CN111235279B - Application of tumor-associated fibroblast specific long non-coding RNA in lung adenocarcinoma prognosis evaluation - Google Patents
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Abstract
The invention discloses a specific lncRNA marker of a tumor-associated fibroblast or a combined marker thereof and application thereof, wherein the lncRNA marker or the combined marker thereof comprises one or more of LINC01614, AC134312.5, LINC01429, LINC02544, AC012213.3 or LINC 01705. The invention discloses that the specific high-expression lncRNA (LINC 01614, AC134312.5, LINC02544 and LINC 01705) of tumor-related fibroblasts is a reliable molecular target related to lung adenocarcinoma prognosis, and can be used for lung cancer prognosis judgment by detecting the expression level of the genes of a patient, guide clinical establishment of an individualized chemotherapy scheme and improve curative effect.
Description
Technical Field
The invention belongs to the field of biotechnology and treatment, and relates to application of tumor-associated fibroblast specific long non-coding RNA in lung adenocarcinoma prognosis evaluation.
Background
The incidence and mortality of lung cancer has been stable in all malignancies, with lung adenocarcinoma being the more than 2/3 pathological type, with increasing incidence year by year, especially in women, non-smoking patients and young adults. Factors influencing the prognosis of lung adenocarcinoma comprise tumor stage, and early patients can effectively control the recurrence and metastasis of tumors through surgical operation. Patients with advanced stages often need to receive chemotherapy, radiotherapy, molecular targeted therapy and immunotherapy, wherein tumor resistance, immune cell depletion in the microenvironment and the like determine the prognosis of patients with advanced stages. More and more studies show that tumor microenvironment cells are of great significance in the prognosis of tumors, and the progression of tumors depends on cancer cells and fibroblasts, endothelial cells, immune cells and matrix components in the microenvironment. Tumor associated fibroblasts (CAFs) are a heterogeneous population of cells from multiple sources, and studies have shown that normal fibroblasts, mesenchymal cells, epithelial cells, pericytes, adipocytes, and endothelial cells may all be converted into CAFs. CAFs are used as the main component of tumor stroma, can promote the invasion and metastasis of tumor cells by the modes of direct cell contact or cell factor secretion and the like, and can enhance the chemotherapy drug resistance of cells. The lung adenocarcinoma is one of tumors rich in CAFs, and the CAFs can regulate and control the growth and metastasis of tumor cells, stem cell microenvironment, immunosuppression and chemotherapy resistance, and are closely related to the prognosis of the lung adenocarcinoma.
Long non-coding RNA (lncRNA) is a kind of RNA with the length of more than 200 nucleotides and the quantity of which is far larger than that of coding protein genes, and becomes a research hotspot in recent years because of rich biological functions. Among lung cancers, it has been found that a transcription factor (metastasis associated lung adenocarcinoma metastasis 1, MALAT 1) affects relapse and metastasis in non-small cell lung cancer patients and is an important prognostic indicator; HOX transcription Antisense RNA (HOX Transcript Antisense RNA, HOTAIR) can promote cell proliferation and mediate platinum drug resistance by regulating p 21. In addition, the exosome lncRNA also becomes a molecular target with tumor marker potential. Exosome lncRNA (long non-coding RNA) derived from microenvironment cells transmits different phenotypes to adjacent cells through interaction between the cells, and the tumor development is promoted.
Disclosure of Invention
The purpose of the invention is as follows: the method is characterized by firstly preliminarily screening 195 lncRNA which is highly expressed in lung adenocarcinoma but is not expressed in 51 lung adenocarcinoma cell lines based on RNA-seq data of a cancer gene map (TCGA) and a tumor cell line encyclopedia (CCLE) database. And (3) performing correlation analysis on the 195 lncRNAs in TCGA data by combining cell subset markers in lung adenocarcinoma single cell sequencing data to obtain 20 microenvironment high-expression lncRNAs of potential lung cancer, wherein 6 lncRNAs are in high-medium correlation with CAFs cell subsets (LINC 01614, AC134312.5, LINC01429, LINC02544, AC012213.3 and LINC 01705). These 6 lncRNAs are a group of potential CAFs-Derived high-expression lncRNAs, which we named as tumor Associated fibroblast-Derived lncRNAs (Cancer Associated fibroblast Derived LncRNAs 1-6, CAFDL 1-6).
All the above 6 lncRNAs are closely related to CAFs. Fresh tissues of 5 patients with lung adenocarcinoma were obtained, primary cultured CAFs cells and paracancerous normal fibroblasts (normal fibroblasts, NFs), and fluorescent quantitative PCR showed that: all 6 lncRNAs were expressed only in CAFs, significantly higher than NFs cells. The tissue samples of 50 patients with lung adenocarcinoma are obtained, and the expression of 6 lncRNA is detected, so that the expression in the lung adenocarcinoma is obviously higher than that in normal tissues. Correlation analysis is carried out on 6 lncRNA and TCGA lung adenocarcinoma patient survival time, and LINC01614, AC134312.5, LINC02544 and LINC01705 expression can be used for lung adenocarcinoma prognosis evaluation, and the higher the expression is, the worse the patient prognosis is.
The invention establishes effective postoperative prognostic evaluation markers on the basis of LINC01614, AC134312.5, LINC02544 and LINC01705, and provides a new approach and theoretical basis for developing individualized treatment targets.
The technical scheme is as follows: in order to solve the above technical problems, the present invention provides a lncRNA marker or a combined marker thereof specific to tumor-associated fibroblasts, wherein the lncRNA marker or the combined marker thereof comprises one or more of LINC01614, AC134312.5, LINC01429, LINC02544, AC012213.3 or LINC 01705.
Wherein the nucleotide sequences of LINC01614, AC134312.5, LINC01429, LINC02544, AC012213.3 and LINC01705 are respectively shown in SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 5 and SEQ ID NO: and 6.
In some embodiments, the invention provides reagents for detecting the expression levels of lncRNA as described above.
In some specific embodiments, the reagent is a reagent for detecting the expression level of lncRNA by a fluorescent quantitative PCR method.
Specifically, the reagents may comprise a reverse transcription kit and a fluorescent quantitative PCR kit. The reverse transcription kit includes the necessary elements for polymerase chain reaction. The fluorescent quantitative PCR kit comprises 6 pairs of primers specific to lncRNA. Each primer is a nucleic acid having a nucleic acid sequence specific to the gene, and has a length of about 20bp and an amplification product of about 200 bp. Wherein the primer pairs corresponding to LINC01614, AC134312.5, LINC01429, LINC02544, AC012213.3 and LINC01705 are respectively SEQ ID NO: 7 and SEQ ID NO: 8. SEQ ID NO: 9 and SEQ ID NO: 10. SEQ ID NO: 11 and SEQ ID NO: 12. SEQ ID NO: 13 and SEQ ID NO: 14. SEQ ID NO: 15 and SEQ ID NO: 16. SEQ ID NO: 17 and SEQ ID NO: 18, respectively.
In addition, the kit may further comprise a nucleic acid sequence primer for an internal reference gene.
Wherein, in the lung cancer prognosis evaluation of the kit, when the expression level of the tumor tissue tumor-associated fibroblast specific lncRNAs (CAFDLs) is higher than the cutoff value corresponding to the specific lncRNA markers, the possibility of poor prognosis of the subject is indicated, otherwise, the possibility of good prognosis of the subject is indicated; specifically, cutoff values corresponding to LINC01614, AC134312.5, LINC02544 and LINC01705 in the specific lncRNA marker are 1.12, 5.97, 5.80 and 6.06 respectively.
Wherein, the fluorescent quantitative PCR kit can comprise a PCR reaction tube, a pre-mixed reaction solution and sterile water.
In some specific embodiments, the lncrnas of the invention are highly expressed in TCGA lung adenocarcinoma tissue.
In some embodiments, lncRNA of the invention is low or absent in lung adenocarcinoma cell line and normal lung fibroblast cell line (salt) in CCLE database.
In some specific embodiments, lncrnas of the invention correlate in lung adenocarcinoma single cell sequencing data with high-counts of CAFs cell subpopulations.
In some embodiments, lncrnas of the invention are associated with CAFs in the NJLCC non-small cell lung cancer cohort transcriptome sequencing data.
In some specific embodiments, the lncrnas of the invention are highly expressed in primary lung adenocarcinoma CAFs and less expressed in NFs.
In some specific embodiments, the lncrnas of the invention are associated with patient prognosis in TCGA database lung adenocarcinoma tissue, exhibiting higher expression and poorer prognosis. Based on the above, the lncRNA gene expression detection can be applied to lung adenocarcinoma prognosis evaluation and individual treatment guidance.
In some specific embodiments, the present invention uses the above tumor-associated fibroblast-specific lncRNA markers for the prognostic evaluation of lung cancer.
In some specific embodiments, the invention also applies the tumor-associated fibroblast specific lncRNA combined marker to the lung cancer prognosis evaluation.
In some specific embodiments, the invention also provides application of a detection reagent of the tumor-associated fibroblast-specific lncRNA marker or a combined marker thereof in preparation of a kit for the prognosis evaluation of lung cancer.
The invention also relates to a reagent and/or a kit for lung cancer prognosis evaluation, wherein the kit comprises a reverse transcription kit and/or a fluorescent quantitative PCR kit.
In some specific embodiments, the fluorescent quantitative PCR kit comprises a primer pair specific for lncRNA.
In some specific embodiments, the kit may further comprise a nucleic acid sequence primer directed against an internal reference gene.
In some specific embodiments, the fluorescent quantitative PCR kit further comprises a PCR reaction tube, a pre-mixed reaction solution, and sterile water.
Has the advantages that: compared with the prior art, the invention has the advantages that: the invention discloses that the specific high-expression lncRNA (LINC 01614, AC134312.5, LINC02544 and LINC 01705) of tumor-related fibroblasts is a reliable molecular target related to lung adenocarcinoma prognosis, and can be used for early diagnosis or prognosis judgment of lung cancer by detecting the expression level of genes of a patient, guide clinical establishment of an individualized chemotherapy scheme and improve curative effect.
Drawings
FIG. 1 is a screening process of high expression lncRNA from CAFs cell source in lung adenocarcinoma microenvironment;
FIG. 2 is a screening process of high expression lncRNA from CAFs cell source in lung adenocarcinoma microenvironment; the codes in fig. 2 represent: N1-NCIH441, N2-NCIH22, N3-NCIH2228, N4-SKLU1, N5-HCC44, N6-HCC78, N7-RELFCAD 1, N8-NCIH1703, N9-NCIH1395, N10-NCIH1781, N11-MORPP, N12-NCIH1563, N13-NCIH1358, N14-NCIH1650, N15-NCIH1437, N16-NCIH1792, N17-NCIH1355, N17-NCIH 17, N17-NCIH 2347, N17-NCIH 2087, N17-NCIH 2085, N17-NCIH 15772-NCIH 1653, N17-NCIH 1651, N17-NCIH 1656, N17-NCIH 364172, N364172-NCIH 36413, N17-NCIH 364172, N364172-NCIH 36413, N17-NCDV 17-NCIH 36413, N17-NCIH 364172-NCIH 36413, N17-NCIH 36413, N36
N33-NCIH1623,N34-NCIH1755,N35-NCIH1693,N36-CALU3,N37-NCIH2009,N38-HCC4006,N39-NCIH2405,N40-NCIH2126,N41-HCC827,N42-LXF289,N43-HCC2108,N44-NCIH650,N45-NCIH854,N46-CORL105,N47-HCC2279,N48-HCC1833,N49-NCIH3255,N50-HCC364,N51-HCC827GR5;
FIG. 3 is a high-moderate relative high expression lncRNA screening of microenvironment subgroup cells;
FIG. 4 NJLCC transcriptome sequencing verifies the correlation of 6 CAFs-derived lncRNA;
FIG. 5 is a representation of the CAFs and NFs cell identification;
FIG. 6 shows the expression of CAFDLs in CAFs and NFs cells
FIG. 7 shows the expression of CAFDLs in lung adenocarcinoma tissue;
FIG. 8 is a graph of the relationship between CAFDLs and prognosis of lung adenocarcinoma; the TCGA database suggests that the high expression of CAFDLs is related to the poor prognosis of lung adenocarcinoma;
FIG. 9 shows the relationship between CAFDLs and prognosis of lung adenocarcinoma; the qPCR result indicates that the high expression of CAFDLs is related to the poor prognosis of lung adenocarcinoma;
FIG. 10 is a graph of the relationship of the combined detection of CAFDL1, CAFDL2, CAFDL4 and CAFDL6 to the prognosis of lung adenocarcinoma; the qPCR result indicates that the high expression of the combined detection value is closely related to the poor prognosis of the lung adenocarcinoma.
Detailed Description
The present invention is further illustrated by the following specific examples, it should be noted that, for those skilled in the art, variations and modifications can be made without departing from the principle of the present invention, and these should also be construed as falling within the scope of the present invention. The experimental procedures in the following examples are conventional unless otherwise specified.
Example 1
1. Primary cell culture
Selecting 100 mg of fresh lung adenocarcinoma tumor tissue or paracancer normal tissue, cleaning with PBS for 3 times, and cutting the tissue into 1.5-2.0 mm pieces3And (4) placing the mixture into pancreatin containing 0.02% of EDTA for digestion for 20 min. After the serum had stopped digesting, it was screened through a 400 mesh screen. PBS washing, centrifugation at 1000 rpm for 5 min, and discarding the supernatant. Repeat 3 times. DMEM medium containing 10% fetal bovine serum, 0.37% sodium bicarbonate, 0.1 mM non-essential amino acids, 100. mu.g/ml streptomycin was placed at 37 ℃ in 5% CO2Culturing in an incubator. Fluid changes were made every 3-4 days and the cells were passaged when they reached 90% confluence. And 3-6 generation cells after isolated culture are used for experiments.
2. Lung adenocarcinoma tissue sample collection
The lung adenocarcinoma tissues with prognosis and the paired normal tissues of 50 cases are applied and obtained from a biological sample library of a tumor hospital in Jiangsu province, all samples are immediately put into liquid nitrogen for freezing after being isolated, and the whole operation and preservation process follows an enzyme-free principle.
3. RNA extraction
Trizol lysed primary cultured cell tissue. Total RNA was extracted using a cell/tissue RNA extraction kit (TIANGEN Co.), and the detailed procedures are described in the specification.
4. Mass analysis of RNA samples
The concentration and purity of RNA are detected by NanoDrop2000, the OD260/280 range interval is 1.8-2.2, Agilent technologies 2100 Bioanalyzer detects the concentration, RIN value, 28S/18S and fragment size of total RNA, RNA integrity index is qualified (RIN value >7, 28S/18S ratio >1.5, sample peak pattern base line is smooth, 18S and 28S can see clear single peak), RNA concentration is more than or equal to 20 ng/mul, and mRNA library construction is carried out.
5. Reverse transcription and fluorescent quantitative PCR
Unifying the RNA quantification, preparing the first strand cDNA according to the instruction of TaKaRa reverse transcription kit (PrimeScript RT reagent kit with gDNA Eraser):
1) removal reaction of genomic DNA:
2) removal reaction procedure of genomic DNA: reacting at 42 ℃ for 2 minutes; storing at 4 deg.C;
3) reverse transcription reaction:
4) the procedure is as follows: reacting at 37 ℃ for 15 minutes; reaction at 85 ℃ for 5 seconds; reacting at 4 ℃ for 5 minutes;
5) the cDNA sample obtained by reverse transcription and the remaining RNA sample were stored in a refrigerator at-80 ℃ until use.
6) Fluorescent quantitative PCR
10 μ l Real-Time PCR System:
the primer sequence is as follows:
7) PCR amplification procedure: at 95 ℃ for 3 minutes;
8) PCR results and calculations
β -actin is used as an internal reference gene, △ Ct value = target gene Ct value-internal reference gene Ct value, △△ Ct value = cancer tissue △ Ct value-paracancer tissue △ Ct value, 2 is added-△△CtThe expression level of the target gene in the sample is expressed. And by adopting independent sample t test, P is less than 0.05, so that the method has statistical significance.
6. Immunohistochemical staining of cells
5 × 104Cells/ml were seeded on cell slides, incubated for 2 days, fixed with 4% paraformaldehyde for 30 minutes, washed with PBS, blocked for 30 minutes, primary antibody added, and incubated overnight at 4 ℃. After three PBS washes, secondary antibody was added and incubated for 2 hours at room temperature. After three washes with PBS, nuclei were counterstained with DAPI. And (6) sealing and observing.
7. TCGA and CCLE data retrieval
Human lung adenocarcinoma RNA-seq data and prognosis were downloaded from the TCGA data website (https:// genome-cancer. ucsc. edu /). Among these, RNA-seq data was available from 433 samples (including 32 paired tumor normal samples) from 398 patients. FC according to the statistical difference in mRNA expression between normal tumor and paracancerous tissue>2,p<823 strips of differentially expressed lncRNA were screened on the 0.01 principle. Downloading RNA-seq data of human lung adenocarcinoma cell strains and normal lung fibroblast cell strains from a CCLE data website (https:// ports. branched. organ. org/CCLE /), and screening 195 lncRNA according to the principle that TCGA is highly expressed and CCLE is not expressed.
8. Statistical analysis
Data statistics and results analysis were performed using SPSS19.0 and graphpadprism6.0 software and results are expressed as mean ± sem. Tumor and paracarcinoma were analyzed by t-test on independent samples when compared. The difference between P <0.05 and P < 0.01 is statistically significant.
9. The experimental results are as follows:
1) CAFs-related lncRNA screening
Based on the RNA-seq data of the American cancer gene map (TCGA) and the encyclopedia of tumor cell lines (CCLE) database, 195 IncRNAs which are highly expressed in the lung adenocarcinoma and are not expressed in 51 tumor cell lines are preliminarily screened (FIGS. 1 and 2). Further correlation analysis was performed in combination with lung adenocarcinoma single cell sequencing markers in TCGA data to obtain 20 potential lung adenocarcinoma microenvironment cell high-expression lncrnas, of which 6 lncrnas were highly-moderately correlated with CAFs cell subsets (LINC 01614, AC134312.5, LINC01429, LINC02544, AC012213.3, LINC 01705) (fig. 3). As shown by verification in NJLCC cohort transcriptome sequencing data published by the team, the above 6 lncRNAs were found to be closely related to CAFs (FIG. 4), suggesting that these 6 lncRNAs are a group of potential CAFs-Derived high-expression lncRNAs, and we named them as tumor-Associated fibroblast-Derived lncRNAs (Cancer Associated fibroblast Derived LncRNAs 1-6, CAFDL 1-6).
2) Primary cell culture and identification
Fresh tissues of 5 lung adenocarcinoma patients are obtained, and primary CAFs and NFs are cultured by a digestion method, so that the cells are in a fusiform shape, abundant in cytoplasm, and circular or elliptical in shape, wherein nuclei are located in the centers of the cells. The cells grow faster and are arranged in a radial or vortex pattern. Immunohistochemical staining shows that CAFs cells express the mesenchymal marker protein Vimentin and the activated fibroblast marker alpha SMA, but do not express the epithelial marker protein cytokeratin. NFs cells expressed negatively for α SMA and Cytokeratin proteins and positively for Vimentin protein (FIG. 5).
3) CAFDL is highly expressed in CAFs and lung adenocarcinoma tissues
The qPCR detection result shows that CAFDL1-6 is highly expressed in CAFs, is low expressed in NF, has P <0.05 and has statistical difference (figure 6). In 50 pairs of lung adenocarcinoma tissues, CAFDL1-6 expression was higher than that of paired normal paracarcinoma tissues, P <0.05 (FIG. 7).
4) High expression of LINC01614, AC134312.5, LINC02544 and LINC01705 is associated with patient prognosis
Performing survival analysis on 6 CAFDLs in a TCGA lung adenocarcinoma database, and calculating an optimal cutoff value according to the FPKM value of each lncRNA, wherein: CAFDL 1: 0.46, CAFDL 2: 0.08, CAFDL 4: 0.19, CAFDL 6: 0.06. high expression is defined as FPKM values greater than cutoff and low expression is defined as below cutoff. The dead patient is marked as 1, otherwise 0. The survival curves of the patients in the high-expression and low-expression groups were calculated by using the K-M method. The results show that the higher the expression of four lncRNA of CAFDL1, CAFDL2, CAFDL4 and CAFDL6 is, the worse the prognosis of lung adenocarcinoma patients is, and the average survival time is obviously shorter than that of the low-expression group. (FIG. 8).
Further, survival validation was performed in 50 versus lung adenocarcinoma tissue, where CAFDL1 cutoff values: 1.12, CAFDL2cutoff value: 5.97, CAFDL4 cutoff value: 5.80, CAFDL6 cutoff value: 6.06. the results show that the expression groups with high CAFDL1, CAFDL2, CAFDL4 and CAFDL6 are obviously reduced when the survival time of the patients is lower (FIG. 9).
Finally, the four lncRNA joint assays CAFDL1, CAFDL2, CAFDL4 and CAFDL6 were used for prognostic comparisons. Each sample expression value was calculated as Score = 2.492 × CAFDL1 + 2.408 × CAFDL2 + 3.288 × CAFDL4 + 2.534 × CAFDL6, combined cutoff value was 1.59. The results show that the higher the expression of the combined detection values of CAFDL1, CAFDL2, CAFDL4 and CAFDL6, the worse the prognosis of the patient, the more significant the statistical difference (P < 0.001), and the combined detection is more helpful for the prognosis judgment of the lung adenocarcinoma patient (FIG. 10).
Sequence listing
<110> tumor hospital in Jiangsu province
<120> application of long non-coding RNA (ribonucleic acid) specific to tumor-associated fibroblasts in lung adenocarcinoma prognosis evaluation
<160>20
<170>SIPOSequenceListing 1.0
<210>1
<211>648
<212>DNA
<213>LINC01614(CAFDL1)
<400>1
gggaaaaggg ctgagggggt agaggtataa aaatcagata gctcagcact tcgctccaaa 60
ggagctgaag gagagaggat tccactgcgt gtatgttttc tctttcacgt ctcacttttg 120
ttctctgcca tcaggaaatg cagtagacat ttgtcaccag cagctctttg gagacttcaa 180
tgttccttcc tcccaggatc cgaaagggga cttcagacac ggagaatcta agacaccagg 240
agctcagaag aatggagtct caagacatca ggcaaatacc gaagagatca gagccaagtt 300
ctaaagggca gccatttccc aaatgtcaac caagagcgaa gccaagagaa ttatgatttc 360
ctggctatac aatgacagga aaagcttgat caaagctgga gtgcaagtta aaaagtgtaa 420
ggtactcaag tgctagggtc tgtgtccaag cccaatcaat tgatcattag ttgtttttaa 480
cttcaacgtt tcttttgcaa cagcacctct tccttaaagt agcaatctta gcagactgcc 540
accttacatg atgcatctta ttctccaagg acaatgaaga ctgaactatc gcacattacc 600
taagaaagat gggaattgac atgcacatca caattgtata cacaacag 648
<210>2
<211>2066
<212>DNA
<213>AC134312.5(CAFDL2)
<400>2
ggatgccgga gccctcccag cgagagccgt gtttatttta ctttacatac attttccaac 60
acggagcggc ttgcacacat gcagctctta ggcccgggcc gcacgtctca gaagccccgt 120
gtgcgacttt gaccgccgca cgatcctctg ccgggggagg tgggcccgct gcgctttggg 180
agcacccgcg cccgacactg aggtctcggt gctgtgttcg gcctcttcgt ccctgcgggt 240
cccctctggg agcagaggcg gtcggaaaac cctggggctg aagtgcaggc ttcgggagga 300
cgcgacctgc caagatcagc tcccggcacg tgatgggagc ctggctcacc ttcccccagc 360
gcacgatggg ccgcagcctc cccggtcggc ctggcctgct ggaaaggagc agctctgttt 420
ccagaggctt ctggcgaagc ccacggcctc ccattgttgg ctgatttata aggaaagagg 480
ggaaaggcca agtgtggatg ccattagcat aacctaatcc agaccccatg acaagtccag 540
gatcctgcag ggagagggca tccttgaacg tgaaggactg gctttggaaa cttggcctcc 600
cggaagaaag gtctccgggc ccacccacac ccaccttgtg gacgcccccg cagtcgaata 660
cactccacag gaagacggac cacaaacagc agcagcctcc ggtgtcggcc cagtgatccg 720
ggagctcaga gttgcagcaa gcttaaactt ccttaaaaac aaacctctgg gaagaggaaa 780
acgaggcaag atgctgagaa aaagtcatct cccactctgc ccttctctgg caaatcgaag 840
tgtcctcttg tgggaggcgg tgggcagcga cactcctgtt ggacgtcagt gctctcggga 900
tgggcccggc caccccggag cagaactcac ttagggcctg gtgcccctgt gcccagttct 960
cagaacgtcg gagtgctcag ttctcagaac gccggagtgc cccattcata acctgcagcc 1020
ccgttcatag cctgcagctc ccctccttcc tctcctcccg gtcgtcaggg agcaccccca 1080
gctcccctcc ccactctcaa accctcctcc tccctcccct ctctcatttc cctgtttgca 1140
ggatctcctc ctgcccactc tccagcccag gtcctcccct ctcatctcca attctgcttc 1200
atgggccatg acagcaggca ggccatggcc tgctctcagc agtttgatcg atggactaag 1260
gagatcactc ctatgagagt gaaaaagtca aatgtgcctg agattcggat ggggagctct 1320
caaagctacg gattgcaaga gtcaacaccg cctcaaaagt acaaggccgg ccgggtgcag 1380
tggctcacac ctgtaatccc agcactttgg gagactgagg cgggtggatc acctgaggtc 1440
aggagctcaa gaccagcctg accaacatgg tgaaatcccg tctctattaa aaatacaaaa 1500
aagattagcc gggtgtggtg gtgcatgctt gtagtcccag ctactcggga ggctgaggca 1560
gggggagcac ttgaacccgg gaggtggagg ttgcagtgag ccgagattgc accattgcac 1620
tccagcctgg gcaacagagt gagaccctgc ctcaaaaaaa taaaaaaata aaagtacgag 1680
gcccggttta aatgcaggag gaatctggtg agcgagtttc actctgatgc aacagaaaag 1740
ccaagccaag ccttggctgt gcctggtctt ggccggggag ccacaggcag ggtctgctgg 1800
tttcggtgtc aggggcacat ccccaggacc acgtctgaga tgggaagtgc cttgatggaa 1860
aactgatacc tttttctttc cgagtaactt tagaacatgt cgcttcaaat ataactaata 1920
tcctgatttc agtggacttg catggaagga aacgcaagtt catttgccag tggggggtga 1980
ggactgggag agtggggagc agggtgggga agaggggagc attccacaag ccgcaagagc 2040
ctggacacct ccagccttca ataaat 2066
<210>3
<211>2326
<212>DNA
<213>LINC01429(CAFDL3)
<400>3
tttctgctgg ctggatgggg atgtggagga gaggaccctt ggaccatggg gatgggagca 60
acgtgccagg catggcagcg caatagaggg tggaacatct tttgaagagc tcagaccctg 120
gacccctgat cttaatgagc tttgcagcct tcagtgacac aacctggatt taaactcatg 180
gatgtcggcc tctgctctgc gttgaattgt atcctcccaa aaaggagatg ttggagctct 240
gagacccagc acctcagaat gtgtttctac ttggaaatag ggtctttacc aagaccctat 300
tccaatagga ctggtgacct gtgggagaaa tgtggacaca gagacagaca gacatagagg 360
gaagatgacg gcagagtcac agggagaaga cggtcatcta cgagccaagg agagaggcct 420
ggcacagatc cttccctcac agcacccaga gagccttcag acaatacatc tgtgctgtgt 480
aagccgctcc gtctgtggct ctctgttaca gcagccccag aaaacaaata gagactctta 540
attgccaagc catgttcctc ccatagtacg atttctcatc agatagtcta tcttcgctgc 600
taaaacaggt atttctggat ggtagaggtc atgtgtagct tgcatattta gtatgaggta 660
tgtgaaagtc acctcataga tattaaatta tttggctaca gctttgagct tggcctctct 720
cttaaggatt accttgccat agggacagga agccatcgtg atataaaggc tgaagtttct 780
gtaagatttt gatttttaat ctaacatgat attaacaact gtgcccgttg ggcgtgatgg 840
ctcacaccta caatcccagc actttgggag gccaaggcgg aggatcactt gaggtcagga 900
gtttgagacc agcctggcca acatggtgaa accccgtctc taccaaaaat acaaaaatta 960
gccgggcttg ggggtgcatg cctgtaatcc cagctacttg ggaggcgaag gcaggagaat 1020
tgcttgaacc caggaggtgg aggttgcagt gagccaagac tgtaccactg cgctccagcc 1080
tcggcaacag agtgagactc tgtctcaaaa cagacaaaca aacaaaataa taactgtacc 1140
tatacaaggt ccccaagata attaaaaggc tcacttgagc agagtaactg aggagagtct 1200
tttggggttc tttgtttttt ttttttagag acagtctcac tctgtcgccc agtctagagt 1260
gcagaggcgc aatcttggct cactgcaacc tccgcctccc aggttcaaga gatcctcctg 1320
cctcggcctc tcaaagtgct gggactacag gcacatgcca ccatgtccgg ctgaggaatg 1380
tttaataaaa ggggtattta ccaagatata agcaaggtat agagaaatca gcagggttga 1440
tgaagcaccc cggggctacc aacaaccaaa agcccttccc tctccaaggc ctccaaaggc 1500
agcaggaggg agtggttatc agaaaatggc aagatccata ggaaaaaggc caccccatgg 1560
gagctgtggg tttcagtaga gggacacagc cactgccaga ccatggccag ccagggagtt 1620
aatagcccag ccccaccttg gccctcccat ctcccactcc tccttattgg ctgaacccag 1680
caagaaacca gatgtcacag aaccaggaga tgtggtcccc actgcccggc ctcttgggca 1740
cagaggaggg aagagaaaat ggagagtgga tctggagggg caaatgaagt attaatattc 1800
tgtacactca gaatgtccac agagtgaaca ctggcttgcc ggctgacccc atttcttgaa 1860
aatcatccta tccctgcagg ttatttatgt tgttaccctt ttgcaagaac cacataaaat 1920
attttacatg cttttaaaac tcataaatgt aatgaagcca agatgtttca gcaaatgtgc 1980
tcaatgggca aatccttcca aagatctgct ctcagctgac gtcaggacaa aaggcacatc 2040
tcacgacctg cagagcgtct gacgttaaag ccaataaatt catccccctg acccacatgg 2100
cttccagaat gtacaagctg tcagtggatc ttaaaaatgt atctattcct tcattcactt 2160
cctgccgttc gtctcttgca agtatttact gctctttaaa aactgaactc caccagctgg 2220
aaatgcatta gtgcagggag atggctgtac tttgttccgg caggaattgt gcatgcaact 2280
tccaatctga cttttctgag acattaataa aaacacatgc gtcctg 2326
<210>4
<211>672
<212>DNA
<213>LINC02544(CAFDL4)
<400>4
ataccgttct tctggtagtg aacaagtctc acaagagctg atggttttat gaagggtttc 60
tacatttgca tcttcctcat tttctcttgc cgccgccaca taagaagtgc ctttcacctc 120
tcatcatgat tctgtggcct ccccagccat gtggaacttt tttctcttgc tgctaccatg 180
taagaagtgc ctttcacctc gtgccatgat tatgagactt ccccagccat gtggaactgt 240
taagtccaat taaacatctt tttcttctta gtcccaggtt gttctcattc gtggctggat 300
cacaccagca agaaggattt taaaggacaa ctccttgggg ccgtccaaca ctgaaaccgc 360
ctgaccgctg ctgtctttgt gcttgcagaa aatcacggtg ggcacaaaat tttggacacg 420
atttcgagag cgtgagagct actgttcacg aatccagggc cagcctcgtg tctgtcttcc 480
tttaaagcac ctggcttggt acctaaaatg ttcctttttt tttttttttt gaaattcaat 540
aacttcagaa caggcgttga tgtagacaga tctccatctt tttggttttt tgagtgagtt 600
tttgctctgt aggctgaatt ctttggaatt tagttctgaa atcaccctat tgttgagtat 660
tcgtttccta gc 672
<210>5
<211>777
<212>DNA
<213>AC012213.3(CAFDL5)
<400>5
attttaatat attatgaact catgtattta aacatatttg atgttttaaa ccattgcagt 60
tctctttatt gatgcttaaa ttgtgtcatc tttaacagtg agaaccaatt caaatgttgg 120
ctcttggatc cttttgagtt gtttttcata gctgccttgt tttccagtat gacaaaatgt 180
tccatgctca tattgtacat ttcctgcccc ataggtgaaa tctcccattt cttcaaggat 240
ttttagttcc ttttagtagg aaatgggagt gtagagatca cattctgggc attactggat 300
tggtcattgt ttctgggcct tttcggtaaa caaagctaga ttttgttgct gttttgtagc 360
tttatttaag ataaagtact ttaggagttc atactgatac ttccaattca agcccagaat 420
ttacttaact tcactaatct tacatgtata ctgccttctt ggattttttt ctgatcacct 480
cagccagaag tgatcactct tcctttgaac cacatcaccc acttcaacac ttttatagta 540
atcaacacat taacttttga tttattgtta tttctatata tgtctgatat tctctaccaa 600
atggtaagga atttgaggat agaatccatc ctttgttacc ctttgttaaa taaagataaa 660
attcctaaag tgctgatata tttcctgaca tataaatacc actttctctt tgtattcctt 720
taaaaatgga accatattat acatgtacta aaaattaata aatgagtgtt aaatgaa 777
<210>6
<211>873
<212>DNA
<213>LINC01705(CAFDL6)
<400>6
ctctttgcct gctgcaatcc atgtaagatg tgacttgttc ctccttgcct tctgccatga 60
ttgtgaggcc tccacagcca tgcggaactg ttgaaataca cactacctga agctggataa 120
ctcatgaact ctgttgacaa ttatattctt tgtcttttta attaactcat ttggagttgg 180
tgtcctgagg acctaagcct aagaatcttc aagatggaag agtagagtga acaatgaatg 240
agccagaggg ctcttgtcct ctgtgtgcag ctctgggcag ccagcaggtg gatatgggac 300
ttggcctgct ggaggggccc tggcaagagg agctggcagg agcagatggc aaagttcact 360
cactctggga gacactggct tccctgttgt gaggacactc aagcagccct attcagaggt 420
ccatgtggta aggtacgaag gcctcctgtc aattctagtg agaaacctag gtttcttgct 480
aacagctatg tgagtgagcc atcttggaag cagattctcc caccccagat gagccttcgg 540
atgactggag ttctggctga catcttgact gcaatctcat cagagacctc aagccaaaat 600
taccccactt ccaaattcct gaccctacag aaactgtgtg agataatata tgcttattgt 660
ttaagccact aagttgttct tttggtgggg gggatgggtg atttgttatg caacaacaga 720
aaactaatac agaaattata cctgccatta tcaaagaggc ctccagtgag ggttgatggt 780
atccttgaat atttagggaa actaagcatg ttaagtgtaa atacagaaac tgtctttgtc 840
aaaactaaat aaagcaaggg aaatggaact tga 873
<210>7
<211>20
<212>DNA
<213>CAFDL1-F(Artificial Sequence)
<400>7
<210>8
<211>20
<212>DNA
<213>CAFDL1-R(Artificial Sequence)
<400>8
<210>9
<211>20
<212>DNA
<213>CAFDL2-F(Artificial Sequence)
<400>9
<210>10
<211>20
<212>DNA
<213>CAFDL2-R(Artificial Sequence)
<400>10
<210>11
<211>20
<212>DNA
<213>CAFDL3-F(Artificial Sequence)
<400>11
<210>12
<211>20
<212>DNA
<213>CAFDL3-R(Artificial Sequence)
<400>12
<210>13
<211>20
<212>DNA
<213>CAFDL4-F(Artificial Sequence)
<400>13
<210>14
<211>20
<212>DNA
<213>CAFDL4-R(Artificial Sequence)
<400>14
<210>15
<211>21
<212>DNA
<213>CAFDL5-F(Artificial Sequence)
<400>15
accacatcac ccacttcaac a 21
<210>16
<211>22
<212>DNA
<213>CAFDL5-R(Artificial Sequence)
<400>16
acaaagggta acaaaggatg ga 22
<210>17
<211>20
<212>DNA
<213>CAFDL6-F(Artificial Sequence)
<400>17
<210>18
<211>20
<212>DNA
<213>CAFDL6-R(Artificial Sequence)
<400>18
<210>19
<211>23
<212>DNA
<213>β-actin-F(Artificial Sequence)
<400>19
gtcattccaa atatgagatg cgt 23
<210>20
<211>20
<212>DNA
<213>β-actin-R(Artificial Sequence)
<400>20
Claims (7)
1. The detection reagent of the lncRNA combined marker specific to the tumor-associated fibroblasts is characterized in that the lncRNA combined marker comprises LINC01614, AC134312.5, LINC02544 and LINC01705, and the detection reagent is used for lung adenocarcinoma prognosis evaluation.
2. The use of the detection reagent for the tumor-associated fibroblast-specific lncRNA combined marker of claim 1 in the preparation of a kit for the prognostic evaluation of lung adenocarcinoma.
3. The application of a detection reagent of a tumor-associated fibroblast-specific lncRNA marker in preparing a kit for the prognosis evaluation of lung adenocarcinoma is characterized in that the lncRNA marker is selected from LINC01614, AC134312.5, LINC02544 or LINC 01705.
4. The use according to claim 2, wherein the nucleotide sequences of LINC01614, AC134312.5, LINC02544 and LINC01705 are set forth in SEQ ID NO: 1 and SEQ ID NO: 2 and SEQ ID NO: 4 and SEQ ID NO: and 6.
5. A kit for prognostic assessment of lung adenocarcinoma, characterized in that said kit comprises a reverse transcription kit or a fluorescent quantitative PCR kit comprising primer pairs specific for incrna markers including LINC01614, AC134312.5, LINC02544 and LINC01705, the corresponding primer pairs of which are SEQ ID NOs: 7 and SEQ ID NO: 8. SEQ ID NO: 9 and SEQ ID NO: 10. SEQ ID NO: 13 and SEQ id no: 14. SEQ ID NO: 17 and SEQ ID NO: 18, respectively.
6. The kit of claim 5, further comprising a nucleic acid sequence primer directed against an internal reference gene.
7. The kit of any one of claims 5 to 6, wherein in the prognosis evaluation of lung adenocarcinoma, when the expression level of tumor tissue tumor associated fibroblast specific lncRNAs is higher than the cutoff value, the prognosis of the subject is poor, otherwise the prognosis of the subject is good, and the cutoff values corresponding to LINC01614, AC134312.5, LINC02544 and LINC01705 in the specific lncRNA markers are 1.12, 5.97, 5.80 and 6.06 respectively.
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