried out after PCR product glue being recycled target fragment.
2. result
It expresses in Tca8113 cells of 2.1 circRNF13 after cisplatin treated and significantly increases
After cis-platinum (P) processing, we are had detected by real-time fluorescence quantitative PCR in cell Tca8113 cells
The expression of circRNF13 finds that circRNF13 expressions are significantly raised (Fig. 1, left) compared with negative control (NC);We
In order to which that confirm real-time fluorescence quantitative PCR detection is strictly circRNF13, we carry out real-time fluorescence quantitative PCR product
Sequencing analysis finds that amplified fragments are strictly that the 8 exons 3 ' end of RNF13 and 2 exons 5 ' end are spliced, strictly
One new circRNA molecule.
2.2 we expand sequencing and confirm the full length sequence of circRNF13
Because circRNF13 is a new circular RNA molecule, specific sequence is especially expressed in Dendritic cell
Transcript sequence need further confirmation.We design two pairs of primers and by PCR successful clones nested twice
The full length sequence of circRNF13, it was confirmed that circRNF13 is strictly by the RNF13 genes on the regions chromosome 3q25.1
The 2-8 exons cyclisation of (ring finger protein 13, NM_183381.2) is spliced to form (Fig. 2), and overall length is
716bp (such as SEQ NO:Shown in 1).
Embodiment 2, circRNF13 inhibit Tca8113 cells Cycle Arrest, inducing cell apoptosis
1. materials and methods
1.1 reagents and kit
Restriction enzyme Cla I and Sac II and T4DNA ligases etc. are purchased from TakaRa companies;TRIZOLTM
Reagent(Invitrogen);Plasmid extraction kit, plastic recovery kit (OMEGA);Reverse Transcriptase kit (Promega);
Proteinase K, DNase I, RNAsin, RNase A (GBICOL companies);Methyl thiazoly tetrazolium assay (MTT, Sigma);Antibiotic
G418(Ameresc);Cell cycle detection kit (Invitrogen), cell apoptosis detection kit (Invitrogene).
The Fiber differentiation of 1.2 Dendritic cell mdr cells
In cell culture medium, the cis-platinum of low dosage is added, and cis-platin concentrations are gradually increased, is trained by long-time induction
It supports, is finally obtained the drug-resistant cell strain being resistant to cis-platinum.
The structure of 1.3circRNF13 carrier for expression of eukaryon
We are inserted into upstream and downstream in pcDNA3.1 carriers (deriving from Invitrogen companies) at multiple cloning sites first
Loop-forming sequences, i.e. GTGCTGGGATTACAGGTGTGAGCTACCACCCCCGGCCCACTTTTT such as SEQ NO:Shown in 10,
GAAAAGAATTAGGCTCGGCACGGTAGCTCACACCTGTAATCCCAGCA such as SEQ NO:Shown in 11) it is used to help be inserted into piece
Section transcription is processed as circular rna;Here it is circular rna eukaryotic expression blank expression vectors;Specific building process is as follows:
According to the fundamentum of the Forming Mechanism of circRNA and eucaryote RNA montages, design suitable for circRNA
The loop-forming sequences of expression;According to the sequence information of commercial carrier pcDNA3.1, suitable multiple cloning sites are designed, to obtain
The complete DNA sequence dna realized circRNA and be overexpressed:
GTGCTGGGATTACAGGTGTGAGCTACCACCCCCGGCCCACTTTTTCTTAAGCTTGGTACCGAGCTCGGATCCACATC GATTGGTGGAATTCTGCAGATATCCACCGCGGTGGCGGCCGCTCGAGTCTAGAGAAAAGAATTAGGCTCGGCACGGT
AGCTCACACCTGTAATCCCAGCA, such as SEQ NO:Shown in 12;
Intermediate underscore part is multiple cloning sites, and both ends are respectively upstream and downstream loop-forming sequences;
Above-mentioned synthesis is realized that NheI restriction enzyme site sequences, the other end are added in DNA sequence dna one end that circRNA is overexpressed
Addition ApaI restriction enzyme site sequences obtain pcCirc blank by being built into pcDNA3.1 carriers after NheI and ApaI digestions
Plasmid (sequence such as SEQ NO:Shown in 13), sequencing confirms that plasmid is correct.
In order to build circRNF13 over-express vectors.We select Cla I and Sac II restriction enzyme sites for digestion
PcCirc empty plasmids (the i.e. above-mentioned pcDNA3.1 carriers for inserting upstream and downstream loop-forming sequences built), and will
Between the upstream and downstream loop-forming sequences of circRNF13 sequence insertion vectors (Fig. 3, left).
Building circRNF13 over-express vectors (i.e. carrier for expression of eukaryon), steps are as follows:
1) using Tca8113 cells Tca8113cDNA as template, TaKaRa LA are utilizedEnzyme carries out PCR amplification overall length
CircRNF13 sequences.CircRNF13 full length sequence amplimers are as follows:
Sense primer:5 '-GTGATTTTACAACGAGAT-3 ' such as SEQ NO:Shown in 14;
Downstream primer:5 '-CTTTCTTGAATTTATGTA-3 ' such as SEQ NO:Shown in 15;
In upstream and downstream, 5 ' ends of primer add restriction enzyme Cla I and Sac II recognition sites and protection alkali respectively
After base, primer sequence is as follows:
Upstream:5’-AGGAATCGAT GTGATTTTACAACGAGAT-3 ' (underscore part is Cla I recognition sites) is such as
SEQ NO:Shown in 16;
Downstream:5’-ATGCCCGCGG CTTTCTTGAATTTATGTA-3 ' (underscore part is Sac II recognition sites)
Such as SEQ NO:Shown in 17;
2) PCR amplification, circRNF13 full length sequences, PCR reaction conditions are as follows:
PCR reaction steps
5 return to the 2nd step, carry out 39 secondary response cycles altogether;
3) by through Cla I and Sac II double digestion rear electrophoresis, glue returns again after PCR product electrophoresis, glue recycling target fragment
It receives;
4) pcCirc empty plasmids recycle target fragment through Cla I and Sac II double digestion rear electrophoresis glue;
3) and 4) 5) with T4 DNA ligases connection step glue recovery product, you can obtain can be used for eukaryotic expression
The vector plasmid of circRNF13;
6) by the eukaryon expression plasmid transformed competence colibacillus large intestine bar comprising circRNF13 full length sequences that 5) step obtains
Bacterium, to expand plasmid.
1.4 circRNF13 tiny RNAs interference sequences (siRNA)
We for circRNF13 splice site devise specificity siRNA (siRNA, Fig. 4, a left side are shown
The position of siRNA) sequence is as follows:
5-AGAAAGGUGAUUUUACAACGA-3 such as SEQ NO:Shown in 18,
Control of the simultaneous selection in human genomic sequence there is no the Scramble sequences of target spot as siRNA:
Scramble sequences are as follows:
5’-GACACGCGACUUGUACCAC-3’.Such as SEQ NO:Shown in 19,
The sequence is synthesized by Invitrogen companies.
1.5 prepare the coated nano silicon particles of poly-D-lysine
The coated nano silicon particles of poly-D-lysine are carried out with OP-10/ hexamethylenes/ammonia microemulsion self-assembling technique
The synthesis of nano silicon particles (silica nanoparticle, SiNP), and using the surface energy of nano silicon particles and pass through ion
Electrostatic interaction prepares the nano silicon particles of polylysine modification;The nano particle can be prepared by following methods:
1) OP-10 (nonylphenol polyoxyethylene ether), hexamethylene and ammonium hydroxide are mixed, positive silicic acid is added after being stirred at room temperature uniformly
Different ester (TEOS), continue stirring to polymerization complete, be added equal-volume acetone, ultrasonic disperse, centrifugation, distilled water wash three times, from
Heart collection is deposited in 80 DEG C of dryings, finely ground to obtain nano silicon particles (SiNP, particle size range 10-50nm).Wherein H2O and OP-10 and
H2The molar ratio of O and TEOS is 2~10, ammonia concn is 1.6~28%, molar concentrations of the TEOS in hexamethylene be 0.1~
3mol/L。
2) SiNP is resuspended in 0.6M NaCO by 0.1~10mg/ml3In solution, supernatant is abandoned in ultrasonic disperse, centrifugation, then
Sediment is resuspended in by 0.1~10mg/ml in PBS (pH 7.4), ultrasonic disperse, add polylysine (final concentration of 4~
15nmol/mL), it mixes well, room temperature is mixed to shake;Centrifugation, abandons supernatant, and precipitation is resuspended in distilled water by 0.1~10mg/ml, is obtained
To the nano silicon particles of polylysine modification.
3) by modified nano silicon particles ultrasonic disperse, 10~50ug circRNF13 mistakes are added in every milliliter of nano particle suspension
Expression vector or 10-100pmol siRNA, mixing, are stored at room temperature and make it combine.
1.6 cell culture and transfection
The good Tca8113 cells Tca8113 and Cal27 of growth conditions or mdr cell are pressed 2 × 105A cells/well
It is inoculated in 6 orifice plates, 6 orifice plates is placed in 37 DEG C, 5%CO2In incubator, cell growth to be cultivated to 50-70% density
Start circRNF13 over-express vectors or the transfection of siRNA;Transfection process is as follows:
The poly that carrying circRNF13 eukaryon expression plasmids or siRNA that 100 μ l are prepared are added in sterile EP tube relies
The nano silicon particles suspension of propylhomoserin modification, with the 100 mild mixings of μ l serum free mediums;Cell is washed with D-Hank's liquid 3 times;
800 μ l serum free mediums (antibiotic-free) will be added in said mixture, 1 hole in 6 orifice plates is added after mild mixing;It will
6 orifice plates are placed in CO2In incubator, 37 DEG C are cultivated 6 hours, and supernatant is then abandoned, and complete medium is added and continues overnight incubation.With sky
The nano silicon particles of carrier or the polylysine modification of Scramble sequences are as experiment contrast.1.7 real time fluorescent quantitative
PCR detects the expression of intracellular circRNF13
After cell processing, in suitable time point collecting cell, extracted total RNA, 1 μ g RNA after reverse transcription is at cDNA,
Carry out real-time fluorescence quantitative PCR.CircRNF13 forward primers are 5-GTCCAGGATAGACATAGAGC-3, such as SEQ NO:2 institutes
Show and reverse primer 5-GTGTAGACTTGTGTGGCTGA-3.Such as SEQ NO:Shown in 3.
GAPDH forward primers for control are 5 '-ACCACAGTCCATGCCATCAC-3 ' such as SEQ NO:Shown in 4, and
Reverse primer 5 '-TCCACCACCCTGTTGCTGTA-3 ', such as SEQ NO:Shown in 5.
Real-time fluorescence quantitative PCR reaction system
Real-time fluorescence quantitative PCR reaction step
5 Plate read
6 82℃ 30sec
7 Plate read
8 Go to step 2for more 39times
9 55.0 DEG C of from of Perform melting curve 95.0 DEG C of to, 0.2 DEG C of read every, hold
for 1sec.
The amplification curve and melting curve of real-time fluorescence quantitative PCR, the expression intensity root of each gene are confirmed after reaction
After CT values (threshold cycle values), reference gene (GAPDH) markization, is examined and calculated using group t-test
P values.
1.8 MTT cell proliferation experiments
1) digest cell obtained in the previous step, cell counted with cell counter, will transfection circRNF13 and
The cell inoculation of pcDNA3.1 empty carriers is inoculated with 1000 cells per hole, as a result each 5 hole of cell inoculation takes in 96 orifice plates
Its mean value.
2) 37 DEG C, 5%CO2Incubator culture 6 hours adds 20 μ l of MTT liquid (5mg/ml) after cell is adherent per hole.After
It is continuous to be incubated 4 hours, culture is terminated, culture solution is discarded.150 μ l DMSO are added per hole, vibrate 10 minutes, crystal is made to dissolve.
3) 490nm wavelength is selected, zeroing hole is concurrently set, on enzyme-linked immunosorbent assay instrument, measures each hole absorbance value simultaneously
Record result.
4) it is same as above every 24 hours repetition steps, detects 6 days altogether.Using absorbance value as ordinate, interval time is horizontal seat
Plotting MTT curves.
5) it tests in triplicate.Using each time point as abscissa, absorbance value is ordinate, draws cell growth curve.
1.9 flow cytometry analysis cell cycles
1) trypsin digestion cell when culture cell reaches 85% fusion, 1200rpm/min centrifuge 5min, and it is heavy to collect cell
It forms sediment.
2) cell is resuspended in 1xPBS, and 1200rpm/min centrifuges 5min, collects cell.Repeat this step 2 time.
3) cell is resuspended in 1xPBS, and 70% ethyl alcohol that precooling is added fixes cell pellet overnight.
4) 1000rcf/min centrifuges 5min, collects cell precipitation.
5) PH7.4PBS washs cell 1 time, PBS is added after centrifugation, cell is resuspended, and adjust cell concentration to Ix 106/
ml。
6) propidium iodide (PI) dyeing liquor (PI containing 50mg/L, 1g/L Triton X-100,100g/L RNase) is added
Mixing, 4 DEG C are protected from light incubation 30min.
7) flow cytometer FACStar (U.S. company BD) is detected.Received signal is handled through Cellquest softwares, right
The fluorescence intensity of detection cell is analyzed.Experiment is repeated 3 times.
1.10 flow cytometry analysis natural death of cerebral cells rates
1) it when culture cell reaches 85% fusion, digests each group cell with pancreatin and is collected in centrifuge tube, be collected simultaneously
Each group supernatant suspension cell.Pay attention to gently blowing and beating cell, pancreatin is avoided excessively to digest.
2) merge each group cell respectively and be transferred in centrifuge tube, 1000rpm centrifuges 5min, abandons supernatant and collects cell, PBS
After being gently resuspended, cell count.
3) it takes 5~100,000 resuspension cells, 1000rpm to centrifuge 5min, abandon supernatant, 195ul Annexin V-FITC knots is added
It closes liquid and cell is gently resuspended.
4) 5ul Annexin V are added, gently mixing.It is protected from light incubation at room temperature 10min.
5) 1000rpm centrifuges 5min, abandons supernatant, 190ul Annexin V-FITC combination liquid is added, cell is gently resuspended.
6) 10ul PI dyeing liquors are added, gently mixing, ice bath are protected from light.
7) flow cytomery is carried out immediately, and Annexin V-FITC are green fluorescence, and PI is red fluorescence.
2. result
2.1 circRNF13 inhibit the growth of Tca8113 cells
After transfecting circRNF13 carrier for expression of eukaryon, we are had detected by real-time fluorescence quantitative PCR in cell
The expression of circRNF13 finds that circRNF13 expressions are significantly raised (Fig. 3);And transfect the small dry of targeting circRNF13
After disturbing RNA (siRNA), the expression of intracellular circRNF13 obviously lowers (Fig. 4);
Compared with the cell of transfection empty carrier, it is overexpressed the life of the Tca8113 cells Tca8113 and Cal27 of circRNF13
Long speed significantly slows down, and after striking the expression of low circRNF13 using siRNA interference sequences, vitro growth rates accelerate (figure
5)。
2.2 circRNF13 inhibit the growth of Tca8113 cells by arresting cell cycle, inducing cell apoptosis.
Flow cytometry analysis shows after transfecting circRNF13 in Tca8113 cells that G2/M phase cell proportions significantly increase
Add, and S phase cell proportions are reduced, and show that circRNF13 can block Tca8113 cells in the G2/M phases, make its cell division slow
Speed (Fig. 6).Meanwhile apoptotic cell ratio obviously increases (Fig. 7) after transfection circRNF13 in Tca8113 cells, this is also
CircRNF13 inhibits one of the reason of Tca8113 cells growth.And it strikes low circRNF13 and has then obtained opposite result.
2.3 circRNF13 express downward in Dendritic cell mdr cell
We by some months are cultivated by being stepped up cis-platin concentrations in the medium, and successfully induction obtains
Tca8113 and Cal27 has detected the expression of circRNF13 in cell to the cell of cisplatin resistance, real-time quantitative PCR, sends out
Existing expression of the circRNF13 in drug-resistant cell strain has significantly lowered (Fig. 8) compared with initial cell strain.
2.4 overexpression circRNF13 can reverse Tca8113 cells drug-resistant phenotype
The expression (si-circRNF13) of low circRNF13 is struck in original Tca8113 cells (NC) or in mdr cell
(CR) after being overexpressed circRNF13 (OE-circRNF13) in, with the cisplatin treated cell of same concentrations, then flow cytometer
Apoptosis situation is detected, the tolerance (apoptosis reduction) that the expression of low circRNF13 can increase cell to cis-platinum is struck in discovery, and
The drug resistance (Fig. 9) of mdr cell can then be significantly reduced by being overexpressed circRNF13.
Embodiment 3, quantitative real-time PCR detection confirm that circRNF13 expresses downward in Dendritic cell
1. materials and methods:
Collect by 28 Dendritic cells and 28 Dendritic cell tissues, extracted total RNA, 1 μ g RNA after reverse transcription is at cDNA, into
Row real-time fluorescence quantitative PCR.CircRNF13 forward primers are 5-GTCCAGGATAGACATAGAGC-3, such as SEQ NO:Shown in 2,
With reverse primer 5-GTGTAGACTTGTGTGGCTGA-3.Such as SEQ NO:Shown in 3.
GAPDH forward primers for control are 5 '-ACCACAGTCCATGCCATCAC-3 ' such as SEQ NO:Shown in 4, and
Reverse primer 5 '-TCCACCACCCTGTTGCTGTA-3 ', such as SEQ NO:Shown in 5.
Real-time fluorescence quantitative PCR reaction system
Real-time fluorescence quantitative PCR reaction step
5 Plate read
6 82℃ 30sec
7 Plate read
8 Go to step 2for more 39times
9 55.0 DEG C of from of Perform melting curve 95.0 DEG C of to, 0.2 DEG C of read every, hold
for 1sec
The amplification curve and melting curve of real-time fluorescence quantitative PCR, the expression intensity root of each gene are confirmed after reaction
After CT values (threshold cycle values), reference gene (GAPDH) markization, is examined and calculated using group t-test
P values.
2. result
CircRNF13 expressed in control tissue by cancer it is higher, and in Dendritic cell tissue expression significantly reduce P<0.01
(Figure 10).
Embodiment 4, in situ hybridization detection find that low expressions of the circRNF13 in Dendritic cell is related to patient's poor prognosis
1. MATERIALS METHODS
1.1 design and synthesize hybridization probe
In order to detect the expression of circRNF13 using in-situ hybridization method, we are for circRNF13 cyclisation splicings
Site (namely 2 exon of RNF13 genes and 8 exon stitching portions) devises oligonucleotide probe 1, and is used as
The in situ hybridization oligonucleotide probe of positive control 3.
Oligonucleotide probe in situ hybridization detection circRNF13 expression:
TCGTTGTAAAATCACCTTTCTTGAATTTAT is such as:SEQ NO:Shown in 20,
Positive control probe (detection house-keeping gene GAPDH):
GAPDH probes 1:5 '-CCACTTTACCAGAGTTAAAAGCAGCCCTGG-3 ', such as SEQ NO:Shown in 21,
GAPDH probes 2:5 '-CAGTAGAGGCAGGGATGATGTTCTGGAGAG-3 ', such as SEQ NO:Shown in 22,
GAPDH probes 3:5 '-GTCAGAGGAGACCACCTGGTGCTCAGTGTA-3 ', such as SEQ NO:Shown in 23.
Each gene specific oligonucleotides probe sequence of above-mentioned design is synthesized using chemical synthesis process.
1.2 oligonucleotide probe labelling kits and in situ hybridization detection reagent
Digoxin oligonucleotides tailing reagent (Dig Oligonucleitide Tailing Kit 2ndGeneration,
Roche companies), anti-digoxin-horseradish peroxidase complex detection kit (Anti-Digoxigenin-POD, Fab
Fragments, Roche company), enhance the TSA signal amplifying systems (TSA of detection of expression signal in situTMBiotin System,
NEL700 kits, PerkinElmer companies), DAB staining kits (Beijing Zhong Shan companies), 20x sodium citrate buffers
(saline sodium citrate, SSC), dextran sulfate (Dextran sulphate), deionized formamide
(Deionized Formamide), polyadenylic acid (polyadenylic acid, Poly A), poly deoxyadenylic acid
(polydeoxyadenylic acid, Poly dA) is denaturalized frog essence DNA (the denatured and sheared of shearing
Salmon sperm DNA, ssDNA), yeast transfer RNA (yeast t-RNA, tRNA), dithiothreitol (DTT) (DTT), 50x Deng Han
Family name's buffer solution (Denhardts ' s solution), phosphate buffer (PBS buffer), pepsin K, bovine serum albumin(BSA)
(BSA), triethanolamine (TEA), TNB Buffer (0.1M Tris-HCl, pH7.5,0.15M NaCL, 0.5%Blocking
Reagent), TNT Buffer (0.1M Tris-HCl, pH7.5,0.15M NaCL, 0.05%Tween 20), acetic anhydride, resistance
Disconnected reagent (Blocking reagent agent, Roche companies).
1.3 other main agents and material
Absolute ethyl alcohol, 90% alcohol, 70% alcohol, 50% alcohol, turpentine oil, distilled water, PBS buffer solution (pH7.2~
7.4, NaCl 137mmol/L, KCl 2.7mmol/L, Na2HPO44.3mmol/L, KH2PO41.4mmol/L);3% methanol-
Hydrogen peroxide solution (80% methanol and the configuration of 30% hydrogen peroxide);0.01mol/L citrate buffers (citrate buffer,
CB, pH6.0 ± 0.1,9ml 0.1M citric acid solutions and 41ml 0.1M sodium citrate solutions are added interim in 450ml distilled water
With postponing correction work liquid pH value again);0.1% trypsase;Haematoxylin;1% hydrochloride alcohol (1ml concentrated hydrochloric acid+99ml70% wine
Essence configuration);Mounting glue (PTS Cure Mount II);Special coverslip (480 × 240mm2) customize in Zhengzhou Glassware Factory.
Leica low melting points (58 DEG C) paraffin, domestic beeswax, absolute alcohol, dimethylbenzene, 10% neutrality paraformaldehyde (0.01mol/L,
PH7.4, DEPC distilled water and PBS buffer solution are prepared), haematoxylin, Yihong, neutral mounting natural gum, coverslip, glass slide.
1.4 label probe
Oligonucleotide probe label is carried out using 3-tailing DIG Olignucleutide Kit, reaction system is such as
Under.
100pmol oligonucleotide+ddH2O=9 μ l (control:control oligonucleutide 5μ
l+ddH2O4μl)
Mixing slightly centrifuges.37 DEG C of water-bath 30min add 2 μ l EDTA (0.2M, PH 8.0) stopped reactions.
It is purified after 1.5 oligonucleotide probes label
In order to increase the purity of label probe, marked probe need to be purified, concrete operations are as follows:
1) 100% cold ethyl alcohol (- 20 DEG C) of+2.5 μ l 4M LiCL+75 μ l of probe reaction mixture (22 μ l)
2) -70 DEG C of precipitations 60min, or-20 DEG C of 2h.
3) 13.000 × g, 4 DEG C of centrifugation 15min.
4) supernatant is abandoned, is washed with 70% ice-cold (V/V) ethyl alcohol of 50 μ l.
5) 4 DEG C of 13.000xg centrifuge 5min.
6) supernatant, 4 DEG C of dryings of vacuum are abandoned.
7) with the molten probe of aseptic double-distilled water weight.
1.6 in situ hybridizations detection achieves the expression of circRNF13 in paraffin section
Paraffin section hybridizes pre-treatment
1) paraffin section of 4 DEG C of preservations is placed in 58 DEG C of roasting piece 30min, melted surface paraffin.
2) dimethylbenzene dewaxes 3 × 5min successively.
3) step ethanol wash, 100% 1 × 5min of the alcohol of 2 × 2min of alcohol → 95% alcohol of 1 × 5min → 70% →
50% 1 × 5min of alcohol → 2 × 3min of DEPC water washings → DEPC-PBS washs 2 × 5min.
4) 300 μ l pepsins K (10 μ g/ml) are added dropwise on slice, 37 DEG C digest 20min.
5) it is sliced and washs 1min, stopped reaction into PBS (0.1M PBS+2mg/ml glutamic acid).
6) it is sliced into 0.2N HCL, reacts 20-30min in 37 DEG C, increase the permeability of tissue.
7) slice fixes 10min, room temperature afterwards with 4% paraformaldehyde (0.1M PBS dissolvings).
8) in order to increase tissue positive intensity for hybridization, acetyl processing is carried out to slice.It is sliced into 0.25% acetic anhydride
Buffer I (0.1M triethanolamines), room temperature 10min.
9) 1M PBS wash 2 × 5min.
Prehybridization and hybridization
Prehybridization:The prehybridization solution of -20 DEG C of preservations is first placed in 37 DEG C of incubation 60min, and the dosage of prehybridization solution is 50 μ l,
Parafilm is carried out lid and is sliced, prehybridization 2 hours in 37 DEG C of wet box.(prehybridization solution composition includes:2XSSC, 10%Dextran
Sulphate, 1X Denhardt ' s solution, 50mM Phosphate Buffer (PH 7.0), 50mM DTT, 250 μ l,
100 μ g/ml poly A, 5 μ g/ml poly dA, 250 μ g/ml yeast t-RNA, 500 μ g/ml ssDNA, 47%
Deionized formamide)。
1) parafilm is removed, prehybridization solution is got rid of, slice is placed in 5min in 2 × SSC.
2) hybridization reaction:37 DEG C of hybridized overnights (18-20h).Each slice is added 250 μ l hybridization solutions and is carried out with parafilm
Lid.Corresponding probe is added in prehybridization solution just becomes hybridization solution.Hybridization solution is prepared in prehybridization, is placed 37 DEG C of incubations, is made
Probe is completely dissolved in hybridization solution, this experiment is mixed with a plurality of oligonucleotide probe, is matched by each probe 500ng/ml concentration
Probe hybridization solution is made.Digoxin tailing labelling kit label probe concentration calculation basis:The concentration of each probe by its with
Colour developing is compared and the label reaction theory spy of the naked probe of 30 bases of 100pmol when detection reaction when the positive quantifies probe
Needle yield is that two kinds of standards of 900ng carry out the concentration that COMPREHENSIVE CALCULATING goes out label probe.
3) post-hybridization washing, slice immerse 2 × SSC, 10min, throw off parafilm.It is washed successively in shake on shaking table, 2 ×
SSC (0.5%SDS), 2 × 15min → 0.25 × SSC (0.5%SDS), 2 × 15min.
Color developing detection is reacted after hybridization
1) Anti-Digoxigenin-POD detection digoxigenin-probes and mRNA combination compounds are used;TSA amplification systems
Enhance the positive signal of in situ hybridization reaction solution reaction, DAB colour developings.
2) slice is gone in TNT buffer solutions, 3 × 5min.
3) TNB is added dropwise and blocks buffer solution, 300 μ l/TMAs, room temperature, 30min.
4) extra blocking agent is sucked, 1:100 diluted Anti-Digoxigenin-POD (TBS+0.1%Triton X-
100+1% blocking agents), room temperature 4 hours.
5) TNT Buffer (0.1M Tris-CL, pH7.5,0.15M NaCL, 0.05%Tween 20) are washed,
3x5min。
6) signal amplification reagent Biotinyl Tyamid, 300 μ l/TMAs, (Biotinyl Tyramid are added dropwise on slice
Store liquid:Biotinyl Tyramid are dissolved in 0.2ml DMSO, Biotinyl Tyramid working solutions:1 × dilution, 1:50
Dilute Biotinyl Tyramid and store liquid), room temperature 10 minutes.
7) TNT is washed, 3 × 5min.
8) SA-HRP (strepto- avidin-horseradish peroxidase), 300 μ l/TMAs, room temperature 30min are added dropwise in slice.
9) TNT is washed, 3 × 5min.
10) distilled water washing, 1 × 1min.
11) DAB develops the color, and chromogenic reaction is controlled under microscope.
12) haematoxylin is redyed,
13) alcohol step is dehydrated, chip drying.
14) mounting glue, the coverslip cover plate of dimension, crosslinking slice 1min under ultraviolet lamp is added dropwise.
1.7 results judge and standard
Application Optics microscope is observed under low power and high power lens respectively, looks first at the positive expression of target RNA
The signal positioning intracellular in object observing:Positioned at nucleus, cytoplasm or cell membrane.
It is carried out respectively with two kinds of standards of the intensity of the detection rna expression position positive signal and the cell number of positive expression again
Comprehensive score, criterion are:(1) judge according to positive cell dyeing intensity:A. cell dye-free remembers 0 point;B. cell is dyed
Light brown is weakly positive, remembers 1 point;C. cell dyes brown and dyes dark-brown without background coloration or cell and have the light brown back of the body
Scape is moderate positive, remembers 2 points;D. cell dyes dark-brown and is strong positive without background coloration, remembers 3 points.(2) according to positive cell
Express number score:A. no positive cell expression, remembers 0 point;B. positive expression cell number≤25% remembers 1 point;C.25% < is positive thin
Born of the same parents' number < 50% remembers 2 points;D. positive expression cell number >=50% remembers 3 points.
In order to reduce the subjective factor of appraisal result as possible, one of above-mentioned standard is pressed respectively respectively by two pathology experts
Judged and scored, then the two is scored and is multiplied, result is:1. 0 point of person is finally calculated as 0 point, it is believed that feminine gender expression;2. 1 point
It is finally calculated as 1 point with 2 points of persons, it is believed that weakly positive is expressed;3. 3 points and 4 points of persons are finally calculated as 2 points, it is believed that moderate positive is expressed;④
6, which assign to 9 points of persons, is finally calculated as 3 points, it is believed that strong positive is expressed.
1.8 analyses and statistical software
Statistical analysis is carried out to experimental result using SPSS13.0 statistical softwares, compares use χ two-by-two2Test or Fisher
Exact test, correlation analysis use Spearmen correlation methods;P < 0.05 are that difference is statistically significant.
Survivorship curve analysis uses Kaplan-Meier method and log-rank test;Multi-variables analysis uses Cox ' s
proportional hazards model;P < 0.05 are that difference is statistically significant.
2 results
Expression of 2.1 circRNF13 in Dendritic cell raising more notable than expression in normal control tissue
CircRNF13 is not expressed in Dendritic cell tissue or expression is relatively low, and the normal control by Dendritic cell cancer
There is expression (Figure 11) in tissue, there is apparent significant difference between the two.
Dendritic cell patient's prognosis of 2.2 circRNF13 low expressions is worse
Effect of follow-up visit by telephone has been carried out to 88 Dendritic cell patients, inquired in detail they start time, treatment, whether there is or not
Recurrence, whether there is or not suffering from other diseases, recurrence and death time etc. again, and register life span and state, and to Dendritic cell tissue
The survival analysis that the expression of middle circRNF13 and the life span of patient and state carry out finds not express in Dendritic cell tissue
The patient of circRNF13 is significantly shorter than circRNF13 the mean survival times and expresses higher patient (Figure 12).Explanation
CircRNF13 is one and the relevant molecular labeling of Dendritic cell prognosis, and circRNA expression is low or does not express, patient's poor prognosis.
Sequence table
<110>Central South University
<120>Promote application of the reagent of circular rna circRNF13 expression on preparing treatment Dendritic cell drug
<160> 23
<170> SIPOSequenceListing 1.0
<210> 1
<211> 716
<212> RNA
<213>Homo sapiens (Homo sapiens)
<400> 1
gugauuuuac aacgagaugc ugcucuccau agggaugcuc augcugucag ccacacaagu 60
cuacaccauc uugacugucc agcucuuugc auucuuaaac cuacugccug uagaagcaga 120
cauuuuagca uauaacuuug aaaaugcauc ucagacauuu gaugaccucc cugcaagauu 180
ugguuauaga cuuccagcug aagguuuaaa ggguuuuuug auuaacucaa aaccagagaa 240
ugccugugaa cccauagugc cuccaccagu aaaagacaau ucaucuggca cuuucaucgu 300
guuaauuaga agacuugauu guaauuuuga uauaaagguu uuaaaugcac agagagcagg 360
auacaaggca gccauaguuc acaauguuga uucugaugac cucauuagca ugggauccaa 420
cgacauugag guacuaaaga aaauugacau uccaucuguc uuuauuggug aaucaucagc 480
uaauucucug aaagaugaau ucacauauga aaaagggggc caccuuaucu uaguuccaga 540
auuuagucuu ccuuuggaau acuaccuaau ucccuuccuu aucauagugg gcaucugucu 600
caucuugaua gucauuuuca ugaucacaaa auuuguccag gauagacaua gagcuagaag 660
aaacagacuu cguaaagauc aacuuaagaa acuuccugua cauaaauuca agaaag 716
<210> 2
<211> 20
<212> DNA
<213>Unknown (Unknown)
<400> 2
gtccaggata gacatagagc 20
<210> 3
<211> 20
<212> DNA
<213>Unknown (Unknown)
<400> 3
gtgtagactt gtgtggctga 20
<210> 4
<211> 20
<212> DNA
<213>Unknown (Unknown)
<400> 4
accacagtcc atgccatcac 20
<210> 5
<211> 20
<212> DNA
<213>Unknown (Unknown)
<400> 5
tccaccaccc tgttgctgta 20
<210> 6
<211> 24
<212> DNA
<213>Unknown (Unknown)
<400> 6
gctagaagaa acagacttcg taaa 24
<210> 8
<211> 24
<212> DNA
<213>Unknown (Unknown)
<400> 8
ctaatgaggt catcagaatc aaca 24
<210> 8
<211> 20
<212> DNA
<213>Unknown (Unknown)
<400> 8
aatgttgatt ctgatgacct 20
<210> 9
<211> 21
<212> DNA
<213>Unknown (Unknown)
<400> 9
agattgtgta gacttgtgtg g 21
<210> 10
<211> 45
<212> DNA
<213>Unknown (Unknown)
<400> 10
gtgctgggat tacaggtgtg agctaccacc cccggcccac ttttt 45
<210> 11
<211> 47
<212> DNA
<213>Unknown (Unknown)
<400> 11
gaaaagaatt aggctcggca cggtagctca cacctgtaat cccagca 47
<210> 12
<211> 177
<212> DNA
<213>Unknown (Unknown)
<400> 12
gtgctgggat tacaggtgtg agctaccacc cccggcccac tttttcttaa gcttggtacc 60
gagctcggat ccacatcgat tggtggaatt ctgcagatat ccaccgcggt ggcggccgct 120
cgagtctaga gaaaagaatt aggctcggca cggtagctca cacctgtaat cccagca 177
<210> 13
<211> 5515
<212> DNA
<213>Unknown (Unknown)
<400> 13
gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60
ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120
cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180
ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240
gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300
tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360
cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420
attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480
atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540
atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600
tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660
actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720
aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780
gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840
ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 900
gtgctgggat tacaggtgtg agctaccacc cccggcccac ttttttttaa acttaagctt 960
ggtaccgagc tcggatccac atcgattggt ggaattctgc agatatccac cgcggtggcg 1020
gccgctcgag tctagagaaa agaattaggc tcggcacggt agctcacacc tgtaatccca 1080
gcagggcccg tttaaacccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct 1140
gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt 1200
tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg 1260
ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg 1320
gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc tagggggtat 1380
ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg 1440
accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc 1500
gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga 1560
tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt 1620
gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat 1680
agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat 1740
ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa 1800
tttaacgcga attaattctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct 1860
ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa 1920
agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa 1980
ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt 2040
ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct 2100
ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc 2160
tcccgggagc ttgtatatcc attttcggat ctgatcaaga gacaggatga ggatcgtttc 2220
gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat 2280
tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt 2340
cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac 2400
tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg 2460
tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc 2520
aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa 2580
tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc 2640
gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg 2700
aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg 2760
acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa 2820
atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg 2880
acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct 2940
tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc 3000
ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag cgacgcccaa 3060
cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat 3120
cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt 3180
cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac 3240
aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat 3300
caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg 3360
gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc 3420
cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc 3480
gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat 3540
cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac 3600
tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt 3660
aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca 3720
gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc 3780
ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact 3840
ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct 3900
gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag 3960
ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca 4020
cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa 4080
cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc 4140
gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag 4200
aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg 4260
tagctcttga tccggcaaac aaaccaccgc tggtagcggt ttttttgttt gcaagcagca 4320
gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga 4380
cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat 4440
cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga 4500
gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg 4560
tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga 4620
gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc 4680
agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac 4740
tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc 4800
agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc 4860
gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc 4920
catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt 4980
ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc 5040
atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg 5100
tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag 5160
cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat 5220
cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc 5280
atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa 5340
aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta 5400
ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa 5460
aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtc 5515
<210> 14
<211> 18
<212> DNA
<213>Unknown (Unknown)
<400> 14
gtgattttac aacgagat 18
<210> 15
<211> 18
<212> DNA
<213>Unknown (Unknown)
<400> 15
ctttcttgaa tttatgta 18
<210> 16
<211> 28
<212> DNA
<213>Unknown (Unknown)
<400> 16
aggaatcgat gtgattttac aacgagat 28
<210> 17
<211> 28
<212> DNA
<213>Unknown (Unknown)
<400> 17
atgcccgcgg ctttcttgaa tttatgta 28
<210> 18
<211> 21
<212> RNA
<213>Unknown (Unknown)
<400> 18
agaaagguga uuuuacaacg a 21
<210> 19
<211> 19
<212> RNA
<213>Unknown (Unknown)
<400> 19
gacacgcgac uuguaccac 19
<210> 20
<211> 30
<212> DNA
<213>Unknown (Unknown)
<400> 20
tcgttgtaaa atcacctttc ttgaatttat 30
<210> 21
<211> 30
<212> DNA
<213>Unknown (Unknown)
<400> 21
ccactttacc agagttaaaa gcagccctgg 30
<210> 22
<211> 30
<212> DNA
<213>Unknown (Unknown)
<400> 22
cagtagaggc agggatgatg ttctggagag 30
<210> 23
<211> 30
<212> DNA
<213>Unknown (Unknown)
<400> 23
gtcagaggag accacctggt gctcagtgta 30
Claims (4)
1. promoting application of the reagent of circular rna circRNF13 expression on preparing treatment Dendritic cell drug, the ring-type
The sequence of RNA circRNF13 such as SEQ NO:Shown in 1.
2. application according to claim 1, which is characterized in that the examination for promoting circular rna circRNF13 expression
Agent is circRNF13 over-express vectors.
3. application according to claim 1, which is characterized in that the building process of circRNF13 over-express vectors is as follows:
(1) it according to the fundamentum of the Forming Mechanism of circRNA and eucaryote RNA montages, designs suitable for circRNA tables
The loop-forming sequences reached;According to the sequence information of commercial carrier pcDNA3.1, multiple cloning sites are designed, to obtain complete reality
The DNA sequence dna that existing circRNA is overexpressed is as follows:
GTGCTGGGATTACAGGTGTGAGCTACCACCCCCGGCCCACTTTTTCTTAAGCTTGGTACCGAGCTCGGATCCA CATCGATTGGTGGAATTCTGCAGATATCCACCGCGGTGGCGGCCGCTCGAGTCTAGAGAAAAGAATTAGGCTCGGCA
CGGTAGCTCACACCTGTAATCCCAGCA, intermediate underscore part are multiple cloning sites, and both ends are respectively upstream and downstream cyclization
Sequence;
(2) above-mentioned synthesis is realized that NheI restriction enzyme site sequences, the other end are added in DNA sequence dna one end that circRNA is overexpressed
Addition ApaI restriction enzyme site sequences obtain pcCirc blank by being built into pcDNA3.1 carriers after NheI and ApaI digestions
Plasmid, sequencing confirm that plasmid is correct, sequence such as SEQ NO:Shown in 13;
(3) selection Cla I and Sac II restriction enzyme sites are used for digestion pcCirc empty plasmids, and circRNF13 sequences are inserted into
Between the upstream and downstream loop-forming sequences of carrier.
4. application according to claim 3, which is characterized in that the detailed process of step (3) is as follows:
1) using Tca8113 cells Tca8113cDNA as template, TaKaRa LA are utilizedEnzyme carries out PCR amplification overall length
CircRNF13 sequences;CircRNF13 full length sequence amplimers are as follows:
Sense primer:5’-GTGATTTTACAACGAGAT-3’;
Downstream primer:5’-CTTTCTTGAATTTATGTA-3’;
After 5 ' ends of upstream and downstream primer add restriction enzyme Cla I and Sac II recognition sites and protection base respectively,
Primer sequence is as follows:
Sense primer:5’-AGGAATCGATGTGATTTTACAACGAGAT-3 ', underscore part are Cla I recognition sites;
Downstream primer:5’-ATGCCCGCGGCTTTCTTGAATTTATGTA-3 ', underscore part are Sac II recognition sites;
2) PCR amplification, circRNF13 full length sequences, PCR reaction conditions are as follows:
PCR reaction steps
5 return to the 2nd step, carry out 39 secondary response cycles altogether;
3) by through Cla I and Sac II double digestion rear electrophoresis, glue recycles again after PCR product electrophoresis, glue recycling target fragment;
4) pcCirc empty plasmids recycle target fragment through Cla I and Sac II double digestion rear electrophoresis glue;
5) with the connection of T4DNA ligases 3) and 4) step glue recovery product to get to the vector plasmid for being overexpressed circRNF13;
6) by the eukaryon expression plasmid transformed competence colibacillus Escherichia coli comprising circRNF13 full length sequences that 5) step obtains, with
Expand plasmid.
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CN110093419A (en) * | 2019-04-26 | 2019-08-06 | 北京大学深圳医院 | Application, kit and the pharmaceutical composition of circular rna and its application |
CN110859861A (en) * | 2019-12-04 | 2020-03-06 | 武汉工程大学 | Application of plant pathogenic microorganism in killing cancer cells or AIDS virus |
CN112574992A (en) * | 2020-12-17 | 2021-03-30 | 湖南丰晖生物科技有限公司 | Circular RNA over-expression cyclization vector DNA sequence and construction method and application thereof |
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110093419A (en) * | 2019-04-26 | 2019-08-06 | 北京大学深圳医院 | Application, kit and the pharmaceutical composition of circular rna and its application |
CN110093419B (en) * | 2019-04-26 | 2023-10-27 | 北京大学深圳医院 | Application of circular RNA, kit and pharmaceutical composition and application thereof |
CN110859861A (en) * | 2019-12-04 | 2020-03-06 | 武汉工程大学 | Application of plant pathogenic microorganism in killing cancer cells or AIDS virus |
CN112574992A (en) * | 2020-12-17 | 2021-03-30 | 湖南丰晖生物科技有限公司 | Circular RNA over-expression cyclization vector DNA sequence and construction method and application thereof |
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