CN107674917A - A kind of UCR sequences, kit and detection method for detecting the high expression in B cell lymphoma tissue - Google Patents
A kind of UCR sequences, kit and detection method for detecting the high expression in B cell lymphoma tissue Download PDFInfo
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Abstract
The invention discloses a kind of UCR sequences, kit and detection method for detecting the high expression in B cell lymphoma tissue.The present invention has found to attack the specific one group of related UCRs of transfer to B cell lymphoma, as a result screens the UCR, i.e. uc.189 of a significantly high expression in B cell lymphoma tissue after the UCR express spectras of system exploration B cell lymphoma.The present invention explores the super conserved genetic sequences 189 related to B cell lymphoma occurrence and development specificity, the application as B cell lymphoma medicine.
Description
Technical field
The invention belongs to oncomolecularbiology field, and in particular to one kind detection high table in B cell lymphoma tissue
UCR sequences, kit and the detection method reached.
Background technology
Malignant lymphoma is one of ten big malignant tumour of China, and incidence and mortality is located at tumour forefront.According to clinic
Pathological characteristic is divided into Hodgkin lymphoma and NHL, and Hodgkin lymphoma is single tumour cell disease, ECDC
Reason, which effectively treats most cases, to cure, and NHL has height heterogeneity, is made up of many hypotypes, its
Middle B cell lymphoma is a kind of lymthoma with Highly invasive and recurrence, is that B cell lymphoma Endodontic failure and patient are dead
The main reason for dying.Research for lymthoma molecular mechanism, scientific research person is always in ceaselessly exploring, such as in hair tonic
There is transposition in the bcl-6 gene 3q27 chromosomes of heart B cell, cause cell differentiation to suppress, multiplication capacity is most strong；50% it is non-
There occurs high frequency mutation, this genoid control for proto-oncogene (pim-1, myc, RhoH/TTF and PAX5) in Hodgkin lymphoma cell
Growth of tumour cell, propagation, apoptosis and transfer processed.The mutation of other p53 genes, the missing of p16 genes, rel, myc gene
Amplification have impact on the occurrence and development of B lymphoma cells.
Uc.189 is super conserved genetic sequences (UCRs) a member, is a kind of conservative length of absolute altitude in biological evolution
Chain non-coding RNA (LncRNA), its gene order keep high homology in the higher organism such as, mouse and rat.Study table
The bright UCRs for being transcribed into RNA plays its distinctive biological function, i.e., by adjusting tables of other RNA so as to adjusting function gene
Reach, participate in the biological processes such as the growing of tumour cell, Apoptosis, cell cycle and invasion and attack transfer, currently into
For tumor diagnosis and treatment and the new focus of research.By today, oneself has more UCRs to be proved straight including carcinoma of urinary bladder, breast cancer, knot
Having differences property is expressed and performs important adjusting function in class disease including intestinal cancer and IBD etc..It is but thin on B
UCR expression and its functional research of index of correlation in born of the same parents' lymthoma yet there are no document report.
The content of the invention
In view of above-mentioned technical problem, the present invention provides a kind of UCR sequences for detecting the high expression in B cell lymphoma tissue
Row, kit and detection method.The present invention has found and B cell lymph after the UCR express spectras of system exploration B cell lymphoma
One group of related UCRs of knurl invasion and attack transfer specificity, as a result screens a significantly high expression in B cell lymphoma tissue
UCR, i.e. uc.189.The present invention explores the super conserved genetic sequences 189 related to B cell lymphoma transfer specificity, makees
For the application of B cell lymphoma medicine.
Solve above-mentioned technical problem technical scheme be：
In order to which system research and B cell lymphoma occur with developing closely related new UCRs, 20 provided by invention
Individual B cell lymphoma and 15 normal structure samples of pairing, frozen after obtaining fresh specimens with being used after in liquid nitrogen container, collecting neat
The detection of Arraystar Human T-UCR chips 2.0 of upper Haikang into biological Co., Ltd filters out two in B cell lymphoma
The UCR of high expression, the significantly high expression of wherein uc.189 in tissue.Its gene order is as shown in sequence table SEQ ID NO.1.Later stage
By totally 132 pairs of B cell lymphomas with finding that UCR expression is aobvious in 106 pairs of samples with normal tissue sample qRT-PCR checkings
Write up-regulation.The UCR is expected to turn into the mark of Lymphoma Diagnosis and Index for diagnosis, while is also provided newly for the treatment of lymthoma
Target spot.
It is an object of the invention to provide a kind of uc.189 sequences for detecting the high expression in B cell lymphoma tissue, have
Nucleotide sequence as shown in SEQ ID NO.1 in sequence table.
Present invention also offers the detection method of the uc.189 sequences, prepared according to the sequence for B cell lymphoma
The preparation of auxiliary diagnosis or outcome prediction.Explore its application in the medicine of B cell lymphoma is prepared.
3 pairs of primers are used to detect uc.189 sequences the present invention according to the uc.189 sequences Designs.
The sense primers of Pair 1：SEQ ID NO.2
The anti-sense primers of Pair 1：SEQ ID NO.3
The sense primers of Pair 2：SEQ ID NO.4
The anti-sense primers of Pair 2：SEQ ID NO.5
The sense primers of Pair 3：SEQ ID NO.6
The anti-sense primers of Pair 3：SEQ ID NO.7
The present invention is according to the uc.189 sequences Designs and synthesizes the detection primer group for qRT-PCR.The primer
Group is applied to SYBR Green, TaqMan probe, molecular beacon, double cross probe, combined probe etc. and detected.
Being preferably used in the primer sets that dye class qRT-PCR is detected is respectively：
The sense primers of Pair 2：SEQ ID NO.4
The anti-sense primers of Pair 2：SEQ ID NO.5
The invention provides a kind of dye class qRT-PCR kits of detection UCR expressions, component are as follows：Specificity
Sense primer primer sequence, specific Down Stream primer sequence, DNA profiling, fluorescent dye, wherein qRT-PCR reaction solutions, institute
The specific forward primer and anti-sense primer stated include SEQ ID NO.4 and SEQ ID NO.5.
The qRT-PCR reaction solutions include buffer buffer solutions, Taq enzyme, Mg2+And dNTP.
For the preferred SYBR Green II of fluorescent dye, the preferred thermal starting enzyme of Taq enzyme.
The invention also discloses extraction, the sample of the detection method of UCR in B cell lymphoma a kind of, including sample total serum IgE
CDNA preparation, uc.189 amplification.Particular content is as follows：
1) extraction of sample total serum IgE：According to the TRIZOL 8. reagent (article No.s of ThermoFisher Scientific companies
15596018) reagent needed for and step extraction B cell lymphoma tissue or the total serum IgE of tumour；NanoDrop ND 1000 are used again
Micro-ultraviolet-visible spectrophotometer it is quantitative (NanoDrop Technologies, Wilmington,
Delaware) quantitative extracted RNA purity and concentration.
2) sample cDNA preparation：Using TaKaRa kit PrimeScript First Strand cDNA
Total serum IgE reverse transcriptions of the synthesis (article No. 6110A) to extraction synthesizes cDNA.
Reaction system and condition are as follows：
| Reagent | Usage amount |
| Template RNA/Primer Mixture | 2.0μl |
| Total RNA | 0.5μg |
| RNase Free dH20 | Upto10μl |
| Cumulative volume | 10μl |
By above-mentioned component it is well mixed after 37 DEG C of 85 DEG C 5 seconds after 15 minutes, that is, obtain cDNA.
3) uc.189 amplification：Carried out using the PrimeScriptTM RT Master Mix kits of Takara companies
Real-time fluorescence quantitative PCR.QRT-PCR amplifications are carried out by template of the cDNA of reverse transcription.
Reaction system and condition are as follows：
QRT-PCR programs：95 DEG C of 30s pre-degenerations, connect 40 circulations：95℃5s,60℃30-60s.
By the detection to positive, it is found that dye class fluorescence quantitative kit Detection accuracy of the present invention exists
More than 82%-87%, continuous 10 repetitions are tested, and experimental result is stable.
Present invention is alternatively directed to uc.189 sequences Designs and synthesize its specific RNA interference sequence：siRNA1(SEQ ID
NO.8), siRNA2 (SEQ ID NO.9), the interference sequence can significantly strike the expression (figure of uc.189 in low tumour cell
8), and the invasive ability of tumour cell can be caused substantially to weaken (Fig. 9) than before.
Beneficial effects of the present invention：
1st, the UCR express spectras for system exploration B cell lymphoma and discovery and B cell lymphoma occurrence and development specificity phase
The one group of UCRs closed, the present invention screen one in B cell lymphoma tissue using long-chain non-coding RNA-UCR chip technologies
The UCRs of significantly high expression, compare UCR genome databases and find that uc.189 is located in No. 6 chromosome positive-sense strands of chromosomoid
Between 23027-23599 bases, full length gene is about 573.
2nd, compared with normal tissue, the UCRs significantly high expression in B cell lymphoma tissue, and it is big in the later stage
Expression of the uc.189 in B cell lymphoma tissue is further confirmed in the real-time qRT-PCR experiments of sample tissue, is significantly higher than and matches somebody with somebody
Normal tissue.
3rd, the qRT-PCR kits of detection uc.189 expressions of the present invention.The qRT-PCR kits are suitable for
Presently, there are all types fluorescence quantitative gene extender of in the market, high sensitivity, it is quantitative quick and precisely, stability it is good, tool
There is good application prospect.
4th, the present invention has paid close attention to the UCR express spectras of B cell lymphoma tissue, this kind of new gene regulation factor
By the research for the Tumorigenesis for being expected to further enrich and improving including B cell lymphoma, also examined for discovery tumour
Disconnected and Index for diagnosis mark, new cancer target bring hope.
Brief description of the drawings
Fig. 1 .UCR chips illustrate
Display uc.189 expresses rise (control group is 15), differential expression multiple in 20 B cell lymphoma tissues
Up to 2.741 times, light color represents expression and declined, and dark color represents expression rise.
Fig. 2 specific primer the selection result figures
For row agarose gel electrophoresis test primer after 3 couples of specific primer PCR amplifications of uc.189 sequences Design
Effect.
The preliminary qRT-PCR testing results (2 of uc.189 of first 40 B cell lymphoma clinical samples of Fig. 3-△CtMake
Figure).
The uc.189 of Fig. 4 90 B cell lymphoma clinical samples of second batch verifies qRT-PCR testing results (2 again-△CtMake
Figure).
Totally 130 sample uc.189 analyze (2 to Fig. 5 two batches in cancer and cancer beside organism differential expression qRT-PCR detection systems-△△CtValue compares, * p ＜ 0.05, * * p ＜ 0.01).
Fig. 6 .qRT-PCR detections uc.189 expression and the correlation analysis (2 of patient clinical by stages-△△CtValue compares, * p
＜ 0.05, * * p ＜ 0.01).
Fig. 7 .uc.189 express the correlation analysis (2 with B cell lymphoma lymphatic metastasis-△△CtValue compares, * p ＜
0.05, * * p ＜ 0.01).
Fig. 8 .qRT-PCR detections siRNA strikes drop uc.189 efficiency.
Fig. 9 .Transwell experiment detections siRNA strikes the invasive ability of tumour cell after drop uc.189.
(uc.189 strikes low group for the growth ability of tumour cell after Figure 10 nude mices lotus knurl experiment detection siRNA strikes drop uc.189
With nude mice of control group growth of transplanted human curve map and nude mice model tumor tissue).
Embodiment
With reference to specific embodiment, the present invention is expanded on further, is only used for explaining the present invention, and it is not intended that to this
The limitation of invention.Member of ordinary skill in the art is appreciated that:Can be with the case where not departing from the principle and objective of the present invention
These embodiments are carried out with a variety of change, modification, replacement and modification, the scope of the present invention is limited by claim and its equivalent
It is fixed.The experimental method of unreceipted actual conditions in the following example, generally according to normal condition or according to the bar proposed by manufacturer
Part examinations.
Embodiment 1B Lymphocytes tumor tissue and the UCR chip differences expression analysis with normal tissue
First, material and method
1st, material
Tissue samples come from inpatient's surgery excision sample of 20 B cell lymphoma patients (numbering 1-20),
Wherein 15 pairs are the B cell lymphoma tissue of pairing and swollen peri- tumorous normal tissues.
2nd, method
2.1 tumor tissues and the extraction with normal tissue total serum IgE
Extract B cell lymphoma disease tumor tissues and the total serum IgE with normal tissue is tried by the RNA extractions of Qiagen companies
Agent box (RNeasyMicroKit, #74004) specification step operation, the kit include RNase-FreeDNase I
(lyophilized) DNA in genome, can effectively be removed.Quantified again with the nucleic acid quantification instrument of NanoDrop ND 1000
(NanoDrop Technologies, Wilmington, Delaware) quantitative extracted RNA purity and concentration.
2.2 couples of sample RNA carry out fluorescence labeling (CRNA expands labelling kit, catalog number：360060-
10)。
2.2.1 reverse transcription synthesizes the first chain cDNA
Using Total RNA as starting, T70ligo (dT) Primer containing T7 promoter sequences is primer, is used
CbcScr ipt the first chains of enzymatic synthesis cDNA.
2.2.2 2nd strand cDNA are synthesized
The RNA in heterozygosis chain is cut into short-movie section with RNase H, DNA Polymerase prolong using RNA short-movie sections as primer
Stretch, synthesize 2nd strand cDNA, and purify double-strand cDNA.
2.2.3 in-vitro transcription synthesizes cRNA
Using cDNA as template, cRNA is synthesized using T7Enzyme Mix；Then it is pure with RNA Clean-up K it (MN)
Change.
2.2.4 random primed reverse transcription
5ug cRNA are taken, with CbcScr ipt II enzymes, Random Prime carry out reverse transcription, reverse transcription product PCR
NucleoSpinExtract II Kit (MN) are purified.
2.2.5cDNA marked with KLENOW enzymes
Above-mentioned reverse transcription product is taken, KLENOW enzyme marks, marked product PCR are carried out by primer of Random Primer
NucleoSp inExtract II Kit (MN) are purified, and are drained after purification.(Cy5-dCTP or Cy 3-dCTP(GE Heal
thcare)。
2.3 hybridization and cleaning
The DNA of mark is dissolved in hybridization solution (2 × GEx Hyb Buf f er (HI-RPM), 25% formamide), in 45 DEG C
Hybridized overnight.After hybridization terminates, first contain 0.2%SDS at 42 DEG C or so, 5min washed in 2 × SSC liquid, then 0.2 ×
Room temperature washes 5min in SSC.Slide can be used to scan after drying.
2.4 chip scanning
Chip sweep taking out with lncRNA-TUCR Scanner, obtains hybridizing picture.
The collection and data analysis of 2.5 chip images
Chip image is carried out using Feature Extraction image analysis softwares and GeneSpr ing GX softwares
Analysis, picture signal is converted into data signal, carries out differential gene screening.
2nd, result
TUCR chip differential genes screening diagram on B cell lymphoma is shown in Fig. 1.Chip examination is found in a plurality of expression
The TUCR that mediation expression is lowered, wherein uc.189 show to express in tumor tissues and significantly raised, in view of it may be in B cell
Exist specific expressed in lymphoma tissue, the present invention by following examples enters row index in batches using extensive sample
Repeated authentication.
The real time fluorescent quantitative qRT-PCR preliminary identifications uc.189 of embodiment 2 is in B cell lymphoma tissue and matches by cancer just
The often differential expression in tissue
First, experiment material
Other 40 couple (numbering 21-60) B cell lymphoma tissues and pairing Carcinoma side normal tissue are chosen again, to uc.189
Differential expression carry out qRT-PCR first time case verifications.
2nd, experimental method and result
1st, primer specificity screening and identification
Transcript base according to uc.189 gene locis from UCSC Genome Brower databases extraction uc.189 correlations
Primer is designed to uc.189 because of sequence, and with according to the primer-design softwares of Primer 5；
(1) primer after designing is evaluated with Oligo7, obtains the primer sequence of 3 pairs of designs；
First pair：Sense primer SEQ ID NO.2
Anti-sense primer SEQ ID NO.3
Second pair：Sense primer SEQ ID NO.4
Anti-sense primer SEQ ID NO.5
3rd pair：Sense primer SEQ ID NO.6
Anti-sense primer SEQ ID NO.7
Through regular-PCR and agarose gel electrophoresis experimental verification, screen for dye class qRT-PCR detections primer sets such as
Under (Fig. 2):
Second pair of sense primer：SEQ ID NO.4
Anti-sense primer：SEQ ID NO.5
(2) by B cell lymphoma tissue with matching Carcinoma side normal tissue according to ThermoFisher Scientific companies
TRIZOL 8. reagent needed for reagent (article No. 15596018) and step extraction B cell lymphoma tissue or tumour total serum IgE；Again
With the micro-ultraviolet-visible spectrophotometers of NanoDrop ND 1000 (NanoDrop Technologies, Wilmington,
Delaware) quantitative extracted RNA purity and concentration.
(3) sample cDNA preparation：Using TaKaRa kit PrimeScript First Strand cDNA
Total serum IgE reverse transcriptions of the synthesis (article No. 6110A) to extraction synthesizes cDNA.
Reaction system and condition are as follows：
| Reagent | Usage amount |
| Template RNA/Primer Mixture | 2.0μl |
| Total RNA | 0.5μg |
| RNase Free dH20 | Upto10μl |
| Cumulative volume | 10μl |
By above-mentioned component it is well mixed after 37 DEG C of 85 DEG C 5 seconds after 15 minutes, that is, obtain cDNA.
Using the cDNA of synthesis as template, set reaction groups for 3 pairs of primers respectively and be not added with the negative right of cDNA templates
According to group, primer dimer is possible except, then the primer designed by step (2) enters performing PCR reaction；
(4) electrophoresis detection, from Marker DL1000 (TaKaRa).Specific qRT-PCR is chosen according to electrophoresis detection result
Primer, its selection standard is as follows：A. amplified fragments size is identical with expection；B. amplified production only has one, clearly to expand
The specificity of product.As a result show that in three pairs of primers optimal primer pair is second pair of primer (Fig. 2), upstream and downstream it is special
Property primer sequence, its sequence table SEQ ID NO.4 and SEQ ID NO.5.
2nd, the extraction of 40 sample total serum IgEs：
Using liquid nitrogen grinding method, according to ThermoFisher Scientific companiesReagent (article No.
15596018) reagent needed for and step extraction B cell lymphoma tissue or the total serum IgE of tumour.Main operational steps are as follows：
(1) sample quick freeze after in vitro is put into tissue in the mortar of own precooling in liquid nitrogen, during extracting RNA is carried out
Grinding, the liquid feeding nitrogen in grinding, whole process all not make liquid nitrogen volatilization dry.
(2) after tissue sample abrasive flour shape, 1.5ml TRIZOL examinations are added when liquefied ammonia volatilizees substantially, in each mortar
Agent, being transferred to containing TRIZOL mixed liquors in 2ml centrifuge tube after mixing, room temperature 8min.
(3) add 200ul chloroform, be aggressively shaken 30 seconds with hand, room temperature 8 minutes, 4 DEG C of centrifugations, 12000g × 5min.
(4) supernatant 600ul being gone into new centrifuge tube, adds 0.5ul isopropanols, precipitation at room temperature 8 minutes, 4 DEG C centrifuge,
12000g×5min。
(5) supernatant is abandoned, unnecessary supernatant is drawn with small pipette tips, adds 1ml75% ethanol to clean RNA, is vibrated in a moment,
12000g × 5 minute, carefully abandon supernatant.
(6) 5-15min) is stored at room temperature, dries RNA precipitate, adds the dissolving of 20ul DEPC water.
(7) after using spectrophotometer RNA concentration.Part uses or is placed in liquid nitrogen to be preserved for a long time.
3rd, take above-mentioned 40 pairs of B cell lymphoma tissues and match Carcinoma side normal tissue total serum IgE, using according to
ThermoFisher Scientific companiesReagent needed for reagent (article No. 15596018) and step extraction B cell
The total serum IgE of lymphoma tissue or tumour；Quantified again with the micro-ultraviolet-visible spectrophotometers of NanoDrop ND 1000
(NanoDrop Technologies, Wilmington, Delaware) quantitative extracted RNA purity and concentration.
(1) sample cDNA preparation：Using TaKaRa kit PrimeScript First Strand cDNA
Total serum IgE reverse transcriptions of the synthesis (article No. 6110A) to extraction synthesizes cDNA.
Reaction system and condition are as follows：
| Reagent | Usage amount |
| Template RNA/Primer Mixture | 2.0μl |
| Total RNA | 0.5μg |
| RNase Free dH20 | Upto10μl |
| Cumulative volume | 10μl |
By above-mentioned component it is well mixed after 37 DEG C of 85 DEG C 5 seconds after 15 minutes, that is, obtain cDNA.
(2) uc.189 amplification：Carried out using the PrimeScriptTM RT Master Mix kits of Takara companies
Real-time fluorescence quantitative PCR., qRT-PCR amplifications are carried out by template of the cDNA of reverse transcription.
Reaction system and condition are as follows：
QRT-PCR programs：95 DEG C of 30s pre-degenerations, connect 40 circulations：95℃5s,60℃30-60s.
According to qRT-PCR relative freight volume formula：2-△Ct, uc.189 is calculated respectively in B cell lymphoma patient tumors
Tissue is (T) and as shown in Figure 3 with the expression in normal tissue (N), comparative result：QRT-PCR stable amplification results, its
Middle uc.189 is between the expression in normal tissue is concentrated mainly on 0.000-0.001, and in tumor tissues
Uc.189 expression quantity is concentrated mainly between 0.002-0.150, hence it is evident that higher than with normal tissue, these results suggest that this refers to
Universal high expression is marked in tumor tissues.According still further to relative expression quantity T-N>0 is defined as the index up-regulated expression；T N<0 definition
Express and lower for the index.Then this experimental result is shown：Uc.189 is in 40 B cell lymphomas with matching somebody with somebody in normal tissue
Mileometer adjustment is up to 33, according still further to formula：Up-regulated expression number of cases/total detection number of cases x100% defines the positive rate of the index, then
The positive rate of the index is 82.5%.
Embodiment 3qRT-PCR further verifies uc.189 in the tumor tissues of B cell lymphoma and with normal tissue
Differential expression
1st, qRT-PCR kit forms
1.1 dye class uc.189PCR kit forms：
(1) sense primer：SEQ ID NO.4
(2) anti-sense primer：SEQ ID NO.5；
Other reagents are with reference to SYBR Premix Ex TaqTMII (Tli RNaseH Plus) fluorescence quantitative kit
(Code No.RR820A)。
2.uc.189 detection
The preparation of 2.1 total serum IgEs
Choose the tumor tissues of other 90 pairs of B cell lymphoma diseases (numbering 61-130) and with normal tissue, according to
ThermoFisher Scientific companiesReagent needed for reagent (article No. 15596018) and step extraction are total
RNA, referring specifically to specification.The micro-ultraviolet-visible spectrophotometers of NanoDrop ND 1000 (NanoDrop Techno are used again
Logies, Wilmington, Delaware) quantitative extracted RNA purity and concentration.
2.2cDNA synthesis
It is right using TaKaRa kit PrimeScript First Strand cDNA synthesis (article No. 6110A)
The total serum IgE of above-mentioned extraction carries out reverse transcription reaction.
2.3qRT-PCR detection
QRT-PCR instrument uses Applied Biosystems 7500Real-Time PCR system (Applied
Biosystems；Thermo Fisher Scientific,Inc.)；Fluorescent dye in qRT-PCR response procedures such as embodiment two
Class qRT-PCR detects uc.189 expression quantity.
3 testing results
As a result show：Choose the tumor tissues of 90 pairs of B cell lymphoma diseases (numbering 61-130) and match somebody with somebody normal tissue
Enlarged sample amount verifies that qRT-PCR detects index up-regulated expression in lymphoma tissue, Positive rate in 76 pairs of samples
Reach 84.4% (Fig. 4).Result above demonstrates again that the index universal high expression in tumor tissues.We enter to above-mentioned sample
Row is repeated 3 times qRT-PCR inspections, and as a result repeatability shows that the repeatability of kit of the present invention and stability are preferable up to 100%.
Example IV uc.189 analyzes in the potential value of B cell lymphoma diagnosis and Index for diagnosis
On the basis of high expression, with reference to the clinical and pathological data of patient, enter have detected uc.189 in B cell lymphoma
One step analyzes correlation between uc.189 expression and different pathological and clinical stages (referring in particular to American
Joint Committee on Cancer criteria standards), inquire into uc.189 and examined in B cell lymphoma with disease
It is disconnected, including possessed by the selection etc. of the relation of its expression and clinic/pathological staging, Index for diagnosis, therapeutic scheme it is potential
Value.
Use SPSS 16.0software package (SPSS Inc., Chicago, IL, USA) statistical software pair
Uc.189 rna expression and the correlation of B cell lymphoma clinicopathologic features carry out statistical procedures, and related data passes through T-
Test or chi-square criterion and data analysis, P<0.05 thinks that the differential expression of the index is statistically significant.Statistical result shows
Uc.189 rna expression level is significantly higher than with normal tissue (Fig. 5, P in 130 pairs of B cell lymphoma samples<0.01)；
Uc.189 expression and lymphatic metastasis are closely related (Fig. 7)：Lymphatic metastasis group is significantly higher than non-lymphatic metastasis group patient
(P<0.05)；Uc.189 expression and clinical stages height correlation (Fig. 6)：Uc.189 in III- IV phase B cell lymphoma patients
Expression, be significantly higher than I-II phase patients (P<0.05).
Uc.189 have detected in B cell lymphoma tissue on the basis of high expression, design RNA interfering (siRNA) sequence
After row, the RNA interfering (siRNA) that is synthesized by LifeTechnology companies:siRNA1(SEQ ID NO.8)、siRNA2(SEQ
ID NO.9), the expression (Fig. 9) for dropping the index, tumour can be significantly struck in B cell lymphoma cell line A20 and DOHH2
The invasive ability of cell substantially weakens (Fig. 9) than before.In nude mouse in transplantable tumor experiment, uc.189-siRNA1 will be contained
B cell lymphoma cell line DOHH2 and control group be expelled to the growth of nude mice dorsal sc lotus knurl, results expression strikes low uc.189
The growth (Figure 10) of transplantable tumor can be suppressed after expression.
In a word, it is significantly high in B cell lymphoma tissue to express and with modulate tumor cell growth and transfer ability
Index, it is expected to turn into the biomarker for participating in Help B Cells Lymphoma Diagnosis, treatment and prognosis evaluation correlation, has very
Important clinical value.Given birth to as the later stage further progressively illustrates uc.189 modulate tumor cells in B cell lymphoma
Long and forwarding function mechanism of action, uc.189 can not only turn into the biomarker of diagnosis and Index for diagnosis correlation, more be expected to
As new B cell lymphoma therapy target to improve, improve clinical B cell lymphoma therapeutic effect.
Sequence table
<110>Yangzhou University
<120>A kind of UCR sequences, kit and detection method for detecting the high expression in B cell lymphoma tissue
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 573
<212> DNA
<213>Artificial sequence ()
<400> 1
agatggttgt actgatggct tgtttttcat tttttttgtg ctttttggtc catctattaa 60
taaaaatgaa ccccgttaca gagtcaccat catgtctctt ctcaccaccc tctgaatctg 120
cattagccag tcaactagcc ctttcagcgt catgtgacca gcgcgcccca ttcagcttgg 180
ctggtgtcgt ttcacatgac ccaggctggc cagtcgtcag gttgcaccgc cctttggttc 240
ccgagcatgc tgttttctct cagccttctc tccaacctta accaaatcgg cagcagccac 300
ctcgaccgcc cacacattcc tggccaatca gctcagctgt ttatttacca aatgtcttca 360
caacaactac agcagcagcc ttcggctaac aaaaaagcag gaaaaatcca caacaccccc 420
ttcgccaacc aactaaatcc aacgcaacat ctggcaaaac cttttcagca aattcttcct 480
ggccgtcagt ccggcagcct cacctcacca tttctagctt gttgaaaccc aaaactagta 540
agtttttcct gcttatacag tttactgctg gtt 573
<210> 2
<211> 18
<212> DNA
<213>Artificial sequence ()
<400> 2
gatggttgta ctgatggc 18
<210> 3
<211> 18
<212> DNA
<213>Artificial sequence ()
<400> 3
tggtcacatg acgctgaa 18
<210> 4
<211> 18
<212> DNA
<213>Artificial sequence ()
<400> 4
cacacattcc tggccaat 18
<210> 5
<211> 18
<212> DNA
<213>Artificial sequence ()
<400> 5
ccagatgttg cgttggat 18
<210> 6
<211> 18
<212> DNA
<213>Artificial sequence ()
<400> 6
ggttgtactg atggcttg 18
<210> 7
<211> 18
<212> DNA
<213>Artificial sequence ()
<400> 7
aggttacccc tgtttcag 18
<210> 8
<211> 22
<212> DNA
<213>Artificial sequence ()
<400> 8
aaccttaacc aaatcggcag ca 22
<210> 9
<211> 22
<212> DNA
<213>Artificial sequence ()
<400> 9
aactagccct ttcagcgtca tg 22
Claims (8)
1. a kind of UCR sequences for detecting the high expression in B cell lymphoma tissue, it is characterised in that the UCR sequences have such as
Nucleotide sequence in sequence table shown in SEQ ID NO.1.
2. a kind of dye class qRT- PCR detection kits of detection UCR expressions, it is characterised in that including according to nucleosides
Acid sequence is that SEQ ID NO.1 long-chain non-coding RNA designs and synthesizes out the specificity for being specifically used for qRT- PCR detections
Sense primer and anti-sense primer.
3. PCR detection kit according to claim 2, it is characterised in that the specificity for qRT- PCR detections is drawn
Thing includes 3 pairs, is respectively：
The sense primers of Pair 1：SEQ ID NO. 2
The anti-sense primers of Pair 1：SEQ ID NO. 3
The sense primers of Pair 2：SEQ ID NO. 4
The anti-sense primers of Pair 2：SEQ ID NO. 5
The sense primers of Pair 3：SEQ ID NO. 6
The anti-sense primers of Pair 3：SEQ ID NO. 7.
4. PCR detection kit according to claim 3, it is characterised in that the specificity for qRT- PCR detections is drawn
Thing is as shown in sequence table SEQ ID NO.4 and SEQ ID NO.5.
5. PCR detection kit according to claim 2, it is characterised in that the kit also includes DNA templates, glimmering
Photoinitiator dye, qRT-PCR reaction solutions, the qRT-PCR reaction solutions include buffer buffer solutions, Taq enzymes, Mg2+And dNTP.
6. PCR detection kit according to claim 5, it is characterised in that the fluorescent dye is SYBR Green II,
Taq enzymes are thermal starting enzyme.
7. UCR detection method in a kind of B cell lymphoma, it is characterised in that the extraction including sample total serum IgE, sample cDNA
Preparation, uc.189 amplification；Particular content is as follows：
1）The extraction of sample total serum IgE：Extract B cell lymphoma tissue or the total serum IgE of tumour；
2）Sample cDNA preparation：CDNA is synthesized to the total serum IgE reverse transcription of extraction using TaKaRa kits；
Reaction system and condition are as follows：The μ of 2. 0 μ l, Total RNA of Template RNA/Primer Mixture 0.5
G, RNase Free dH2The μ l of 0 Up to 10, by above-mentioned component it is well mixed after 37 DEG C of 85 DEG C 5 seconds after 15 minutes, produce
To cDNA；
3) uc.189 amplification：QRT-PCR amplifications are carried out by template of the cDNA of reverse transcription；
Reaction system and condition are as follows：SYBR® Premix Ex Taq II(Tli RNaseH Plus)（2×）Mixture
10 μ l, the μ l of sense primer 0.5,0.5 μ l, cDNA solution of anti-sense primer 2.0 μ l, ddH2The μ l of 0 Up to 20,
QRT-PCR programs：95 DEG C of 30s pre-degenerations, connect 40 circulations：95℃ 5s, 60℃ 30-60s.
A kind of 8. specific RNA interference sequence of the UCR sequences described in claim 1：The interference sequence such as sequence table SEQ
Shown in ID NO.8 and SEQ ID NO.9.
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