CN108977536A - Predict the biomarker long-chain non-coding RNA LINC01504 and kit of lung cancer for prognosis - Google Patents

Predict the biomarker long-chain non-coding RNA LINC01504 and kit of lung cancer for prognosis Download PDF

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CN108977536A
CN108977536A CN201810805941.0A CN201810805941A CN108977536A CN 108977536 A CN108977536 A CN 108977536A CN 201810805941 A CN201810805941 A CN 201810805941A CN 108977536 A CN108977536 A CN 108977536A
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lung cancer
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田田
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First Affiliated Hospital of Zhengzhou University
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Abstract

The present invention provides a kind of biomarker long-chain non-coding RNA LINC01504 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 LINC01504 expression, also 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 LINC01504 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

Predict biomarker long-chain non-coding RNA LINC01504 and the examination of lung cancer for prognosis Agent box
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 LINC01504 and kit.
Background technique
Lung cancer is to seriously endanger one of the malignant tumour of human health.Now with our people's life style transformation, The exacerbation of air pollution, lung cancer morbidity rate and the death rate also increase sharply, and it is highest to have become China's disease incidence for lung cancer at present Malignant tumour and China lead to the first cause of cancer related mortality.The statistical data of China's National Cancer Center in 2015 It has been shown that, 2012 National urban Incidence first be lung cancer, about 380,000 people of annual new hair, dead about 310,000 people, Middle male lung cancer disease incidence and the equal shelter of the death rate have first of malignant tumour；Female lung cancer disease incidence occupies second, but the death rate Occupy first.In rural area, men and women's lung cancer morbidity and the equal shelter of the death rate have malignant tumour first place, annual new cases about 33 Ten thousand, dead about 260,000 people.Currently, clinically there are no ideal molecular indexes come the risk assessing prognosis and shift.Cause This, finding new prognosis biomarker is current urgent problem.
In recent years, the development of two generation sequencing technologies is greatly promoted the progress of biology.In the Human Genome Project Afterwards, the genome of a large amount of species is sequenced.It is researcher's very concern to the annotation of these genomes.Center method It is proposed then has pushed the development of modern molecular biology, the bridge that RNA is transmitted as hereditary information between DNA and protein significantly Beam is constantly subjected to the highest attention of scientist.With the reach of science, distinct has now been found, functional diversities RNA.The function of RNA has run far deeper than the effect in traditional understanding as hereditary information transmitting intermediary, and the RNA of many types divides Sub (such as Microrna, long-chain non-coding RNA etc.) can be functioned directly.It has now been found that, 80% or more human genome can To be transcribed into RNA, and only 2% or so region can be translated as albumen, and most of transcript is non-coding RNA, Which results in the interest of numerous researchers.How effectively to identify these non-coding RNAs and determine their function, is present A major challenge of research work.More and more researches show that non-coding RNA is not that transcription generates " rubbish ", they are turning Record, translational control etc. played an important role.
LINC01504 is a kind of newfound non-coding RNA, and transcript length is greater than 200 nucleotide.Currently, The important function that LINC01504 is played during normal cell development or tumor development is also in further research. Whether LINC01504 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 LINC01504 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 LINC01504 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 LINC01504, the nucleic acid sequence of long-chain non-coding RNA LINC01504 is as shown in SEQ NO:1.
Application of the long-chain non-coding RNA LINC01504 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 that the real-time fluorescence quantitative PCR detection kit of lung cancer for prognosis includes for testing goal gene The specific primer of LINC01504 expression, primer sequence are SEQ NO:2 and SEQ NO:3.
For predicting that the real-time fluorescence quantitative PCR detection kit of lung cancer for prognosis includes the spy of 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 also 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 LINC01504 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 LINC01504 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 LINC01504 in normal tissue and lung cancer.
The LINC01504 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 LINC01504, the long-chain non-coding RNA The nucleic acid sequence of LINC01504 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 for testing goal gene LINC01504 expression, and primer sequence is 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 Shown in SEQ NO:4 and SEQ NO:5.
The kit that the reagent of preparation detection LINC01504 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 LINC01504 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 LINC01504 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 LINC01504
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, should not be dried Point, it otherwise can reduce the solubility of RNA.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 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 LINC01504 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 LINC01504
Specific primer is synthesized in Sangon Biotech (Shanghai) Co., Ltd., including for detecting LINC01504 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
(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 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 A circulation；E. melting curve analysis (optional): 95 DEG C of 15sec, 60 DEG C of 15sec, 95 DEG C of 15sec；F. fixed using fluorescence It measures the software that PCR instrument provides and 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 this increased step Rapid totally 40 circulations.
5, the data analysis of LINC01504 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 LINC01504 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 LINC01504)-Ct(reference gene TUBA1A)；Then, by each group The △ Ct of each target gene of tumor sample LINC01504 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 a result as shown in Figure 1, the expression quantity ratio of LINC01504 is being just in the tumor tissues of lung cancer Often tissue is high, 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 LINC01504 expression is higher than normal control tissue in tumor tissues is defined as LINC01504 high expression Group, remaining is low expression group.By Kaplan-Meier survival analysis, LINC01504 high expresses the life cycle ratio of patient The patient of LINC01504 low expression group is substantially reduced, and prognosis is worse, difference have it is statistically significant (p< 0.05).Therefore, LINC01504 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 LINC01504 and kit of lung cancer for prognosis are predicted
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<160> 5
<170> PatentIn version 3.5
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aattagtgct tttatttgct tgtcctgcta agaccgtagg caaaggccat cctcctccca 180
ttgacagttt agagtggaaa caaccacggt taccctagaa gtaaatccag agctgcggga 240
cgctggagga caaaaccaac cttccaggga agccgaggag aagtcatgcc ttcaaaaggt 300
gtctgccttt ttttttttca ccctctctcc tctaacctgt ttgtaactgg gaaatgaaat 360
agttttggtt tgcaagctgt cgccagggag tggagcaaga ctttgtaata aacaagagca 420
actgccagag cccccagcga gcctctccaa ggccagactg ccaagaaaag ctggcagtgt 480
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ccagaggggg taccccccag tctcaccttg gggaagagta cagcctccta ttagtgagct 600
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Claims (7)

1. the biomarker for predicting lung cancer for prognosis is long-chain non-coding RNA LINC01504, the long-chain non-coding RNA The nucleic acid sequence of LINC01504 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 LINC01504 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 LINC01504 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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