CN108998522A - Predict the biomarker long-chain non-coding RNA LINC01725 and kit of colon cancer prognosis - Google Patents
Predict the biomarker long-chain non-coding RNA LINC01725 and kit of colon cancer prognosis Download PDFInfo
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Abstract
The present invention provides a kind of biomarker long-chain non-coding RNA LINC01725 and kit for predicting colon cancer prognosis, for predicting colorectal cancer patients prognosis life cycle.The preparation for predicting colon cancer prognosis is real-time fluorescence quantitative PCR detection kit, kit includes the specific primer of the specific primer and reference gene TUBA1A expression for testing goal gene LINC01725 expression, also include real time fluorescent quantitative SYBR dyestuff, the water without RNA enzyme, real time fluorescent quantitative SYBR dyestuff in kit, target gene LINC01725 specific primer, reference gene TUBA1A specific primer, water without RNA enzyme volume ratio be 10:1:1:6.The present invention is related with the survival rate of patient by quantitative fluorescent PCR and the survivorship curve analysis LINC01725 for finding colon cancer tissue source, and content is higher, and survival rate is higher.
Description
Technical field
The invention belongs to biomedicine technical fields, and in particular to the non-volume of biomarker long-chain of prediction colon cancer prognosis
Code RNA LINC01725 and kit.
Background technique
Colon cancer is one of most common malignant tumour of human gut, and disease incidence has the tendency that rising year by year.According to stream
Row disease learns investigation display, and the morbidity and mortality of colon cancer occupy the third position of malignant tumour in western countries；In China, knot
The disease incidence of intestinal cancer rises to the 4th by the 5th, and the death rate rises to the 5th by the 6th, about there is 200,000 people every year
Colon cancer crouching 1 is died of, the first cause of colorectal cancer patients death is hepatic metastases closely related 1.Colon carcinogenesis DISTANT METASTASES IN organ it is suitable
Sequence is successively liver, lung, bone etc..Colon cancer with hepatic metastases high frequency occur be colon cancer a notable feature, clinically get over
More to attract attention.About 25% patient has occurred that hepatic metastases when making a definite diagnosis, and the patient in 80% or more colon cancer advanced stage goes out
Existing hepatic metastases.Although continuously improving screening method and supplemental treatment regimens, synchronous and the different time hepatic metastases is still one
A serious problem, thus patient's prognosis is very poor.Currently, clinically there are no ideal molecular indexes to assess prognosis and generation
The risk of transfer.Therefore, finding new prognosis biomarker is current urgent problem.
Human genome analysis after the sequencing is completed the result shows that, encode the DNA of albumen in whole mankind's genomic DNA only
Account for a little, however coding protein, people were not once thinking that this part DNA was " rubbish once to overwhelming majority DNA
DNA".Recent studies indicate that these so-called junk DNAs, which can be also transcribed, generates RNA, and it is found that many this
RNA is not so-called transcription noise, although they cannot directly participate in gene coding and albumen synthesis, but in gene
Transcriptional control, RNA shearing and modification etc. have the function of important, and vital work is played in many vital movements
With generation, diagnosis and treatment with disease have close relationship.The research of non-coding RNA is achieved not in recent years
Few achievement, but major part all concentrates on small non-coding RNA.Research achievement about tiny RNA is also often cited as the world
Medicine breakthrough.However, the research achievement for long-chain non-coding RNA is comparatively also seldom, belongs at present and grind
Study carefully than less clear one of transcription product.Long-chain non-coding RNA is the non-coding RNA that length is greater than 200 nucleotide.Long-chain
The expression that non-coding RNA corresponds to gene is generally more lower than the expression of encoding egg white gene, this illustrates long-chain non-coding
RNA may primarily serve the effect of regulation.Recent studies indicate that long-chain non-coding RNA epigenetic regulation, transcription and
Gene expression regulation, transcriptional activation, transcription interference, cell differentiation regulation, cell cycle regulating and agent on post-transcriptional level
It plays an important role in numerous vital movements such as amount compensating effect.
LINC01725 is a kind of newfound non-coding RNA, and transcript length is greater than 200 nucleotide.Currently,
The important function that LINC01725 is played during normal cell development or tumor development is also in further research.
Whether LINC01725 can be used as the new tumor markers of one kind for predicting that colorectal cancer patients prognosis not yet has been reported that.Therefore,
We illustrate for the first time with LINC01725 is new colon carcinoma marker the feasibility for predicting patient's prognosis.
Summary of the invention
The purpose of the present invention is to provide a kind of biomarker long-chain non-coding RNAs for predicting colon cancer prognosis
LINC01725 and kit, for predicting colorectal cancer patients prognosis life cycle.
To achieve the above object, the present invention adopts the following technical scheme: the biomarker of prediction colon cancer prognosis is
Long-chain non-coding RNA LINC01725, the nucleic acid sequence of long-chain non-coding RNA LINC01725 is as shown in SEQ NO:1.
Application of the long-chain non-coding RNA LINC01725 biomarker in the preparation of preparation prediction colon cancer prognosis.
The preparation of the prediction colon cancer prognosis is real-time fluorescence quantitative PCR detection kit.
For predicting the real-time fluorescence quantitative PCR detection kit of colon cancer prognosis, comprising being used for testing goal gene
The specific primer of LINC01725 expression, primer sequence are SEQ NO:2 and SEQ NO:3.
For predicting that the real-time fluorescence quantitative PCR detection kit of colon cancer prognosis includes reference gene TUBA1A expression
Specific primer, primer sequence are SEQ NO:4 and SEQ NO:5.
Described is used to predict that the real-time fluorescence quantitative PCR detection kit of colon cancer prognosis also includes real time fluorescent quantitative
SYBR dyestuff, the water without RNA enzyme.
Real time fluorescent quantitative SYBR in the real-time fluorescence quantitative PCR detection kit for predicting colon cancer prognosis
The volume ratio of dyestuff, target gene LINC01725 specific primer, reference gene TUBA1A specific primer, water without RNA enzyme
For 10:1:1:6.
The beneficial effects of the present invention are: the present invention has found colon cancer group by quantitative fluorescent PCR and survivorship curve analysis
The LINC01725 for knitting source is related to the survival rate of patient, and content is higher, and survival rate is higher.This method is prediction colorectal cancer patients
Prognosis life cycle analysis provide strong technical support, help to improve the postoperative life quality of colorectal cancer patients, make
Aftertreatment scheme is ordered, survival rate is improved, there is far-reaching clinical meaning.
Detailed description of the invention
Fig. 1 is that real-time fluorescence quantitative PCR analyzes differential expression of the LINC01725 in normal tissue and colon cancer.
The prognosis of the LINC01725 expression height to colorectal cancer patients in the tracing analysis colon cancer tissue source for survival Fig. 2
It influences.
Specific embodiment
Present invention will be further explained below with reference to the attached drawings and specific embodiments.
Embodiment 1
The biomarker for predicting colon cancer prognosis is long-chain non-coding RNA LINC01725, the long-chain non-coding RNA
The nucleic acid sequence of LINC01725 is as shown in SEQ NO:1.
The preparation for predicting colon cancer prognosis is real-time fluorescence quantitative PCR detection kit, predicts the real-time of colon cancer prognosis
Fluorescent quantificationally PCR detecting kit includes the specific primer for testing goal gene LINC01725 expression, and primer sequence is
SEQ NO:2 and SEQ NO:3, real-time fluorescence quantitative PCR detection kit also include the specificity of reference gene TUBA1A expression
Primer, primer sequence are SEQ NO:4 and SEQ NO:5.
The reagent of preparation detection LINC01725 expression quantity is used to prepare the kit of colorectal cancer patients prognosis (for 50 times
Reaction), required reagent includes: 3 μM of 50 μ L of target gene LINC01725 specific primer, 3 μM of reference gene TUBA1A special
50 μ L of specific primer, 500 μ L of SYBR Green qPCR Mix, the 300 μ L of water without RNA enzyme.
The extracted total RNA agents useful for same from the tumour of colon cancer or normal control tissue, including Trizol l20mL, inhibition
RNA degradation solvent 40mL, chloroform 80mL, isopropanol 80mL, DEPC water 10mL.
It by LINC01725 reverse transcription is cDNA agents useful for same, including 10 × random primer and Oligo dT that total serum IgE, which is template,
100 μ L of mixture, 5 × reverse transcription reaction buffer, 150 μ L, the 10mM of primer contain Mg2+Triphosphoric acid base deoxynucleotide
100 μ L of dNTP, 50 μ L of 200U/ μ L M-MLV reverse transcriptase.
Embodiment 2
The detection of tissue samples LINC01725
1, colon cancer tumours tissue or normal control tissue to be measured are collected, after physiological saline cleans up, is put into and fills inhibition RNA
It degrades in the cryopreservation tube of solvent, puts spare to -80 DEG C of refrigerators.
2, the extracting of RNA in organizing
(1) liquid nitrogen is first added in mortar, then tissue is cut into small pieces and is clayed into power in liquid nitrogen, taken with the spoon of Liquid nitrogen precooler
100mg organizes powder to be added in the EP pipe for the Trizol liquid for having filled 1ml.Organize total powder volume no more than Trizol used
The 10% of volume is sufficiently mixed uniformly.
(2) 5 minutes are placed at room temperature for, the chloroform of 200 μ L is then added, EP is covered tightly and manages and acutely sway 0.5 minute.12000
Rev/min centrifugation 10 minutes.
(3) it takes upper strata aqueous phase in a new EP pipe, 500 μ L isopropanols is added, are mildly mixed by inversion.It is placed at room temperature for 10 points
Clock, 12000 revs/min are centrifuged 10 minutes.
(4) liquid is carefully discarded supernatant, 75% ethyl alcohol of 1ml is added, is vortexed and mixes, 12000 revs/min of centrifugations 5 at 4 DEG C
Minute.Repetitive operation is primary.
(5) discard supernatant liquid, as far as possible remove residual liquid, room temperature or vacuum drying 5~10 minutes.With 50 μ L DEPC
Processed water dissolves RNA.
(6) RNA concentration mensuration: being measured with nucleic acid concentration analyzer, is inhaled 1 μ L RNA sample and is added to sample well, according to
Reading directly determines the concentration of RNA.The OD260/OD280 ratio of the measurement of nucleic acid concentration analyzer, ratio is between 1.8-2.0
Think that RNA purity is fine.Finally, to be stored in -80 DEG C of refrigerators spare by RNA.
3, the reverse transcription of LINC01725 RNA
Use the Reverse Transcriptase kit (D7170S) of the green skies Bioisystech Co., Ltd in Shanghai.Specific step is as follows:
(1) it removes genomic DNA: template Total RNA, 5 × gDNA Eraser Buffer is thawed on ice, 5 × RT
Buffer, 10 × RT Primer Mix, DEPC-treated Water thaw at (15-25 DEG C) of room temperature, are immediately placed in after defrosting
On ice.Every kind of solution is mixed before use and of short duration centrifugation is so that all liq is settled down to tube bottom.The denaturation of RNA, RNA sample
Product after thermal denaturation 5 minutes, are immediately placed in ice water cooling under the conditions of 65 DEG C.According to the form below ingredient is in the mixing of preparation reaction on ice
Then liquid is dispensed into each reaction tube again, is eventually adding RNA sample.Reaction system and condition are as shown in table 1.
Table 1
(2) in PCR instrument or in water-bath, 37 DEG C are incubated for 2 minutes.Be immediately placed in place on ice it is spare.
(3) reverse transcription system is prepared: being carried out reaction solution preparation on ice, is carried out reverse transcription reaction immediately after soft mixing.
Mixed liquor is prepared according to the reverse transcription reaction system of following table.Reaction system and condition are as shown in table 2.
Table 2
(4) 42 DEG C are incubated for 60 minutes to carry out reverse transcription reaction, and subsequent 80 DEG C of incubations are put in for 10 minutes after inactivation reverse transcriptase
On ice.Reverse transcription temperature can be improved to 50 DEG C, to enhance reverse transcription effect in complicated for secondary structure or high GC content template
Rate.
(5) cDNA obtained can immediately or -80 DEG C freeze after for subsequent real-time fluorescence quantitative PCR, cDNA was preferably avoided
More multigelations.
4, real-time quantitative PCR is carried out using the specific primer of LINC01725
Specific primer is synthesized in Sangon Biotech (Shanghai) Co., Ltd., including for detecting LINC01725 expression
Specific primer, primer sequence is the specific primer that SEQ NO:2 and SEQ NO:3 and reference gene TUBA1A are expressed,
Primer sequence is SEQ NO:4 and SEQ NO:5.The other reagents of real-time quantitative PCR utilize the green limited public affairs of skies biotechnology in Shanghai
The BeyoFast SYBR Green qPCR Mix (2 ×) of department, the specific steps are as follows:
(1) various solution needed for melting and mixing PCR reaction, BeyoFast SYBR Green qPCR Mix melt completely
And it mixes and is placed in ice chest.
(2) PCR reaction system (by taking 96 orifice plates as an example) are set on ice bath, reaction system and condition are as shown in table 3.
Table 3
| Reagent | Usage amount |
| BeyoFast™ SYBR Green qPCR Mix (2X, Low ROX) | 10μl |
| Specific primer (3 μM of concentration) | 2μl |
| Template DNA | 2μl |
| Water without RNA enzyme | 6μl |
| Total volume | 20μl |
(3) the usually amount of DNA profiling with 1-10ng cDNA is with reference to dosage, and final concentration of 0.2-0.5 μM of primer when can be obtained
Good detection effect is obtained, the final concentration of primer can also be according to circumstances adjusted.If it is necessary, gradient dilution can be carried out to template,
With the optimal template usage amount of determination.When the cDNA that reverse transcription PCR reacts is directly as template, additive amount is not exceeded
PCR reacts the 10% of total volume.The recommendation response system of 96 orifice plates is 20 μ l, can also be expanded in proportion according to actual experiment demand
Big or diminution reaction system.
(4) mixing is gently blown and beaten with pipettor or slight Vortex is mixed, and room temperature is centrifuged the several seconds, and liquid is made to accumulate in pipe
Bottom.(5) the PCR reaction tube set or PCR reaction plate are placed on fluorescence quantitative PCR instrument, start PCR reaction. (6)PCR
Response procedures: the initial denaturation of template is carried out before real-time fluorescence quantitative PCR reacts, is typically set at 95 DEG C 2 minutes.Using such as
Under PCR program, this program is by taking ABI 7900HT fluorescence quantitative PCR instrument as an example: a. initial denaturation: 95 DEG C of 2min；B. become
Property: 95 DEG C of 15sec；C. annealing/extension: 60 DEG C of 15-30sec；D. step b and step c is repeated, in total 40 circulations；
E. melting curve analysis (optional): 95 DEG C of 15sec, 60 DEG C of 15sec, 95 DEG C of 15sec；F. quantitative fluorescent PCR is used
The software that instrument provides analyzes result.Three-step approach need to only add 72 DEG C of 30sec of a step after annealing/extension, then repeat step b, c
And the step for increased, recycles for 40 totally.
5, the data analysis of LINC01725 expression quantity
This experimental data is included in 60 colorectal cancer patients and its normal control tissue.The interpretation of result of real-time quantitative PCR uses phase
To quantitative approach, that is, 2^-△△CtMethod.It is specific as follows: firstly, all gene C t values that will once test are put in order, later with each
The Ct value of target gene LINC01725 in group tumor sample subtracts the Ct value of itself reference gene TUBA1A, and obtained number is just
It is △ Ct；Changing formula into is exactly: △ Ct=Ct(target gene LINC01725)-Ct(reference gene TUBA1A)；It then, will be each
The △ Ct of group each target gene of tumor sample LINC01725 is calculated.With the △ Ct of tumor tissues sample in this experiment
The △ Ct of normal control tissue group sample is subtracted, and opposite number is taken to all results simultaneously, the result which obtains is exactly-
△△Ct.Finally, p- △ △ Ct carries out 2 power operation, i.e. 2^-△△CtJust show that the multiple of expression quantity changes.In triplicate, sharp
It is examined with nonparametric t- for statistical analysis.As a result as shown in Figure 1, in the tumor tissues of colon cancer LINC01725 expression quantity
It is higher than normal tissue, difference have statistical difference (p< 0.05).
By 60 colorectal cancer patients follow-up statistics being included in above-mentioned experiment, including patient's First episode when
Between, treatment condition, recurrence status and death time etc., follow up time is at least 12 months.In selected colorectal cancer patients,
The expression value for choosing fluorescence real-time quantitative PCR analysis is reference standard, and the corresponding normal tissue of acquired results compares, and is tied
Fruit is as shown in Figure 2.The patient that LINC01725 expression is higher than normal control tissue in tumor tissues is defined as LINC01725 high table
Up to group, remaining is low expression group.By Kaplan-Meier survival analysis, LINC01725 high expresses the life cycle ratio of patient
The patient of LINC01725 low expression group is substantially reduced, and prognosis is worse, difference have it is statistically significant (p< 0.05).Therefore,
LINC01725 can be used as the specificity molecular marker of colorectal cancer patients prognosis.
The above is the preferred embodiment of the present invention, it is noted that for those skilled in the art, this hair
It is bright to be not restricted to the described embodiments, under the premise of not departing from general idea of the present invention, several variations can also be made and changed
Into these also should be considered as protection scope of the present invention.
SEQUENCE LISTING
<110>the first affiliated hospital of Zhengzhou University
<120>the biomarker long-chain non-coding RNA LINC01725 and kit of colon cancer prognosis are predicted
<130> 1
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 723
<212> DNA
<213>people
<400> 1
cgagggcaga gcgggaagct ccgcccctcg cggaaccccg gcctgtgcgc agttcccgtg 60
ccctaggagt tggcggcggg gcccagccga gaatcttctt cccttctggt ctgactagga 120
aagccagagg gagatggtga aggagacaca gagagagtaa aagaacagac catgcccagc 180
ctctccactg caggagcttg gaatcagtga agcagagttg ccagccatct atggatccta 240
actaaattgg tccttgtaat actcggtatt atttgccaag atattgaaaa acctctaaac 300
agtgagaagc tcccagcccc ttttctgtgt ctctcatcac cagctgctgt gccctgagga 360
cacccaggca gcctaaggag aggcccacag gtgagaaact gcatgtgtga gctgagcttg 420
gaagtggatc ttctatgttt tgccaacagc cacgtgacta gttttggaag cagattctcc 480
agccccagct gcacctgtgc cagaggcacc cattaagccg cacctagtta aattcctgac 540
ccacaaagac tttgaaatca taaatgtttg ttgttttaag ctgctaagtt ttgaagtaat 600
ctgccatgta gcaacatgaa actacttgga ccaaatatgt gtttttctaa gcacaggaat 660
attcacactt tattaaactt gagtattcca acaataggat ggaagtccaa gttttatttc 720
ttt 723
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<213>artificial synthesized
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gcccagccga gaatcttctt 20
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<213>artificial synthesized
<400> 3
tggtccaagt agtttcatgt tgc 23
<210> 4
<211> 20
<212> DNA
<213>artificial synthesized
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tgcttctgca cggtctcttt 20
<210> 5
<211> 20
<212> DNA
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ccccaatctg cccagagaag 20
Claims (7)
1. the biomarker for predicting colon cancer prognosis is long-chain non-coding RNA LINC01725, the long-chain non-coding RNA
The nucleic acid sequence of LINC01725 is as shown in SEQ NO:1.
2. application of the biomarker as described in claim 1 in the preparation of preparation prediction colon cancer prognosis.
3. application as claimed in claim 2, it is characterised in that: the preparation of the prediction colon cancer prognosis is real time fluorescent quantitative
PCR detection kit.
4. for predicting the real-time fluorescence quantitative PCR detection kit of colon cancer prognosis, it is characterised in that: comprising for detecting mesh
Gene LINC01725 expression specific primer, primer sequence be SEQ NO:2 and SEQ NO:3.
5. as claimed in claim 4 for predicting the real-time fluorescence quantitative PCR detection kit of colon cancer prognosis, feature exists
In: the real-time fluorescence quantitative PCR detection kit includes the specific primer of reference gene TUBA1A expression, and primer sequence is
SEQ NO:4 and SEQ NO:5.
6. as claimed in claim 5 for predicting the real-time fluorescence quantitative PCR detection kit of colon cancer prognosis, feature exists
In: the kit also includes real time fluorescent quantitative SYBR dyestuff, without the water of RNA enzyme.
7. as claimed in claim 6 for predicting the real-time fluorescence quantitative PCR detection kit of colon cancer prognosis, feature exists
In: real time fluorescent quantitative SYBR dyestuff, target gene LINC01725 specific primer, reference gene in the kit
TUBA1A specific primer, water without RNA enzyme volume ratio be 10:1:1:6.
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