CN108192975A - Applications of the long-chain non-coding RNA LINC00559 as biomarker in lung squamous cancer prognosis detection preparation is prepared - Google Patents
Applications of the long-chain non-coding RNA LINC00559 as biomarker in lung squamous cancer prognosis detection preparation is prepared Download PDFInfo
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- CN108192975A CN108192975A CN201810174127.3A CN201810174127A CN108192975A CN 108192975 A CN108192975 A CN 108192975A CN 201810174127 A CN201810174127 A CN 201810174127A CN 108192975 A CN108192975 A CN 108192975A
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Abstract
The invention discloses applications of the long-chain non-coding RNA LINC00559 as biomarker in lung squamous cancer prognosis detection preparation is prepared.Expressions of the long-chain non-coding RNA LINC00559 in Lung Squamous Carcinoma Patients can be detected, strong molecular biology mechanism is provided for Lung Squamous Carcinoma Patients prognosis prediction, there is far-reaching clinical meaning and generalization.
Description
Technical field
The invention belongs to oncomolecularbiology fields, and in particular to long-chain non-coding RNA LINC00559 is as biology
Application of the marker in lung squamous cancer prognosis detection reagent is prepared.
Background technology
Lung squamous cancer, that is, squamous cell carcinoma is one of most important lung cancer histological type, derives from bronchial epithelial cell
Canceration, case load account for 30% of non-small cell lung cancer or so.The conventional treatments of lung squamous cancer mainly include operative treatment, chemistry
Treatment and molecular targeted therapy, 5 years survival rates of patient less than 15%, the whole world every year about more than 40 ten thousand people because its is lethal.At present,
The prognosis of Lung Squamous Carcinoma Patients is judged, without reference to standard, also without the index of specificity, to adapt to suffer from lung squamous cancer
Person carries out the demand of prognosis judgement.Therefore, Lung Squamous Carcinoma Patients prognosis is judged, to select therapeutic regimen, significantly
Survival is improved, becomes thoracic surgery field important topic urgently to be resolved hurrily.
Long-chain non-coding RNA (long noncoding RNA) is the general designation of non-coding RNA of the length more than 200nt.Most
First research thinks that it is transcript profile " noise ", but life science nearly ten years finds that they take part in multinomial life
Object regulation process.Some researches show that long-chain non-coding RNA can be used as a kind of very important epigenetic tune in human genome
The factor is controlled, is methylated by the number of mechanisms regulating DNA such as epigenetic, transcriptional control and post-transcriptional control, histone modification
Or chromatin reconstruct makes gene silencing or activation.In recent years, long-chain non-coding RNA as a kind of important regulatory molecule and its
The application value of clinical diagnosis, chemosensitivity and prognostic evaluation etc. gradually obtains the attention of researcher.There is research
Show that expression of the HOTAIR in primary tumor of breast and metastatic tumor can all increase, the expression of HOTAIR can in primary tumo(u)r
To be used for effectively predicting metastasis of cancer and death.Long-chain non-coding RNA PCA3 is highly overexpressed in prostate cancer, this long-chain
Non-coding RNA is found in urine, quite convenient for clinical detection.Long-chain non-coding RNA LALR1 by activate Wnt/ β-
Catenin signals, accelerate cell cycle progression, promote hepatocyte growth.Long-chain non-coding RNA is intervened using medicine target
LALR1 induced livers regenerate, it is possible to be beneficial to hepatic failure and Liver Transplantation for Treatment.It is examined it can be seen that long-chain non-coding RNA is used as
The molecular marker of disconnected, prognosis and treatment has great potential.
At present, the prognosis of Lung Squamous Carcinoma Patients is judged without reference to standard, also without the index of specificity, far can not fitted
Lung Squamous Carcinoma Patients should be carried out with the demand of prognosis judgement.Therefore, Lung Squamous Carcinoma Patients prognosis is judged, to select most preferably to control
Treatment scheme, significantly improves survival, becomes thoracic surgery field important topic urgently to be resolved hurrily.
Invention content
The present invention is directed to overcome the deficiencies of the prior art and provide long-chain non-coding RNA LINC00559 as biological marker
Application of the object in lung squamous cancer prognosis detection preparation is prepared.
Long-chain non-coding RNA LINC00559 is positioned at chromosome 13q31.3, GeneBank accession number：NR-047489.1
(long-chain non-coding RNA LINC00559 sequences are shown in SEQ NO:1).In previous research work, inventor is in lung squamous cell carcinoma cancers
RNA is extracted, the expression of real-time fluorescence quantitative PCR analysis long-chain non-coding RNA LINC00559 is carried out after reverse transcription, is found：It is long
Chain non-coding RNA LINC00559 up-regulated expressions in lung squamous cell carcinoma cancers, with the significantly (P of the differential expression in Normal Lung tissue<
0.0001) (Fig. 1).And in the different Lung Squamous Carcinoma Patients of long-chain non-coding RNA LINC00559 expressions prognosis exist it is apparent
Difference, the Lung Squamous Carcinoma Patients for finding long-chain non-coding RNA LINC00559 high expression through Kaplan-Meier survival analysis are survived
Rate is significantly lower than the patient (P=0.0041) (Fig. 2) of low expression, and ratio risk model prognostic analysis finds long-chain non-coding RNA
The relative risk of LINC00559<1 (HR=0.2414), show long-chain non-coding RNA LINC00559 high expression patient's prognosis compared with
Patient's poor prognosis of low expression, the high expression of long-chain non-coding RNA LINC00559 is that Lung Squamous Carcinoma Patients prognostic risk is predicted
Independent hazard factor.Therefore, long-chain non-coding RNA LINC00559 can be as the criterion of lung squamous cancer prognosis, as biology
Marker is used to prepare the prognosis preparation of Lung Squamous Carcinoma Patients, is further capable of providing a kind of cost-effective, application easy to spread
Lung squamous cancer prognosis prediction kit.
The lung squamous cancer prognosis detection kit includes long-chain non-coding RNA LINC00559 real-time fluorescence quantitative PCRs
Specific primer：Forward primer：5′-GGCTGTGATATGAAAGTTGGCTC-3′(SEQ ID NO.2)；Reverse primer：5′-
CAACTACATATCAAGCCAGGC-3′(SEQ ID NO.3)。
Preferably, ACTB internal reference real-time fluorescence quantitative PCR specific primers are further included in the kit：Forward primer：
5′-GAACCCCAAGGCCAACCGCGAG-3′(SEQ ID NO.4)；Reverse primer：5′-
CAGTGGTACGGCCAGAGGCGTAC-3′(SEQ ID NO.5)。
Long-chain non-coding RNA LINC00559 is as follows for the detection method of Lung Squamous Carcinoma Patients prognosis：(1) it collects to be measured
The lung squamous cell carcinoma cancers of the postoperative excision of individual, extracted total RNA；(2) it is template by long-chain non-coding RNA LINC00559 using total serum IgE
Reverse transcription is cDNA；(3) real-time fluorescence quantitative PCR amplification is carried out with long-chain non-coding RNA LINC00559 specific primers, obtained
Obtain relative expression quantity 2ΔΔCT, when 2ΔΔCT>Long-chain non-coding RNA LINC00559 is prompted to be expressed to be high when 2.92.
To sum up, long-chain non-coding RNA LINC00559 can be detected in Lung Squamous Carcinoma Patients using the detection preparation of the present invention
In expression, provide strong molecular biology mechanism for Lung Squamous Carcinoma Patients prognosis prediction, have far-reaching clinic
Meaning and generalization.
Description of the drawings
Fig. 1 analyzes long-chain non-coding RNA LINC00559 in lung squamous cell carcinoma cancers and normal lung group for real-time fluorescence quantitative PCR
Differential expression in knitting；
Fig. 2 is the relationship of long-chain non-coding RNA LINC00559 and 35 Lung Squamous Carcinoma Patients prognosis.
Specific embodiment
Embodiment 1 prepares long-chain non-coding RNA LINC00559 for lung squamous cancer prognosis detection kit (50 secondary response)
1.RNA stablizing solutions 50ml
2. isopropanol 100ml
3. chloroform 100ml
4.Trizol 50ml
5. without enzyme water 10ml
6.1 μM random reverse transcriptase primer 50ul
7.5 × RT Buffer 200ml
8.10mM triphosphoric acid base deoxynucleotides 100ul
9.40U/ μ l RNase inhibitors 500ul
10.200U/ μ l MMLV reverse transcriptases 50ul
11.Premix Ex Taq 50ul
12.10 μM of long-chain non-coding RNA LINC00559 real-time fluorescence quantitative PCR specific primers：
Forward primer 5 '-GGCTGTGATATGAAAGTTGGCTC-3 ' (SEQ ID NO.2),
Reverse primer 5 '-CAACTACATATCAAGCCAGGC-3 ' (SEQ ID NO.3),
13.10 μM of internal reference ACTB real-time fluorescence quantitative PCR specific primers：
Forward primer is 5 '-GAACCCCAAGGCCAACCGCGAG-3 ' (SEQ ID NO.4),
Reverse primer is 5 '-CAGTGGTACGGCCAGAGGCGTAC-3 ' (SEQ ID NO.5).
Long-chain non-coding RNA LINC00559 is detected in 2 lung squamous cell carcinoma cancers of embodiment
1st, the preservation of lung squamous cell carcinoma cancers：Lung squamous cell carcinoma cancers to be measured are collected to deposit in the cryopreservation tube for filling RNA stablizing solutions,
It puts spare to -80 DEG C of refrigerators.
2nd, the extracting of RNA in organizing：Appropriate sample is taken to add in liquid nitrogen grinding mark in the mortar after 180 DEG C are toasted 6-8h
This, be ground to it is powdered after adding in 1ml Trizol mortar samples in mortar, be ground into it is liquid after with moving to centrifuge tube pipe,
Chlorination imitates 200 μ l/ml in centrifuge tube, shakes 15-30s with hand, places 5min on ice, and 4 DEG C of 12000g centrifuge 15min；Carefully
Upper strata aqueous phase is taken to enter in new centrifuge tube, adds in the isopropanol 0.5ml mixings of precooling, -20 DEG C of refrigerators stand 20min, 4 DEG C of 12000g
Centrifuge 10min；Supernatant is abandoned, adds in the diluted ethyl alcohol 1-2ml mixings of 75% nuclease free water, 4 DEG C of 7500g centrifuge 5min, abandon as possible
Supernatant, drying at room temperature 5-10min add in nuclease free water 10-20 μ l dissolvings RNA.Spectrophotometric measures the concentration and quality of RNA,
OD260/280 ratios are between 1.8-2.0, -80 DEG C of preservations.
3rd, long-chain non-coding RNA LINC00559 is transcribed：Use the Reverse Transcriptase kit of Thermo companies.20 μ L reverse transcriptions
The system of reaction such as table 1：
Table 1
| Ingredient | Dosage/pipe |
| Random reverse transcriptase primer (1uM) | 1ul |
| RNA samples | 2ug |
| Nuclease free water | To 12ul |
Reverse transcription first step condition：65 DEG C 5 minutes, such as table 2：
Table 2
| Ingredient | Dosage/pipe |
| 5 × reverse transcription buffers | 4μl |
| Triphosphoric acid base deoxynucleotide (10mM) | 2μl |
| RNase inhibitor (40U/ μ l) | 1μl |
| MMLV reverse transcriptases (200U/ μ l) | 1μl |
| The product of first step PCR | 12μg |
Reverse transcription second step program：25 DEG C 5 minutes, 42 DEG C 60 minutes, 70 DEG C 5 minutes.
4th, long-chain non-coding RNA LINC00559 specific primers carry out real-time quantitative PCR：Long-chain non-coding RNA
LINC00559 specific primer sequences are synthesized by Shanghai Sheng Gong bioengineering Co., Ltd.
Transcription product dilutes 5 times, mixing.20 μ L reaction systems such as tables 3：
Table 3
Real-time fluorescence quantitative PCR response procedures：95 DEG C 3 minutes, 40 cycle, 95 DEG C 10 seconds, 60 DEG C 30 seconds.
Long-chain non-coding RNA LINC00559 specific primer sequences are：
Forward primer 5 '-GGCTGTGATATGAAAGTTGGCTC-3 ' (SEQ ID NO.2),
Reverse primer 5 '-CAACTACATATCAAGCCAGGC-3 ' (SEQ ID NO.3),
5、-2ΔΔCTThe measure of index：This experimental data uses the analysis method of relative quantification, ACTB as reference gene,
Data are analyzed using software GraphPad Prism.Analysis is found, with long-chain non-coding RNA LINC00559 normal lung groups
Expression in knitting is compared, the apparent up-regulation of long-chain non-coding RNA LINC00559 expression in 35 Lung Squamous Carcinoma Patients, and significant
Difference (P<0.0001).
6th, Index for diagnosis
By 35 Lung Squamous Carcinoma Patients or family members' follow-up used by experiment, we have inquired these patients for the first time in detail
The time of morbidity, treatment, recurrence status and death time etc., follow up time are 1-60 months.In selected lung squamous cancer
In patient, the expression value for choosing quantitative fluorescent PCR analysis is reference standard, is higher than median after the arrangement of acquired results descending
Long-chain non-coding RNA LINC00559 high is expressed, totally 18, other are long-chain non-coding RNA LINC00559 low expressions, and totally 17
Example.Through Kaplan-Meier survival analysis, long-chain non-coding RNA LINC00559 high expresses the non-volume of life cycle longer chain of patient
The patient of code RNA LINC00559 low expressions is short, poor prognosis.Difference is statistically significant (P=0.0041).Ratio risk model
Prognostic analysis finds the relative risk of long-chain non-coding RNA LINC00559<1 (HR=0.2414), shows long-chain non-coding RNA
Patient's prognosis of LINC00559 high expression is compared with patient's poor prognosis of low expression, the high expression of long-chain non-coding RNA LINC00559
It is the independent hazard factor of Lung Squamous Carcinoma Patients prognostic risk prediction.
Above research is it is also shown that long-chain non-coding RNA LINC00559 can be as the specificity of Lung Squamous Carcinoma Patients prognosis point
Sub- marker.
SEQUENCE LISTING
<110>Xiangya Hospital, Central-South China Univ.'s hair is super
<120>Long-chain non-coding RNA
Applications of the LINC00559 as biomarker in lung squamous cancer prognosis detection preparation is prepared
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 3867
<212> DNA
<213>It is artificial synthesized
<400> 1
gaaattctaa ccccaactac cacacagagt gaccttattt ggaaatagag tctttatgaa 60
gacaattaag atgtggtcat tacagtgggc tctaatacga tttgactggt gtccttgtaa 120
gaaggggaaa tttgaacata gacacataca tagataggga aggaaatgcg aagacacaca 180
gaaagatgac catcttacaa gccaacaaga gaggtcttaa gcagatcctc tcacaatctc 240
agaaggcacc aacactgcca acatttcact ctctgacttc cagcctccag aagtgtgaga 300
cataattttc tgtttaaggc gcctgattaa taataatttg ttgcagcagc cacggcaaac 360
ggaaacattt gggtttctaa agacggaagc agctgggcgc ggagactcag gactgtaatc 420
ccagcacttt gggaggccga ggcaggcgaa tcatgaggtc aggagatcca ggaccatcct 480
ggataacaca gtgaaacccc atctctacta aaaatacaaa atattagctg ggcatggtgg 540
cgggcgcctg cctgtagtct cagttactct ggaggctgac gcaggagaat cacttaaacc 600
agggaggcgg aggttgcagt gagctgagat cacaccactg cactccagct agcatgacag 660
agcgagactc cgtctcaaaa agaaataata ataaaataaa gaaggatgct ctgaaaaggg 720
aaaggagaaa agggaggcat ttgtggagta ggtgctcatc actttctcaa ggatatttac 780
aaatgtaatc atatttaccg cacagaatcc tcgtaaaagt gacttaactt cctgacagag 840
catgagcacc atcatctctg acaaacactc cctttctagc ctcacgcatt tcaaggaaat 900
cacttctaac tacaagcagc cagaaaagag cagacagtaa aacacagaga tcaggataga 960
attgctgttt gacatctctc ccaacttcct tatgctctcc ggagcagaac acttcacagc 1020
agttatcaat cccaggggga ggatccaaac acttcttcct attgcatctt ggaactggag 1080
tcactaagaa aaatgcaaca tggtcaagca ctgggtagaa caacacactt tacttacaga 1140
gagaagaaac agaacaagat aagcttaata gagtgctgtg atcccttatg gctagcacgt 1200
cacccaggca ggtcacccag gcaggtggct gagaatgggc aattggccta cacgcacccc 1260
tgtcgggctg aagcagaaga acccaacccc tcctccatgg agtctgaaat agtaaaaact 1320
gggtatgttc ctgaggacca ttgacttacg agcttaaacg gaacaaagga gtgcaccctg 1380
agcttataag aaaagatact tgcatataag acaataggcc ctgcagaggc tatctgggct 1440
tttgatgttt tgcaataaaa aatttctagg atcaaggccc atttttatgc agtcaactgg 1500
gggtggaaag accccacaaa ctacctttcc caacagcctg tcttccttgt tgagaaaatt 1560
ctctcaaggt atttaccagc agtaagttgt ttgatcctct agaatgtcta atgtcatctt 1620
gatgttggat tattgtgtta ccaagctaac acaagtgatg ccaagaatag cctgagaaat 1680
gcagcagtaa gatggggatt tatgactgac ccaccccctt gctggcgggt gagaggatgc 1740
catctctgtt ggaaggtgag gctcgaatca ttcttggcac aaagtcccag gtcagtcatc 1800
tgccagttgg tgttagccag tgtcaaagga caagactaca atacatttag ttaatgatat 1860
aactggcttt ctttcatgat tcatgaattg gggtagcatt ctattctgta gaataagagc 1920
ttccctgagc tgagcagaaa aggatggctt cataggcaga aaaagactga agaaagaaat 1980
aatgaatgga aagtgaattg cttgttccag ggttactttc cttacagaat taaaacgagg 2040
ggacttcctt attatgctaa attgaattga ctggaatctt gcttgttttt cttcttgaaa 2100
ttttgcccat ttcaaagttc agtttgatta agtggcactt atcaggagtg actccattct 2160
ggtttggtct ggtctactga ggcctagtgc aggaaggcag ttcaagacaa tgacctccca 2220
taaaatttgg ttaacatcat gctttattat cagtcacatg aaaactgcct gaattgtcac 2280
agaatggagt ggcaaagttg gagactacat gtggatctaa gagcttagac gcttaattat 2340
aatagctgat ctagctactc ctgcctctga aagaccaact tgtcaacctt taatatgaat 2400
atcccttaaa ggaacctaaa ggacacttgg tggtaaattg actatattga gcttctccct 2460
tccacgaagg tcgtggatac ccattctggt tataagtctg ctttgccacc cttgagtctg 2520
agccaaaatc cttcactggt gcttatggta tgcctgatac acagcgtagc ttctgatcag 2580
gaaaccctct tgacagtgaa ggatgtgagg ggcaaagccc aaacccatgg gcttcaatca 2640
ttgtatcaca cacagaccca tccataagca gctggtatca caaggtgttg gaagagcttc 2700
taaatgcacc ctcctcaagt ctcaggtgta agacaataat caaaaaggat gtggtatttc 2760
ataggaagaa aaaagttgtt catcattaga aagaacagaa ggtcttgaaa ccaaggagga 2820
gaaaaacgag taactccact tactacactt cctattgatt taatgaagga tttgcttccc 2880
atctctgcaa atctgggttc atcagcttga aggtcctgtt cctcaaagct ggtgtatttt 2940
ggtgactaga agcagcaagt gtcccaatgg actaccattt atgactacca gggaagttca 3000
gactccttgt gcccaaagtt taccaggaag aaaggcctgc agatacaata gaaaagcctt 3060
cacacaaatc tgctggagaa gtagatccgg gtgaaacagc aggaaaactg tcgtaaaaat 3120
gcatatctca gatctcctac tagtagggtc ataagcaacc catgactcca gctgctgagg 3180
tctaaatctg tcactactta tgctcggaga ctatttcctg tggaggaagt attcttaacc 3240
aattactgca tttggctgtg atatgaaagt tggctcagtc ctgctggatg gaggctgagg 3300
caaatgtcac agatgatgga gacatgacca gggtgatggg aatggcgttg aagagaagtt 3360
gcctggcttg atatgtagtt gaaaagtaga acttacagga cttgctaata gtctacagaa 3420
ccagccatca tgccaaagtg actttcagca ttgaactaca cctcacaata cctgcattct 3480
cttttttact acaacttgct gcagatttct tttttttttt tttttctggt agcagaaatc 3540
cccatttgat gccaattcct catcatcata agggaatgta atcaaggatt ctttcctaat 3600
aatgtgaata gatgtgcagt gactgacaga ggtggtacag cgggtttatt cttcttatgg 3660
cagtgatctt ctgactgtaa cccaagggtg aaagctcatg cactcttagc aacaagtaaa 3720
ttgtacgaac atgccccaga tatttcttca tttatttgta aattatatac agtttagtct 3780
attctgaaaa tatacagttt tttaaaaatt gtaaaacaaa tatgtttttc tttactgtgt 3840
aaaattaaat atcactgaga gtaaaaa 3867
<210> 2
<211> 23
<212> DNA
<213>It is artificial synthesized
<400> 2
ggctgtgata tgaaagttgg ctc 23
<210> 3
<211> 21
<212> DNA
<213>It is artificial synthesized
<400> 3
caactacata tcaagccagg c 21
<210> 4
<211> 22
<212> DNA
<213>It is artificial synthesized
<400> 4
gaaccccaag gccaaccgcg ag 22
<210> 5
<211> 23
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<213>It is artificial synthesized
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cagtggtacg gccagaggcg tac 23
Claims (4)
1. applications of the long-chain non-coding RNA LINC00559 as biomarker in lung squamous cancer prognosis detection preparation is prepared.
2. long-chain non-coding RNA LINC00559 answering in lung squamous cancer prognosis detection kit is prepared as biomarker
With.
3. a kind of lung squamous cancer prognosis detection kit, which is characterized in that the kit includes long-chain non-coding RNA
LINC00559 real-time fluorescence quantitative PCR specific primers：
Forward primer：5′-GGCTGTGATATGAAAGTTGGCTC-3′；
Reverse primer：5′-CAACTACATATCAAGCCAGGC-3′.
4. kit as claimed in claim 3, which is characterized in that ACTB internal reference real-time fluorescences are further included in the kit and are determined
Measure PCR specific primers：
Forward primer：5′-GAACCCCAAGGCCAACCGCGAG-3′；
Reverse primer：5′-CAGTGGTACGGCCAGAGGCGTAC-3′.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108728546A (en) * | 2018-07-09 | 2018-11-02 | 郑州大学第附属医院 | Application, kit and the detection method of lung cancer for prognosis novel molecular marker non-coding RNA LINC00963 |
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