CN105256017A - Application method of BTRC gene - Google Patents

Application method of BTRC gene Download PDF

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CN105256017A
CN105256017A CN201510649041.8A CN201510649041A CN105256017A CN 105256017 A CN105256017 A CN 105256017A CN 201510649041 A CN201510649041 A CN 201510649041A CN 105256017 A CN105256017 A CN 105256017A
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btrc
expression
gene
nasopharyngeal carcinoma
cell
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李桂源
曾朝阳
熊炜
晏其佳
李小玲
李夏雨
张文玲
范松青
龚朝建
石磊
周鸣
向波
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Central South University
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Central South University
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • 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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    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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    • G01—MEASURING; TESTING
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Abstract

The invention discloses an application method of a BTRC gene. The application method is used for preparing a preparation for predicting recurrence and metastasis of the nasopharynx cancer. Research shows that the expression level of the BTRC gene in nasopharynx cancer tissue is negatively correlated with invasion, metastasis and prognosis of the nasopharynx cancer, recurrence and metastasis more easily occur to a patient with the low expression level of the BTRC gene in the nasopharynx cancer tissue, and then the survival time of the patient is shorter than a patient with the high expression level of the BTRC gene. It is shown that the BTRC gene can serve as a molecular marker for auxiliary diagnosis, curative effect prediction, prognosis and the like of the nasopharynx cancer, and a special fluorescence real-time quantitative PCR primer, a special in-situ hybridization probe, an immunohistochemistry detection kit and the like are designed for the BTRC gene, and used for detecting the expression level of the BTRC gene in the nasopharynx cancer tissue to be expected to provide a reference for prediction of recurrence and metastasis of the nasopharynx cancer clinically. In addition, the application method can also be used for preparing the preparation for preventing recurrence and metastasis of the nasopharynx cancer, and a new path is provided for prevention and treatment of the nasopharynx cancer.

Description

The application method of BTRC gene
Technical field
The invention belongs to oncomolecularbiology field, be specifically related to BTRC gene for the preparation of prediction recurrent nasopharyngeal carcinoma and transfer, and prevent the application method of preparation of recurrent nasopharyngeal carcinoma and transfer.
Background technology
Nasopharyngeal carcinoma (NasopharyngealCarcinoma, NPC) be the common head-neck malignant tumor in south China area, by nasopharyngeal mucosal epithelium generation vicious transformation, great majority are PDSCC, grade malignancy is high and site of pathological change is hidden, the patient's early symptom particularly occurring in pharyngeal recess and nasopharynx top is not obvious, is thus difficult to early discovery, and sing misdiagnosis and mistreatment rate is higher; Nasopharyngeal carcinoma very easily shifts in addition, and first visit patient metastasic cervical lymph nodes rate is up to 80%.Radiotherapy is the most frequently used clinically treatment means of current nasopharyngeal carcinoma, and the patient of 60 ~ 70% can obtain good curative effect, but still has the patient of 20 ~ 30% to there will be recurrence and transfer.Recurrence and transfer are one of principal elements causing Nasopharyngeal Carcinoma Patients death clinically, and chemicotherapy is to the recurrence for the treatment of nasopharyngeal carcinoma, transfer effect is undesirable, therefore, screen the molecular marked compound that the early stage Metastasis and prognosis of new nasopharyngeal carcinoma judges, clinical treatment for nasopharyngeal carcinoma has important directive significance, the chemicotherapy means of simultaneously comparing traditional, the method of biotherapy is more suitable for the treatment of recurrence and transition nasopharyngeal carcinoma, wherein gene therapy is to the recurrence preventing and treating nasopharyngeal carcinoma, transfer has more important and wide potential applicability in clinical practice, the target spot of Screening of Nasopharyngeal Carcinoma gene therapy is significant too.
We, by biochip technology, have screened BTRC gene (GenebankGeneID:8945; Reference sequences: nM_033637.3) lower at nasopharyngeal carcinoma.There is the relation of developing effect about BTRC and nasopharyngeal carcinoma up to now and all there is no bibliographical information in the early screening, auxiliary diagnosis or outcome prediction etc. of nasopharyngeal cancer patient.We are shown by research, Invasion and Metastasis and the prognosis of the expression level of BTRC in tissues of nasopharyngeal carcinoma and nasopharyngeal carcinoma are closely related, the patient that in tissues of nasopharyngeal carcinoma, BTRC gene expression dose is lower is more easily recurred and is shifted, and the patient that thus survival time is high compared with BTRC gene expression dose is shorter.Show that BTRC gene can as the molecule marker of nasopharyngeal carcinoma auxiliary diagnosis, outcome prediction and Index for diagnosis etc., for BTRC design specialized fluorescence real-time quantitative PCR primer, in situ hybridization probe and Immunohistochemical detection test kit etc., be expected to provide reference for the prediction of recurring nasopharyngeal carcinoma clinically and shift for detecting the expression level of BTRC in tissues of nasopharyngeal carcinoma.
In addition, the BTRC Gene interfere sequence (siRNA) of our transfection synthetic in nasopharyngeal carcinoma cell is to suppress the expression of BTRC gene, and the expression confirming to lower BTRC gene in human nasopharyngeal epithelioma 1 can promote the Invasion and Metastasis of nasopharyngeal carcinoma cell; By designing and building the over-express vector of BTRC gene, successfully in nasopharyngeal carcinoma cell, express BTRC gene, effectively can suppress the invasion inhibition ability of nasopharyngeal carcinoma cell; By a series of research, applicant further demonstrate that the E3 ubiquitin protein ligase (E3ubiquitinproteinligase) of BTRC genes encoding one β-TrCP (beta-transducinrepeatcontainingE3ubiquitinproteinligase) by name, β-TrCP is one of intracellular protein key enzyme in ubiquitination pathway degradation process, substrate β-catenin and the Snail that can regulate and control it more specifically degrade through ubiquitination pathway, thus maintain the content of these two albumen of β-catenin and Snail in cell.β-catenin, Snail are cell epithelia-interstitial conversion (epithelial-mesenchymaltransition, EMT) key regulator in process, and epithelial-mesenchymal conversion is the most key the first step of tumour cell generation Invasion and Metastasis.Therefore, applicant confirms BTRC down-regulated expression in nasopharyngeal carcinoma cell by research, β-TrCP the protein content causing it to encode reduces, substrate β-the catenin of β-TrCP and the degraded of Snail are slowed down, cause β-catenin and Snail at intracellular accumulation, promoted the epithelial-mesenchymal conversion of nasopharyngeal carcinoma cell, made nasopharyngeal carcinoma cell possess stronger invasion inhibition ability, thus show as the relapse and metastasis of Nasopharyngeal Carcinoma Patients, finally cause death.In nasopharyngeal carcinoma cell, again express BTCR by engineered means, then can suppress the invasion inhibition ability of nasopharyngeal carcinoma cell significantly.Therefore, cell epithelia-interstitial that BTRC gene and its downstream comprise β-catenin and Snail changes the potential target spot that associated signal paths can become again nasopharyngeal carcinoma gene therapy, and particularly BTRC over-express vector has important application prospect in the gene therapy of nasopharyngeal carcinoma.To the nano silicon particles of polylysine modification, Nanoparticulate Carriers for Gene Delivery is made by carrier loaded for BTRC gene overexpression; the nano silicon particles of polylysine modification can protect BTRC gene overexpression carrier from nuclease degradation; extend action time, and have higher transfection efficiency.And then provide new approach for preparing the preparation of recurrence and transfer preventing and treating nasopharyngeal carcinoma.
Summary of the invention
The object of this invention is to provide the application method of BTRC gene.
The application method of BTRC gene, for the preparation of the preparation predicting recurrent nasopharyngeal carcinoma and transfer.
The expression of described BTRC gene and recurrent nasopharyngeal carcinoma and translate into negative correlation.
Described prediction recurrent nasopharyngeal carcinoma and the preparation of transfer comprise: PCR detection reagent, in situ hybridization detection reagent or immunohistochemical reagents.
PCR detection reagent is fluorescence real-time quantitative PCR reagent.
The primer of fluorescence real-time quantitative PCR reagent place comprises:
BTRCforward,5’-CCCCTTCTCGAACATACACCT-3’,
BTRCreverse,5’-AGTCTCAAAGCCCTGCTCCT-3’
GAPDHforward,5’-AACGGATTTGGTCGTATTGG-3’,
GAPDHreverse,5’-TTGATTTTGGAGGGATCTCG-3’。
The oligonucleotide probe of in situ hybridization detection reagent and the oligonucleotide probe of positive control, sequence is as follows:
BTRC probe 1:5'-GTCTAAGTGAATTCTTCTCTGGTATTATCT-3'
BTRC probe 2:5'-GTACTTGTGTTCCATACCTTTATAGTTCTA-3'
BTRC probe 3:5'-ATCTCCAATTAGATTCTATTGTCTCAATGT-3'
GAPDH probe 1:5'-CCACTTTACCAGAGTTAAAAGCAGCCCTGG-3'
GAPDH probe 2:5'-CAGTAGAGGCAGGGATGATGTTCTGGAGAG-3'
GAPDH probe 3:5'-GTCAGAGGAGACCACCTGGTGCTCAGTGTA-3'.
The application method of BTRC gene, can also for the preparation of the preparation preventing recurrent nasopharyngeal carcinoma and transfer.Specifically for the preparation of the preparation of the substrate β-catenin of BTRC gene expression product β-TrCP and the degraded of Snail.More specifically for the preparation of suppression epithelial-mesenchymal conversion preparation.Said preparation promotes that the expression of BTRC gene can make the expression of epithelial mark ZO-1, E-cadherin and Claudin-1 raise, and the expression of mark ZEB1, N-cadherin, Vimentin and Slug of mesenchymal cell reduces.
The present invention's research shows, Invasion and Metastasis and the prognosis of the expression level of BTRC in tissues of nasopharyngeal carcinoma and nasopharyngeal carcinoma become negative correlation, the patient that in tissues of nasopharyngeal carcinoma, BTRC gene expression dose is lower is more easily recurred and is shifted, and the patient that thus survival time is high compared with BTRC gene expression dose is shorter.Show that BTRC gene can as the molecule marker of nasopharyngeal carcinoma auxiliary diagnosis, outcome prediction and Index for diagnosis etc., for BTRC design specialized fluorescence real-time quantitative PCR primer, in situ hybridization probe and Immunohistochemical detection test kit etc., be expected to provide reference for the prediction of recurring nasopharyngeal carcinoma clinically and shift for detecting the expression level of BTRC in tissues of nasopharyngeal carcinoma.
The expression that the present invention also confirms to lower BTRC gene in human nasopharyngeal epithelioma 1 can promote the Invasion and Metastasis of nasopharyngeal carcinoma cell; By designing and building the over-express vector of BTRC gene, successfully in nasopharyngeal carcinoma cell, express BTRC gene, effectively can suppress the invasion inhibition ability of nasopharyngeal carcinoma cell.And then provide new approach for preparing the preparation of recurrence and transfer preventing and treating nasopharyngeal carcinoma.
Accompanying drawing explanation
Fig. 1 utilizes biochip technology to screen the difference expression gene collection of illustrative plates obtained from 6 routine normal nasopharyngeal epithelium and 10 routine tissues of nasopharyngeal carcinomas;
Altogether screening obtains difference expression gene 2461, the gene wherein raised at nasopharyngeal carcinoma and 1677, and the gene lowered in nasopharyngeal carcinoma has 784; N represents normal nasopharyngeal epithelium, and T represents tissues of nasopharyngeal carcinoma.
Fig. 2 is the expression of BTRC gene in normal nasopharyngeal epithelium and tissues of nasopharyngeal carcinoma in microarray data, and the expression of BTRC gene in tissues of nasopharyngeal carcinoma is obviously lowered (P=0.001);
N represents normal nasopharyngeal epithelium, and T represents tissues of nasopharyngeal carcinoma.
Fig. 3 utilizes the expression of fluorescence real-time quantitative PCR technical identification BTRC gene in normal nasopharyngeal epithelium and tissues of nasopharyngeal carcinoma, and the expression of BTRC gene in tissues of nasopharyngeal carcinoma is obviously lowered (P<0.05);
N represents normal nasopharyngeal epithelium (totally 9 examples), and T represents tissues of nasopharyngeal carcinoma (totally 28 examples).
Fig. 4 is that immunohistochemical method detects the expression of BTRC in nasopharyngeal carcinoma and normal nasopharyngeal epithelium;
BTRC expression level higher (positive) in normal nasopharyngeal epithelium (Non-tumorNPE), and in 106 routine nasopharyngeal carcinoma (NPC), having 48 examples the low expression (Low) of BRTC to be detected, all the other 58 examples are high expression level (high).
Fig. 5 is that in situ hybridization detects the expression of BTRC in nasopharyngeal carcinoma and normal nasopharyngeal epithelium;
The result of in situ hybridization and immunohistochemistry results have very high consistence.
Fig. 6 is the expression of BTRC in nasopharyngeal carcinoma and the relation of Nasopharyngeal Carcinoma Patients prognosis;
In nasopharyngeal carcinoma the expression of BTRC and the prognosis of Nasopharyngeal Carcinoma Patients closely related, BTRC high expression level (High) though patient's disease free survival time (Diseasefreesurvival, DFS, left) or total survival time (Overallsurvival, OS, right) all obviously to be longer than the patient of the low expression of BTRC (Low).
Fig. 7 is import BTRC over-express vector (BTRCOE) and RNA interference sequence (siBTRC) in nasopharyngeal carcinoma cell HNE2,5-8F and C666-1 after, real time fluorescence quantifying PCR method have detected the expression (mRNA level in-site) of BTRC in nasopharyngeal carcinoma cell, after importing BTRC over-express vector, the expression of BTRC gene significantly raises on (left side), and after importing RNA interference sequence, the expression of BTRC gene significantly reduces on (right side), and NC is negative control (negativecontrol).
Fig. 8 is import BTRC over-express vector (BTRCOE) and RNA interference sequence (siBTRC) in nasopharyngeal carcinoma cell HNE2,5-8F and C666-1 after, Westernbloting method have detected the expression (protein level) of BTRC in nasopharyngeal carcinoma cell, after importing BTRC over-express vector, the expression of BTRC gene significantly raises on (left side), and the expression of BTRC gene significantly reduces (right side) after importing RNA interference sequence, NC is negative control (negativecontrol).
Fig. 9 is import BTRC over-express vector (BTRCOE) and RNA interference sequence (siBTRC) in nasopharyngeal carcinoma cell HNE2,5-8F and C666-1 after, cell-penetrating (transwell) experiment confirms, after the expression of artificial promotion BTRC, significantly can reduce through the nasopharyngeal carcinoma cell number of matrix glued membrane, show cell invasion reduced capability, on the contrary, after the expression of artificial reduction BTRC, significantly can increase through the nasopharyngeal carcinoma cell number of matrix glued membrane, show that cell invasion ability strengthens, NC is negative control (negativecontrol).
Figure 10 is at nasopharyngeal carcinoma cell HNE2, after importing BTRC over-express vector (BTRCOE) and RNA interference sequence (siBTRC) in 5-8F and C666-1, cell scratch experiment confirms, after the expression of artificial promotion BTRC, from cut both sides toward cut, central authorities' travelling speed obviously slows down nasopharyngeal carcinoma cell, the time lengthening of cut healing, show that cell movement transfer ability reduces, on the contrary, after the expression of artificial reduction BTRC, from cut both sides toward cut, central authorities' travelling speed significantly improves nasopharyngeal carcinoma cell, the time shorten of cut healing, show that cell movement transfer ability improves, NC is negative control (negativecontrol).
In order to import BTRC over-express vector (BTRCOE) and RNA interference sequence (siBTRC) in nasopharyngeal carcinoma cell HNE2 and 5-8F, we detect the β TrCP albumen of BTRC genes encoding and the expression of substrate β-catenin and Snail to Figure 11 afterwards, the degraded of β-catenin and Snail is accelerated after the expression of artificial promotion BTRC, in cell, the expression amount of β-catenin and Snail reduces, otherwise the degraded of β-catenin and Snail reduces after the expression of the artificial BTRC of reduction, in cell, the expression amount of β-catenin and Snail strengthens; (epithelial-mesenchymaltransition is changed with epithelial-mesenchymal, EMT) expression of associated protein also there occurs corresponding change, the expression of mark ZO-1, E-cadherin and Claudin-1 of the expression epithelium posterius cell of artificial promotion BTRC raises, and the expression of mark ZEB1, N-cadherin, Vimentin and Slug of mesenchymal cell reduces, show that BTRC can suppress epithelial-mesenchymal to be changed; Otherwise after the expression of artificial reduction BTRC, epithelial cell marker expression reduces, and the marker expression of mesenchymal cell raises, and after showing the low expression of BTRC, cell is changed to interstitial like cell by epithelium, and Invasion and Metastasis ability strengthens.
Figure 12 is the mechanism of action figure of BTRC gene in nasopharyngeal carcinoma generation evolution, the expression of key molecule β-catenin and Snail in BTRC gene controllable epithelial-mesenchymal switching process, during BTRC high expression level, β-catenin and Snail easily degrades, cell maintains Epithelial state, and Invasion and Metastasis is less likely to occur, when BTRC down-regulated expression, β-catenin and Snail is not easy degraded, cell is changed to interstitial sample by Epithelial, easily Invasion and Metastasis occurs, causes patient comparatively fast dead.
Embodiment
The present invention is further illustrated below in conjunction with embodiment, and unrestricted the present invention.
Embodiment 1, utilizes biochip technology to screen and finds that BTRC gene is lowered at nasopharyngeal carcinoma
1. materials and methods:
1) gene chip:
Gene chip used is Agilent company SurePrintG3HumanGeneExpressionv38x60KMicroarray (article No.: G4851C), comprises the probe of the mankind's 27,958 knowns.
2) main agents:
3) purifying of total serum IgE
Collect 6 routine normal nasopharyngeal epithelium and 10 routine tissues of nasopharyngeal carcinomas, by Trizol (invitrogen Products) extracted total RNA, if the purity of total serum IgE is not high, labeling effciency and the chip hybridization results of probe can be affected.So use QIAGEN kit purifying total serum IgE, detailed principle of operation and method are shown in RNeasyMiniProtocol.
1. get total serum IgE≤100 μ g to be dissolved in 100 μ lRNasefree water, add 350 μ lBufferRLT and fully mixing.
2. add 250 μ l dehydrated alcohols, Tip head fully mixes.
3. to proceed to amounting to 700 μ l containing the solution of total serum IgE in the RNeasy pillar that is enclosed within 2ml centrifuge tube, centrifugal 30 seconds of >=8000g, discards filtered solution.
4. draw in 500 μ lBufferRPE to RNeasymini pillars, >=8000g centrifuge washing 30 seconds, discard filtered solution, use 500 μ lBufferRPE at >=8000g centrifuge washing 2min again, discard the sleeve pipe of filtered solution and 2ml, RNeasymini pillar is proceeded in a new 1.5mlEppendorf pipe.
5. the water of 40 μ lRNasefree is drawn, the centrifugal wash-out 1min of >=8000g.
6. repeating step 5. once.
4) cDNA first chain and the second chain one-step synthesis method
1. get 0.2 μ gRNA in 0.2ml centrifuge tube, configure reaction soln as follows:
2. 65 DEG C are incubated 10 minutes, ice bath 5 minutes, note: shift to an earlier date 5 × FirstStrandB μ ffer at 80 DEG C of preheating 3-4 minute
3. following cDNA synthetic system is configured:
4. above-mentioned 4.7 μ lmix are added in the RNA of ice bath after sex change.
5. with the mixing of rifle head, centrifugal afterwards.
6. in PCR instrument:
40℃2hour
70℃15min
movetoice5min
5) fluorescent mark cRNA synthesizes
1. Transcriptionmix is configured as follows:
2. add 6 μ lTranscriptionmix and mix
3. in PCR instrument: 40 DEG C of 2hours.
6) cRNA purifying (QIAGEN miniKit)
Purify cRNA with QIAGENRNeasyMinikit, concrete grammar step is as follows:
1. add 84 μ lRNasefree water, add 350 μ lBufferRLT and fully mixing.
2. add 250 μ l dehydrated alcohols, Tip head fully mixes.
3. to proceed to amounting to 700 μ l containing the solution of total serum IgE in the RNeasy pillar that is enclosed within 2ml centrifuge tube, the centrifugal 15-30sec of >=8000g, discards filtered solution.
4. draw in 500 μ lBufferRPE to RNeasymini pillars, >=8000g centrifuge washing 15-30sec, discard filtered solution, use 500 μ lBufferRPE at >=8000g centrifuge washing 2min again, discard the sleeve pipe of filtered solution and 2ml, RNeasymini pillar is proceeded in a new 1.5mlEppendorf pipe.
5. draw the water of 30 μ lRNasefree, leave standstill the centrifugal wash-out 1min of 1min, >=8000g.
6. repeating step 5 once.
7) cRNA concentration determination
1. cRNA Quality Control: by spectrophotometric analysis RNA concentration.Need to measure in 260 and 280nm concentration and the purity that absorbancy determines sample, A260/A280 should close to 2.0 (between 1.9-2.1)
2. calculate and adjust the content of cRNA:
8) fluorescence molecule concentration and incorporation efficiency calculate:
Cy3-concentration (pmol/ μ l)=A552/0.15
Cy3-incorporation efficiency (pmol/ μ g)=Cy3-concentration/cRNA concentration (μ g/ μ l)
9) cRNA sample fragment and chip hybridization
1. according to the form below preparation fragmentation mixed solution, then carries out fragmentation, ice bath 1min at 60 DEG C of temperature bath 30min
2. 2XGExHybridizationBuffer mixing is added
3. go up chip, 65 DEG C 17 hours, 10rpm rolls hybridization
10) chip washing
1. take out chip to wash 1 minute in washing lotion 1
2. again chip is put into washing lotion 2 and wash 1 minute (37 DEG C)
11) chip scanning: scan in Agilent scanner, resolving power is 3 μm/5 μm, and scanner is automatically with 100% run-down.
12) data analysis: after (Normalize) process being normalized to gene chip raw data with the reference gene in gene chip, the difference expression gene in normal nasopharyngeal epithelium and tissues of nasopharyngeal carcinoma is screened, with the expression map of Genesis Software on Drawing difference expression gene with SAM software (SignificanceAnalysisofMicroarrays).
2. result
Utilize biochip technology, altogether screening obtains difference expression gene 2461 between nasopharyngeal carcinoma and normal nasopharyngeal epithelium, the gene wherein raised at nasopharyngeal carcinoma and 1677, and the gene lowered in nasopharyngeal carcinoma has 784 (Fig. 1).
Wherein BTRC gene is expressed and is significantly lowered (P=0.001, Fig. 2) in tissues of nasopharyngeal carcinoma.
Embodiment 2, quantitative real-time PCR detects and confirms that BTRC gene is lowered at nasopharyngeal carcinoma
1. materials and methods:
Collect 9 routine normal nasopharyngeal epithelium and 28 routine tissues of nasopharyngeal carcinomas, by Trizol (invitrogen Products) extracted total RNA, after 2 μ gRNA SuperscriptFirst-StrandSynthesisSystem Reverse Transcriptase kit (Invitrogen Products) reverse transcriptions become cDNA, carry out with QuantiTectSYBRGreenPCR test kit (Qiagen Products) expression that real-time fluorescence quantitative PCR detects BTRC and reference gene GAPDH.BTRC and GAPDH gene primer is by the synthesis of Invitrogen company, and sequence is as follows:
BTRCforward,5’-CCCCTTCTCGAACATACACCT-3’,
BTRCreverse,5’-AGTCTCAAAGCCCTGCTCCT-3’
GAPDHforward,5’-AACGGATTTGGTCGTATTGG-3’,
GAPDHreverse,5’-TTGATTTTGGAGGGATCTCG-3’
Real-time fluorescence quantitative PCR reaction system
Real-time fluorescence quantitative PCR reactions steps
Reaction terminates amplification curve and the melting curve of rear confirmation real-time fluorescence quantitative PCR, the expression intensity of each sample BTRC gene, according to after CT value (thresholdcyclevalues), reference gene (GAPDH) markization, adopts groupt-test inspection to calculate P value.
2. result
BTRC gene is expressed higher in normal control tissue, and in most of tissues of nasopharyngeal carcinoma down-regulated expression P<0.05 (Fig. 3).
Embodiment 3, immunohistochemical method detects the expression of β TrCP albumen in nasopharyngeal carcinoma of BTRC coding
1. materials and methods
1.1 immunologic combined detection reagent kits and other main agents
BTRC antibody is purchased from CellSignaling company; S-P two step method instant against murine or anti-rabbit (Two-Step tManti-MouseorRabbitDetectionReagent, HRP) immunologic combined detection reagent kit (NovocastraLaboratories, Ltd.); Normal nonimmune rabbit and sheep blood serum are purchased from Beijing Zhong Shan biotech company.PBS damping fluid (pH7.2-7.4, NaCl137mmol/L, KCl2.7mmol/L, Na 2hPO 44.3mmol/L, KH 2pO 41.4mmol/L); 0.01mol/L citrate buffer; 0.1% trypsin solution; 3% methyl alcohol-H 2o 2solution; Micro-array tissue is cut into slices special mounting glue.RPMI1640, DMEM, Lipofectamine, Trizol, ssDNA (Invitrogen); DEPC; Taq enzyme, Proteinase K, RNase (DNasefree), pancreatin (Beijing is magnificent).
1.2 immunohistochemical methodss detect the protein expression of β TrCP albumen in micro-array tissue
1) tissue paraffin section de is after experiment pre-treatment, dewaxing, aquation.
2) PBS washes, 5min × 3.
3) cut into slices into 3%H 2o 2in, block endogenous peroxydase, room temperature 30min.
4) PBS washes, 5min × 3.
5) antigen retrieval: according to position and the antibody specification sheets recommendation restorative procedure of detected albumen, use microwave or enzymic digestion antigen retrieval.During microwave antigen retrieval, TMAs section is placed in 0.01mol/L citrate buffer, after damping fluid boiling, is adjusted to low fire screen, repairs section 20min.During enzymic digestion antigen retrieval, drip 0.1% trypsin solution in section, at 37 DEG C, digest 25min.
6) PBS washes, 5min × 3.
7) normal goats or the rabbit anteserum that drip 300 μ l/TMAs sheets are closed, and room temperature 30min, gets rid of surplus liquid.Drip instant or concentrated primary antibodie (press 1:50 or 1:100,1:200 dilution proportion not etc. by antibody specification sheets) 300 μ l/TMAs, keep section in the wet box sealed, overnight incubation in 4 DEG C of refrigerators.
8) PBS washes, 5min × 3.
9) drip instant two to resist, 300 μ l/TMAs sheets, room temperature (more than 25 DEG C) 1h.
10) PBS washes, 5min × 3.
11) DAB colour developing 5 ~ 20min, controls dye levels under microscope.
12) enter distilled water stopped reaction, distilled water rinses 10min.
13) bush uniformly dyeing redyes 2min, and warm water breaks up.
14) distilled water rinses 10 ~ 15min.
15) dehydration, transparent, mounting rubber seal sheet.
The foundation of 1.3 results observations and detected result database
Under opticmicroscope, the β TrCP protein expression in each routine tissue sample is observed.Two pathology experts carry out result score by following a kind of judging criterion respectively: (1) judges according to positive staining intensity: cell dye-free is 0 point; Cell is light brown, remembers 1 point; Cell is dyed brown, there is no background stainings, or dark-brown, but background is light brown, is moderate positive, remembers 2 points; Cell is dark-brown, without background coloration, is strong positive, remembers 3 points.(2) express number score according to cell positive: no positive cell expressing, count 0 point; Positive expression cell≤25%, counts 1 point; 25% < positive cell number≤50%, counts 2 points; Positive expression cell count more than 50% is strong positive, counts 3 points.Final both score are scored result product score.Result is 0 ~ 3 point and is defined as low expression, and final score is 4 ~ 9 points, is high expression level.
2 results
The β TrCP protein expression level higher (positive) of BTRC coding in normal nasopharyngeal epithelium (Non-tumorNPE), and in 106 routine nasopharyngeal carcinoma (NPC), having 48 examples the low expression (Low) of β TrCP to be detected, all the other 58 examples are high expression level (high).There were significant differences (P<0.05, Fig. 4) in the expression of β TrCP albumen in normal nasopharyngeal epithelium and nasopharyngeal carcinoma.
Embodiment 4, in-situ hybridization method detects and confirms that BTRC gene to be lowered and relevant to Nasopharyngeal Carcinoma Patients poor prognosis at nasopharyngeal carcinoma
1. MATERIALS METHODS
1.1 design and synthesize hybridization probe
In order to the expression adopting in-situ hybridization method to detect BTRC gene, we devise and detect the oligonucleotide probe of BTRC expression and the in situ hybridization oligonucleotide probe of positive control (GAPDH) in situ hybridization, and sequence is as follows:
BTRC probe 1:5'-GTCTAAGTGAATTCTTCTCTGGTATTATCT-3'
BTRC probe 2:5'-GTACTTGTGTTCCATACCTTTATAGTTCTA-3'
BTRC probe 3:5'-ATCTCCAATTAGATTCTATTGTCTCAATGT-3'
GAPDH probe 1:5'-CCACTTTACCAGAGTTAAAAGCAGCCCTGG-3'
GAPDH probe 2:5'-CAGTAGAGGCAGGGATGATGTTCTGGAGAG-3'
GAPDH probe 3:5'-GTCAGAGGAGACCACCTGGTGCTCAGTGTA-3'
Adopt chemical synthesis process to synthesize each gene specific oligonucleotides probe sequence of above-mentioned design, in building-up process middle probe sequence, uridylic has marked vitamin H (bio-U).
1.2 oligonucleotide probe labelling kits and in situ hybridization detection reagent
Digoxin oligonucleotide tailing reagent (DigOligonucleitideTailingKit2 ndgeneration, Roche company), anti-digoxin-horseradish peroxidase complex detection kit (Anti-Digoxigenin-POD, Fabfragments, Roche company), strengthen the TSA signal amplifying system (TSA of expressed in situ detection signal tMbiotinSystem, NEL700 test kit, PerkinElmer company), DAB staining kit (Beijing Zhong Shan company), 20x sodium citrate buffer (salinesodiumcitrate, SSC), T 500 (Dextransulphate), deionized formamide (DeionizedFormamide), polyadenylic acid (polyadenylicacid, PolyA), poly deoxyadenylic acid (polydeoxyadenylicacid, PolydA), the frog essence DNA (denaturedandshearedsalmonspermDNA that sex change is sheared, ssDNA), yeast transfer RNA (yeastt-RNA, tRNA), dithiothreitol (DTT) (DTT), Deng 50x Han Shi damping fluid (Denhardts ' ssolution), phosphoric acid buffer (PBSbuffer), stomach en-K, bovine serum albumin (BSA), trolamine (TEA), TNBBuffer (0.1MTris-HCl, pH7.5, 0.15MNaCL, 0.5%BlockingReagent), TNTBuffer (0.1MTris-HCl, pH7.5, 0.15MNaCL, 0.05%Tween20), acetic anhydride, block reagent (Blockingreagentagent, Roche company).
1.3 other main agents and materials
Dehydrated alcohol, 90% alcohol, 70% alcohol, 50% alcohol, turps, distilled water, PBS damping fluid (pH7.2 ~ 7.4, NaCl137mmol/L, KCl2.7mmol/L, Na 2hPO 44.3mmol/L, KH 2pO 41.4mmol/L); 3% methyl alcohol-hydrogen peroxide solution (80% methyl alcohol and the configuration of 30% hydrogen peroxide); 0.01mol/L citrate buffer (citratebuffer, CB, pH6.0 ± 0.1,9ml0.1M citric acid solution and 41ml0.1M sodium citrate solution to add in 450ml distilled water after provisional configuration again correction work liquid pH value); 0.1% trypsinase; Hematorylin; 1% hydrochloride alcohol (configuration of 1ml concentrated hydrochloric acid+99ml70% alcohol); Mounting glue (PTSCureMount II); Special cap slide (480 × 240mm 2) customize in Zhengzhou Glassware Factory.Leica low melting point (58 DEG C) paraffin, domestic beeswax, raw spirit, dimethylbenzene, 10% neutral paraformaldehyde (0.01mol/L, pH7.4, DEPC distilled water and PBS buffer), Hematorylin, Yihong, neutral mounting natural gum, cover glass, slide glass.1.4 label probe
Utilize 3-tailingDIGOlignucleutideKit to carry out oligonucleotide probe mark, reaction system is as follows.
100pmololigonucleotide+ddH 2O＝9μl(control:controloligonucleutide5μl+ddH 2O4μl)
Mixing, slightly centrifugal.37 DEG C of water-bath 30min, add 2 μ lEDTA (0.2M, PH8.0) stopped reactions.
Purifying after 1.5 oligonucleotide probe marks
In order to increase the purity of label probe, need carry out purifying to the probe marked, concrete operations are as follows:
1) probe reaction mixture (22 μ l)+2.5 cold ethanol of μ l4MLiCl+75 μ l100% (-20 DEG C).
2)-70 DEG C of precipitations 60min, or-20 DEG C of 2h.
3) 13.000 × g4 DEG C of centrifugal 15min.
4) supernatant is abandoned, by 70% (V/V) washing with alcohol that 50 μ l are ice-cold.
5) 13.000xg4 DEG C, centrifugal 5min.
6) supernatant is abandoned, vacuum 4 DEG C of dryings.
7) with the heavy molten probe of aseptic double-distilled water.
1.6 in situ hybridizations detect the expression of EBV-miR-BART10 in file paraffin section
Paraffin section hybridization pre-treatment
1) 4 DEG C of paraffin sections preserved are placed in 58 DEG C of roasting sheet 30min, melted surface paraffin.
2) dimethylbenzene dewaxes 3 × 5min successively.
3) step ethanol wash, 100% alcohol 2 × 2min → 95% alcohol 1 × 5min → 70% alcohol 1 × 5min → 50% alcohol 1 × 5min → DEPC water washing 2 × 3min → DEPC-PBS washs 2 × 5min.
4) 300 μ l stomach en-K (10 μ g/ml) are dripped in section, 37 DEG C of digestion 20min.
5) cut into slices and wash 1min, stopped reaction into PBS (0.1MPBS+2mg/ml L-glutamic acid).
6) cut into slices into 0.2NHCL, in 37 DEG C of reaction 20-30min, increase the permeability of tissue.
7) section fixes 10min, room temperature afterwards with 4% paraformaldehyde (0.1MPBS dissolving).
8) organize positive intensity for hybridization to increase, acetyl process is carried out to section.Cut into slices into 0.25% diacetyl oxide Buffer I (0.1M trolamine), room temperature 10min.
9) 1MPBS washs 2 × 5min.
Prehybridization and hybridization
Prehybridization :-20 DEG C of prehybridization solutions preserved, be first placed in 37 DEG C and hatch 60min, the consumption of prehybridization solution is 50 μ l, and Parafilm carries out lid section, prehybridization 2 hours in 37 DEG C of wet boxes.(prehybridization solution composition comprises: 2XSSC, 10%Dextransulphate, 1XDenhardt ' ssolution, 50mMPhosphateBuffer (PH7.0), 50mMDTT, 250 μ l, 100 μ g/mlpolyA, 5 μ g/mlpolydA, 250 μ g/mlyeastt-RNA, 500 μ g/mlssDNA, 47%Deionizedformamide).
1) remove Parafilm, get rid of prehybridization solution, section is placed in 2 × SSC 5min.
2) hybridization: 37 DEG C of hybridized overnight (18-20h).Each section adds 250 μ l hybridization solutions and carries out lid with Parafilm.Add corresponding probe in prehybridization solution and just become hybridization solution.Hybridization solution is prepared when prehybridization, and place 37 DEG C and hatch, probe is fully dissolved in hybridization solution, and this experiment is mixed with hybridization solution by 500ng/ml concentration and probe concentration.
3) post-hybridization washing, section immersion 2 × SSC, 10min, throws off Parafilm.Shake washing on shaking table successively, 2 × SSC (0.5%SDS), 2 × 15min → 0.25 × SSC (0.5%SDS), 2 × 15min.
Color developing detection reaction after hybridization
1) Anti-Digoxigenin-POD is adopted to detect digoxigenin-probe with mRNA in conjunction with mixture; TSA amplification system strengthens the positive signal of in situ hybridization reaction solution reaction, and DAB develops the color.
2) section goes in TNT damping fluid, 3 × 5min.
3) drip TNB and block damping fluid, 300 μ l/TMAs, room temperature, 30min.
4) unnecessary blocker is sucked, the Anti-Digoxigenin-POD (TBS+0.1%TritonX-100+1% blocker) of 1:100 dilution, room temperature 4 hours.
5) TNTBuffer (0.1MTris-CL, pH7.5,0.15MNaCL, 0.05%Tween20) washing, 3x5min.
6) section drips signal and amplify reagent BiotinylTyamid, 300 μ l/TMAs, (BiotinylTyramid stock solution: BiotinylTyramid is dissolved in 0.2mlDMSO, BiotinylTyramid working fluid: 1 × diluent, 1:50 dilutes BiotinylTyramid stock solution), room temperature 10 minutes.
7) TNT washes, 3 × 5min.
8) section drips SA-HRP (strepto-avidin-horseradish peroxidase), 300 μ l/TMAs, room temperature 30min.
9) TNT washes, 3 × 5min.
10) aquae destillata washing, 1 × 1min.
11) DAB colour developing, controls color reaction under microscope.
12) Hematorylin is redyed,
13) alcohol step dehydration, chip drying.
14) mounting glue is dripped, the cover glass cover plate of dimension, crosslinked section 1min under ultraviolet lamp.
1.7 results judge and standard
Applied optics microscope is observed respectively under low power and high power lens, and first the positive expression signal of object observing RNA is in the intracellular location of object observing: be positioned at nucleus, cytoplasm or cytolemma.
Carry out comprehensive grading with cell count two kinds of standards of the intensity of this detection rna expression position positive signal and positive expression respectively again, judging criterion is: (1) judges according to positive cell dyeing intensity: a. cell dye-free, remembers 0 point; B. cell dyes light brown is the weak positive, remembers 1 point; C. cell is dyed brown and without background coloration, or cell is dyed dark-brown and has light brown background to be moderate positive, remembers 2 points; D. cell is dyed dark-brown and is strong positive without background coloration, remembers 3 points.(2) express number score according to positive cell: the no positive cell expressing of a., remember 0 point; B. positive expression cell count≤25%, remembers 1 point; C.25% < positive cell number < 50%, remembers 2 points; D. positive expression cell count >=50%, remembers 3 points.
In order to reduce the subjective factor of appraisal result as far as possible, carried out separately judging and marking by one of above-mentioned standard respectively by two pathology experts, then both scorings be multiplied, result is: 1. 0 point of person finally counts 0 point, thinks negative and expresses; 2. 1 point and 2 points of persons finally count 1 point, think weak positive expression; 3. 3 points and 4 points of persons finally count 2 points, think that moderate positive is expressed; 4. 6 assign to 9 points of persons and finally count 3 points, think that strong positive is expressed.
1.8 analyze and statistical software
Application SPSS13.0 statistical software carries out statistical analysis to experimental result, compares between two and uses χ 2test or Fisherexacttest, correlation analysis adopts Spearmencorrelation method; P < 0.05 i.e. difference has statistical significance.Survival curve analysis adopts Kaplan-Meiermethod and log-ranktest; Multivariate analysis adopts Cox ' sproportionalhazardsmodel; P < 0.05 i.e. difference has statistical significance.
2 results
Expression in the expression ratio normal control tissue of 2.1BTRC gene in nasopharyngeal carcinoma is significantly lowered
The β TrCP protein expression level higher (positive) of BTRC coding in normal nasopharyngeal epithelium (Non-tumorNPE), and in 106 routine nasopharyngeal carcinoma (NPC), having 48 examples the low expression (Low) of β TrCP to be detected, all the other 58 examples are high expression level (high).There were significant differences (P<0.05, Fig. 5) in the expression of β TrCP albumen in normal nasopharyngeal epithelium and nasopharyngeal carcinoma.The result that in situ hybridization detects is consistent with immunohistochemical result.
The Nasopharyngeal Carcinoma Patients prognosis of the low expression of 2.2BTRC gene is poor
The survival analysis that we carry out the expression of BTRC gene in tissues of nasopharyngeal carcinoma and the survival time of patient and state, find that the expression of BTRC and the prognosis of Nasopharyngeal Carcinoma Patients are closely related in nasopharyngeal carcinoma, namely BTRC high expression level (High) though patient's disease free survival time (Diseasefreesurvival, DFS) or total survival time (Overallsurvival, OS) all obviously to be longer than the patient (Fig. 6) of the low expression of BTRC (Low).
Embodiment 5, the invasion inhibition of process LAN BTRC gene inhibition nasopharyngeal carcinoma in nasopharyngeal carcinoma cell, and the invasion inhibition of nasopharyngeal carcinoma, its mechanism are by its substrate β-catenin and the conversion of Snail regulating cell epithelial-mesenchymal to disturb the expression of BTRC gene to promote
1. MATERIALS METHODS
1.1 reagent and test kit
Restriction enzyme NheI and EcoRI and T4DNA ligase enzyme etc. are purchased from TakaRa company; TRIZOL tMreagent (Invitrogen); Plasmid extraction test kit, glue reclaim test kit (OMEGA); Reverse Transcriptase kit (Promega); Proteinase K, DNaseI, RNAsin, RNaseA (GBICOL company).
The structure of 1.2pcDNA3.1 – BTRC carrier for expression of eukaryon
We select pcDNA3.1 empty vectors (deriving from Invitrogen company) to build the over-express vector of BTRC.We select NheI and EcoRI restriction enzyme site cut pcDNA3.1 carrier for enzyme and BTRC sequence is inserted this site.
Build pcDNA3.1 – BTRC eukaryotic vector step as follows:
1) with the cDNA of nasopharyngeal carcinoma cell HNE2 for template, utilize TaKaRaLA enzyme carries out pcr amplification total length BTRC coding region (CDS) sequence (see sequence table SEQ NO:1).BTRC coding region sequence amplimer is as follows:
Upstream primer: 5 '-ATGGACCCGGCCGAGGCGGT-3 '
Downstream primer: 5 '-TTATCTGGAGATGTAGGTGTATGTT-3 '
After 5 ' end of upstream and downstream primer adds restriction enzyme NheI and EcoRI recognition site and protection base respectively, primer sequence is as follows:
Upstream: 5 '-AGGA gCTAGCaTGGACCCGGCCGAGGCGGT-3 ' (underscore part is NheI recognition site)
Downstream: 5 '-ATGC gAATTCtTATCTGGAGATGTAGGTGTATGTT-3 ' (underscore part is EcoRI recognition site)
2) pcr amplification, BTRC coding region sequence, PCR reaction conditions is as follows:
PCR reactions steps
3) PCR primer electrophoresis, glue reclaimed through NheI and EcoRI double digestion rear electrophoresis after object fragment, glue reclaims again.
4) pcDNA3.1 plasmid reclaims object fragment through NheI and EcoRI double digestion rear electrophoresis glue.
5) 4 are connected with T4DNA ligase enzyme) and 5) step glue recovery product, the vector plasmid that can be used for eukaryotic expression BTRC can be obtained.
6) by the 5th) the pcDNA3.1 eukaryon expression plasmid transform competent E. coli comprising BTRC coding region sequence that obtains of step, with the plasmid that increases.
The RNA interference sequence (siBTRC) of 1.3BTRC gene is as follows:
5'-CCCAGGGACUGGCGCACUCdTdT-3'
Synthesized by chemical synthesis by Invitrogen company.
1.4 nano silicon particles preparing poly-lysine bag quilt
The nano silicon particles of poly-lysine bag quilt uses OP-10/ hexanaphthene/ammonia microemulsion self-assembling technique to carry out nano silicon particles (silicananoparticle, SiNP) synthesis, and utilize nano silicon particles surface energy and by ion electrostatic interaction, prepare the nano silicon particles of polylysine modification; Described nano particle can be prepared by following methods:
1) OP-10 (polyoxyethylene nonylphenol ether), hexanaphthene and ammoniacal liquor are mixed, the different ester of positive silicic acid (TEOS) is added after stirring at room temperature is even, continue to be stirred to polymerization to complete, add equal-volume acetone, ultrasonic disperse, centrifugal, distilled water washs three times, centrifugal collecting precipitation is in 80 DEG C of dryings, and porphyrize obtains nano silicon particles (SiNP, particle size range 10-50nm).Wherein H 2o and OP-10 and H 2the mol ratio of O and TEOS is 2 ~ 10, ammonia concn is 1.6 ~ 28%, the volumetric molar concentration of TEOS in hexanaphthene is 0.1 ~ 3mol/L.
2) SiNP is resuspended in 0.6MNaCO by 0.1 ~ 10mg/ml 3in solution, ultrasonic disperse, centrifugal, abandon supernatant, then be resuspended in PBS (pH7.4) by throw out by 0.1 ~ 10mg/ml, ultrasonic disperse, add poly-lysine (final concentration is 4 ~ 15nmol/mL), fully mix, room temperature is mixed shakes; Centrifugal, abandon supernatant, precipitate and be resuspended in distilled water by 0.1 ~ 10mg/ml, obtain the nano silicon particles of polylysine modification.
3) by modified nano silicon particles ultrasonic disperse, every milliliter of nano particle suspension adds the RNA interference sequence (siBTRC) of 10 ~ 50ugBTRC expression vector or BTRC, mixing, and room temperature leaves standstill and makes it combine.
1.5 cell cultures and transfection
Nasopharyngeal Carcinoma Cell Line HNE2,5-8F and C666-1 are purchased from Central South University's cell centre, and cell cultures RPMI1640 used trains base and foetal calf serum, and peptic cell trypsinase used is U.S. Gibco Products.
By good for growth conditions nasopharyngeal carcinoma cell HNE2,5-8F and C666-1 by 2 × 10 5individual cells/well is inoculated in 6 orifice plates, 6 orifice plates is placed in 37 DEG C, 5%CO 2in incubator, treat that culturing cell grows to the transfection that 50-70% density can start BTRC expression vector or RNA interference sequence; Transfection process is as follows:
In aseptic EP pipe, add the nano silicon particles suspension carrying the polylysine modification of BTRC eukaryon expression plasmid or siBTRC that 100 μ l prepare, mix with 100 μ l serum free medium gentlenesses; With D-Hank's liquid washed cell 3 times; 800 μ l serum free mediums (antibiotic-free) will be added in said mixture, after gentle mixing, add 1 hole in 6 orifice plates; 6 orifice plates are placed in CO 2in incubator, cultivate 6 hours, then abandon supernatant for 37 DEG C, add perfect medium and continue overnight incubation.With carrying the nano silicon particles of polylysine modification of pcDNA3.1 empty carrier as experiment contrast.
1.6 real-time quantitative PCRs are detected the effect expressed or disturb BTRC and express:
After cell transfecting success, extracted total RNA, reverse transcription, quantitative real-time PCR detects BTRC and expresses, and method and step are with embodiment 2.
1.7Westernblotting is detected and expresses or disturb the effect of BTRC expression and the expression of downstream molecules thereof:
Westernblotting detects the β TrCP albumen of BTRC coding, the substrate β-catenin of β TrCP and Snail, epithelial-mesenchymal conversion associated molecule, comprise epithelial mark ZO-1, E-cadherin, Claudin-1, mark ZEB1, N-cadherin, Vimentin, Slug of mesenchymal cell, and the antibody that reference gene GAPDH expresses is purchased from CellSignalingTechnology company.Collecting cell after nasopharyngeal carcinoma cell transfection BTRC carrier for expression of eukaryon or RNA interference sequence, the total protein in extracting cell, conventional Westernblotting method detects the expression of above-mentioned albumen, and GAPDH is as reference gene.
1.8 cell-penetrating experiments
((transwell) experiment is the experimental technique of checking tumor cell invasion ability to cell-penetrating.Transwell cell (8 μm, aperture) and matrigel (Matrigel) purchased from American BD company, 4% paraformaldehyde stationary liquid, Viola crystallina dye liquor (0.1%g/ml) available from Sigma.Matrigel is pressed 1:8 dilution, be coated on the face, upper room of Transwell cell bottom film, put 37 DEG C and make Matrigel aggregate into gel in 30 minutes.Matrigel film water is carried out by BD company specification sheets before using.
Serum free medium and 1 × 10 is added on each Transwell cell upper strata 5the individual transfection nasopharyngeal carcinoma cell of BTRC over-express vector or interference sequence (siBTRC), with the cell of transfection empty vectors in contrast.The substratum containing 20% foetal calf serum is added in Transwell cell lower floor.Cell continued cultivation after 36 hours, fixes, violet staining, dab off the non-migrating cell in upper strata, wash 3 times with PBS with cotton swab with 4% paraformaldehyde stationary liquid.Examine under a microscope the nasopharyngeal carcinoma cell through matrix glued membrane.
1.9 cell scratch experiments
Cell scratch experiment is the experimental technique of checking tumor cell migration ability.The nasopharyngeal carcinoma cell of transfection BTRC over-express vector or interference sequence (siBTRC) is inoculated in 6 orifice plates, with the cell of transfection empty vectors in contrast.When cell density reaches 90%, in each 6 orifice plates, (cut) is drawn a straight line with 200ulpipet, cut healing state is examined under a microscope subsequently at each time point (depending on different cell migration ability) such as 0,8,12,16,24,32,48 hour, take pictures, and calculate each group of cell migration rates.
2. result
2.1 in nasopharyngeal carcinoma cell successfully process LAN or disturb the expression of BTRC gene
Real-time fluorescence quantitative PCR detects and confirms, in nasopharyngeal carcinoma cell HNE2,5-8F and C666-1 after transfection BTRC over-express vector, the expression of BTRC gene in nasopharyngeal carcinoma cell significantly raises (Fig. 7 is left), and in nasopharyngeal carcinoma cell HNE2,5-8F and C666-1 transfection BTRC interference sequence after, the expression of BTRC gene in nasopharyngeal carcinoma cell significantly reduces (Fig. 7 is right), shows BTRC over-express vector or RNA interference sequence transfection success.
Further, after we use Westernblotting technology for detection process LAN BTRC process LAN or interference BTRC genetic expression, the expression of the albumen β TrCP of its coding, consistent (Fig. 8) that result and real-time fluorescence quantitative PCR detect.
2.2 cell invasion ability reductions after process LAN BTRC in nasopharyngeal carcinoma cell, after interference BTRC expresses, cell invasion ability strengthens
For import BTRC over-express vector (BTRCOE) and RNA interference sequence (siBTRC) in nasopharyngeal carcinoma cell HNE2,5-8F and C666-1 after, cell-penetrating (transwell) experiment confirms, after the expression of artificial promotion BTRC, significantly can reduce through the nasopharyngeal carcinoma cell number of matrix glued membrane, show cell invasion reduced capability, on the contrary, after the expression of artificial reduction BTRC, significantly can increase through the nasopharyngeal carcinoma cell number of matrix glued membrane, show that cell invasion ability strengthens (Fig. 9).
2.3 cell migration ability reductions after process LAN BTRC in nasopharyngeal carcinoma cell, after interference BTRC expresses, cell migration ability strengthens
For at nasopharyngeal carcinoma cell HNE2, after importing BTRC over-express vector (BTRCOE) and RNA interference sequence (siBTRC) in 5-8F and C666-1, cell scratch experiment confirms, after the expression of artificial promotion BTRC, from cut both sides toward cut, central authorities' travelling speed obviously slows down nasopharyngeal carcinoma cell, the time lengthening of cut healing, show that cell movement transfer ability reduces, on the contrary, after the expression of artificial reduction BTRC, from cut both sides toward cut, central authorities' travelling speed significantly improves nasopharyngeal carcinoma cell, the time shorten of cut healing, show that cell movement transfer ability improves (Figure 10).
2.4 in nasopharyngeal carcinoma cell model, we confirm that BTRC passes through its substrate β-catenin and Snail and regulates and controls epithelial-mesenchymal conversion, thus affect Invasion and Metastasis and the prognosis of nasopharyngeal carcinoma
In nasopharyngeal carcinoma cell HNE2 and 5-8F, importing BTRC over-express vector (BTRCOE) and RNA interference sequence (siBTRC), we detect the β TrCP albumen of BTRC genes encoding and the expression of substrate β-catenin and Snail afterwards, the degraded of β-catenin and Snail is accelerated after the expression of artificial promotion BTRC, in cell, the expression amount of β-catenin and Snail reduces, otherwise the degraded of β-catenin and Snail reduces after the expression of the artificial BTRC of reduction, in cell, the expression amount of β-catenin and Snail strengthens; (epithelial-mesenchymaltransition is changed with epithelial-mesenchymal, EMT) expression of associated protein also there occurs corresponding change, the expression of mark ZO-1, E-cadherin and Claudin-1 of the expression epithelium posterius cell of artificial promotion BTRC raises, and the expression of mark ZEB1, N-cadherin, Vimentin and Slug of mesenchymal cell reduces, show that BTRC can suppress epithelial-mesenchymal to be changed; Otherwise after the expression of artificial reduction BTRC, epithelial cell marker expression reduces, and the marker expression of mesenchymal cell raises, and after showing the low expression of BTRC, cell is changed to interstitial like cell by epithelium, and Invasion and Metastasis ability strengthens (Figure 11).
Thus, we tentatively depict the mechanism of action figure (Figure 12) of BTRC gene in nasopharyngeal carcinoma generation evolution, the expression of key molecule β-catenin and Snail in BTRC gene controllable epithelial-mesenchymal switching process, during BTRC high expression level, β-catenin and Snail easily degrades, cell maintains Epithelial state, Invasion and Metastasis is less likely to occur, when BTRC down-regulated expression, β-catenin and Snail is not easy degraded, cell is changed to interstitial sample by Epithelial, easy generation Invasion and Metastasis, causes patient comparatively fast dead.

Claims (10)

  1. The application method of 1.BTRC gene, is characterized in that, for the preparation of the preparation predicting recurrent nasopharyngeal carcinoma and transfer.
  2. 2. application method according to claim 1, is characterized in that, the expression of described BTRC gene and recurrent nasopharyngeal carcinoma and translate into negative correlation.
  3. 3. application method according to claim 1 and 2, is characterized in that, described prediction recurrent nasopharyngeal carcinoma and the preparation of transfer comprise: PCR detection reagent, in situ hybridization detection reagent or immunohistochemical reagents.
  4. 4. application method according to claim 3, is characterized in that, PCR detection reagent is fluorescence real-time quantitative PCR reagent.
  5. 5. application method according to claim 4, is characterized in that, the primer of fluorescence real-time quantitative PCR reagent place comprises:
    BTRCforward,5’-CCCCTTCTCGAACATACACCT-3’,
    BTRCreverse,5’-AGTCTCAAAGCCCTGCTCCT-3’
    GAPDHforward,5’-AACGGATTTGGTCGTATTGG-3’,
    GAPDHreverse,5’-TTGATTTTGGAGGGATCTCG-3’。
  6. 6. application method according to claim 3, is characterized in that, the oligonucleotide probe of in situ hybridization detection reagent and the oligonucleotide probe of positive control, and sequence is as follows:
    BTRC probe 1:5'-GTCTAAGTGAATTCTTCTCTGGTATTATCT-3'
    BTRC probe 2:5'-GTACTTGTGTTCCATACCTTTATAGTTCTA-3'
    BTRC probe 3:5'-ATCTCCAATTAGATTCTATTGTCTCAATGT-3'
    GAPDH probe 1:5'-CCACTTTACCAGAGTTAAAAGCAGCCCTGG-3'
    GAPDH probe 2:5'-CAGTAGAGGCAGGGATGATGTTCTGGAGAG-3'
    GAPDH probe 3:5'-GTCAGAGGAGACCACCTGGTGCTCAGTGTA-3'.
  7. The application method of 7.BTRC gene, is characterized in that, for the preparation of the preparation preventing recurrent nasopharyngeal carcinoma and transfer.
  8. 8. the application method of BTRC gene according to claim 7, is characterized in that, for the preparation of the preparation of the substrate β-catenin of BTRC gene expression product β-TrCP and the degraded of Snail.
  9. 9. application method according to claim 8, is characterized in that, for the preparation of suppression epithelial-mesenchymal conversion preparation.
  10. 10. application method according to claim 9, it is characterized in that, promote that the expression of BTRC gene can make the expression of epithelial mark ZO-1, E-cadherin and Claudin-1 raise, and the expression of mark ZEB1, N-cadherin, Vimentin and Slug of mesenchymal cell reduces.
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