CN109022577A - Predict the biomarker long-chain non-coding RNA LINC01366 and kit of lung cancer for prognosis - Google Patents
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Abstract
The present invention provides a kind of biomarker long-chain non-coding RNA LINC01366 and kit for predicting lung cancer for prognosis, for predicting patients with lung cancer prognosis life cycle.The preparation for predicting lung cancer for 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 LINC01366 expression, further includes real time fluorescent quantitative SYBR dyestuff, 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 LINC01366 for finding cancerous lung tissue source, and content is higher, and survival rate is higher.This method helps to improve the postoperative life quality of patients with lung cancer, works out aftertreatment scheme, improve survival rate, have far-reaching clinical meaning to predict that the prognosis life cycle analysis of patients with lung cancer provides strong technical support.
Description
Technical field
The invention belongs to biomedicine technical fields, and in particular to predict the biomarker long-chain non-coding of lung cancer for prognosis
RNA LINC01366 and kit.
Background technique
Lung cancer is the highest malignant tumour of disease incidence in global range, and disease incidence, the death rate rise year by year in recent years, is had become
For the primary cause of disease of tumour associated death.Lung cancer be to human life threaten it is huge, disease incidence is higher and higher, has occupy cancer
First of associated death reason.The estimation of China's lung cancer is increased year by year with prediction death toll.The natural growth of the population, population are aged
The aggravation of change process, urbanization of villages, cities and towns process of industrialization are also being accelerated, and China's smoking size of population is also increasing year by year, on
It states the reason is that China's lung cancer morbidity principal element that linear formula rises in recent years.According to the statistical data in the U.S. in 2009, life in 5 years
The rate of depositing only has 16%.Due to lacking the effective technology and means of early detection and early diagnosis, most of lung cancer is in when finding
Middle and advanced stage loses radical cure chance, treats the poor prognosis only based on palliative treatment.Recently as the fast development of medicine,
Many new technologies are emerged, new Clinics provide possibility to cure lung cancer, but clinical test results are not still in recent years
It can be entirely satisfactory.Therefore, finding new prognosis biomarker is current urgent problem.
Long-chain non-coding RNA is the RNA that a kind of transcript length is more than 200nt, non-coding protein.Long-chain non-coding
RNA participates in the different phase of the various vital movements of cell, the every single step reaction being almost related in life cycle, such as gene and turns
Record, the montage of mRNA, RNA decay and translation etc..In the disease of some complexity, relevant signal is typically derived from genome
Non-coding region, more and more researchs also indicate that the unconventionality expression of long-chain non-coding RNA is associated with mankind's major disease.
In addition, more and more result of study discoveries, numerous long-chain non-coding RNAs have participated in the occurrence and development of cancer.Currently,
Research confirms the long-chain non-coding RNA of unconventionality expression, all exists in numerous malignant tumours, the significant up-regulation having, what is had is significant
It lowers.Wherein representative research is found in breast cancer, liver cancer and prostate cancer.The research of long-chain non-coding RNA has
There is critically important researching value, especially in clinical research and on drug development, it can be not only our numerous diseases
Research, including tumour and cardiovascular disease and other very refractory disease provides new foundation and target spot at present, and
Facilitate people and recognizes complicated life regulation process.Future life science field will be to the research of long-chain non-coding RNA
Center of gravity and focus facilitate the pathogenesis for more thoroughly understanding mankind's major disease, for disease early diagnosis and control in time
It treats and biomarker and corresponding drug target is provided.
LINC01366 is a kind of newfound non-coding RNA, and transcript length is greater than 200 nucleotide.Currently,
The important function that LINC01366 is played during normal cell development or tumor development is also in further research.
Whether LINC01366 can be used as the new tumor markers of one kind for predicting that patients with lung cancer prognosis not yet has been reported that.Therefore, I
To be illustrated for the first time with LINC01366 be new lung cancer marker the feasibility of 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 lung cancer for prognosis
LINC01366 and kit, for predicting patients with lung cancer prognosis life cycle.
To achieve the above object, the present invention adopts the following technical scheme: the biomarker of prediction lung cancer for prognosis is long
Chain non-coding RNA LINC01366, the nucleic acid sequence of long-chain non-coding RNA LINC01366 is as shown in SEQ NO:1.
Application of the long-chain non-coding RNA LINC01366 biomarker in the preparation of preparation prediction lung cancer for prognosis.
The preparation of the prediction lung cancer for prognosis is real-time fluorescence quantitative PCR detection kit.
For predicting the real-time fluorescence quantitative PCR detection kit of lung cancer for prognosis, comprising being used for testing goal gene
The specific primer of LINC01366 expression, primer sequence are SEQ NO:2 and SEQ NO:3.
For predicting the real-time fluorescence quantitative PCR detection kit of lung cancer for prognosis, include 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 lung cancer for prognosis further includes real time fluorescent quantitative
SYBR dyestuff, the water without RNA enzyme.
Real time fluorescent quantitative SYBR dye in the real-time fluorescence quantitative PCR detection kit for predicting lung cancer for prognosis
Material, target gene LINC01366 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 cancerous lung tissue by quantitative fluorescent PCR and survivorship curve analysis
The LINC01366 in source and the survival rate of patient are related, and content is higher, and survival rate is higher.This method is the pre- of prediction patients with lung cancer
Life cycle analysis afterwards provides strong technical support, helps to improve the postoperative life quality of patients with lung cancer, works out postoperative
Therapeutic scheme improves survival rate, has far-reaching clinical meaning.
Detailed description of the invention
Fig. 1 is that real-time fluorescence quantitative PCR analyzes differential expression of the LINC01366 in normal tissue and lung cancer.
The LINC01366 expression height in the tracing analysis cancerous lung tissue source for survival Fig. 2 is to the influence prognosis of patients with lung cancer.
Specific embodiment
Present invention will be further explained below with reference to the attached drawings and specific embodiments.
Embodiment 1
The biomarker for predicting lung cancer for prognosis is long-chain non-coding RNA LINC01366, the long-chain non-coding RNA
The nucleic acid sequence of LINC01366 is as shown in SEQ NO:1.
The preparation for predicting lung cancer for prognosis is real-time fluorescence quantitative PCR detection kit, real-time fluorescence quantitative PCR detection reagent
Box includes the specific primer expressed for testing goal gene LINC01366, primer sequence such as SEQ NO:2 and SEQ NO:3
Shown, real-time fluorescence quantitative PCR detection kit also includes the specific primer of reference gene TUBA1A expression, and primer sequence is such as
Shown in SEQ NO:4 and SEQ NO:5.
The kit that the reagent of preparation detection LINC01366 expression quantity is used to prepare patients with lung cancer prognosis is (anti-for 50 times
Answer), required reagent includes: 500 μ L of SYBR Green qPCR Mix, 3 μM of target gene LINC01366 specific primers
50 μ L, 3 μM of 50 μ L of reference gene TUBA1A specific primer, the 300 μ L of water without RNA enzyme.
The extracted total RNA agents useful for same from the tumour of lung cancer or normal control tissue, comprising: Trizol l20mL, inhibit
RNA degradation solvent 40mL, chloroform 80mL, isopropanol 80mL, DEPC water 10mL.
It by LINC01366 reverse transcription is cDNA agents useful for same by template of total serum IgE, comprising: 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 LINC01366
1, lung cancer tumor tissue or normal control tissue to be measured are collected, after physiological saline cleans up, is put into and fills inhibition RNA drop
In the cryopreservation tube for solving solvent, put 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 nucleic acid concentration analyzer measurement, ratio are recognized between 1.8-2.0
It is fine for RNA purity.Finally, to be stored in -80 DEG C of refrigerators spare by RNA.
3, the reverse transcription of LINC01366 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 LINC01366
Specific primer is synthesized in Sangon Biotech (Shanghai) Co., Ltd., including for detecting LINC01366 expression
Specific primer, primer sequence be SEQ NO:2 and be SEQ NO:3 and reference gene TUBA1A expression specificity draw
Object, primer sequence are SEQ NO:4 and are SEQ NO:5.The other reagents of real-time quantitative PCR have using the green skies biotechnology in Shanghai
The BeyoFast SYBR Green qPCR Mix (2 ×) of limit company, 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, as shown in table 3.
Table 3
(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 get
Good detection effect can also according to circumstances adjust the final concentration of primer.If it is necessary, gradient dilution can be carried out to template, with
Determine optimal template usage amount.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 DEG C
2min；B. it is denaturalized: 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. using glimmering
The software that Fluorescent Quantitative PCR instrument provides analyzes result.Three-step approach need to only add 72 DEG C of 30sec of a step after annealing/extension, then weight
Multiple step b, c and it is increased the step for totally 40 circulations.
5, the data analysis of LINC01366 expression quantity
This experimental data is included in 60 patients with lung cancer and its normal control tissue.The interpretation of result of real-time quantitative PCR is using opposite
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 group
The Ct value of target gene LINC01366 in tumor sample subtracts the Ct value of itself reference gene TUBA1A, and obtained number is exactly
△Ct；Changing formula into is exactly: △ Ct=Ct(target gene LINC01366)-Ct(reference gene TUBA1A)；Then, by each group
The △ Ct of each target gene of tumor sample LINC01366 is calculated.Subtracted with the △ Ct of tumor tissues sample in this experiment
The △ Ct of normal control tissue group sample is removed, 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, it utilizes
Nonparametric t- examines for statistical analysis.As shown in Figure 1, the expression quantity of LINC01366 is than normal group in the tumor tissues of lung cancer
Knit height, difference have statistical difference (p< 0.05).
By 60 patients with lung cancer 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 patients with lung cancer, choosing
The expression value for taking fluorescence real-time quantitative PCR to analyze is reference standard, and the corresponding normal tissue of acquired results compares, as a result
As shown in Figure 2.The patient that LINC01366 expression is higher than normal control tissue in tumor tissues is defined as LINC01366 high expression
Group, remaining is low expression group.By Kaplan-Meier survival analysis, LINC01366 high expresses the life cycle ratio of patient
The patient of LINC01366 low expression group is substantially reduced, and prognosis is worse, difference have it is statistically significant (p< 0.05).Therefore,
LINC01366 can be used as the specificity molecular marker of patients with lung cancer 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 LINC01366 and kit of lung cancer for prognosis are predicted
<130> 2
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 306
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<213>people
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tcatgaccag agggatctgt tcaacaccct gtgggtccca gcacctatct caggaagcag 120
ggtgcatctg tccaccacca agtctcaggg tggctgggtg agcgctcatc agtcccagac 180
agcaggttgg cacaaaacga cactgcccac aaaaccacag gaagaagaga aactccgact 240
gtctttcagc acacagaaga cactgtactg gacccggaca ttaggcagac acccacgcct 300
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ggcgaggatg ctgaggac 18
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aaccagttcc cccaccaaag 20
Claims (7)
1. the biomarker for predicting lung cancer for prognosis is long-chain non-coding RNA LINC01366, the long-chain non-coding RNA
The nucleic acid sequence of LINC01366 is as shown in SEQ NO:1.
2. application of the biomarker as described in claim 1 in the preparation of preparation prediction lung cancer for prognosis.
3. application as claimed in claim 2, it is characterised in that: the preparation of the prediction lung cancer for prognosis is real time fluorescent quantitative
PCR detection kit.
4. for predicting the real-time fluorescence quantitative PCR detection kit of lung cancer for prognosis, it is characterised in that: comprising being used for testing goal
The specific primer of gene LINC01366 expression, primer sequence are SEQ NO:2 and SEQ NO:3.
5. as claimed in claim 4 for predicting the real-time fluorescence quantitative PCR detection kit of lung cancer for 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 lung cancer for prognosis, feature exists
In: the kit further 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 lung cancer for prognosis, feature exists
In: real time fluorescent quantitative SYBR dyestuff, target gene LINC01366 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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Application publication date: 20181218 |