CN109022576A - Predict the biomarker long-chain non-coding RNA LINC02447 and kit of colon cancer prognosis - Google Patents
Predict the biomarker long-chain non-coding RNA LINC02447 and kit of colon cancer prognosis Download PDFInfo
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Abstract
The purpose of the present invention is to provide a kind of biomarker long-chain non-coding RNA LINC02447 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 LINC02447 expression, also includes real time fluorescent quantitative SYBR dyestuff and the water without RNA enzyme.The present invention is related with the survival rate of patient by quantitative fluorescent PCR and the survivorship curve analysis LINC02447 for finding colon cancer tissue source, and content is higher, and survival rate is higher.This method helps to improve the postoperative life quality of colorectal cancer patients, works out aftertreatment scheme, improve survival rate, have far-reaching clinical meaning to predict that the prognosis life cycle analysis of colorectal cancer patients provides strong technical support.
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 LINC02447 and kit.
Background technique
Colon cancer is one of most common malignant tumour of gastrointestinal tract, and its disease incidence is constantly in lasting rising level, with
The factors such as dietary structure change, living standard raising, aging of population are related.Colon cancer is in the disease incidence of male malignancy
The 3rd is arranged, the 2nd is arranged in the disease incidence of female malignant.West Europe and North America are respectively 43/,100,000 and 45/,100,000.Colon
Cancer is 13/,100,000 in China's disease incidence.According to existing relevant clinical investigation result, the at present postoperative life in 5 years of colorectal cancer patients
The rate of depositing is about 50%, and the death rate is about 80.The primary treatment regimen of colon cancer is operative treatment at present, to occurring shifting no operation
Refer to that the patient of disease carries out chemotherapy.In order to improve patients ' life quality and overall survival, Most patients need to carry out art
Auxiliary chemotherapy afterwards.But most of destiny for being finally still unable to escape relapse and metastasis, the recurrence and transfer of tumour are still that patient is dead
The main reason for dying.Currently, clinically there are no ideal molecular indexes come the risk assessing prognosis and shift.Therefore,
Finding new prognosis biomarker is current urgent problem.
In recent decades, more and more research discovery RNA Noncoding gene segments, which have, applies in diagnosing and treating weight
Potential commercial value in terms of big disease.Noncoding gene segment includes the noncoding region of non-coding RNA and coding protein mRNA
Section.Non-coding RNA is the general designation of the rna transcription sheet of all not coding proteins, and representative is Microrna and long non-coding RNA.
It is riboswitch in non-coding section representative present on codified protein mRNA.Microrna is a kind of length between 18 ~ 25
The non-coding RNA of nucleotide, research shows that the mankind encode albumen gene in about 30% by miRNAs regulate and control.They participate in base
The expression and regulation of cause, RNA are processed and the biological processes such as shearing and protein translation, regulates and controls growth and the hair of biology strongly
It educates.Long non-coding RNA is the non-coding RNA that a kind of length is greater than 200 nucleotide, the expression and regulation of their participation genes,
RNA processing and editor, protein translation, imprinting and the regulation of telomere system etc..
LINC02447 is a kind of newfound non-coding RNA, and transcript length is greater than 200 nucleotide.Currently,
The important function that LINC02447 is played during normal cell development or tumor development is also in further research.
Whether LINC02447 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 LINC02447 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
LINC02447 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 LINC02447, the nucleic acid sequence of long-chain non-coding RNA LINC02447 is as shown in SEQ NO:1.
Application of the long-chain non-coding RNA LINC02447 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 LINC02447 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.
For predicting that the real-time fluorescence quantitative PCR detection kit of colon cancer prognosis also includes real time fluorescent quantitative SYBR dye
Material, the water without RNA enzyme.
Real time fluorescent quantitative SYBR dyestuff in real-time fluorescence quantitative PCR detection kit for predicting colon cancer prognosis,
Target gene LINC02447 specific primer, reference gene TUBA1A specific primer, water without RNA enzyme volume ratio be 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 LINC02447 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 LINC02447 in normal tissue and colon cancer.
The prognosis of the LINC02447 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 LINC02447, the long-chain non-coding RNA
The nucleic acid sequence of LINC02447 is as shown in SEQ NO:1.
The preparation for predicting colon cancer prognosis is real-time fluorescence quantitative PCR detection kit, real-time fluorescence quantitative PCR detection examination
Agent box includes the specific primer expressed for testing goal gene LINC02447, primer sequence such as SEQ NO:2 and SEQ
Shown in NO:3.
For predicting that the real-time fluorescence quantitative PCR detection kit of colon cancer prognosis also includes reference gene TUBA1A expression
Specific primer, primer sequence is as shown in SEQ NO:4 and SEQ NO:5.
The reagent of preparation detection LINC02447 expression quantity is used to prepare the kit of colorectal cancer patients prognosis (for 50 times
Reaction), required reagent includes: that 500 μ L of SYBR Green qPCR Mix, 3 μM of target gene LINC02447 specificity are drawn
50 μ L of object, 3 μM of 50 μ L of reference gene TUBA1A specific primer, the 300 μ L of water without RNA enzyme.
Extracted total RNA agents useful for same includes: Trizol l20mL, inhibits from the tumour of colon cancer or normal control tissue
RNA degradation solvent 40mL, chloroform 80mL, isopropanol 80mL, DEPC water 10mL.
It is cDNA agents useful for same by LINC02447 reverse transcription by template of total serum IgE include: 10 × random primer and Oligo
100 μ L of mixture, 5 × reverse transcription reaction buffer, 150 μ L, the 10mM of dT 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 LINC02447
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) liquid is discarded supernatant, room temperature or vacuum drying 5~10 minutes.RNA is dissolved with 50 μ L DEPC processed water.
(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 LINC02447 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 LINC02447
Specific primer is synthesized in Sangon Biotech (Shanghai) Co., Ltd., including for detecting LINC02447 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 such as table
Shown in 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 following PCR program, this program is by taking ABI 7900HT fluorescence quantitative PCR instrument as an example: a. initial denaturation:
95℃ 2min；B. it is denaturalized: 95 DEG C of 15sec；C. annealing/extension: 60 DEG C of 15-30sec；D. step b and step are repeated
C, 40 recycle in total；E. melting curve analysis (optional): 95 DEG C of 15sec, 60 DEG C of 15sec, 95 DEG C of 15sec； f.
Result is analyzed using the software that fluorescence quantitative PCR instrument provides.Three-step approach need to only add 72 DEG C of 30sec of a step after annealing/extension,
Then repeat step b, c and it is increased the step for totally 40 circulation.
5, the data analysis of LINC02447 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 LINC02447 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 LINC02447)-Ct(reference gene TUBA1A)；It then, will be each
The △ Ct of group each target gene of tumor sample LINC02447 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 LINC02447 expression quantity
It is higher than normal control 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 LINC02447 expression is higher than normal control tissue in tumor tissues is defined as LINC02447 high table
Up to group, remaining is low expression group.By Kaplan-Meier survival analysis, LINC02447 high expresses the life cycle ratio of patient
The patient of LINC02447 low expression group is substantially reduced, and prognosis is worse, difference have it is statistically significant (p< 0.05).Therefore,
LINC02447 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 LINC02447 and kit of colon cancer prognosis are predicted
<130> 1
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 219
<212> DNA
<213>people
<400> 1
ggcagggcgg ggccggggca ctggtgattg gaagcctggt gggcggggcc caggacctcc 60
ctgccctctc cgccattaac tcggagcccg gcgcagtgga aagcgggcca agaagcagag 120
gtccaggttc caactcttga ttctatcaca aaacactgca cccgaggcag ccaggcagct 180
tccgaagacc tcattctcct aaacacatcc attcctgtt 219
<210> 2
<211> 20
<212> DNA
<213>artificial synthesized
<400> 2
gcactggtga ttggaagcct 20
<210> 3
<211> 24
<212> DNA
<213>artificial synthesized
<400> 3
acaggaatgg atgtgtttag gaga 24
<210> 4
<211> 20
<212> DNA
<213>artificial synthesized
<400> 4
gtcctccatc actgcttccc 20
<210> 5
<211> 20
<212> DNA
<213>artificial synthesized
<400> 5
cagctctctg taccttggcc 20
Claims (7)
1. the biomarker for predicting colon cancer prognosis is long-chain non-coding RNA LINC02447, the long-chain non-coding RNA
The nucleic acid sequence of LINC02447 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 LINC02447 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 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 LINC02447 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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