CN111118157B - Long-chain non-coding RNA lncBCBMAT and application thereof as breast cancer brain metastasis molecular marker - Google Patents
Long-chain non-coding RNA lncBCBMAT and application thereof as breast cancer brain metastasis molecular marker Download PDFInfo
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Abstract
The invention discloses a lncRNA molecule and application thereof as a breast cancer brain metastasis molecular marker, wherein the base sequence of the lncRNA molecule is shown as SEQ ID NO:1 is shown in the specification; the lncRNA molecule can be used as a marker of the brain metastasis of the breast cancer, and is used for predicting and diagnosing the brain metastasis of the breast cancer; the accuracy and specificity of the breast cancer brain metastasis diagnosis can be obviously improved; the expression of the long-chain non-coding RNA lncBCBMAT in a malignant breast cancer tissue specimen is higher than that of benign breast tumor, and the expression level in the plasma of a breast cancer brain transfer patient is obviously higher than that of a common breast cancer patient; the long-chain non-coding RNA lncBCBMAT can be applied to molecular markers for predicting and diagnosing the brain metastasis risk of the breast cancer, can be used as a target point for developing novel medicaments for the brain metastasis of the breast cancer, provides basis for diagnosis and treatment of patients with the brain metastasis of the breast cancer, and has important application significance.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to a long-chain non-coding RNA lncBCBMAT and application thereof as a breast cancer brain metastasis molecular marker.
Background
The breast cancer is the second solid malignant tumor which causes brain metastasis, about 25 percent of patients with advanced breast cancer finally develop the brain metastasis, and the tumor metastasis is the main cause of death of the malignant tumor, so the brain metastasis of the breast cancer is the difficulty of clinical diagnosis and treatment. High risk factors for brain metastasis of breast cancer include young patients, poorly differentiated tumors (high grade neoplasias), hormone receptor negative, four and more lymph node metastases. Her2 positive and triple negative breast cancers are also risk factors for brain metastases, with incidence rates of 30% -40%. The mortality rate of the breast cancer brain metastasis is high, the prognosis is very poor, the median survival time of a patient is only 6 months, and the survival rate of 1 year is lower than 20%. Similar to primary breast cancer, patients with triple negative breast cancer brain metastases have the worst prognosis. A retrospective study of 1256 patients with breast cancer brain metastases by Niikura et al showed that Luminal-type tumor patients had an Overall Survival (OS) of 9.3 months (95% Confidence Interval (CI): 7.2-11.3), patients who were Her2 positive had an OS of 16.5 months (95% CI:9.1-13.8), and patients who were triple negative had an OS of only 4.9 months (95% CI: 3.9-5.9). Early detection and treatment of breast cancer brain metastasis is therefore critical to patient cure.
Long non-coding RNA (lncRNA) is an RNA molecule with a length of 200-100000 nt. The mammalian protein coding gene accounts for 1 percent of the total RNA, and the proportion of lncRNA in the total RNA can reach 4 to 9 percent. IncRNAs have conserved secondary structure, some form of cleavage and subcellular localization. The main characteristics of lncRNA include: first, lncRNAs are usually longer, have mRNA-like structures, have polyA tail and promoter structures after splicing, and have dynamic expression and different splicing modes in the differentiation process; secondly, the tissue specificity and the space-time specificity exist, the lncRNA expression quantity of different tissues is different, and the lncRNA expression quantity of the same tissue or organ is also changed at different growth stages; thirdly, the diversity of regulation and control, the lncRNA can realize the regulation and control of gene expression from various aspects such as chromatin remodeling, transcription regulation and post-transcription processing and the like; the lncRNAs promoter can also be combined with a transcription factor, and local chromatin histones also have characteristic modification modes and structural characteristics; fourth, the sequence conservation is low, and only about 12% of lncRNA can be found in organisms other than human; there are characteristic expression patterns in tumors and other diseases. And since lncRNA participates in processes such as X chromosome silencing, chromosome modification and genome modification, transcriptional activation, transcriptional interference, intranuclear transport and the like, lncRNA is a hotspot molecule in clinical medical research. It can regulate the expression of coding gene through different molecular biological mechanisms, and participate in signal passage related to diseases, thus having important effect on the occurrence, development and treatment of diseases. A large number of researches show that the expression level of certain specific lncRNA in tumor cells is abnormally changed, and the abnormally expressed lncRNA can be used as a molecular marker and a potential drug target for tumor diagnosis. However, long-chain non-coding RNA markers related to breast cancer brain metastasis are only reported at present.
Therefore, it is urgently needed to screen and obtain suitable lncRNA molecules expressed abnormally by the breast cancer brain metastasis, which can be used as molecular markers for predicting and diagnosing the risk of the breast cancer brain metastasis, and even can be used as targets for developing novel medicaments for breast cancer brain metastasis.
Disclosure of Invention
In order to solve the problems, the invention aims to provide a long-chain non-coding RNA lncBCBMAT and application thereof as a breast cancer brain metastasis molecular marker.
In order to achieve the purpose, the invention is realized by the following technical scheme:
an lncRNA molecule, the base sequence of which is shown in SEQ ID NO:1, named lncBCBMAT.
The nucleotide sequence of the specific primer of the lncRNA molecule is shown as SEQ ID NO: 2 and SEQ ID NO: 3 is shown in the specification; the nucleotide sequence of the specific primer of the reference gene ACTB is shown in SEQ ID NO.4 and SEQ ID NO. 5.
The application of the preferred lncRNA molecule in the prediction and diagnosis of breast cancer brain metastasis.
The application of the preferred lncRNA molecule in the research and development of medicines for transferring breast cancer brain.
The application of the preferred lncRNA molecule in preparing a breast cancer brain metastasis detection kit.
The invention also comprises a breast cancer brain metastasis detection kit which consists of an lncBCBMAT reaction liquid system and an internal reference gene ACTB reaction liquid system;
wherein the lncBCBMAT reaction liquid system consists of the following components:
carrying out reverse transcription on the obtained cDNA template, carrying out real-time fluorescence quantitative PCR reaction solution, an lncBCBMAT specific primer, ROX Reference Dye and sterilized distilled water;
the reference gene ACTB reaction liquid system consists of the following components:
the kit comprises a cDNA template obtained by reverse transcription, real-time fluorescence quantitative PCR reaction liquid, an internal Reference gene ACTB specific primer, ROX Reference Dye and sterilized distilled water.
Preferably, the real-time fluorescent quantitative PCR reaction solution is TB Green Premix Ex Taq II.
Further preferably, the lncBCBMAT reaction liquid system comprises the following components:
2 mul of cDNA template obtained by reverse transcription;
TB Green Premix Ex Taq II(2×)10μl；
IncBCBMAT specific primers PCR Forward Primer (10. mu.M) 0.8. mu.l;
IncBCBMAT specific primers PCR Reverse Primer (10. mu.M) 0.8. mu.l;
ROX Reference Dye(50×)0.4μl，
and sterilized distilled water 6. mu.l;
the reference gene ACTB reaction liquid system consists of the following components:
2 mul of cDNA template obtained by reverse transcription;
TB Green Premix Ex Taq II(2×)10μl；
0.8 mu l of Primer PCR Forward Primer (10 mu M) specific to the reference gene ACTB;
0.8 mu l of Primer PCR Reverse Primer (10 mu M) specific to the internal reference gene ACTB;
ROX Reference Dye(50×)0.4μl，
and sterilized distilled water 6. mu.l.
The invention also comprises a use method of the breast cancer brain metastasis detection kit, which comprises the following steps:
extracting total RNA of breast cancer brain metastasis cells, tissues or blood plasma, performing real-time fluorescence quantitative PCR (polymerase chain reaction) by using a breast cancer brain metastasis detection kit after reverse transcription, and performing statistical analysis by using ACTB as an internal reference gene and the relative expression quantity of a target gene.
Compared with the prior art, the invention has the following advantages:
the expression level of the long-chain non-coding RNA lncBCBMAT in the breast cancer brain metastasis cells is obviously higher than that of the breast cancer cells and normal cells in a control group, and the long-chain non-coding RNA lncBCBMAT can be used as a breast cancer brain metastasis molecular marker and can obviously improve the accuracy and specificity of breast cancer brain metastasis diagnosis; the expression of the long-chain non-coding RNA lncBCBMAT in a malignant breast cancer tissue specimen is higher than that of benign breast tumor, and the expression level in the plasma of a breast cancer brain transfer patient is obviously higher than that of a common breast cancer patient; the long-chain non-coding RNA lncBCBMAT can be applied to molecular markers for predicting and diagnosing the brain metastasis risk of the breast cancer, can be used as a target point for developing novel medicaments for the brain metastasis of the breast cancer, provides basis for diagnosis and treatment of patients with the brain metastasis of the breast cancer, and has important application significance.
Drawings
FIG. 1 is a volcano plot of differential expression of breast cancer brain metastatic cells 231-BR (BCBM) and their parental non-specific metastatic cells MDA-MB-231 (BC);
FIG. 2 is a graph showing relative expression levels of IncBCBMAT in breast cancer brain metastasis cell line 231-BR and other breast cancer and normal cell lines detected by real-time fluorescence quantitative PCR;
FIG. 3 is a graph showing the difference in expression of lncBCBMAT in pathological tissue specimens of benign and malignant breast tumors by real-time fluorescence quantitative PCR detection;
FIG. 4 is a graph showing the difference in plasma expression of lncBCBMAT in patients with brain metastasis, breast cancer and healthy control population by real-time fluorescence quantitative PCR detection.
Detailed Description
The invention aims to provide application of long-chain non-coding RNA lncBCBMAT as a breast cancer brain metastasis molecular marker, which is realized by the following technical scheme:
an lncRNA molecule, the base sequence of which is shown in SEQ ID NO:1, designated lncbbmat, with transcript ID LNCipedia of lnnc-SRCIN 1-1:5, and entembl transcript ID of ENST00000617990, with a length of 702bp, the corresponding parent gene is located at chr17: 38450394-38452399.
The nucleotide sequence of the specific primer of the lncRNA molecule is shown as SEQ ID NO: 2 and SEQ ID NO: 3 is shown in the specification; the nucleotide sequence of the specific primer of the reference gene ACTB is shown in SEQ ID NO.4 and SEQ ID NO. 5.
SEQ ID NO: 2 is tccctcccat tccaccatcc;
SEQ ID NO: 3 is gtggtcccaa caccgaatcc;
SEQ ID NO: 4 is gcactcttcc agccttcctt;
SEQ ID NO: 5 is ctgtgttggc gtacaggtct;
the application of the preferred lncRNA molecule in the prediction and diagnosis of the brain metastasis of the breast cancer can be used as a molecular marker, for example, the expression difference of lncBCBMAT in the brain metastasis of the breast cancer and the expression difference of the breast cancer and normal cells can be identified; can identify the expression difference of lncBCBMAT in the pathological tissue specimen of the breast tumor benign and malignant patient; the expression difference of lncBCBMAT in the breast cancer brain metastasis and the plasma of a breast cancer patient can be identified, and the specific method comprises the following steps:
extracting total RNA of breast cancer brain metastasis cells, tissues or blood plasma, identifying expression difference of lncBCBMAT in the breast cancer brain metastasis cells and breast cancer and normal cells through real-time fluorescence quantitative PCR after reverse transcription, identifying expression difference of lncBCBMAT in pathological tissue specimens of breast cancer benign and malignant patients, and identifying expression difference of lncBCBMAT in the breast cancer brain metastasis and blood plasma of the breast cancer patients.
The application of the preferred lncRNA molecule in the research and development of the medicine for breast cancer brain metastasis can be used as a target point for the research and development of a novel medicine for breast cancer brain metastasis.
The application of the preferred lncRNA molecule in preparing a breast cancer brain metastasis detection kit.
The invention also comprises a breast cancer brain metastasis detection kit, which comprises a cDNA template obtained by reverse transcription, real-time fluorescence quantitative PCR reaction liquid, a specific (amplification) primer, ROX Reference Dye and sterilized distilled water;
the lncBCBMAT reaction system is concretely as follows:
10. mu.l of real-time fluorescent quantitative PCR reaction solution TB Green Premix Ex Taq II (2X);
IncBCBMAT specific amplification Primer PCR Forward Primer (10. mu.M) 0.8. mu.l;
lncBCBMAT specific amplification Primer PCR Reverse Primer (10. mu.M) 0.8. mu.l;
ROX Reference Dye(50×)0.4μl；
2 mul of cDNA template obtained by reverse transcription;
and sterilized distilled water (dH)2O)6μl。
The ACTB reaction system is specifically as follows:
10. mu.l of real-time fluorescent quantitative PCR reaction solution TB Green Premix Ex Taq II (2X);
ACTB specific amplification primers PCR Forward Primer (10. mu.M) 0.8. mu.l;
ACTB specific amplification Primer PCR Reverse Primer (10. mu.M) 0.8. mu.l;
ROX Reference Dye(50×)0.4μl；
2 mul of cDNA template obtained by reverse transcription;
and sterilized distilled water (dH)2O)6μl。
Wherein the real-time fluorescent quantitative PCR reaction solution can be selected from TB Green Premix Ex Taq II of TAKARA, including fluorescent dye, dNTPs, Mg2+Taq DNA polymerase and a buffer, the fluorescent dye is preferably TB Green. The nucleotide sequence of the specific primer of lncBCBMAT is shown in SEQ ID NO: 2 and SEQ ID NO: 3 is shown in the specification; the nucleotide sequence of the specific primer of the reference gene ACTB is shown in SEQ ID NO.4 and SEQ ID NO. 5.
The invention also comprises a use method of the breast cancer brain metastasis detection kit, which comprises the following steps:
extracting total RNA of breast cancer brain metastasis cells, tissues or blood plasma, performing real-time fluorescence quantitative PCR (polymerase chain reaction) by using a breast cancer brain metastasis detection kit after reverse transcription, and performing statistical analysis by using ACTB as an internal reference gene and the relative expression quantity of a target gene.
The invention is further described with reference to specific examples.
The main experimental materials mentioned in the following examples, and the rest materials not mentioned are all commercially available products.
Cell lines: the breast cancer brain metastasis cell line 231-BR and the breast cancer parental cell line MDA-MB-231 were gifted by the national cancer institute of National Institute of Health (NIH). Breast cancer cell lines BT-474, T47D and human embryonic kidney cell line 293T were purchased from chinese academy of sciences cell banks.
Primer: all primers of the present invention were synthesized by Biopsis (Shanghai) GmbH.
Primary reagent
1. Cell culture reagents: DMEM medium, fetal bovine serum, and trypsin were purchased from Hyclone.
RNA extraction, pooling and high throughput sequencing reagents: rRNA Removal kit was purchased from Epicentre, RNA Library Prep kit from NEB, and AMPure XP system from Beckman Coulter.
RNA extraction, reverse transcription and PCR reagents: RNA extraction kits were purchased from Qiagene, TRIzol from ThermoFisher, and reverse transcription and real-time fluorescent quantitative PCR reagents from TAKARA.
Example 1 screening of lncRNA
The method adopts high-throughput sequencing to screen lncRNA molecules with high expression of breast cancer brain transfer cells, and comprises the following specific steps:
RNA extraction and quantification: culturing 231-BR cells and MDA-MB-231 cells, and extracting total RNA of the cells by using TRIzol;
the RNA purity was checked using a NanoPhotometer;
the RNA concentration was measured using a Qubit2.0 Fluorometer;
RNA integrity was checked using Bioanalyzer 2100;
preparation and sequencing of lncRNA library: rRNA Removal Kit was used to remove rRNA and the Library was sequenced to generate the RNA Library Prep Kit, the main steps were as follows:
disruption was performed in NEBNext first strand synthesis reaction buffer (5X) (NEB, USA). Synthesizing first strand cDNA by using random hexamer primer and M-MuLV reverse transcriptase; second strand cDNA synthesis is then performed with DNA polymerase I and RNase H;
in a reaction buffer solution, dUTP is used for replacing dNTPs containing dTTP, end repair is carried out through exonuclease/polymerase activity, and after the A tail is added to a DNA fragment, NEBNext adaptor is connected with a hairpin structure for hybridization; purifying the library fragment by using an AMPure-XP system, and screening a cDNA fragment with the length of 150-200 bp; the second strand of the U-containing cDNA was degraded using USER enzyme, PCR was performed using Phusion high fidelity DNA polymerase, universal PCR primers, and Index primers (NEB, USA) and a library was obtained. After the library is constructed, firstly, carrying out primary quantification by using Qubit2.0, diluting the library to 1ng/ul, then, detecting the insert size of the library by using Agilent 2100, and accurately quantifying the effective concentration of the library by using a Q-PCR method (the effective concentration of the library is more than 2nM) after the insert size meets the expectation; and after the library is qualified, sequencing different libraries according to the effective concentration and the requirement of the target offline data volume, wherein an Illumina Hiseq 2500 platform is used for sequencing, and the sequencing reading length is 125 bp.
Analysis of data: after obtaining the original sequencing data, performing data quality control, and performing comparative analysis on the filtered reads by using HISAT 2;
using HTSeq software to carry out quantitative analysis on known type genes on each sample of the species, and obtaining the expression condition of each type of gene in the sample according to expression quantity statistics;
merging transcripts obtained by splicing the samples by using Cuffmerge software, removing the transcripts with uncertain chain directions to obtain complete transcriptome information of the sequencing, then screening IncRNA from a merged transcript set to obtain IncRNA transcripts, quantitatively analyzing the transcripts by using StringTie-eB software, and performing IncRNA differential expression analysis by using edgeR.
As shown in FIG. 1, the volcanic map of differential expression of the breast cancer brain metastatic cells 231-BR (BCBM) and its parent non-specific metastatic cells MDA-MB-231(BC) shows that 421 lncRNA were up-regulated and 318 lncRNA were down-regulated in the breast cancer brain metastatic cells, compared to the parent breast cancer cells.
Among 421 lncRNA molecules highly expressed in breast cancer brain metastatic cells, lnc-SRCIN1-1:5 expression was 22.81-fold upregulated in breast cancer brain metastatic cells compared to parental breast cancer cells (adjusted P ═ 0.001), the base sequence of which is shown in SEQ ID NO:1, and we named lncBCBMAT (lncRNAreverse cancer brain tissue associated transcript) for follow-up studies.
Example 2 use of a breast cancer brain metastasis assay kit for detecting the expression level of long non-coding RNA incbcbmat in cells, comprising the steps of:
respectively extracting total RNA in breast cancer brain transfer cells, other breast cancer cells and normal cells, carrying out real-time fluorescence quantitative PCR by a breast cancer brain transfer detection kit after reverse transcription, adopting ACTB as an internal reference gene, and carrying out statistical analysis by the relative expression quantity of a target gene. The method comprises the following specific steps:
differential expression analysis of lncBCBMAT in breast cancer brain transfer cells and other breast cancer and normal cells
And detecting the differential expression level of the long-chain non-coding RNA lncBCBMAT in the breast cancer brain transfer cells and other breast cancer and normal cells by adopting real-time fluorescent quantitative PCR. The method comprises the following specific steps:
firstly, RNA extraction: extraction of cellular total RNA using TRIzol:
the method comprises the following steps: culturing the cells until the fusion degree is 70% -80%, discarding the cell culture medium, washing with PBS 3 times, adding into TRIzol (1 ml/well) of 6-well plate, and standing at room temperature for 5 min; transferring the lysate into a 2ml centrifuge tube, adding 200 mu l of chloroform, violently shaking for 15sec, and standing at room temperature for 2-3 min; centrifuging at 12,000 Xg for 15min at 4 deg.C, and carefully transferring the upper aqueous phase containing RNA to a new centrifuge tube; adding 500 μ l isopropanol into the centrifuge tube, gently mixing, standing at room temperature for 10min, centrifuging at 4 deg.C and 12,000 Xg for 15 min; taking out the centrifuge tube, discarding the supernatant, washing the precipitate with 75% ethanol prepared from DEPC water, centrifuging at 4 deg.C and 7,500 × g for 5 min; and (4) opening the centrifugal tube, standing, naturally air-drying, and adding a proper amount of DEPC (diethylpyrocarbonate) water to dissolve the RNA precipitate.
The reverse transcription is carried out: the reaction was prepared on ice as follows:
after mixing, the reaction is carried out according to the following reaction conditions: 15min at 37 ℃; 85 ℃ for 5 sec. And (4) after the reaction is finished, storing the product at-20 ℃ until real-time fluorescent quantitative PCR detection is carried out.
The real-time fluorescent quantitative PCR:
lncbbmat reaction solution was prepared on ice as follows:
after the reaction solution is mixed uniformly, the reaction is carried out according to the following reaction conditions: 30s at 95 ℃; 95 ℃ for 5s, 60 ℃ for 34s, 45 cycles.
The ACTB reaction was prepared on ice as follows:
after the reaction solution is mixed uniformly, the reaction is carried out according to the following reaction conditions: 30s at 95 ℃; 95 ℃ for 5s, 60 ℃ for 34s, 45 cycles.
Wherein ROX Reference Dye is produced by TAKARA, PCR Forward Primer and PCREverse Primer are designed for themselves and delivered to Producer organism (Shanghai) GmbH for synthesis. Nucleotide sequences of lncbbmat specific primers (lncbbmat PCR Forward Primer and lncbbmat PCR Reverse Primer) are SEQ ID NOs: 2 and SEQ ID NO: 3; the nucleotide sequences of specific primers (ACTB PCR Forward Primer and ACTB PCR Reverse Primer) of the internal reference gene ACTB are respectively SEQ ID NO.4 and SEQ ID NO. 5.
(the prepared reaction solution can be used as an effective component of a breast cancer brain metastasis detection kit for the next detection).
Fourthly, statistical analysis: relative expression amount of target Gene 2-ΔΔCtAnd (4) showing. Statistical analysis ANOVA analysis was used.
As shown in fig. 2, lncbbmat was expressed at relatively low levels in breast cancer parental cells MDA-MB-231(P ═ 0.005) and other breast cancer cells T47D (P ═ 0.004), BT-474(P ═ 0.001), and human embryonic kidney cell 293T cell (P ═ 0.001), relative to breast cancer brain metastatic cells, with the differences statistically significant (P < 0.05). Indicating that lncBCBMAT is highly expressed in the breast cancer brain transfer cells.
Example 3 application of a kit for detecting brain metastasis of breast cancer in detecting expression level of long non-coding RNA lncBCBMAT in pathological tissue specimen of breast cancer patient
The differential expression level of the long-chain non-coding RNA lncBCBMAT in a pathological tissue specimen of a breast tumor benign and malignant patient is detected by adopting real-time fluorescent quantitative PCR, and the specific steps are as follows:
RNA extraction: half of the mung bean sized tissue was placed in a 2ml centrifuge tube, 1ml of Trizol was added, and the tissue was sufficiently crushed using a tissue homogenizer. Tissue lysates the following steps for extraction of total cellular RNA using TRIzol reference example 2.
Reverse transcription and real-time fluorescent quantitative PCR step refer to example 2. ACTB serves as an internal reference gene.
Thirdly, statistical analysis: expression level of the target gene is 2ΔCT. Δ CT ═ CT (reference gene) -CT (target gene). Statistical analysis used the t-test.
The results are shown in fig. 3, the relative expression level of lncbbmat in malignant breast cancer tissues is high relative to benign breast cancer brain tissues, and the difference is statistically significant (P ═ 0.027), which indicates that lncbbmat is highly expressed in malignant breast cancer tissues.
Example 4 use of a Breast cancer brain metastasis assay kit for detecting the expression level of Long non-coding RNA lncBCBMAT in plasma samples of patients
And detecting the differential expression level of the long-chain non-coding RNA lncBCBMAT in the plasma of a breast cancer brain metastasis patient, a breast cancer patient and a healthy control population by adopting real-time fluorescent quantitative PCR. Total RNA was extracted from plasma samples using TRIzol, RNA extraction, reverse transcription and real-time fluorescent quantitative PCR steps were referred to example 2. ACTB serves as an internal reference gene. Analysis of expression level of target Gene statistical analysis was performed by ANOVA analysis, as described in example 3.
The results are shown in fig. 4, where lncbbmat was low relative to plasma of breast cancer brain metastases patients (P0.010) and healthy control populations (P <0.001), and the difference was statistically significant (P < 0.05). The high expression of lncBCBMAT in the breast cancer brain metastasis plasma is shown, and the further suggestion is that the lncBCBMAT can be used as a molecular marker for predicting and diagnosing the breast cancer brain metastasis risk and can be used as a target point for developing novel medicaments for breast cancer brain metastasis.
Sequence Listing information
1. Base sequence of lncBCBMAT
SEQ ID NO：1
agcagatgcagctccccttccctcccattccaccatccactgtccccagcaagaacctgcgggagggtggcccaatggggagaaaactaaggattcggtgttgggaccactcctgccctgacctgccctgtgactccgtcatactctccaaaggccagaccctcctagaccagctggaaccaccatcaagatgtccccagccatgtcagactctggggccccaggcggagggcaaccagatgtcttcagctccaagtctggcctctcctcccagcaagcagccaactgcagagaccttggaaaggatcaaccatatacaatgtccatttcctgccctctaacctggcaggggagcaaggcccagccaaggagttacagaaactgaggcttggccaggcgtggtggctcacacctgcaatctcagcactgggaggccaaggtgggcagatcgcttgagcccaggagtttgagaccagcccgaggaacatggcgaaaccccatctctacaaaaaatacagaaattagccaagtgtggtggcacgtgtctgtagtttcagctactcaggaggcttaggtgggaggatcacctgagcctgggaggtagaggttgcaatgagcagagattgctctccagccagggagacagagtgagaccctgtcccaaaataaataaataaataagaataaaatttaaaaaagaaa；
IncBCBMAT specific Primer PCR Forward Primer
SEQ ID NO: 2 is tccctcccat tccaccatcc;
lncBCBMAT specific Primer PCR Reverse Primer
SEQ ID NO: 3 is gtggtcccaacaccgaatcc;
4. primer PCR Forward Primer specific to reference gene ACTB
SEQ ID NO: 4 is gcactcttcc agccttcctt;
5. primer PCR Reverse Primer specific to ACTB of internal reference gene
SEQ ID NO: and 5 is ctgtgttggc gtacaggtct.
Sequence listing
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<120> long-chain non-coding RNA lncBCBMAT and application thereof as breast cancer brain metastasis molecular marker
<141> 2020-01-17
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<212> DNA
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agcagatgca gctccccttc cctcccattc caccatccac tgtccccagc aagaacctgc 60
gggagggtgg cccaatgggg agaaaactaa ggattcggtg ttgggaccac tcctgccctg 120
acctgccctg tgactccgtc atactctcca aaggccagac cctcctagac cagctggaac 180
caccatcaag atgtccccag ccatgtcaga ctctggggcc ccaggcggag ggcaaccaga 240
tgtcttcagc tccaagtctg gcctctcctc ccagcaagca gccaactgca gagaccttgg 300
aaaggatcaa ccatatacaa tgtccatttc ctgccctcta acctggcagg ggagcaaggc 360
ccagccaagg agttacagaa actgaggctt ggccaggcgt ggtggctcac acctgcaatc 420
tcagcactgg gaggccaagg tgggcagatc gcttgagccc aggagtttga gaccagcccg 480
aggaacatgg cgaaacccca tctctacaaa aaatacagaa attagccaag tgtggtggca 540
cgtgtctgta gtttcagcta ctcaggaggc ttaggtggga ggatcacctg agcctgggag 600
gtagaggttg caatgagcag agattgctct ccagccaggg agacagagtg agaccctgtc 660
ccaaaataaa taaataaata agaataaaat ttaaaaaaga aa 702
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
<210> 3
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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<400> 5
Claims (1)
1. The application of a reagent for detecting the expression level of lncRNA molecules in preparing a breast cancer brain metastasis detection kit is characterized in that: the name of the lncRNA molecule is lncBCBMAT, and the nucleotide sequence of the lncRNA molecule is shown in SEQ ID NO:1 is shown.
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| JAK2-binding long noncoding RNA promotes breast cancer brain metastasis;Shouyu Wang;《the Journal of clinical investigation》;20171231;第127卷(第12期);第4498-4515页 * |
| Transcript:AC244153.1-201 ENST00000617990.4;Ensembl release 98;《Achive Ensembl》;20191231;第1页 * |
| 长链非编码RNA与乳腺癌侵袭转移相关性研究进展;苏薇等;《天津医药》;20151231;第43卷(第05期);第566-569页 * |
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