CN106222170B - Circular rna circ-CCNY and application thereof - Google Patents
Circular rna circ-CCNY and application thereof Download PDFInfo
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Abstract
The present invention provides a kind of circular rna circ-CCNY and application thereof, the nucleotide sequence of the circ-CCNY gene is as shown in SEQ ID NO:1；The present invention has found that its expression is substantially reduced by circ-CCNY expression conditions in detection hepatocarcinoma patient；The liver cancer cells for being overexpressed circ-CCNY gene compared with compareing liver cancer cells, are it is found that its growth rate significantly slows down by the present invention.The marker of circ-CCNY gene and its expression product as diagnosing liver cancer keeps diagnosing cancer of liver more accurate, quick, and the target gene as preparation treatment liver-cancer medicine provides new therapy target and therapy approach for treatment liver cancer.
Description
Technical field
The invention belongs to molecular biology and oncology, and in particular to a kind of circular rna circ-CCNY and its use
On the way.
Background technique
Primary hepatoma (Hepatocellular carcinoma, HCC, hereinafter referred to as liver cancer) is in global range
One of most common malignant tumour.There are about 740,000 patients to be newly diagnosed as hepatocellular carcinoma every year in the whole world, while there are about 690,000 patients
Die of liver cancer.Wherein China has accounted for the 55% of global annual new cases, becomes most high-incidence one of the area of whole world liver cancer.
Circular rna (ciucular RNA, circRNA) is the RNA family newcomer for being different from conventional linear RNA, is not had
There are the end 5' cap and the end 3' poly (A) tail and forms the non-coding RNA molecule of ring structure with covalent bond.It is newest to grind
Study carefully and show that circRNA has closed hoop structure, is mainly generated by the processing of atypia variable sheer, be widely present in various lifes
In object cell, there is stable structure, it is difficult to which good by conservative between RNA enzyme degradation, gene expression abundance height, species, expression has tissue
And the features such as Space-time speciality, these features have circRNA in the development and application of New Type of Diseases Clinics and Practices method
It holds out broad prospects.
CCNY is the member of a kind of cyclin family found recently.The CCNY assignment of genes gene mapping in No. 10 chromosomes,
Overall length 3968bp has 10 exons, and 9 intrones encode the albumen of 341 amino acid, and molecular weight of albumen is about 39KD.It grinds
Study carefully and shows the high expression in non-small cell lung cancer tumor tissues and lung cancer cell line of CCNY gene；Inhibited using RNA perturbation technique
The expression of CCNY gene can significantly inhibit proliferation of lung cancer cells.The proliferation of CCNY gene and brain glioblastoma cell is closely related, suppression
CCNY expression processed can equally significantly inhibit the growth of Glial cells oncocyte.In addition, also found CCNY1 by yeast two-hybrid
Can be in conjunction with another kind cyclin kinase PFTK1 (CDK14), this is close with DISTANT METASTASES IN and the capilary transfer of tumour
It is related；In addition, CCNY can be obviously promoted the activity of transcription factor C-myc.Transcription factor C-myc is in many tumours in activity
Adjust, during the various biologicals such as cell cycle variation, cell Proliferation and apoptosis from important adjustment effect, prompt CCNY to exist
The generation of kinds of tumors, development play an important role in transfer.
Circ-CCNY (CircBase ID:hsa_circ_0000235), positioning in the genome are as follows: chr10:
35805450-35819171, corresponding glm gene are CCNY (NM_145012), and cyclization sequence has 315 bases, includes
4th to the 7th exon of CCNY gene.There has been no any reports about circ-CCNY function at present.
Present invention applicant's early-stage study find the RNA of CCNY gene in hepatic tissue there are cyclic arrangement, and circular rna
Circ-CCNY is significantly lowered in Expression In Hepatocellular Carcinoma, is overexpressed circ-CCNY and is inhibited hepatoma cell proliferation, prompts circ-
CCNY is likely to become the new way of diagnosing cancer of liver and treatment.
Summary of the invention
It is an object of the invention to according to inventor to expression of the circular rna circ-CCNY in human liver cancer and its to liver
The research of the adjustment effect of cancer cell biology function provides the examining in liver cancer based on circ-CCNY gene and its expression product
Disconnected and treatment aspect purposes.
To achieve the above object, the technical solution used in the present invention are as follows: a kind of circular rna circ-CCNY, the ring
The nucleotide sequence of the gene of shape RNAcirc-CCNY is as shown in SEQ ID NO:1.The structure of the circular rna circ-CCNY
It is the end to end circular rna structure as being formed after the transcription of the nucleotide sequence as shown in SEQ ID NO:1 through montage.It is described
The mature body sequence of circular rna circ-CCNY is as shown in SEQID NO:2.
The present invention also provides a kind of pharmaceutical composition, described pharmaceutical composition includes the upper circular rna circ-
CCNY。
The present invention also provides a kind of diagnosing cancer of liver kit, the kit includes that can expand ring-type described above
The primer of RNA circ-CCNY.
Preferably, the amplimer is made of the DNA sequence dna as shown in SEQ ID NO:3 and SEQ ID NO:4
Primer pair or the primer pair being made of the DNA sequence dna as shown in SEQ ID NO:5 and SEQ ID NO:6.
The present invention provides the purposes of circular rna circ-CCNY described above in the drug of preparation treatment liver cancer.
Purposes the present invention provides circ-CCNY gene as target gene in the drug of preparation treatment liver cancer, it is described
The nucleotide sequence of circ-CCNY gene is as shown in SEQ ID NO:1.
The present invention provides purposes of the circ-CCNY gene activation in the drug of preparation treatment liver cancer, the circ-
The nucleotide sequence of CCNY gene is as shown in SEQ ID NO:1.
Compared with prior art, the beneficial effects of the present invention are:
(1) present invention carries out RT-PCR by designing special circular rna primer first, a generation is sequenced, RNA enzyme degradation is real
Verify the objective reality of reality circ-CCNY gene；
(2) present invention has found that its expression obviously drops by circ-CCNY expression conditions in detection hepatocarcinoma patient
It is low, therefore, it can be used as the diagnostic markers of liver cancer；
(3) present invention has carried out the research of cell in vitro function assessment to circ-CCNY gene, by by circ-CCNY base
Because being building up on the dedicated slow virus carrier of circular rna, to establish the stable cell line for being overexpressed the gene.It is overexpressed circ-
Compared with control cell strain, growth rate significantly slows down the hepatoma cell strain of CCNY gene.Circ-CCNY gene and its expression
Product can be applied in the drug of preparation treatment liver cancer.
The marker of circ-CCNY gene and its expression product as diagnosing liver cancer keeps diagnosing cancer of liver more accurate, fast
Speed, the target gene as preparation treatment liver-cancer medicine provide new therapy target and therapy approach for treatment liver cancer.
Detailed description of the invention
Fig. 1 is the agarose gel electrophoresis figure of circ-CCNY gene expression product in the embodiment of the present invention 1.
Fig. 2 is to reflect that RNaseR is on the histogram of circ-CCNY and GAPDH influence situation in the embodiment of the present invention 2.
Fig. 3 is that QPCR detects circ-CCNY expression of results comparison diagram in liver cancer tissue in the embodiment of the present invention 2.
Fig. 4 is stable cell line 97L-circ-CCNY and the LM3-circ-CCNY mistake built in the embodiment of the present invention 3
Express the comparative result figure of circ-CCNY.
Fig. 5 is the hepatoma cell strain 97L and LM3 and control cell lines that circ-CCNY is overexpressed in the embodiment of the present invention 4
Cell Proliferation curve synoptic diagram, wherein abscissa is number of days, and ordinate is the 490nm light absorption value of microplate reader detection.
Specific embodiment
The present invention passes through design first can expand the specific primer of circular rna, and PCR amplification comes out the ring-type of CCNY gene
RNA, and the accurate cyclisation site come circular rna circ-CCNY is determined by the method that a generation is sequenced, then carry out
A kind of RNaseR degradation experiment, it is determined that ring being made of 315 nucleotide, with closed hoop structure of CCNY gene expression
Shape RNA molecule.
Further, the present invention is by using the method detection circ-CCNY gene of quantitative fluorescent PCR in liver cancer sample
Differential expression, expression of the circ-CCNY gene in liver cancer tissue is significantly lower than the expression in cancer beside organism as the result is shown
It is horizontal.Therefore the kit for detecting the changes in gene expression can be made for diagnosing liver cancer.
Then, we have carried out the research of cell in vitro function assessment to circ-CCNY gene, pass through the side of lentivirus mediated
Method building is overexpressed the stable cell line of circ-CCNY gene.Be overexpressed circ-CCNY gene hepatoma cell strain with compare liver
Cancer cell line is compared, and growth rate significantly reduces.Therefore, circ-CCNY gene and its expression product, which can be applied, is preparing
It treats in the drug of liver cancer
Technology according to the present invention is molecular cloning conventional technical means, the enzyme being directed to, primer, reagent and
Reaction condition can reasonably select in the case where not specified (NS) according to the experience of those skilled in the art, be directed to try
Agent consumptive material belongs to commercially available common product, and the detection means and instrument being directed to also are well known to the skilled person
And it skillfully grasps.
Below by embodiment and test example, the following further describes the technical solution of the present invention, but should not be construed as pair
Limitation of the invention.
Expression of the embodiment 1:RT-PCR reaction detection circ-CCNY gene in liver cancer tissue.
Specific experiment scheme is as follows:
1.RNA is extracted
1) it tissue treatment: takes 10mg or so tissue that 1mlTrizol is added, is homogenized with refiner；Centrifugation 15 minutes,
12000g takes supernatant.
2) 200ul chloroform is added into supernatant, is firmly mixed by inversion half a minute up and down, stands 3 minutes.
3) 4 DEG C, 12000g is centrifuged 15 minutes, and visible lysate divides three layers at this time: upper layer is the RNA of water phase；Middle layer is
DNA, lipid etc.；Lower layer is cell residue, albumen, polysaccharide etc..
4) it takes in supernatant to new EP pipe；Isometric isopropanol is added, mixes, stands after ten minutes, 4 DEG C, 12000g
Centrifugation 10 minutes.
5) carefully remove supernatant, be careful not to lose RNA precipitate, 75% ethyl alcohol of 1ml is added, turns upside down, make to precipitate block
It is resuspended.
6) 4 DEG C, 12000g is centrifuged 10 minutes, is carefully removed supernatant, is blotted the liquid of tube wall as far as possible, is careful not to lose
RNA precipitate, if precipitating, which loosens, to be centrifuged again.It dries about 15 minutes, until tube wall no liquid.
7) be added appropriate volume (20-30ul) DEPC water dissolve RNA, 58 DEG C water-bath 10 minutes.
8) it is quantitative to take out 2ul, measures buffer:10mM TrisCl (pH7.8), reverse transcription is carried out according to quantitative result.
(1A260=40 μ g/ml, A260/A280=1.8~2.1).
2.cDNA reverse transcription
1) experimental system
M-MLV Reverse Transcriptase:
3. primer: circular rna primer is reverse primer, while designing a pair of of forward primer and compareing, and when RT-PCR sets up
One group of gDNA Template Controls, it was demonstrated that circRNA comes from post transcription cleavage, rather than Gene Fusion etc. is mutated.It detects simultaneously linear
Gene GAPDH is as negative control.The primer used is listed below:
| SEQ ID NO:3 | circ-CCNY_F divergent | ACAACCCAGAGCAGAAGCAGA |
| SEQ ID NO:4 | circ-CCNY_R divergent | CTGTATTTCCTTGCTATTTGTCCTG |
| SEQ ID NO:5 | circ-CCNY_F convergent | GGCTGAATGTGCCATCGTC |
| SEQ ID NO:6 | circ-CCNY_R convergent | GTCCTCCACCGTGATGTCTT |
| SEQ ID NO:7 | hsaGAPDH convergent_F | GAGTCAACGGATTTGGTCGT |
| SEQ ID NO:8 | hsaGAPDH convergent_R | GACAAGCTTCCCGTTCTCAG |
| SEQ ID NO:9 | hsaGAPDH divergent_F | TCCTCACAGTTGCCATGTAGACCC |
| SEQ ID NO:10 | hsaGAPDH divergent_R | TGCGGGCTCAATTTATAGAAACCGGG |
4.PCR: using GAPDH as internal reference.The reaction system of PCR: be separately added into each reaction tube 2 μ l 10 ×
Buffer, is separately added into 2 μ l dNTP, 1 μ l forward primer, 1 μ l reverse primer, 1 μ l cDNA template, and 0.2 μ l Taq enzyme adds water
To 20 μ l.PCR reaction condition is as follows: 94 DEG C, 5 minutes initial denaturations；It 94 DEG C, is denaturalized within 30 seconds；It 55 DEG C, anneals within 30 seconds；72 DEG C, 30 seconds
Extend；35 recycle, agarose gel electrophoresis detection pcr amplification product, the result is shown in Figure 1, and circular rna primer is reversed in Fig. 1
Primer (black triangle symbol), while designing a pair of of forward primer and compareing (white triangles pictograph number), when RT-PCR, set up
One group of gDNA Template Controls, it was demonstrated that circRNA comes from post transcription cleavage, rather than Gene Fusion etc. is mutated.It detects simultaneously linear
Gene GAPDH is as negative control.
Embodiment 2:QPCR detects the expression of circ-CCNY in liver cancer
1.RNA is extracted: with embodiment 1；
2.cDNA reverse transcription: with embodiment 1；
3.QPCR amplification experiment
1) experimental system:
Select the circ-CCNY divergent primer of embodiment 1 for expanding circular rna circ-CCNY, embodiment 1
HsaGAPDH convergent primer for expanding reference gene.
2) reaction condition:
Step 1: 95 DEG C of 2min
Second step (40 circulation): 95 DEG C 3 seconds, 60 DEG C 30 seconds
60-95 DEG C of solubility curves of third step
3) machine carries out target gene amplification on
4) qPCR relative quantification result
The relative expression quantity calculation formula of target gene are as follows: 2- △ △ Ct=2-[(△ Ct) Test- (△ Ct)
Control].Ct purpose is target gene Ct value, and house keeper Ct is house-keeping gene Ct value.△ Ct=Ct purpose-Ct house keeper indicates each
Opposite Ct value of the sample target gene with respect to house-keeping gene, △ △ Ct=(△ Ct) Test- (△ Ct) Control, expression processing
Group relative comparison group is normalized, and 2- △ △ Ct indicates the relative expression quantity of processing group relative comparison group, indicates target gene
Relative fold expression.
Total RNA is added RNaseR in the ratio of 3U/ug and is digested, and QPCR detection adds and is not added RNaseR pairs
The influence of circ-CCNY and GAPDH, is as a result shown in Fig. 2, and RNaseR digestion confirms that circ-CCNY is insensitive to RNA enzyme.RNaseR
Linear rna can be digested by being one, but not have influential RNA enzyme to circular rna, after total RNA is digested with RNaseR again into
Row QPCR detection as the result is shown plus with RNaseR is not added has not significant impact circ-CCNY expression, but glm gene
GAPDH expresses significant decrease after RNaseR digests.
QPCR detects the expression of circ-CCNY and GAPDH in liver cancer and corresponding adjacent tissues, Fig. 3 is as a result seen, 80
Expression to circ-CCNY is detected in liver cancer clinical tissue sample, circ-CCNY is significantly lowered in cancerous tissue as the result is shown.
N: cancer beside organism, T: liver cancer tissue.
Embodiment 3: circ-CCNY slow virus and its stable cell lines building are overexpressed
1. being overexpressed the building of circ-CCNY slow virus carrier: the synthesis linear complete sequence of circ-CCNY, sequence is by annealing
At double chain DNA fragment, it is inserted into LV-Circ carrier by multiple cloning sites, recombinant plasmid is identified by sequencing,
Control negative control is the LV-Circ empty carrier for being not inserted into sequence.
2. slow virus is packed
(1) before transfection for 24 hours, it with the 293T cell of pancreatin digestion logarithmic growth phase, passes on and arrives 10cm Tissue Culture Dish, 37
DEG C, 5%CO2Culture in incubator.It can be used to transfect when cell density is up to 70%~80% for 24 hours.Cell state is for disease
Poison packaging is most important, it is therefore desirable to guarantee good cell state and less passage number.
(2) cell culture medium is changed to serum free medium before transfection.
(3) prepared each DNA solution (10 μ of LV-Circ-CCNY/LV-Circ carrier is added into a sterile centrifugation tube
5 μ g of g, pGag/Pol carrier, 5 μ g of pRev carrier, 5 μ g of pVSV-G carrier), it is uniformly mixed, adjusts with the Opti-MEM of respective volume
Whole total volume is 1.5ml.
(4) 2000 reagent of Lipofectamine is softly shaken up, takes 60 μ l Lipofectamine, 2000 reagent another
One Guan Zhongyu 1.5ml Opti-MEM mixing, incubates 5 minutes at room temperature.
(5) DNA after dilution is mixed with the Lipofectamine 2000 after dilution, is lightly mixed by inversion,
It not vibrate.
(6) it after mixing, incubates 20 minutes at room temperature, to form DNA and 2000 dilution of Lipofectamine
Transfection composite.
(7) DNA and 2000 mixed liquor of Lipofectamine are transferred in the culture solution of 293T cell, are mixed, in 37
DEG C, 5%CO2It is cultivated in cell incubator.
(8) culture medium containing transfection mixture is sucked after cultivating 6 hours, is added in every bottle of cell thin containing 10% serum
Born of the same parents culture medium 10ml, in 37 DEG C, 5%CO2Continue culture 48 hours in incubator.
3. the harvest and concentration of virus
(1) the 293T cell supernatant of 48 hours and 72 hours (transfection can be counted for 0 hour) is collected after transfecting.
(2) in 4 DEG C, 4000g is centrifuged 10min, removes cell fragment.
(3) with 0.45 μm of filter filtering supernatant in 50ml centrifuge tube.
(4) viral crude extract sample is added in filter cup (most 19ml), closed the lid.Filter cup is inserted into filtration
In liquid collecting pipe.
(5) after combining, balance is carried out, is placed in rotary head.
(6) it is centrifuged in 5000 × g, until the viral concentration volume needed.The time usually required is 10-15 minutes.
(7) after being centrifuged, in filter cup is viral concentration liquid.
(8) viral concentration liquid is removed, is stored in after packing in viral pipe, can saved one week at 4 DEG C, or -80 DEG C long-term
It saves.Take a wherein progress viral biology titer determination.
4. slow virus infected cell:
(1) cell is collected by centrifugation to the serum-free medium for then using 100-200ul according to the amount of cell in 1.5ml pipe
Diluting cells precipitating, cell of being subject to are totally submerged in the medium.
(2) it draws the circ-CCNY virus liquid being overexpressed to be added in cell, 1.5ml pipe is placed in 37 DEG C of degree incubators
It is incubated for 30 minutes.Separately LV-Circ empty vector control virus infection is taken to do control cell lines.
(3) mixed solution suction in pipe is added in culture dish or in hole.
(4) fresh medium of sufficient amount is added.
Liquid is changed after (5) 12 hours.
2ug/ml puromycin is added after (6) 48 hours and carries out stable cell line screening.
5. stable cell line is identified: the stable cell line built being collected part cell QPCR detection, as a result such as Fig. 4 institute
Show, it was demonstrated that be overexpressed circ-CCNY cell strain LM3-circ-CCNY, 97L-circ-CCNY and cellular control unit strain LM3NC,
97L NC is compared, and the expression quantity of circ-CCNY dramatically increases.
Embodiment 4: it is overexpressed the hepatoma cell proliferation ability measurement of circ-CCNY gene
1) cell strain for being overexpressed circ-CCNY gene and cellular control unit are digested to single cell suspension, counted, adjustment
Cell concentration is 1 × 105A/ml, assigns to 96 orifice plates, every hole 100ul, i.e., every hole cell is 1 × 104It is a, each 9 hole.
2) MTS reagent, ratio 1:10, i.e. 100 μ l culture solutions is added in point (for 24 hours, 48h, 72h) in different times respectively
10 μ l detection liquid is added.
3) after 37 DEG C of incubation 4h, microplate reader detects 490nm light absorption value.
As a result see that Fig. 5, the hepatoma cell strain growth rate of circ-CCNY expression up-regulation slow down, be overexpressed circ-CCNY's
The cell Proliferation curve synoptic diagram of hepatoma cell strain 97L and LM3 and control cell lines, wherein abscissa is number of days, and ordinate is
The 490nm light absorption value of microplate reader detection.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than protects to the present invention
The limitation of range is protected, although the invention is described in detail with reference to the preferred embodiments, those skilled in the art should
Understand, it can be with modification or equivalent replacement of the technical solution of the present invention are made, without departing from the essence of technical solution of the present invention
And range.
Claims (7)
1. a kind of circular rna-circ-CCNY, which is characterized in that the cDNA sequence such as SEQ of the circular rna-circ-CCNY
Shown in ID NO:1, the structure of the circular rna circ-CCNY is transcribed as the nucleotide sequence as shown in SEQ ID NO:1
The circular rna structure being connected by the first place that montage is formed.
2. circular rna-circ-CCNY according to claim 1, which is characterized in that the circular rna-circ-CCNY's
Mature body sequence is as shown in SEQ ID NO:2.
3. a kind of pharmaceutical composition for inhibiting hepatoma cell proliferation, which is characterized in that described pharmaceutical composition includes as right is wanted
Circular rna-circ-CCNY described in asking 1.
4. a kind of diagnosing cancer of liver kit, which is characterized in that the kit includes that can expand ring as described in claim 1
The primer of shape RNA-circ-CCNY.
5. diagnosing cancer of liver kit according to claim 4, which is characterized in that the amplimer is by SEQ ID NO:
DNA sequence dna shown in 3 and SEQ ID NO:4 forms primer pair or as the DNA as shown in SEQ ID NO:5 and SEQ ID NO:6
Sequence forms primer pair.
6. purposes of the circular rna-circ-CCNY as described in claim 1 in the drug of preparation treatment liver cancer.
7. purposes of the circular rna-circ-CCNY gene as target gene in the drug of preparation treatment liver cancer, which is characterized in that
The nucleotide sequence of the circular rna-circ-CCNY gene is as shown in SEQ ID NO:1.
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| CN201610638032.3A CN106222170B (en) | 2016-08-05 | 2016-08-05 | Circular rna circ-CCNY and application thereof |
| PCT/CN2017/087832 WO2018024033A1 (en) | 2016-08-05 | 2017-06-10 | Cyclic rna circ-ccny and use thereof |
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| CN106222170B (en) * | 2016-08-05 | 2019-05-07 | 广州永诺生物科技有限公司 | Circular rna circ-CCNY and application thereof |
| CN106834290B (en) * | 2017-01-23 | 2020-05-22 | 中山大学附属肿瘤医院 | Circular RNA and application thereof |
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| CN107254519B (en) * | 2017-06-06 | 2020-10-09 | 重庆市肿瘤研究所 | Screening method of triple negative breast cancer specific circular RNA |
| CN107663539B (en) * | 2017-09-29 | 2020-12-01 | 中山大学附属第三医院 | Use of circular RNA circ-PTGR1 |
| CN110846391B (en) * | 2019-07-23 | 2024-03-19 | 中山大学孙逸仙纪念医院 | Method for detecting full-length sequence of circular RNA |
| CN112301109B (en) * | 2019-07-29 | 2024-02-13 | 中国科学院昆明动物研究所 | Method for detecting RNA cyclization efficiency of genotype |
| CN111020036B (en) * | 2020-01-06 | 2023-03-28 | 南京脑科医院 | Application of human circ-STXBP5L and related product |
| CN113122638B (en) * | 2021-03-15 | 2023-08-25 | 内蒙古医科大学 | Application of hsa-novel_circ_0006787 molecule in liver cancer treatment |
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