CN108728546A - Application, kit and the detection method of lung cancer for prognosis novel molecular marker non-coding RNA LINC00963 - Google Patents
Application, kit and the detection method of lung cancer for prognosis novel molecular marker non-coding RNA LINC00963 Download PDFInfo
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Abstract
The present invention relates to a kind of application, kit and the detection methods of lung cancer for prognosis novel molecular marker non-coding RNA LINC00963, it is related with the survival rate of patient by real-time fluorescence quantitative PCR and the survivorship curve analysis non-coding RNA LINC00963 for finding cancerous lung tissue source, content is higher, survival rate is higher, this method provides strong technical support to predict that the prognosis life cycle of patients with lung cancer is analyzed, help to improve the postoperative life quality of patients with lung cancer, work out aftertreatment scheme, survival rate is improved, there is far-reaching clinical meaning.
Description
Technical field
The invention belongs to oncomolecularbiology fields, and in particular to a kind of lung cancer for prognosis novel molecular marker non-coding
Application, kit and the detection method of RNA LINC00963.
Background technology
Lung cancer is still the first factor in tumour associated death reason in world wide.In patients with lung cancer, non-cellule type
Patients with lung cancer accounts for 85%, is most common Lung Cancer Types.In recent decades, China's lung cancer morbidity is in notable ascendant trend, the whole world
The patients with lung cancer for having more than 1/3 comes from China.Operative treatment is still the main means of lung cancer in non-cellule type at present, however,
Due to early diagnosis difficulty, postoperative easy transfer and relapse and to chemicotherapy relative insensitivity etc., patient's prognosis is in the past few decades
It is not significantly improved inside.Currently, clinically there are no ideal molecular indexes come the risk assessing prognosis and shift.Cause
This, it is current urgent problem to find new prognosis biomarker.
Also include 30,000 recently the study found that in addition to including the mRNA of about more than 20,000 codified albumen in human transcription group
The non-coding RNA of item or so.It can be classified as two major classes, the first kind, less than 200nt's according to the bases longs of non-coding RNA
Short chain non-coding RNA, such as it is primarily involved in the miRNA of RNA degradations or Translational repression after transcription, and participate in turning for mRNA translations
Transport RNA；Second class is more than long-chain non-coding RNA, that is, long-chain non-coding RNA of 200nt, participates in a variety of bioprocess, such as
It is a variety of important to participate in chromatin modification, transcriptional control, miRNA function point analysis, mRNA stability and protein function adjusting etc.
Regulatory mechanism hinders.Studies have shown that non-coding RNA plays great work(during normal cell development and tumor development
Energy.LINC00963 is a kind of newfound non-coding RNA, and transcript length is more than 200 nucleotide.Currently,
The important function that LINC00963 is played during normal cell development or tumor development is also in further research.
Whether LINC00963 can be as a kind of new tumor markers for predicting that patients with lung cancer prognosis not yet has been reported that.Therefore, I
Illustrate for the first time the feasibility of patient's prognosis predicted for new lung cancer marker with LINC00963.
Invention content
The object of the present invention is to provide a kind of answering for lung cancer for prognosis novel molecular marker non-coding RNA LINC00963
With secondly, the present invention provides a kind of marker non-coding RNA the LINC00963 kit of expression quantity and inspection in cancerous lung tissue
Survey method.
The object of the present invention is achieved like this：
Lung cancer for prognosis novel molecular marker non-coding RNA LINC00963 is preparing the preparation of prediction patients with lung cancer prognosis
Using the nucleic acid sequence such as SEQ NO of non-coding RNA LINC00963:Shown in 1.
The preparation for predicting patients with lung cancer prognosis is real-time fluorescence quantitative PCR detection kit.
Kit includes the specific primer for detecting non-coding RNA LINC00963 expression, the specific primer
Nucleic acid sequence be SEQ NO:2 and SEQ NO:The primer sequence that 3 and reference gene TUBA1A is expressed, the core of the primer
Acid sequence is SEQ NO:4 and SEQ NO:5.
Kit also contains all reagents for extracting RNA from tissue and carrying out reverse transcription and real-time fluorescence quantitative PCR, packet
Include (1) extracted total RNA agents useful for same from the tumour of lung cancer or normal control tissue, including Trizol liquid, chloroform, isopropanol,
Inhibit RNA degradations solvent, 75% ethyl alcohol, DEPC water；(2) it is by non-coding RNA LINC00963 reverse transcriptions by template of total serum IgE
CDNA agents useful for same, including reverse transcription reaction buffer solution, contain triphosphoric acid base deoxynucleotide dNTP, reverse transcription containing Mg2+
The mixture of enzyme M-MLV and random primer and Oligo dT primers；(3) by cDNA real-time fluorescence quantitative PCR agents useful for same, packet
Include LINC00963 real-time fluorescence quantitative PCRs specific primer, TUBA1A internal references Specific PCR primers, real time fluorescent quantitative SYBR
Dyestuff, the water without RNA enzyme.
The detection method of lung cancer for prognosis novel molecular marker non-coding RNA LINC00963, includes the following steps：1）It receives
Collect lung cancer to be measured or normal control tissue；
2) extracting of RNA in organizing；
3）The reverse transcription of LINC00963 RNA, using cell total rna as template, reverse transcription obtains cDNA；
4）Using cDNA as template, real-time fluorescence quantitative PCR expansion is carried out using the specific primer of non-coding RNA LINC00963
Increase, detects non-coding RNA LINC00963 expression quantity.
Step 3）Middle reverse transcription reaction system is 5 × RT Buffer（Containing Mg2+ and dNTP）4 μ l, 10 × RT Primer
Mix（Oligo dT Primer and Random Hexamers mixtures）2 μ l, BeyoRT II M-MLV reverse transcriptases
(RNase H-)（Inhibitor containing RNase）2 μ l, DEPC-treated water, 2 μ l remove the Total RNA after gDNA
10μl。
Step 4）The expression quantity relative quantitation method of middle detection marker non-coding RNA LINC00963.
The beneficial effects of the invention are as follows：Cancerous lung tissue source is found by real-time fluorescence quantitative PCR and survivorship curve analysis
Non-coding RNA LINC00963 it is related to the survival rate of patient, content is higher, and survival rate is higher, this method be prediction lung cancer suffer from
The prognosis life cycle analysis of person provides strong technical support, helps to improve the postoperative life quality of patients with lung cancer, makes
Aftertreatment scheme is ordered, survival rate is improved, there is far-reaching clinical meaning.
Description of the drawings
Fig. 1 is that real-time fluorescence quantitative PCR analyzes differential expressions of the LINC00963 in normal structure and lung cancer；
Prognosis of the non-coding RNA LINC00963 expression height in the tracing analysis cancerous lung tissue sources for survival Fig. 2 to patients with lung cancer
It influences.
Specific implementation mode
The present invention is made the following instructions with reference to embodiment：
Embodiment 1：Lung cancer for prognosis novel molecular marker non-coding RNA LINC00963 is preparing prediction patients with lung cancer prognosis
Preparation application, marker non-coding RNA LINC00963 is used to prepare to the preparation of lung cancer for prognosis, predicts patients with lung cancer
Situation.
Embodiment 2：The reagent for preparing detection non-coding RNA LINC00963 expression quantity is used to prepare patients with lung cancer prognosis
Kit (being used for 30 secondary responses) 1. Trizo l20ml
2. inhibiting RNA degradation solvents 40ml；
3. chloroform 80ml；
4. isopropanol 80ml；
5. DEPC water 10ml；
6. the 100 μ l of mixture of 10 × random primer and Oligo dT primers；
7. 5 × reverse transcription reaction buffer solution, 150 μ l；
8. triphosphoric acid base deoxynucleotide dNTP 100 μ ls of the 10mM containing Mg2+；
9. 50 μ l of 200U/ μ l M-MLV reverse transcriptases；
10. SYBR Green qPCR Mix 500μl；
11. 3 μM of target gene LINC00963 specific primers（Its sequence such as SEQ NO:2 and SEQ NO:Shown in 3） 50μl；
12. 3 μM of reference gene TUBA1A specific primers（Its sequence such as SEQ NO:4 and SEQ NO:Shown in 5） 50μl.
Embodiment 3：The detection of tissue samples non-coding RNA LINC00963
1, lung cancer to be measured or normal control tissue are collected, after physiological saline cleans up, is put into and fills inhibition RNA degradation solvents
In cryopreservation tube, 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, is taken with the spoon of Liquid nitrogen precooler
100mg organizes powder to be added in the EP pipes 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 revs/min
Zhongli's heart 10 minutes；
（3）Take upper strata aqueous phase in a new EP pipes（The intermediate beds of precipitation and subnatant are not mixed into）, 500 μ l isopropanols are added,
Mildly reverse mixing, is placed at room temperature for 10 minutes, and 12000 revs/min centrifuge 10 minutes.
（4）Liquid is carefully discarded supernatant, is added 75% ethyl alcohol of 1ml, vortex mixing, 12000 revs/min of centrifugations 5 at 4 DEG C
Minute, repetitive operation is primary；
（5）Discard supernatant liquid（Residual liquid is removed as possible）, room temperature or vacuum drying 5~10 minutes（It is careful not to dried
Point, it otherwise can reduce the solubility of RNA）, RNA is dissolved with 50 μ l DEPC processed water；
（6）RNA concentration mensurations：It is measured with nucleic acid concentration analyzer, inhales 1 μ l RNA samples and add to sample well, according to reading
Directly determine the concentration of RNA.The OD260/OD280 ratios of the measurement of nucleic acid concentration analyzer, ratio are thought between 1.8-2.0
RNA purity is fine, and finally, it is spare that RNA is stored in -80 DEG C of refrigerators.
3, the reverse transcription of LINC00963 RNA：Use the Reverse Transcriptase kit of the green skies Bioisystech Co., Ltd in Shanghai
（D7170S）, it is as follows：
（1）Remove genomic DNA：Template Total RNA, 5 × gDNA Eraser Buffer are thawed on ice, 5 × RT
Buffer, 10 × RT Primer Mix, DEPC-treated Water are immediately placed in (15-25 DEG C) defrosting of room temperature after defrosting
On ice.Using it is preceding by each solution mixing and it is of short duration centrifugation so that all liq is settled down to tube bottom.The denaturation of RNA, RNA samples
Product thermal denaturation under the conditions of 65 DEG C is immediately placed in ice water cooling after 5 minutes.According to the form below ingredient is in the mixing of preparation reaction on ice
Then liquid is dispensed into each reaction tube, is eventually adding RNA sample again；
（2）In PCR instrument or in water-bath, 37 DEG C are incubated 2 minutes.Be immediately placed in place on ice it is spare；（3）Reverse transcription system is matched
System：Reaction solution preparation is carried out on ice, and reverse transcription reaction is carried out immediately after soft mixing.According to the reverse transcription reaction system of following table
Prepare mixed liquor；
（4）42 DEG C are incubated 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, for the template of secondary structure complexity or high GC content, reverse transcription temperature can be improved to 50 DEG C, to enhance reverse transcription effect
Rate；（5）Obtained cDNA can immediately or -80 DEG C freeze after be used for follow-up real-time fluorescence quantitative PCR, cDNA preferably avoids excessive
Multigelation.
4, real-time fluorescence quantitative PCR is carried out using the specific primer of non-coding RNA LINC00963：Specific primer exists
Raw work bioengineering（Shanghai）Limited liability company synthesizes, and includes for detecting the special of non-coding RNA LINC00963 expression
Property primer, primer sequence be SEQ NO:2 and SEQ NO:The primer sequence that 3 and reference gene TUBA1A is expressed, primer sequence
For SEQ NO:4 and SEQ NO:5.The other reagents of real-time fluorescence quantitative PCR utilize the green skies Bioisystech Co., Ltd in Shanghai
BeyoFast SYBR Green qPCR Mix (2 ×), are as follows：
（1）Melt and mixing PCR reacts required various solution, BeyoFast SYBR Green qPCR Mix melt completely
And mixing is placed in ice chest；（2）PCR reaction systems (by taking 96 orifice plates as an example) are set on ice bath：
(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 result is obtained, the final concentration of primer also can be according to circumstances adjusted.If it is necessary, gradient dilution can be carried out to template,
With the template usage amount that determination is best.When reverse transcription PCR cDNA obtained by the reaction is directly as template, additive amount does not exceed
PCR reacts the 10% of total volume.The recommendation response system of 96 orifice plates is 20 μ l, can also in proportion be expanded according to actual experiment demand
Big or diminution reaction system；
(4) mixing or slight Vortex mixings are gently blown and beaten with pipettor, room temperature centrifuges the several seconds, liquid is made to accumulate in tube bottom；
(5) the PCR reaction tubes set or PCR reaction plates are placed on fluorescence quantitative PCR instrument, start PCR reactions；(6) PCR is anti-
Answer program：The pre-degeneration that template is carried out before real-time fluorescence quantitative PCR reacts, is typically set at 95 DEG C 2 minutes.Using as follows
PCR programs, this program is by taking ABI 7900HT fluorescence quantitative PCR instruments as an example：A. pre-degeneration：95℃ 2min ；B. become
Property：95℃ 15sec；C. annealing/extension：60℃ 15-30sec；D. step b and step c is repeated, in total 40 cycles；
E. melting curve analysis (optional)：95℃ 15sec, 60℃ 15sec, 95℃ 15sec；F. quantitative fluorescent PCR is used
The software analysis result that 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 the step for increased, recycles for 40 totally.
5, the data analysis of non-coding RNA LINC00963 expression quantity：This experimental data be included in 60 patients with lung cancer and its
Normal control tissue.The interpretation of result of real-time quantitative PCR uses relative quantitation method, that is, 2^- △ △ Ct methods.It is specific as follows：It is first
First, all gene C t values once tested are put in order, later with the target gene LINC00963's in each group of tumor sample
Ct values subtract the Ct values of itself reference gene TUBA1A, and obtained number is exactly △ Ct；Changing formula into is exactly：△Ct=Ct（Purpose base
Because of LINC00963）-Ct（Reference gene TUBA1A）；Then, by each group of each target gene of tumor sample LINC00963
△ Ct all calculate.The △ Ct of normal control tissue group sample are subtracted with the △ Ct of tumor tissues sample in this experiment, and same
When opposite number is taken to all results, the result which obtains is exactly-△ △ Ct.Finally, p- △ △ Ct carry out 2 power fortune
It calculates, i.e. 2^- △ △ Ct just show that the multiple of expression quantity changes.In triplicate, it is examined using nonparametric t- for statistical analysis；I
Find, the expression quantity of LINC00963 is higher than normal control in the tumor tissues of lung cancer（See Fig. 1）, difference have statistics it is poor
Different (p < 0.05).
6, by 60 patients with lung cancer follow-up statistics being included in above-mentioned experiment, including patient's First episode when
Between, treatment, recurrence status and death time etc., follow up time is at least 12 months.In selected patients with lung cancer, choosing
It is reference standard to take the expression value that real-time fluorescence quantitative PCR is analyzed, and the corresponding normal structure of acquired results compares.Tumour
Non-coding RNA LINC00963 expression is defined as non-coding RNA LINC00963 higher than the patient of normal control tissue in tissue
High expression group, remaining is low expression group.By Kaplan-Meier survival analysis, non-coding RNA LINC00963 high expression is suffered from
The life cycle of person is substantially reduced than the patient of non-coding RNA LINC00963 low expression groups, and prognosis is worse（See Fig. 2）, difference tool
Have statistically significant (p < 0.05).Therefore, non-coding RNA LINC00963 can be used as the specificity point of patients with lung cancer prognosis
Sub- marker.
Sequence table
<110>The first affiliated hospital of Zhengzhou University
<120>Application, kit and the detection method of lung cancer for prognosis novel molecular marker non-coding RNA LINC00963
<141> 2018-04-27
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ctaacagagc ccccagttag gagcagtccg cgagacagtt ggtaacagtg ctggccttgc 120
ccgggcgccg gaaggttccg tgaggcccag caagtcctga cttggcacag ggcccagcac 180
acggcgcagt aacaatataa ttaccgtatg cagtccccca ggatacaggg tcaaaggaga 240
gcacaaccat cgcagttgga agcccatggg gcagggccca ggacacagtg agcatacaat 300
agacattagc tgctgtggtg tcttgatttc aagcccagtg cagatgcatc tgacttacga 360
aacttcagtg acacctgctc tgtgccagac actgaagatg gagcagtgaa cagcactgac 420
ccagcctgtc ctcctgttgc ctgcaggcca gaagcaaggt ctcgctgtgt taccctggct 480
ggagtgcagt ggctacttac aggcatgatc atagcatact gcagcctcga actcctggac 540
tagagcagtt ctcctgcctc agcctccaga gcagctggga ctgcagacac gcaccacaga 600
gcccagcctg agtcattttg attctgtaac tggcttcctc tcggcgggca ggctcacacc 660
ctccaccccc agctggagat ggaaggagga tgctcaccgc ccattcctca tgcaggccat 720
cgatggaaat gtcagacagt gcgagtccac gcctgaacac ttctggggtc cgctctggga 780
ctgacactgg cagatccatc ggtgagctcc cgcctgccac ctctgggcct gcattgcggt 840
gatagggcca agggcctggg ccttcaattc tccagacttg ccaaaatgct cagcgcaggc 900
ctcgtcggca actgcagaca agcagaggca gcacctgggc tctgcctgtg cctggcacag 960
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acaaaatttc accctaatgt agtgtcttta gaccgtgcaa tgttgtgctc ctgttagtct 1080
gatatcaggg gcttgcagcc tggcaaagga aaaaaaacag gctgaggcac atgatagaag 1140
acaggcaggg atgccacaat cataaccatg gcaaagacca ggatcagaaa ggcccaccat 1200
ctctgacccc aaatccctgg gcagatggtt ccagaactca gaattttccc agttgtagaa 1260
agatggggcc aggtgcagtg gctcacgcct gtaatcccag cattttggga gtacaaggtg 1320
ggcggatcac ctgaggtcag gagttcgaga ccagcctggc caacatggtg aaaccctgtc 1380
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Claims (7)
1. lung cancer for prognosis novel molecular marker non-coding RNA LINC00963 is preparing the preparation of prediction patients with lung cancer prognosis
Using the nucleic acid sequence such as SEQ NO of non-coding RNA LINC00963:Shown in 1.
2. the application of lung cancer for prognosis novel molecular marker non-coding RNA LINC00963 according to claim 1, special
Sign is：The preparation for predicting patients with lung cancer prognosis is real-time fluorescence quantitative PCR detection kit.
3. a kind of kit as claimed in claim 2, it is characterised in that：Including being used to detect non-coding RNA LINC00963
The nucleic acid sequence of the specific primer of expression, the specific primer is SEQ NO:2 and SEQ NO:3 and reference gene
The nucleic acid sequence of the primer sequence of TUBA1A expression, the primer is SEQ NO:4 and SEQ NO:5.
4. kit according to claim 3, it is characterised in that：Also contain and extracts RNA from tissue and carry out reverse transcription
And all reagents of real-time fluorescence quantitative PCR, including (1) from the tumour of lung cancer or normal control tissue used in extracted total RNA
Reagent, including Trizol liquid, chloroform, isopropanol, inhibition RNA degradations solvent, 75% ethyl alcohol, DEPC water；(2) using total serum IgE as mould
Plate by non-coding RNA LINC00963 reverse transcriptions be cDNA agents useful for same, including reverse transcription reaction buffer solution, containing containing Mg2+'s
The mixture of triphosphoric acid base deoxynucleotide dNTP, reverse transcriptase M-MLV and random primer and Oligo dT primers；(3)
It will be in cDNA real-time fluorescence quantitative PCR agents useful for same, including LINC00963 real-time fluorescence quantitative PCRs specific primer, TUBA1A
Join Specific PCR primers, real time fluorescent quantitative SYBR dyestuffs, the water without RNA enzyme.
5. the detection method of lung cancer for prognosis novel molecular marker non-coding RNA LINC00963, it is characterised in that：Including with
Lower step：1）Collect lung cancer to be measured or normal control tissue；
2) extracting of RNA in organizing；
3）The reverse transcription of LINC00963 RNA, using cell total rna as template, reverse transcription obtains cDNA；
4）Using cDNA as template, real-time fluorescence quantitative PCR expansion is carried out using the specific primer of non-coding RNA LINC00963
Increase, detects non-coding RNA LINC00963 expression quantity.
6. the detection method of lung cancer for prognosis novel molecular marker non-coding RNA LINC00963 according to claim 5,
It is characterized in that：Step 3）Middle reverse transcription reaction system is 5 × RT Buffer（Containing Mg2+ and dNTP）4 μ l, 10 × RT
Primer Mix（Oligo dT Primer and Random Hexamers mixtures）2 μ l, BeyoRT II M-MLV are reversed
Record enzyme (RNase H-)（Inhibitor containing RNase）2 μ l, DEPC-treated water, 2 μ l remove the Total after gDNA
RNA 10μ。
7. the detection method of lung cancer for prognosis novel molecular marker non-coding RNA LINC00963 according to claim 5,
It is characterized in that：Step 4）The expression quantity relative quantitation method of middle detection marker non-coding RNA LINC00963.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103981269B (en) * | 2014-05-26 | 2016-05-25 | 中南大学 | The application process of long-chain non-coding RNA CRYM-AS1 |
| CN108192975A (en) * | 2018-03-02 | 2018-06-22 | 中南大学湘雅医院 | Application of long-chain non-coding RNA L INC00559 as biomarker in preparation of lung squamous cell carcinoma prognosis detection preparation |
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2018
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| CN103981269B (en) * | 2014-05-26 | 2016-05-25 | 中南大学 | The application process of long-chain non-coding RNA CRYM-AS1 |
| CN108192975A (en) * | 2018-03-02 | 2018-06-22 | 中南大学湘雅医院 | Application of long-chain non-coding RNA L INC00559 as biomarker in preparation of lung squamous cell carcinoma prognosis detection preparation |
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| 裴振等: "长链非编码RNA LINC00467促进非小细胞肺癌细胞的增殖", 《西安交通大学学报（医学报）》 * |
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Application publication date: 20181102 |