CN108728542A - Detect the preparation and its application process of long-chain non-coding RNA BC200 - Google Patents
Detect the preparation and its application process of long-chain non-coding RNA BC200 Download PDFInfo
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- CN108728542A CN108728542A CN201810636555.3A CN201810636555A CN108728542A CN 108728542 A CN108728542 A CN 108728542A CN 201810636555 A CN201810636555 A CN 201810636555A CN 108728542 A CN108728542 A CN 108728542A
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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- C12Q2600/00—Oligonucleotides characterized by their use
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA
Abstract
The invention discloses a kind of preparations and its application process of detection long-chain non-coding RNA BC200 (Lnc-BC200).I.e. the relevant Lnc-BC200 of Epstein-Barr virus is used to prepare the preparation of the screening of herbal medicine therapy, early diagnosis.It confirms to extract RNA in tissues of nasopharyngeal carcinoma by studying, reverse transcription simultaneously carries out real-time fluorescence quantitative PCR analysis and finds that Lnc-BC200 expressions raise.Compared with 20 normal person's pharynx nasalis tissue Lnc-BC200 expression, Lnc-BC200 expression raising, Δ CT≤7.51, P in 48 patient's tissues of nasopharyngeal carcinoma<0.0001.Therefore, Lnc-BC200 can be as the molecular marker of nasopharyngeal carcinoma diagnosis and treatment.
Description
Technical field
The invention belongs to tumor cells marker and its applied technical fields, and in particular to a kind of detection long-chain non-coding
The preparation and its application process of RNA BC200.
Background technology
Epstein-Barr virus (Eptein-Barr virus, EBV) belongs to a member in gamma herpes viruses, can infect 90% people
Class can convert bone-marrow-derived lymphocyte in vitro, closely related with the occurrence and development of nasopharyngeal carcinoma.Its different phase table in tumor development
Up to various molecules, such as EBNA1-6, three kinds of latent membrane protein 1s, 2A and 2B and miRNAs etc..Epstein-Barr virus Latent membrane protein1
(LMP1) be Epstein-Barr virus main cancer protein, many A signal pathways (e.g., NF-KB, MAPK kinase (ERK, p38 can be induced
And JNK), JAK/STAT), cause the change of epithelial cell form and phenotype, promote Epithelial and stromal conversion (EMT), lead to tumour
Cell invasion shifts.During the occurrence and development of EBV related neoplasms, there is important carcinogenesis.Do not divide in low differentiation and
Change in tissues of nasopharyngeal carcinoma, Epstein-Barr virus latent membrane protein 1 can be almost detected, and more and more results of study show EB diseases
Poison effect along with nasopharyngeal carcinoma occurrence and development.
With the continuous development of sequencing technologies, bioinformatics technique, found according in genome sequencing technical research,
The gene for only having about 20,000 coding albumen in human genome, accounts for the 2% of full gene group sequence.The transcription of gene not only limits
In protein-coding region, but it include whole gene group range.Therefore, the region of 90% or more human genome can all transcribe
At RNA, and it is largely transcribed into non-coding RNA (non-coding RNA, ncRNA), including the most long-chain of transcription
Non-coding RNA (Long noncoding RNAs, lncRNA), this is a kind of RNA for being more than 200 nucleotide, lacks specificity
Complete open reading frame, without encoding histone function, it is generally recognized that LncRNA is transcribed by rna plymerase ii, by one or more
3 ' ends of a introne montage, many lncRNA all have polyadenylic acid (i.e. 3 ' ends are mostly ploy A tails).LncRNA
It is substantially mostly similar to mRNA, however be primarily targeted in nucleus compared to mRNA, LncRNA, have tissue special in vivo
The opposite sex, conservative are poor.According to research reports, secondary structure, tertiary structure, and and albumen are formed after the completion of some LncRNA transcriptions
Matter, DNA or other RNA for playing adjusting function interact；Some LncRNA be trans-acting factor, from transcription site compared with
Far, can regulate and control to close on the expression of gene；Some LncRNA adsorb target molecule with target gene interaction such as sponge, mediate thin
Born of the same parents' signal path；Some LncRNA can be used as subnucleus structure stand, regulation protein transhipment or posttranslational modification；Have
LncRNA its important regulating and controlling in virus infects acts on, and causes antiviral response etc..Therefore, LncRNA being capable of controlling gene table
Reach, dosage compensation, gene imprinting, evolve selection, transcriptional control modification, chromatin modification, telomere biology, subcellular structure group
At transport in, core, a variety of important regulation processes such as Protooncogene Activated are adjusted, with a variety of human diseases, especially tumour
Occurrence and development have closely contact.
The non-coding region proportion highest of the mankind, however virus type biology is minimum.The infection induced cell transcription of virus
Group modification causes the expression of the LncRNA after infecting in host cell and function to change.On the one hand, viral infection, meeting
Cell is caused to express the LncRNA of some viruses, viral LncRNA controls host cell toward the direction for promoting viral gene to stablize
Transcription or translation, cause host also to express this viroid LncRNA, inhibit the antiviral inflammatory reaction of host immune system.
According to pertinent literature, Lnc-BC200 and aging and Alzheimer disease are associated with：Lnc-BC200 expression is with declining
It reduces always, but the up-regulation in Alzheimer disease (AD).In the impacted brain regions of AD, expression with disease severity and
Increase.
Lnc-BC200 there is no research in EBV correlation nasopharyngeal carcinoma.These results of study prompt, in EBV correlation nasopharyngeal carcinoma
In, Lnc-BC200 plays an important role in terms of promoting metastases, prompts Lnc-BC200 in Epstein-Barr virus correlation nasopharyngeal carcinoma
There is great potential in diagnosing and treating.
Invention content
In previous research work, by carrying out RNA-seq chip detections to EBV positive or negative cells, screening is gone on business
The LncRNA of different expression.We have found that thering is co-expression to become in positive 293 cell lines of EBV of the Lnc-BC200 in chip results
Gesture, and it is significantly raised compared with EBV negative cells, which is also confirmed in tissues of nasopharyngeal carcinoma.I.e. in Epstein-Barr virus phase
In closing property tissues of nasopharyngeal carcinoma, Lnc-BC200 overexpressions.Therefore, prompt Lnc-BC200 can be used as EBV correlation nasopharynxs
Molecular target in cancer diagnosis.
The primary purpose of the present invention is that a kind of preparation of detection long-chain non-coding RNA BC200 of offer is preparing nasopharyngeal carcinoma
Application on diagnostic preparation, the sequence of the long-chain non-coding RNA BC200 is as shown in SEQ ID NO.1.
It can be seen that Lnc-BC200 can be examined as one of the molecular marker of Epstein-Barr virus correlation nasopharyngeal carcinoma applied to nasopharyngeal carcinoma
It is disconnected.
According to the application, the preparation of the detection long-chain non-coding RNA BC200 include PCR detection reagents or
In situ hybridization detection reagent.
According to the application, the preparation of the detection long-chain non-coding RNA BC200 is long in detection nasopharyngeal tissue
The reagent of chain non-coding RNA BC200 contents.
According to the application, the non-preparation for compiling RNA BC200 of detection long-chain is RT-PCR reagents.
According to the application, the RT-PCR reagents include：
Lnc-BC200 forward primers：5'AGACCTGCCTGGGCAATATAGC3'
Lnc-BC200 reverse primers：5'GTTGTTGCTTTGAGGGAAGTTACG3'.
But the primer of the present invention that can expand long-chain non-coding RNA BC200 is not limited to the primer of above-mentioned offer.
Further, we have been preferably added to β-actin and have been used as reference, are missed to reduce system present in experimentation
Difference, while also relatively reliable experimental data is provided for research.
According to the application, the RT-PCR reagents include：
β-actin internal reference Specific PCR primers：
Forward primer：5'CTCCATCCTGGCCTCGCTGT 3'
Reverse primer：5'GCTGTCACCTTCACCGTTCC 3'.
Second object of the present invention is to provide a kind of nasopharyngeal carcinoma diagnosis kit, containing can expand long-chain non-coding RNA
The primer of BC200.A kind of new accurately and reliably detection product is provided for nasopharyngeal carcinoma auxiliary diagnosis.
The kit contains the primer that can expand long-chain non-coding RNA BC200：
Lnc-BC200 forward primers：5'AGACCTGCCTGGGCAATATAGC3'
Lnc-BC200 reverse primers：5'GTTGTTGCTTTGAGGGAAGTTACG3'.
The kit also contains primers of the β-actin as internal reference：
Forward primer：5'CTCCATCCTGGCCTCGCTGT 3'
Reverse primer：5'GCTGTCACCTTCACCGTTCC 3'.
It is fixed that the kit also contains total tissue RNA extracts reagent, RNA reverse transcription PCRs reaction reagent and real-time fluorescence
Measure the reagent needed for PCR detections.
The kit specifically includes：(1) the extracted total RNA agents useful for same from tissues of nasopharyngeal carcinoma, including RNA stablizing solutions,
Trizol reagents, chloroform, isopropanol, without enzyme water；(2) it is cDNA by Lnc-BC200 reverse transcriptions by template of total serum IgE.Institute
With reagent, including RT Buffer, triphosphoric acid base deoxynucleotide, RNase inhibitor, reverse transcriptase and Lnc-
Random primer used in BC200；(3) cDNA real-time quantitative PCR agents useful for same, including Lnc-BC200 real-time fluorescence quantitative PCRs is special
Specific primer, β-actin internal references Specific PCR primers, real time fluorescent quantitative SYBR dyestuffs, without enzyme water.
The present invention detects the method that tissues of nasopharyngeal carcinoma Lnc-BC200 is expressed：(1) 68, pharynx nasalis tissue is collected, wherein
48 nasopharyngeal carcinoma (NPC) tissues and 20 normal nasopharyngeal tissues extract total serum IgE；(2) using total serum IgE as template, reverse transcription is
cDNA；(3) with Lnc-BC200 specific primers, the expression of fluorescence quantitative PCR detection Lnc-BC200, β-actin are internal reference base
Cause.Quantitative Δ CT=testing gene Lnc-BC200 and reference gene average delta CTThe difference of value judges Δ CTWhether it is less than or waits
In 7.51, as Δ CTWhen≤7.51, prompt Lnc-BC200 expression positive.
Present invention reverse transcription after extracting RNA in nasopharyngeal carcinoma, quantitative real-time PCR have detected the table of Lnc-BC200
It reaches, as a result shows Lnc-BC200 up-regulated expressions in tissues of nasopharyngeal carcinoma.Lnc-BC200 is found for the first time, is existed just with nasopharyngeal carcinoma
To correlativity, to prompt Lnc-BC200 to can be used as the molecular marker of nasopharyngeal carcinoma auxiliary diagnosis, prediction pharynx nasalis suffers from the wind of cancer
Danger or early diagnosis, to make quick auxiliary diagnosis to Nasopharyngeal Carcinoma Patients.The present invention provides for the auxiliary diagnosis of nasopharyngeal carcinoma
Strong biology tool has far-reaching clinical meaning and important popularizing application prospect.
Description of the drawings
Fig. 1：The lncRNA analyses of significant difference expression are screened in RNA-Seq sequencing datas；
In the lncRNA of differential expression, the lncRNA occurred jointly in all cells is selected to take intersection, and filter out
Co-expression trend, difference and its significant lncRNA, fold change are all shown in positive 293 cell lines of EBV>2, P
<0.05, the lncRNA of differential expression is filtered out, the lncRNA for showing as up-regulation there are 72, and the lncRNA for showing as lowering has 75
Item.
Fig. 2：The lncRNA that EBV 293 cell lines of the positive and EBV negative cells system differential expression significantly raise,
The differential expression lncRNA quantity of EBV 293 cells of the positive and negative 293 cells of EBV：293-EBV be 2319,
293-1/NL is 2364, C22 is 2429, C2089 is 2454, wherein Lnc-BC200 expression highests, black mark
It is denoted as the lncRNA, then selects the gene as research object.
Fig. 3：Differential expressions of the Lnc-BC200 in 293 series of cell.
Compared with negative 293 cell lines of EBV, Lnc-BC200 is significantly raised EBV 293 cell lines of the positive.
Fig. 4：Differential expressions of the Lnc-BC200 in NPC cells,
Compared with EBV Nasopharyngeal Carcinoma Patients with Negative cell lines, Lnc-BC200 is significantly raised EBV positive NPCs cell；EBV feminine gender nasopharynxs
Cancerous cell line：CNE-2, CNE-1；EBV positive nasopharyngeal carcinoma's cell lines：C666-1.
Fig. 5：Differential expressions of the Lnc-BC200 in tissues of nasopharyngeal carcinoma and normal structure,
68, pharynx nasalis tissue, wherein 48 tissues of nasopharyngeal carcinoma and 20 normal nasopharyngeal tissues.Lnc-BC200 is detected at this
Expression in two kinds of tissues, the study found that expressions of the Lnc-BC200 in NPC tissues significantly raises, trend and NPC
It is consistent in cell.These nasopharyngeal carcinoma tumor tissues are from low differentiation or undifferentiated Nasopharyngeal Carcinoma Patients, and these patients pass through
It is determined as Nasopharyngeal Carcinoma Patients after pathological section detection.
Specific implementation mode
In previous research work, by carrying out RNA-seq chip detections to EBV positive or negative cells, screening is gone on business
The LncRNAs (Fig. 1) of different expression, based on the analysis of these RNA sequencing technologies and database, we have detected is built by this laboratory
Four kinds of vertical EBV stablize the differential gene situation in 293 cell lines (293-EBV, 293-1/NL, C22, C2089) of infection, and
With the databases such as DAVID, NCBI, Starbase be used for analyze RNA-seq data, analyze EBV stablize it is metainfective significantly
The gene of variation carries out the enrichment analysis of the functional clusterings such as GO, KEGG.Meanwhile 8 lncRNA of screening carry out RNA-Seq transcript profile surveys
Ordinal number is verified according to expression value.Them are further studied in epithelial tumour cell 293-HEK and nasopharyngeal carcinoma clinical sample
Expression, analysis EBV infection after caused by regulating and controlling effect.Wherein significantly high expression has Lnc-BC200 etc. (Fig. 2).
20 normal person's nasopharyngeal tissues and 48 Nasopharyngeal Carcinoma Patients tissues are collected, total serum IgE is extracted, is carried out after reverse transcription real-time
Quantitative fluorescent PCR analyzes the differential expression of Lnc-BC200, finds：There is Lnc- in the tissues of nasopharyngeal carcinoma of nearly all patient
The expression (Fig. 5) of BC200, it is as a result consistent (Fig. 4) with 293 series of cell (Fig. 3) and the result of NPC cells.As a result it is shown as
Average level and β-actin are standardized as, are examined by Mann-Whitney U and calculate p value, and significance,statistical
It is defined as * p<0.05, * * p<0.01, * * * p<0.0001.These nasopharyngeal carcinoma tumor tissues are from low differentiation or undifferentiated nasopharynx
Cancer patient, and these patients are determined as Nasopharyngeal Carcinoma Patients after pathological section detects.
These results indicate that Lnc-BC200 can be as the molecular marker in Nasopharyngeal Carcinoma Patients diagnosis.
(1) tissue samples RNA extracts (TRIZol)：
Nasopharyngeal Carcinoma Patients tissue using RAN Latter store collecteds and health adult tissue, totally 68.Weigh 5mg groups
Knit, be placed in sterile no enzyme EP pipes, be added 30ul RNA Latter, with the minor operation scissors of sterile no enzyme by tissue as far as possible
It shreds, adds 1mlRNA TRIZol；Also it can mill in liquid nitrogen and be added in 1ml RNA TRIZol after tissue.Room temperature is put
It sets 10-20 minutes, chloroform 0.2ml is added in EP pipes, place on ice 5 minutes, 4 DEG C of centrifugations, 12000rpm, 15 minutes；Carefully
Upper strata aqueous phase is distinguished in new EP pipes, repeats chloroform step；The 0.5ml isopropanols of precooling are added, is mixed evenly, puts
It sets 15 minutes on ice, 4 DEG C, 12000rpm centrifugations after ten minutes, discard supernatant liquor；75% ethyl alcohol is added, 4 DEG C centrifuge,
7600rpm, reduction of speed centrifuge 5 minutes；After drying at room temperature 5-10 minutes, 20ul-50ulDEPC water is added.In instrument
In NanoDrop2000, RNA concentration is measured, OD260/280 ratios are between 1.7-2.0 and carry out EB gel electrophoresis detection RNA matter
Amount, -80 DEG C of preservations.
(2) reverse transcription
Reverse transcription is carried out to the total serum IgE of said extracted, reverse transcription reaction system is with reference to Easy Transcript One-
Step gDNA Removal and cDNA Synthesis Super mixed (Quan Shijin, China) specification, is configured
(20ul)：
(3) Realtime PCR
After reverse transcription is turned five times of object dilution, using reverse transcription product as template, PCR reaction systems (20ul)：
Lnc-BC200 primer sequences：
It is positive：5'AGACCTGCCTGGGCAATATAGC3'
Reversely：5'GTTGTTGCTTTGAGGGAAGTTACG3'
Reference gene β-actin primer sequences used：
It is positive：5'CTCCATCCTGGCCTCGCTGT 3'
Reversely：5'GCTGTCACCTTCACCGTTCC 3'
Realtime PCR reaction systems are with reference to PCR kit for fluorescence quantitative：TransStart Tip Green qPCR
SuperMix (Quan Shijin, China)
It is configured (20ul):
PCR programs：94 DEG C, 30 seconds；1. 94 DEG C, 5 seconds；2. 58 DEG C 15 seconds；3. 72 DEG C 10 seconds；2. 1. 3. carrying out 40-45
Cycle, 4 DEG C terminate reaction.
(4) judgement Δ CT
The measurement of index：This experimental data uses the analysis method of relative quantification, β-actin to be used as reference gene, utilizes
Software GraphPad Prism5 are analyzed.Analysis is found, compared with expression of the Lnc-BC200 in normal nasopharyngeal tissue, 48
The up-regulated expression of Lnc-BC200, Δ C in example Nasopharyngeal Carcinoma PatientsTValue≤7.51, difference have conspicuousness (P<0.0001).
Method described above, (be newly admitted to hospital the doubtful Nasopharyngeal Carcinoma Patients of detection normal person 20 and 48 initial patient, operative treatment
Before) in tissue, the expression of Lnc-BC200.
Compared with 20 normal person's pharynx nasalis tissue Lnc-BC200 expression, Lnc-BC200 tables in 48 patient's nasopharyngeal tissues
Up to raising, Δ CT≤ 7.51, P<0.0001, and these patients are determined as Nasopharyngeal Carcinoma Patients after pathological section detects.
Above studies have shown that Lnc-BC200 can be as the molecular marker of nasopharyngeal carcinoma diagnosis and treatment, as Δ CT≤
When 7.51, the generation of nasopharyngeal carcinoma is indicated.The expression of Lnc-BC200 in nasopharyngeal tissue is detected, there is good stability, operation
Simplicity can be applied to EBV correlation Nasopharyngeal Carcinoma Patients diagnosing and treatings.
Sequence table
<110>Central South University
<120>Detect the preparation and its application process of long-chain non-coding RNA BC200
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 200
<212> DNA
<213>Homo sapiens (Homo sapiens)
<400> 1
ggccgggcgc ggtggctcac gcctgtaatc ccagctctca gggaggctaa gaggcgggag 60
gatagcttga gcccaggagt tcgagacctg cctgggcaat atagcgagac cccgttctcc 120
agaaaaagga aaaaaaaaaa caaaagacaa aaaaaaaata agcgtaactt ccctcaaagc 180
aacaaccccc cccccccttt 200
<210> 2
<211> 22
<212> DNA
<213>Unknown (Unknown)
<400> 2
agacctgcct gggcaatata gc 22
<210> 3
<211> 24
<212> DNA
<213>Unknown (Unknown)
<400> 3
gttgttgctt tgagggaagt tacg 24
<210> 4
<211> 20
<212> DNA
<213>Unknown (Unknown)
<400> 4
ctccatcctg gcctcgctgt 20
<210> 5
<211> 20
<212> DNA
<213>Unknown (Unknown)
<400> 5
gctgtcacct tcaccgttcc 20
Claims (10)
1. detecting application of the preparation of long-chain non-coding RNA BC200 on preparing nasopharyngeal carcinoma diagnosis preparation, the long-chain is non-
The sequence of coding RNA BC200 is as shown in SEQ ID NO.1.
2. application according to claim 1, which is characterized in that the preparation of the detection long-chain non-coding RNA BC200
Including PCR detection reagents or in situ hybridization detection reagent.
3. application according to claim 1 or 2, which is characterized in that the system of the detection long-chain non-coding RNA BC200
Agent is to detect the reagent of long-chain non-coding RNA BC200 contents in nasopharyngeal tissue.
4. application according to claim 1 or 2, which is characterized in that the system of the detection long-chain non-coding RNA BC200
Agent is RT-PCR reagents.
5. application according to claim 4, which is characterized in that the RT-PCR reagents include：
Lnc-BC200 forward primers：5'AGACCTGCCTGGGCAATATAGC3'
Lnc-BC200 reverse primers：5'GTTGTTGCTTTGAGGGAAGTTACG3'.
6. application according to claim 4, which is characterized in that the RT-PCR reagents include：
β-actin internal reference Specific PCR primers：
Forward primer：5'CTCCATCCTGGCCTCGCTGT 3'
Reverse primer：5'GCTGTCACCTTCACCGTTCC 3'.
7. a kind of nasopharyngeal carcinoma diagnosis kit, which is characterized in that contain the primer that can expand long-chain non-coding RNA BC200.
8. kit according to claim 7, which is characterized in that containing drawing for long-chain non-coding RNA BC200 can be expanded
Object：
Lnc-BC200 forward primers：5'AGACCTGCCTGGGCAATATAGC3'
Lnc-BC200 reverse primers：5'GTTGTTGCTTTGAGGGAAGTTACG3'.
9. kit according to claim 7, which is characterized in that kit also contains primers of the β-actin as internal reference：
Forward primer：5'CTCCATCCTGGCCTCGCTGT 3'
Reverse primer：5'GCTGTCACCTTCACCGTTCC 3'.
10. kit according to claim 7, which is characterized in that kit also contains total tissue RNA extracts reagent, RNA
Reagent needed for reverse transcription PCR reaction reagent and real-time fluorescence quantitative PCR detection.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113604566A (en) * | 2021-07-13 | 2021-11-05 | 中山大学孙逸仙纪念医院 | Application of lncRNA BCYRN1 in prognosis and treatment of bladder cancer |
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2018
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113604566A (en) * | 2021-07-13 | 2021-11-05 | 中山大学孙逸仙纪念医院 | Application of lncRNA BCYRN1 in prognosis and treatment of bladder cancer |
| CN113604566B (en) * | 2021-07-13 | 2023-05-16 | 中山大学孙逸仙纪念医院 | Application of lncRNA BCYRN1 in prognosis and treatment of bladder cancer |
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Application publication date: 20181102 |