CN103923982B - Effects of the HOXD AS1 in oesophagus squama cancer diagnosis and treatment - Google Patents
Effects of the HOXD AS1 in oesophagus squama cancer diagnosis and treatment Download PDFInfo
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Abstract
Effects of the HOXD AS1 in oesophagus squama cancer diagnosis and treatment, the present invention relates to a kind of discovery of long-chain non-coding RNA, detection and its application, according to the long-chain non-coding RNA sequence, design and synthesize specific real-time quantitative PCR primer, utilize real-time quantitative PCR preparation, the expression of the long-chain non-coding RNA is detected in cancer of the esophagus clinical case sample, it was found that the long-chain non-coding RNA expresses notable up-regulation in esophageal squamous cell carcinoma, and the clinical stages of its expression and patients with esophageal squamous cell carcinoma and pathology N equal height correlation by stages, specific detection sequence involved in the present invention, which is expected to prepare, is used for cancer of the esophagus auxiliary diagnosis, the preparation of outcome prediction or Index for diagnosis.In addition, design and synthesize specificity and strike two siRNA sequences for dropping long-chain non-coding RNA expression, experiment confirms to strike in Human esophageal squamous cell cancer cell line after the expression for dropping the long-chain non-coding RNA, the transfer number of tumour cell is substantially reduced, therefore siRNA sequence involved in the present invention is expected to turn into the molecular targeted therapy instrument for suppressing the esophageal squamous cell carcinoma cell rate of transform.
Description
Technical field
The invention belongs to oncomolecularbiology field, and in particular to a kind of long-chain non-coding RNA and its application, it is specific and
Speech, the present invention relates to a kind of application of long-chain non-coding RNA in cancer of the esophagus auxiliary diagnosis or prognosis preparation is prepared.
Background technology
The cancer of the esophagus (Esophageal Carcinoma, EC) is the malignant disease for seriously endangering whole mankind's health, global model
Its morbidity and mortality occupies the 8th and the 6th respectively in enclosing.EC mainly has two kinds of histological types：Esophageal squamous cell carcinoma
(Esophageal Squamous Cell Carcinoma, ESCC) and adenocarcinoma of esophagus (Esophageal
Adenocarcinoma, EAC), China's histological type (accounts for more than 90%) based on squamous carcinoma.Announced most according to the World Health Organization
New data shows that newly hair ESCC patient is more than 250,000 people every year for China, and because of the people of patient about 200,000 of death from esophageal carcinoma, the incidence of disease is occupied
All kinds of malignant tumours the 5th in the whole nation, the death rate occupies the 4th, remote super world average level.Although being clinically used to eat at present
The technological means of the inspection of pipe cancer and treatment is being updated, but the overall 5 years survival rates of patient are still extremely low.Nearly half a century with
Come, the research on EC pathogenesis both at home and abroad has achieved a series of achievements in mRNA, protein and miRNA fields, but
Its definite pathogenesis is at present still among positive research and probe.(Jemal A, et al.Global cancer
statistics.CA：A cancer journal for clinicians2011,61：69-90；Enzinger PC, et
Al.Esophageal cancer.N Engl J Med2003,349：2241-2252；Old ten thousand green grass or young crops .2004-2005 China dislikes
Property tumor invasion and dead estimation Chinese Journal of Oncology 2009,31：664-668；He Jie, waits esophageal cancer in China epidemiology
Present situation, diagnosis and treatment present situation and future countermeasure Cancer in China magazines, 2011, (7)：501-504.)
After human genome sequencing plan is completed, it was demonstrated that the protein coding gene received much concern for a long time only accounts for whole turn
The 2% of record group, the transcription product more than 98% is ncRNA (noncoding RNA, ncRNA).With entering that RNA groups are studied
Exhibition, be initially believed to be subgenomic transcription " noise " ncRNA, progressively confirmed it in several species including humans, very
To be plant physiology and pathologic event in, be respectively provided with extensive and diversified function rarely known by the people.Confirm to have at present to adjust
The ncRNAs of control effect is broadly divided into length ＞ 200nt LncRNAs and ＜ 200nt microRNAs, although
MicroRNAs is to study the relatively deep small ncRNAs of a class so far, but then it is found that in each species,
Not only quantity is big by LncRNAs, species is more, and the transcription and translation in gene, cell differentiation and ontogeny, hereditary and apparent
Regulating and controlling effect (Bimey E, et more extensive, fine and complicated compared with microRNAs in the vital movements such as heredity
Al.Identification and analysis of functional elements in1%of the human
genome by the ENCODE pilot project.Nature2007,447：799-816；Wilusz JE, et
al.Long noncoding RNAs：Functional surprises from the RNA world.Genes and
Development2009,23：1494-1504；Prasanth KV,et al.Eukaryotic regulatory RNAs：An
answer to the′genome complexity′conundrum.Genes and Development2007,21：11-42；
Tsai MC, et al.Long intergenic noncoding RNAs：new links in cancer
Progression.Cancer Res2011,71：3-7；Pauli A, et al.Non-coding RNAs regulators
Of embryogenesis.Nature Reviews Genetics2011,12：136-149；Van LeeuwenS, et
al.Long non-coding RNAs：Guardians of development.Differentiation2010,80：175-
183；UA, et al.Long Noncoding RNAs with Enhancer-like Function in Human
Cells.Cell2010,143：46-58；Caley DP, et al.Long noncoding RNAs, chromatin, and
Development.The Scientific World Journal2010,10：90-102.).
Long-chain non-coding RNA (long non-coding RNA, lncRNA) is more than 200 cores of a class transcript length
The non-coding RNA of thuja acid, research in recent years finds that it is the RNA that a class has important biomolecule function, participates in genomic imprinting, dye
A variety of important regulation processes such as colour solid silence, chromatin modification, transcriptional activation, transcription interference, the interior transport of core, in cell differentiation
Important regulating and controlling effect is played with the vital movement such as development, genetic transcription and translation, hereditary and epigenetic.In recent years,
Increasing authority's research confirms that lncRNA plays a part of suppressing or promoting tumour in tumour develops, in regulation and control
In terms of tumor cell proliferation, apoptosis, cell cycle, invasion and attack transfer ability, particularly significant effect is respectively provided with.It is existing at present more
LncRNAs is proved in the mankind including breast cancer, prostate cancer, melanoma, liver cancer, colorectal cancer, carcinoma of urinary bladder etc.
Had differences in kinds of tumors and express and perform important adjusting function (Gupta RA, et al.Long non-coding RNA
HOTAIR reprograms chromatin state to promote cancer metastasis.Nature2010,
464：1071-1076；Cui Z, et al.The prostate cancer-up-regulated long noncoding RNA
PlncRNA-1modulates apoptosis and proliferation through reciprocal regulation
of androgen receptor.Urologic Oncology：Seminars and Original
Investigations2013,31：1117-1123；Khaitan D, et al.The melanoma-upregulated long
Noncoding RNA SPRY4-IT1modulates apoptosis and invasion.Cancer Research2011,
71：3852-3862；DuY, et al.Elevation of Highly Up-regulated in Liver Cancer (HULC)
by Hepatitis B Virus X Protein Promotes Hepatoma Cell Proliferation via Down-
regulating p18.J Biol Chem2012,287：26302-26311；Ling H, et al.CCAT2, a novel
Noncoding RNA mapping to8q24, underlies metastatic progression and chromosomal
instability in colon cancer.Genome Research2013,23：1446-1461；Liu Z, et
Al.Downregulation of GAS5Promotes Bladder Cancer Cell Proliferation, Partly by
Regulating CDK6.PLoS ONE2013,8：9Article Number e73991.).To explore LncRNA in ESCC
Whether there is function, inventor has correlation with current have confirmed in people ESCC tissues and cell line in other tumours
The LncRNA of function carries out pilot study, it was demonstrated that the LncRNA HOTAIR found in breast cancer tissue are not only in ESCC cancers
With with differential expression in normal tissue, and can substantially suppress in ESCC cell lines apoptosis of tumor cells and promote tumour to turn
Move (Chen FJ, et al.Upregulation of the long non-coding rna hotair promotes
esophageal squamous cell carcinoma metastasis and poor prognosis.Molecular
Carcinogenesis2013,52：908-915.).In addition, inventor also confirms that the PlncRNA-1 found in prostate exists
Significantly raised and controllable tumor cell proliferation ability (Wang CM, et al.Upregulation of in ESCC tissues
the Long Non-coding RNA PlncRNA-1Promotes Esophageal Squamous Carcinoma Cell
Proliferation and Correlates with Advanced Clinical Stage, Dig Dis
Sci2013.doi：10.1007/s10620-013-2956-7).
Found however, following the trail of document in addition to seminar where inventor, the lncRNA on esophageal squamous cell carcinoma is expressed
Functional research of spectrum and its index of correlation is also less at present.For system exploration ESCC lncRNA express spectras, find with
One group of related lncRNAs of development specificity occurs for ESCC, and inventor screens one in people's ESCC cancer groups using chip technology
The lncRNA of significantly high expression in knitting, the gene is named as HOXD-AS1 (Ensemble database), its transcript regions position
Between No. 2 177,037,923-177,053,686bp pair of chromosome antisense strands, full length gene is about 4235bp.Inventor grinds
Study carefully confirmation：Compared with normal tissue, the lncRNA significantly high expression in people's ESCC cancerous tissues, and in the large sample in later stage
Organize qRT-PCR (Quantitative Real-time Polymerase Chain Reaction, real-time quantitative PCR) experiments
In further confirm expression of the index in people's ESCC cancerous tissues, be significantly higher than with normal tissue.Studied for ease of the later stage
And discuss, inventor is named as HUESCC-lncRNA5 (highly up-regulated in esophageal
Squamous cell carcinoma, long noncoding RNA5).Inventor has paid close attention to ESCC lncRNA expression
Spectrum, the tumor invasion machine that the new gene regulation factor of this class will be expected to further enrich and improve including esophageal squamous cell carcinoma
The research of system, also to find that mark, the new cancer target of diagnosing tumor and Index for diagnosis bring hope.
The content of the invention
For the lncRNA express spectras of system research people's esophageal squamous cell carcinoma, find with people's esophageal squamous cell carcinoma occur with
The closely related new lncRNAs of development, the 6 pairs of cancer of the esophagus provided by inventor and with normal tissue sample pass through
The RNA extracts kits (RNeasy Micro Kit, article No. 74004) of Qiagen companies are extracted after RNA, using Beijing Bo Aosheng
Brilliant core lncRNA chips V2 (4 × 180K) detection of thing Co., Ltd filters out a significantly high expression in human esophageal carcinoma
LncRNA, is named as HUESCC-lncRNA5, and its gene order is as shown in sequence table SEQ ID NO.1.Later stage is by totally 110 pairs
People ESCC cancers with normal tissue sample qRT-PCR checkings with finding the notable up-regulation of lncRNA expression in 75 pairs of samples.Should
LncRNA is expected to turn into the mark of diagnosing tumor and Index for diagnosis, while also providing new target spot for the treatment of tumour.
It is an object of the invention to provide it is a kind of detect in esophageal carcinoma tissue the lncRNA sequences of high expression and
Its application process.
The lncRNA express spectras of the research esophageal squamous cell carcinoma of present system, find occur with esophageal squamous cell carcinoma
With developing closely related new lncRNA.
It is an object of the invention to provide a kind of HUESCC- for detecting the high expression in esophageal carcinoma tissue
LncRNA5 sequences.
The invention provides the detection application process of the HUESCC-lncRNA5 sequences, prepared according to the sequence for eating
The preparation of pipe cancer auxiliary diagnosis or outcome prediction.
3 pairs of primers are used to detect HUESCC-lncRNA5 sequences the present invention according to the HUESCC-lncRNA5 sequences Designs
Row.
Pair1 sense primers：SEQ ID NO.2
Pair1 anti-sense primers：SEQ ID NO.3
Pair2 sense primers：SEQ ID NO.4
Pair2 anti-sense primers：SEQ ID NO.5
Pair3 sense primers：SEQ ID NO.6
The present invention is according to the HUESCC-lncRNA5 sequences Designs and synthesizes detection primer for real-time quantitative PCR
Group.The primer sets are applied to the inspection of SYBR Green, TaqMan probe, molecular beacon, double cross probe, combined probe etc.
Survey.
Be preferably used in dye class real-time quantitative PCR detection primer sets be respectively：
Pair2 sense primers：SEQ ID NO.4
Pair2 anti-sense primers：SEQ ID NO.5
In the present invention, qRT-PCR, real-time quantitative PCR, quantitative fluorescent PCR are identical concepts, can be used interchangeably.
It is a further object of the present invention to provide a kind of quantitative fluorescent PCR reagent of detection HUESCC-lncRNA5 expressions
Box and application method, all types fluorescent quantitation gene that the PCR kit for fluorescence quantitative is suitable for presently, there are in the market expand
Increase instrument, sensitivity is high, it is quantitative quick and precisely, stability it is good, have a good application prospect.
The present invention is prepared for a kind of dye class real-time quantitative PCR kit of detection lncRNA expressions, and component is as follows：
Specific primer, standard DNA template, fluorescent dye, real-time quantitative PCR reaction solution.Wherein described specific primer includes upper
Primer and anti-sense primer are swum, upstream primer sequence is SEQ ID NO.4, and downstream primer sequence is SEQ ID NO.5.The fluorescence
Quantitative PCR reaction solution includes dNTP, Mg2+, Taq enzyme and buffer buffer solutions.The preferred SYBR Green II of fluorescent dye,
The preferred thermal starting enzyme of Taq enzyme.
It is glimmering the invention also discloses a kind of application method for the dye class PCR kit for fluorescence quantitative for detecting esophageal squamous cell carcinoma
Fluorescent Quantitative PCR system：
Sense primer (10 μm of ol/L) 2 μ L；Anti-sense primer (10 μm of ol/L) 2 μ L；Sample cDNA4 μ L (or standard DNA template
DNA2μL)；50×ROX Reference Dye1μL；2×SYBR Premex Ex Taq II(TIi RNaseH Plus)25μ
L, plus ionized water is to 50 μ L.Quantitative fluorescent PCR program：95 DEG C of 30s pre-degenerations, connect 40 circulations：95 DEG C of 5s, 60 DEG C of 30-34s.
The invention also discloses a kind of detection method of long-chain non-coding RNA, include extraction, the sample of sample total serum IgE
CDNA preparation, HUESCC-lncRNA5 amplification.
The extraction of samples described above total serum IgE, sample cDNA preparation, the specific steps of HUESCC-lncRNA5 amplifications
Including：
1) extraction of sample total serum IgE：According to Life Technologies companiesReagent (article No.
15596026) reagent needed for and step extract esophageal carcinoma tissue or the Total RNA of tumour；NanoDrop is used again
ND-1000 nucleic acid quantifications instrument quantitative (NanoDrop Technologies, Wilmington, Delaware) quantifies what is extracted
RNA purity and concentration, denaturing formaldehyde gel electrophoresis quality inspection ensures the RNA extracted integrality.
2) sample cDNA preparation：Using TaKaRa kits PrimeScriptTM RT reagent Kit with
Total serum IgE reverse transcriptions of the gDNA Eraser (Perfect Real Time) (article No. RR047A) to extraction synthesizes cDNA；The reagent
Box includes RNase-Free DNase, can effectively remove the genomic DNA mixed.
The first step removes genomic DNA reaction, and reaction system and condition are as follows：
| Reagent | Usage amount |
| 5×gDNA Eraser Buffer | 2.0μl |
| gDNA Eraser | 1.0μl |
| Total RNA | 1μg |
| RNase Free dH2O | Add water to 10 μ l |
42 DEG C of reaction 2min after above-mentioned component is mixed；
Second step reverse transcription reaction, reaction system and condition are as follows：
| Reagent | Usage amount |
| The reaction solution of step 1 | 10.0μl |
| PrimeScriipt RT Enzyme Mix I | 1.0μl |
| RT Primer Mix | 1.0μl |
| 5×PrimeScript Buffer2 | 4μl |
| RNase Free dH2O | 4.0μl |
| Cumulative volume | 20μl |
By the well mixed rear 37 DEG C of incubations 15min of above-mentioned component, then 85 DEG C of inactivation 5sec, that is, obtain cDNA.
3) HUESCC-lncRNA5 amplification：Using TAKARA SYBR Premix Ex TaqTM II(TIi RNaseH
Plus) fluorescence quantitative kit, fluorescent quantitative PCR is carried out by template of the cDNA of reverse transcription.
Dye class quantitative fluorescent PCR system：Sense primer (10 μm of ol/L) 2 μ L；Anti-sense primer (10 μm of ol/L) 2 μ L；Sample
Product cDNA4 μ L (or standard DNA template DNA2 μ L)；50 × ROX Reference Dye (or 50 × ROX Reference Dye
II)1μL；2 × SYBR Premex Ex Taq II (TIi RNaseH Plus) 25 μ L, plus ionized water is to 50 μ L.Fluorescent quantitation
PCR programs：95 DEG C of 30s pre-degenerations, connect 40 circulations：95 DEG C of 5s, 60 DEG C of 30-34s.
The present invention also have detected kit sensitivity, and it is 10 as a result to show kit detection range of the present invention7-
102Copies/ μ L, minimum concentrations are 100copies/ μ L.
By the detection to positive, it is found that dye class fluorescence quantitative kit Detection accuracy of the present invention exists
More than 67%-69%, continuous 3 repetitions are tested, and experimental result is stable.
The present invention designs and synthesizes its specific interference sequence for HUESCC-lncRNA5 full length sequences：siRNA1(SEQ
ID NO.8), siRNA2 (SEQ ID NO.9), the interference sequence can significantly strike HUESCC-lncRNA5 in drop tumour cell
Expression, and the invasive ability of tumour cell can be caused substantially to weaken (Figure 10) than before.
Brief description of the drawings
Fig. 1 .LncRNA chip dendrograms
6 pairs of esophageal squamous cell carcinoma cancers of display and the LncRNA chips of cancer beside organism's differential expression are clustered
Fig. 2 specific primer the selection result figures
Draw for row agarose gel electrophoresis test after 3 couples of specific primer PCR amplifications of the sequences Design of the LncRNA
The effect of thing
The preliminary qRT-PCR testing results (2 of HUESCC-lncRNA5 of first 30 ESCC clinical samples of Fig. 3-ΔctMake
Figure)
The HUESCC-lncRNA5 of Fig. 4 80 ESCC clinical samples of second batch verifies qRT-PCR testing results (2 again-Δct
Mapping)
Totally 110 sample HUESCC-lncRNA5 detect system to Fig. 5 two batches in cancer and cancer beside organism differential expression qRT-PCR
Meter analysis (2-ΔΔctValue compares,*P ＜ 0.05,**P ＜ 0.01)
Fig. 6 .qRT-PCR detections HUESCC-lncRNA5 expression and the correlation analysis (2 of patient clinical by stages-ΔΔctValue
Compare,*P ＜ 0.05,**P ＜ 0.01)
Fig. 7 .HUESCC-lncRNA5 are expressed and the correlation analysis (2 of ESCC pathology N by stages-ΔΔctValue compares,*P ＜
0.05,**P ＜ 0.01)
Fig. 8 .qRT-PCR detect expression of the HUESCC-lncRNA5 in esophageal carcinoma cell line
Fig. 9 .qRT-PCR detections siRNA strikes drop HUESCC-lncRNA5 efficiency
Figure 10 .Transwell detections siRNA strikes the invasive ability of tumour cell after drop HUESCC-lncRNA5
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.It will be understood by those skilled in the art that：Can in the case where not departing from the principle and objective of the present invention
So that 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.
The people's esophageal squamous cell carcinoma cancer of embodiment 1 and the lncRNA chip expression analysis with normal tissue
One material and method
1st, material
Tissue samples come from inpatient's surgery excision sample of 6 pairs of patients with esophageal squamous cell, and each pair includes food
Pipe tumor tissues and the normal esophageal tissue of pairing.
2nd, method
2.1 tumor tissues and the extraction with normal tissue total serum IgE
Oesophagus is extracted by RNA extracts kits (RNeasy Micro Kit, article No. 74004) specification of Qiagen companies
Phosphorus carninomatosis human tumour tissue and the total serum IgE with normal tissue, the kit include RNase-Free DNase I
(lyophilized) genomic DNA mixed, can effectively be removed.Quantified again with NanoDrop ND-1000 nucleic acid quantifications instrument
(NanoDrop Technologies, Wilmington, Delaware) quantitative extracted RNA purity and concentration, formaldehyde becomes
Property gel electrophoresis quality inspection ensure extract RNA integrality.
2.2 couples of sample RNA carry out fluorescence labeling (CRNA amplification label kits, catalog number：360060-
10)
2.2.1 reverse transcription synthesizes the first chain cDNA
Using Total RNA as starting, T7Oligo (dT) Primer containing T7 promoter sequences is primer, is used
The first chain of CbcScript enzymatic synthesis cDNA.
2.2.2 2 are synthesizednd-strand cDNA
The RNA in heterozygosis chain is cut into short-movie section with RNase H, DNA Polymerase are prolonged using RNA short-movie sections as primer
Stretch, synthesis 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 purified with RNA Clean-up Kit (MN).
2.2.4 random primed reverse transcription
5ug cRNA are taken, with CbcScript 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
NucleoSpinExtract II Kit (MN) are purified, and are drained after purification.(Cy5-dCTP or Cy3-dCTP(GE
Healthcare)。
2.3 hybridization and cleaning
The DNA of mark is dissolved in hybridization solution (2 × GEx Hyb Buffer (HI-RPM), 25% formamide), miscellaneous in 45 DEG C
Friendship is stayed overnight.After hybridization terminates, first contain 0.2%SDS at 42 DEG C or so, 5min is washed in 2 × SSC liquid, then in 0.2 × SSC
Middle room temperature washes 5min.Slide can be used to scanning after drying.
2.4 chip scanning
Chip is scanned with Agilent G2565CAMicroarray Scanner, obtains hybridizing picture.
The collection and data analysis of 2.5 chip images
2.5.1 fluorescence signal value is extracted
Chip image is analyzed using Feature Extraction image analysis softwares, picture signal is converted into
Data signal.
2.5.2 differential gene is screened
Initial data is input in GeneSpring GX softwares, using percentile shift methods to signal value
It is normalized.Then Absolute Fold change >=2 are used, while Flag is carried out labeled as Detected standard
Differential gene is screened.
Two results
LncRNA chip clusterings on people's esophageal squamous cell carcinoma are shown in Fig. 1.Chip examination is found in a plurality of expression
The lncRNAs that the expression that reconciles is lowered.Wherein HUESCC-lncRNA5 is shown expresses significantly up-regulation in cancerous tissue, in view of it can
Can exist in the cancerous tissue of people's esophageal squamous cell carcinoma it is specific expressed, the present invention by following examples use extensive sample
This carries out the repeated authentication of index in batches.
Embodiment 2qRT-PCR preliminary identifications HUESCC-lncRNA5 is in the cancerous tissue of esophageal squamous cell carcinoma and with aligning
Differential expression in often organizing
First, experiment material
Other 30 are chosen again to the cancerous tissue of the sample of chip testing (be different from) people's esophageal squamous cell carcinoma and with aligning
Often tissue, qRT-PCR preliminary identifications are carried out to HUESCC-lncRNA5 differential expression.
2nd, experimental method and result
1 primer specificity is identified
1.1 the screening of specific primer
(1) the related transcript sequences of HUESCC-lncRNA5 are extracted from Ensemble databases, and with according to transcript
Sequence primer is designed by NCBI design of primers instrument (Primer-BLAST)；
(2) primer after designing is evaluated with Oligo7,3 pairs of primers of every design；
Pair1 sense primers：SEQ ID NO.2
Pair1 anti-sense primers：SEQ ID NO.3
Pair2 sense primers：SEQ ID NO.4
Pair2 anti-sense primers：SEQ ID NO.5
Pair3 sense primers：SEQ ID NO.6
Pair3 anti-sense primers：SEQ ID NO.7
Through RT-PCR and agarose gel electrophoresis experimental verification, the primer detected for dye class real-time quantitative PCR is screened
Group is following (Fig. 2)：
Pair2 sense primers：SEQ ID NO.4
Pair2 anti-sense primers：SEQ ID NO.5
(3) by the cancer of people's esophageal squamous cell carcinoma with matching somebody with somebody normal tissue according to Life Technologies companiesReagent needed for reagent (article No. 15596026) and step extract total serum IgE；NanoDrop ND-1000 nucleic acid is used again
Quantitative the extracted RNA of quantitative instrument quantitative (NanoDrop Technologies, Wilmington, Delaware) purity and
Concentration, denaturing formaldehyde gel electrophoresis quality inspection ensures the RNA extracted integrality.
(4) using TaKaRa kits PrimeScriptTM RT reagent Kit with gDNA Eraser
The total serum IgE reverse transcription of (Perfect Real Time) (article No. RR047A) to extraction synthesizes cDNA, and the kit includes gDNA
Eraser DNase, can effectively remove the genomic DNA mixed.
The first step removes genomic DNA reaction, and reaction system and condition are as follows：
| Reagent | Usage amount |
| 5×gDNA Eraser Buffer | 2.0μl |
| gDNA Eraser | 1.0μl |
| Total RNA | 1μg |
| RNase Free dH2O | Add water to 10 μ l |
42 DEG C of reaction 2min after above-mentioned component is mixed；
Second step reverse transcription reaction, reaction system and condition are as follows：
| Reagent | Usage amount |
| The reaction solution of step 1 | 10.0μl |
| PrimeScript RT Enzyme Mix I | 1.0μl |
| RT Primer Mix | 1.0μl |
| 5×PrimeScript Buffer2 | 4μl |
| RNase Free dH2O | 4.Oμl |
| Cumulative volume | 20μl |
By the well mixed rear 37 DEG C of incubations 15min of above-mentioned component, then 85 DEG C of inactivation 5sec, that is, obtain cDNA.To synthesize
CDNA be template, set reaction groups for 3 pairs of primers respectively and be not added with the negative control group of cDNA templates, with except outer primer
Dimer is possible, then the primer designed by step (2) enters performing PCR reaction；
(5) electrophoresis detection, from Marker DL1000 (TaKaRa).Primer selection standard：A. by Marker to amplification
Clip size carries out entry evaluation, and amplified fragments size is identical with expection；B. amplified production only one of which, that is, ensure primer amplification
Specificity.As a result as shown in Fig. 2 optimal primer pair is Pair2, the specific primer of upstream, its sequence is shown in sequence table SEQ
ID NO.4, the specific primer in downstream, its sequence is shown in sequence table SEQ ID NO.5.
The extraction of 2 sample total serum IgEs：
Using liquid nitrogen grinding method, according to Life Technologies companiesReagent (article No.
15596026) reagent needed for and step extract esophageal carcinoma tissue or the Total RNA of tumour.Main operational steps are such as
Under：
(1) sample quick freeze after in vitro is put into tissue in the mortar of precooling in liquid nitrogen, during extracting RNA is carried out
Grinding, the liquid feeding nitrogen in grinding, whole process should not all make liquid nitrogen volatilization dry；
(2) after tissue sample is ground into powder, when liquid nitrogen is evaporated completely substantially, 2- is added in each mortar
3mlTRIZOL reagents, TRIZOL can be frozen into solid-like after adding, and can continue to grind this solid into powder, with mortar temperature
Degree goes back up to room temperature, and the TRIZOL of solid-like is progressively returned to liquid condition, this TRIZOL agent transfer to glass homogenizer
In, further it is homogenized 3-5min；
(3) TRIZOL agent transfers into 1.5ml centrifuge tubes, every 1 milliliter of TRIZOL reagents add 0.2 milliliter of chlorine
It is imitative.Carefully cover sample cell.Shake pipe energetically with hand 15 seconds, 15-30 DEG C is incubated 2-3 minutes.2-8 DEG C, no more than 12000g centrifugations
15 minutes.After centrifugation, mixture is separated into the colourless aqueous phase on red lower floor (phenol-chloroform phase), interphase and upper strata, and RNA is deposited
It is aqueous phase；
(4) aqueous phase is transferred to a new pipe, and mixing isopropanol from aqueous phase to precipitate RNA, and every 1 milliliter is used for initially same
The TRIZOL reagents of matter are using 0.5 milliliter of isopropanol, 15-30 DEG C of samples of incubation 10 minutes, 2-8 DEG C, no more than 12000g from
The heart 10 minutes；
(5) supernatant is abandoned, RNA precipitate is washed once with 75% ethanol, every milliliter is used for the TRIZOL examinations of initial homogeneity
Agent 2-8 DEG C, is centrifuged 5 minutes using at least 1 milliliter 75% of ethanol, vortex mixed no more than 7500g；
(6) supernatant is abandoned, RNA precipitate (be air-dried or be dried in vacuo 5-10 minutes) is simply dried, with 20-100ul without RNA
The water piping and druming of enzyme dissolves RNA and is stored in -70 DEG C several times, in this process it may be noted that RNA precipitate should not be allowed to be completely dried, because
Its solubility will be substantially reduced for this.
The preparation of 3 standard DNA templates
To specifications, from the cancerous tissue of esophageal squamous cell carcinoma, extracted using Invitrogen RTIZOL kits
Total serum IgE, and further useRNA clean-up kits (MACHEREY-NAGEL, Germany) are right
Total serum IgE carried out post purifying, then carried out reverse transcription reaction, reverse transcription system is：Reverse transcription random primer (2 μm of ol/L) 1 μ L,
μ L, 0.1mol/L DTT2 μ L, the SuperScript RNase H of 4 μ L, dNTP mixture (every kind of 2.5mmol/L) of total serum IgE 4 are reversed
Recording enzyme (200U/ μ L) 2 μ L (TAKARA) reaction condition is：37 DEG C of water-baths 60min, 95 DEG C of 3min.
The cDNA that reverse transcription reaction is obtained carries out Standard PCR, and reaction system and condition are as follows：10×Ex Taq
Buffer10 μ L, dNTP Mixture (each 2.5mmol/L) 4 μ L, Sequence NO.4 (10pmol) 4 μ L, Sequence
The μ L of 45 μ L, Ex Taq archaeal dna polymerases of μ L, cDNA (0.1-2 μ g) of NO.5 (10pmol) 0.5, distilled water polishing to 100uL.Reaction
Condition is 94 DEG C of pre-degeneration 5min；94 DEG C of denaturation 50s, 50 DEG C of annealing 50s, 72 DEG C of extensions 50s, 35cycles；Last 72 DEG C are prolonged
Stretch 10min.
5 μ L are sampled, row agarose gel electrophoresis detection is entered to the PCR products expanded, carry out gel extraction and purify (to reclaim
Use kit：EZ-10Spin Column DNA Gel Extraction Kit), purified product is connected to pGM-T clones
Carrier, is then transformed into DH5 α competent cells.Drawn by sequence for SEQ ID NO.4 and SEQ ID NO.5 specificity
Thing screening positive clone.DNA is extracted after positive colony amplification, DNA uses NanoDrop ND-1000 nucleic acid quantifications
Instrument quantitative (NanoDrop Technologies, Wilmington, Delaware) simultaneously does 10 times and is serially diluted and used as standard items
In the preparation of standard curve, (standard DNA template concentration range is 108-102copies/μl)。
4 sensitivity experiments
Recombinant plasmid is taken to be diluted to 10 in proportion8、107、106、105、104、103、102Individual copy/μ L, carries out fluorescent quantitation
PCR, using the least concentration of test positive as the detection sensitivity of this method.This research institute set up method detection range be
108-102Copies/ μ L, minimum concentrations are 100copies/ μ L.
5 synthesis cDNA templates
Above-mentioned 30 pairs of patients with esophageal squamous cell carcinoma cancerous tissues and the total serum IgE with normal tissue are taken, using TaKaRa kits
PrimeScriptTMRT reagent Kit with gDNA Eraser (Perfect Real Time) (article No. RR047A) are right
The total serum IgE reverse transcription synthesis cDNA of extraction, the kit includes gDNA Eraser DNase, can effectively remove the gene mixed
Group DNA.
The first step removes genomic DNA reaction, and reaction system and condition are as follows：
| Reagent | Usage amount |
| 5×gDNA Eraser Buffer | 2.0μl |
| gDNA Eraser | 1.0μl |
| Total RNA | 1μg |
| RNase Free dH2O | Add water to 10 μ l |
42 DEG C of reaction 2min after above-mentioned component is mixed；
Second step reverse transcription reaction, reaction system and condition are as follows：
| Reagent | Usage amount |
| The reaction solution of step 1 | 10.0μl |
| PrimeScript RT Enzyme Mix I | 1.0μl |
| RTPrimerMix | 1.0μl |
| 5×PrimeScript Buffer2 | 4μl |
| RNase Free dH2O | 4.0μl |
| Cumulative volume | 20μl |
By the well mixed rear 37 DEG C of incubations 15min of above-mentioned component, then 85 DEG C of inactivation 5sec, that is, obtain cDNA.
6 dye class fluorescence quantitative PCR detection HUESCC-lncRNA5 expression quantity
Using TAKARA SYBR Premix Ex TaqTMII (TIi RNaseH Plus) fluorescence quantitative kit (article No.
RR820A).50 μ LqRT-PCR reaction systems include：Sense primer (10 μm of ol/L) 2 μ L；Anti-sense primer (10 μm of ol/L) 2 μ L；
Sample cDNA4 μ L；50×ROX Reference Dye1μL；2×SYBR Premex Ex Taq II(TIi RNaseH
Plus) 25 μ L, plus ionized water is to 50 μ L.Instrument uses Applied Biosystems7500.Quantitative fluorescent PCR program：95℃
30s pre-degenerations, connect 40 circulations：95 DEG C of 5s, 60 DEG C of 30-34s.
According to qRT-PCR relative quantification formula：2-ΔCt, HUESCC-lncRNA5 is calculated respectively in patients with esophageal squamous cell carcinoma
Cancerous tissue (T) and with the expression in normal tissue (N), comparative result is as shown in Figure 3：QRT-PCR stable amplification results,
Wherein HUESCC-lncRNA5 is concentrated mainly in the expression in normal tissue between 0.000-0.001, and cancer group
The HUESCC-lncRNA5 knitted expression quantity is concentrated mainly between 0.002-0.150, hence it is evident that higher than with normal tissue, with
Upper result illustrates the index universal high expression in tumor tissues.Index expression is defined as according still further to relative expression quantity T-N ＞ 0
Up-regulation；T-N ＜ 0 are defined as index expression downward.Then this experimental result is shown：HUESCC-lncRNA5 is in 30 ESCC cancers
With with up-regulated expression in normal tissue 20, according still further to formula：Up-regulated expression number of cases/total detection number of cases x100% defines this and referred to
Target positive rate, then the positive rate of the index is 67%.
Embodiment 3qRT-PCR further verifies cancerous tissues and pairing of the HUESCC-lncRNA5 in esophageal squamous cell carcinoma
Differential expression in normal structure
1st, qRT-PCR kit forms
1.1 dye class HUESCC-lncRNA5qRT-PCR 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.HUESCC-lncRNA5qRT-PCR detection
The preparation of 2.1 total serum IgEs
The cancerous tissue of other 80 couples of esophageal squamous cell carcinoma patients is chosen and with normal tissue, according to Life
Technologies companiesReagent needed for reagent (article No. 15596026) and step extract total serum IgE, specific ginseng
See specification.Again with NanoDrop ND-1000 nucleic acid quantifications instrument it is quantitative (NanoDrop Technologies, Wilmington,
Delaware) quantitative extracted RNA purity and concentration, denaturing formaldehyde gel electrophoresis quality inspection ensures the RNA extracted integrality.
2.2cDNA synthesis
Using TaKaRa kits PrimeScriptTM RT reagent Kit with gDNA Eraser(Perfect
Real Time) (article No. RR047A), it will detect that qualified total serum IgE carries out reverse transcription reaction.
2.3qRT-PCR detection
QRT-PCR uses Applied Biosystems7500 in instrument.It is glimmering in qRT-PCR response procedures such as embodiment 2
Photoinitiator dye class real-time quantitative PCR detects HUESCC-lncRNA5 expression quantity.
3 testing results
As a result show：The cancerous tissue of other 80 pairs of patients with esophageal squamous cell is chosen with matching somebody with somebody normal tissue enlarged sample
Amount checking, real-time quantitative PCR detects index up-regulated expression in cancerous tissue in 55 pairs of samples, and Positive rate reaches 69%
(Fig. 4).Result above demonstrates again that the index universal high expression in tumor tissues.We are repeated 3 times to above-mentioned sample
QRT-PCR is examined, and as a result repeatability shows that the repeatability and stability of kit of the present invention are preferable up to 100%.
Embodiment 4HUESCC-lncRNA5 is diagnosed in ESCC and the potential value of Index for diagnosis is analyzed
HUESCC-lncRNA5 have detected in ESCC on the basis of high expression, with reference to the clinical and pathological data of patient,
Further analyze between HUESCC-lncRNA5 expression and different pathological and clinical stages correlation (referring in particular to
The 7th edition cancer of the esophagus TNM stage standard of new UICC), inquired into HUESCC-lncRNA5 in people's esophageal squamous cell carcinoma with disease
The diagnosis of disease, including its expression in terms of the relation of clinic/pathological staging, Index for diagnosis, the selection of therapeutic scheme with being had
Potential value.
SPSS For Windows20.0 softwares are used qRT-PCR reaction results, and related data passes through Normal test
Examine, examined according to data type from Mann-Whitney and carry out data analysis, P ＜ 0.05 think the differential expression of the index
It is statistically significant.QRT-PCR interpretations of result HUESCC-lncRNA5 expressions in 110 pairs of ESCC samples, which are significantly higher than, matches somebody with somebody
Align often to eat tubing (Fig. 5, P ＜ 2 × 10-6).HUESCC-lncRNA5 expression and clinical stages height correlation (Fig. 6)：
HUESCC-lncRNA5 expression in IIB-IIIC phase ESCC patients, is significantly higher than 0-II A phases patient (P ＜ 0.05).In addition,
The expression of the index and T also height correlation (Fig. 7) by stages：N1-N2HUESCC-lncRNA5 expression is significantly high in phase ESCC patient
In N0Phase patient (P ＜ 0.05).
HUESCC-lncRNA5 have detected in ESCC tissues on the basis of high expression, further analysis is found
HUESCC-lncRNA5 also expresses (Fig. 8) in esophageal cancer cell strain in universal height, using purchased from LifeTechnology companies
Stealth RNAiTMsiRNA：SiRNA1 (SEQ ID NO.8), siRNA2 (SEQ ID NO.9), in Human esophageal squamous cell cancer cell
The expression (Fig. 9) for dropping the index can be significantly struck in strain TE10, the invasive ability of tumour cell substantially weakens (figure than before
10).Obviously, find it is more, be more accurately similar to index as HUESCC-lncRNA5, it is significantly high in ESCC tissues
The index of modulate tumor cell transfer ability is expressed and had, is expected to turn into participation auxiliary ESCC diagnosis, treatment and prognosis evaluation
Related biomarker, is of great practical significance.Further go deep into HUESCC- with later stage result of study
LncRNA5 mechanism of action of modulate tumor cell forwarding function in ESCC will be illustrated progressively, and it can not only turn into diagnosis and pre-
The biomarker of correlation is judged afterwards, is more expected to turn into new ESCC therapy targets to improve, improve clinic ESCC therapeutic effects.
Claims (4)
1. a kind of nucleotides sequence be classified as SEQ ID NO.1 long-chain non-coding RNA preparing for cancer of the esophagus auxiliary diagnosis or
Application in curative effect predication reagent.
2. application according to claim 1, it is characterised in that the cancer of the esophagus is esophageal squamous cell carcinoma.
3. application according to claim 1, it is characterised in that described is used for cancer of the esophagus auxiliary diagnosis or outcome prediction
Reagent be real-time quantitative PCR detection reagent.
4. Stealth RNAi of the nucleotide sequence as shown in SEQ ID NO.8 or SEQ ID NO.9TMSiRNA is preparing suppression
Application in the transcellular reagent of esophageal squamous cell carcinoma.
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| CN107227341B (en) * | 2017-04-27 | 2021-01-15 | 中山大学孙逸仙纪念医院 | Application of LncRNA HOXD-AS1 in prostate cancer diagnosis and drug treatment |
| CN108728545A (en) * | 2018-06-25 | 2018-11-02 | 汕头大学医学院附属肿瘤医院 | Colorectal cancer long-chain non-coding RNA-HOTAIR molecular markers and its application |
| CN109055548B (en) * | 2018-07-25 | 2021-09-21 | 中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所） | Application of gene HES2 in auxiliary diagnosis, prognosis judgment and treatment of esophageal squamous cell carcinoma |
| CN109913552B (en) * | 2019-03-27 | 2020-08-04 | 河北医科大学第四医院 | Esophageal squamous carcinoma diagnosis and treatment target and application |
| CN111088357B (en) * | 2019-12-31 | 2022-09-20 | 深圳大学 | Tumor marker for ESCC and application thereof |
| RU2742468C1 (en) * | 2020-05-12 | 2021-02-08 | федеральное государственное бюджетное учреждение "Национальный медицинский исследовательский центр онкологии" Министерства здравоохранения Российской Федерации | Essc-tipe-1test-system for molecular-genetic typing of squamous oesophageal cancer |
| CN113151469B (en) * | 2021-04-19 | 2022-06-10 | 温州医科大学 | Tumor classification marker combination and application thereof |
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| Long Noncoding RNA HOTAIR Is a Prognostic Marker for;Xiao-bin Lv等;《plos one》;20130531;第8卷(第5期);摘要、第2页右栏第1段-第5页右栏第1段 * |
| TPA: Homo sapiens HOXD-AS1 gene for long non-coding RNA OTTHUMT00000334217.2 antisense;Harrow,J等;《GeneBank accession HG494908》;20131023 * |
| Upregulation of the Long Non-coding RNA HOTAIR Promotes Esophageal Squamous Cell Carcinoma Metastasis and Poor Prognosis;Fang-Jun Chen等;《Molecular Carcinogenesis》;20131130;第52卷(第11期);第914页右栏第2段、第909页右栏第3段 * |
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