CN105861705A - Biological marker for evaluating EB virus-positive nasopharyngeal carcinoma DNA (Deoxyribonucleic Acid) damage repair capacity and radiation therapy prognosis and application thereof - Google Patents
Biological marker for evaluating EB virus-positive nasopharyngeal carcinoma DNA (Deoxyribonucleic Acid) damage repair capacity and radiation therapy prognosis and application thereof Download PDFInfo
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Abstract
The invention discloses a biological marker for evaluating EB virus-positive nasopharyngeal carcinoma DNA (Deoxyribonucleic Acid) damage repair capacity and radiation therapy prognosis and application thereof. The biological marker is phosphorylated DNA-PK (Protein Kinase) and AMPK (Adenosine Monophosphate Protein Kinase), preferably phosphorylated DNA-PK Thr2609 and AMPK Thr172. The biological marker can be used for preparing a detection drug and a kit for evaluating the EB virus-positive nasopharyngeal carcinoma DNA damage repair capacity and the radiation therapy prognosis.
Description
Technical field
The present invention relates to biomarker, specifically will participate in the DNA protein kinase (DNA-activated of DNA damage reaction
Protein kinase, DNA-PK) and the protein kinase (AMP-activated protein kinase, AMPK) of AMP activation
Phosphorylation level is as evaluating Epstein-Barr virus (Epstein Barr virus, EBV) positive nasopharyngeal carcinoma's DNA damage reparation energy
Power and the biomarker of radiotherapy prognosis.
Background technology
DNA damage reaction (DNA damage response, DDR) is detected by a plurality of signal transduction pathway and transmits DNA
Damage signal, active cell cycle check point, carry out DNA damage reparation or active cell apoptosis, maintain stablizing of genome
Property and intracellular stable state.DNA Damage repair ability defect is the important mechanisms of tumor invasion.DNA damage is not repaired
Or repair the unstability that may cause genome mistakenly, lead oncogenic generation；The exception of DNA damage reparation simultaneously relates to
And to the tumour sensitiveness to chemicotherapy.Achievement to the research of DNA damage repair mechanism is being converted into the new target of oncotherapy
Point.
The fracture of DNA double chain is the most serious a kind of DNA damage of eukaryotic, be also ionising radiation to produced by chromosome
Crucial fatal injury.Nonhomologous end restructuring (nonhomologous end-joining, NHEJ) is human somatic cell
The main path of DNA plerosis double-strand break, DNA-PK is the central element of NHEJ.
AMPK is a serine threonine kinases, and its major function is the change of permissive cell energy state, maintains cell
Metabolic homeostasis.Up-to-date research finds that AMPK has important function in DDR, has DNA-PK's and AMPK in kinds of tumors
Unconventionality expression and both interactions.
Nasopharyngeal carcinoma (nasopharyngeal carcinoma, NPC) is the malignant tumour that China is common, and annual morbidity is (10
25)/100,000, the death rate accounts for the 2.81% of the national malignant tumor death rate, wherein Epstein-Barr virus (Epstein Barr
Virus, EBV) positive nasopharyngeal carcinoma accounts for more than 80%.
EBV belongs to mankind's gamma herpes viruses, is first and is proved the DNA tumorigenesis disease directly related with human tumor morbidity
Poison.Ebv infection related neoplasms accounts for the 1.5% of whole world malignant tumour, accounts for the 5.6% of all infection associated tumors, mesh
Before there is no EBV vaccine.Ebv infection is the Important cause of disease of nasopharyngeal carcinoma morbidity.
The memebrane protein EBV-LMP1 that the EBV latent infection phase encodes is important oncogene.EBV-LMP1 mediation host cell
Propagation, invasion and attack are shifted and to anti-apoptotic, remarkably promote the Radioresistance of tumour cell.Illustrate EBV-LMP1 DNA is damaged
The Regulation Mechanism of traumatic response, contributes to finding treatment of nasopharyngeal carcinoma and the novel targets of prognosis.
Summary of the invention
It is contemplated that overcome the deficiencies in the prior art, it is provided that evaluate Epstein-Barr virus positive nasopharyngeal carcinoma's DNA damage repair ability and
The biomarker of radiotherapy prognosis and application.Described biomarker is DNA-PK and AMPK of phosphorylation, preferably phosphorus
The DNA-PK Thr2609 and AMPK Thr172 of acidifying.Described biomarker can be used for preparation and evaluates Epstein-Barr virus positive nose
Pharynx cancer DNA damage repair ability and radiotherapy prognosis detect medicine and kit.
The invention will be further described below:
Present invention determine that the molecular mechanism that EBV-LMP1 regulation and control nasopharyngeal carcinoma DNA damage reaction is new, specify DNA-PK and AMPK
Reducing and EBV positive nasopharyngeal carcinoma's DNA damage repair ability and the correlation of Radiotherapy for Nasopharyngeal Carcinoma prognosis of phosphorylation level,
Evaluate both feasibilities as new biomarker.
The present invention utilizes γ-H2AX analytical technology to detect nasopharyngeal carcinoma cell DNA double chain fracture restoration ability, finds EBV-LMP1
The DNA double chain fracture restoration ability of positive cell is substantially suppressed.
By the central element DNA-PK protein phosphorylation horizontal detection to DNA double chain fracture restoration, find that EBV-LMP1 can
To significantly reduce the DNA Damage repair ability of DNA-PK Thr2609 phosphorylation level and mediation thereof；EBV-LMP1 simultaneously
Reduce the combination of DNA-PK Yu AMPK, make nasopharyngeal carcinoma cell AMPK Thr172 phosphorylation level reduce, after activation AMPK
Can significantly reverse the Radioresistance of EBV-LMP1 positive nasopharyngeal carcinoma's cell.
The γ H2AX horizontal compared with normal nasopharyngeal epithelium in tissues of nasopharyngeal carcinoma significantly raises to use ImmunohistochemistryMethods Methods to find, and
DNA-PK Thr2609 phosphorylation level substantially reduces, and both exist negative correlation, prompting DNA-PK Thr2609 level
Reduce the DNA damage repair ability inhibiting EBV positive nasopharyngeal carcinoma's cell.
Also significantly reduce with the AMPK Thr172 phosphorylation level in ImmunohistochemistryMethods Methods detection tissues of nasopharyngeal carcinoma, DNA-PK
There is negative correlation in all expression with EBV-LMP1 of Thr2609 Yu AMPK Thr172 phosphorylation level；To DNA-PK
5 years survival rates after Thr2609 and AMPK Thr172 and Radiotherapy for Nasopharyngeal Carcinoma carry out correlation analysis discovery, AMPK
5 years survival rates of Thr172 negative patient significantly reduce, and prompting AMPK Thr172 phosphorylation level is to evaluate Nasopharyngeal Carcinoma Patients
One new biomarker of radiotherapy prognosis.
In a word, during the research of the present invention finds nasopharyngeal carcinoma cell, DNA double chain fracture restoration ability is substantially less than in normal nasopharyngeal
Skin, the expression of DNA-PK Thr2609 and AMPK Thr172 simultaneously reduces, and the latent membrane protein encoded with EBV
1 (latent membrane protein 1, LMP1) level is negative correlation.EBV-LMP1 is to DNA-PK Thr2609 phosphoric acid
The suppression changed is the important molecule basis that cell DNA double-strand break repair ability reduces, and is combined DNA-PK with AMPK and presses down
System is one of reason of the low expression of AMPK Thr172, and activation AMPK can reverse the nasopharyngeal carcinoma cell of EBV-LMP1 mediation to be put
Penetrate 5 years survival rates after resistance, AMPK Thr172 level and Patients Treated by Radiotherapy and there is significant positive correlation.Therefore, nasopharynx
In cancerous tissue, the low expression of DNA-PK Thr2609 may be used for assessing DNA double chain fracture restoration ability, AMPK Thr172 phosphorus
Acidifying level may be used for predicting the radiotherapy prognosis of Nasopharyngeal Carcinoma Patients.
Accompanying drawing explanation
Fig. 1 uses Western Blot detection γ-H2AX to evaluate EBV-LMP1 to nasopharyngeal carcinoma cell DNA double chain fracture restoration
The impact of ability, the γ-H2AX level relatively CNE1 cell of A display CNE1-LMP1 cell raises；B shows HNE2-LMP1
γ-H2AX level relatively HNE2 the cell of cell raises；
Fig. 2 uses Western Blot detection stably express LMP1 and knock out the nasopharyngeal carcinoma cell phosphorylated cdna-PK of LMP1
The level of Thr2609, the DNA-PK Thr2609 level relatively CNE1 cell of A display CNE1-LMP1 cell reduces, and knocks out
Raise after LMP1；The DNA-PK Thr2609 level relatively HNE2 cell of B display HNE2-LMP1 cell reduces, and knocks out LMP1
Rear rising；
Fig. 3 uses the combination of nasopharyngeal carcinoma cell DNA-PK Yu AMPK of co-immunoprecipitation method detection DNA damage induction, A
The combination relatively CNE1 Leukopenia of DNA-PK Yu AMPK in display CNE1-LMP1 cell；In B display HNE2-LMP1 cell
The combination of DNA-PK Yu AMPK relatively HNE2 Leukopenia；C display EBV-LMP1 suppression nasopharyngeal carcinoma cell DNA damage induction
AMPK Thr172 phosphorylation；
Fig. 4 uses Clone forming Test, to EBV-LMP1 positive nasopharynx after the survival curve detection activation AMPK of drafting cell
The impact of cancer cell radiosensitivity, A display melbine remarkably promotes the radiosensitivity of CNE1-LMP1 cell；B shows
Show that melbine remarkably promotes the radiosensitivity of HNE2-LMP1 cell；
Fig. 5 uses ImmunohistochemistryMethods Methods detection nasopharyngeal carcinoma and normal nasopharyngeal epithelium EBV-LMP1, γ-H2AX, DNA-PK
The expression of Thr2609 and AMPK Thr172.Wherein case1 is normal nasopharyngeal epithelium, case2 and case3 is respectively
It is low expression EBV-LMP1 and the tissues of nasopharyngeal carcinoma of high expressed EBV-LMP1；
The ImmunohistochemistryResults Results that Fig. 6 expresses according to tissues of nasopharyngeal carcinoma DNA-PK Thr2609 and AMPK Thr172, by patient
It is divided into negative and positive expression group, uses Kaplan Meier method to analyze the relation of itself and 5 years survival rates of patient respectively.
Detailed description of the invention
Nasopharyngeal Carcinoma Cell Line CNE1 cell used by the present invention is that Tumour Inst., Chinese Medical Academy builds and is, CNE1-LMP1,
HNE2, HNE2-LMP1 cell is built by Central South University's tumour.
Embodiment 1 EBV-LMP1 suppresses nasopharyngeal carcinoma cell DNA double chain fracture restoration
H2AX albumen is the member in eukaryotic in H2A histone family, and its carboxyl terminal includes one 139 for silk ammonia
Serine one glutamine one glutamic acid (Ser Gin Glu, the SQE) domain of acid residue, it is possible to by PI3K kinases man
Race includes ATM, ATR, DNA-PK phosphorylation, i.e. γ-H2AX.H2AX Ser139 position phosphorylation is ionising radiation or chemistry
Medicine causes the earliest events that DNA double chain ruptures, and forms γ-H2AX foci at breaking part, recruits DNA damage identification
With reparation albumen.It is now recognized that γ-H2AX is one of detection DNA double chain fracture-sensitive and the highest index.Logical
Cross the detection of the Western Blot to γ-H2AX, specify the EBV-LMP1 shadow to nasopharyngeal carcinoma cell DNA double chain fracture restoration
Ring.
Experimental technique is as follows: nasopharyngeal carcinoma cell is cultivated in 6 orifice plates, when merging to 70～80%, uses the x-ray of 6-MV to shine
Penetrating 4Gy with inducing DNA double-strand break, 0.5h, 1h, 4h and 24h extract total protein of cell after irradiation, are placed in-20 DEG C
Frozen.The Western Blot carrying out γ-H2AX after each time point collecting protein completes analyzes.
Result shows, 0.5h, 1h, 4h and 24h after roentgen radiation x, the γ H2AX in 2 strain EBV-LMP1 positive cells
Level relatively EBV-LMP1 negative cells raises, and prompting EBV-LMP1 can suppress the DNA double chain fracture of nasopharyngeal carcinoma cell to repair
Multiple.Result is shown in Fig. 1.
Embodiment 2 EBV-LMP1 suppresses nasopharyngeal carcinoma cell DNA-PK phosphorylation
The phosphorylation of DNA-PK Thr2609 is its key played a role in DNA double chain fracture restoration, by stable table
Reach LMP1 and knock out the Western Blot detection of DNA-PK Thr2609 in LMP1 nasopharyngeal carcinoma cell, specifying EBV-LMP1
Regulation and control to nasopharyngeal carcinoma cell DNA-PK phosphorylation.
Experimental technique is as follows: nasopharyngeal carcinoma cell is cultivated in 6 orifice plates, the siRNA of EBV-LMP1 positive cell transfection LMP1
Rear continuation is cultivated 48 hours, then uses the roentgen radiation x 4Gy of 6-MV, and 0.5h extracts total protein of cell after irradiation, enters
The Western Blot of row DNA-PK Thr2609 analyzes.
Result shows, in 2 strain EBV-LMP1 positive cells, EBV-LMP1 can suppress the phosphoric acid of DNA-PK Thr2609
Change, use the siRNA of selectively targeted LMP1 to reduce the phosphorylation promoting DNA-PK Thr2609 after it is expressed.Result
See Fig. 2.
The combination of embodiment 3 EBV-LMP1 suppression nasopharyngeal carcinoma cell DNA-PK Yu AMPK, reduces the phosphorylation of AMPK
Use the physical bond of DNA-PK Yu AMPK of co-immunoprecipitation method detection DNA damage induction and EBV-LMP1 pair
The regulation and control of this combination, by the AMPK Thr172 phosphorylation of Western Blot detection DNA damage induction.
Experimental technique is as follows: nasopharyngeal carcinoma cell is cultivated in 10cm culture dish, when cell reaches the fusion of 90%, gives
The roentgen radiation x 4Gy of 6-MV, 0.5h extracts total protein of cell after irradiation, first carries out pre cleaning and splits to remove holoprotein
Solve the nonspecific proteins that can combine in liquid with pearl: 1. take A-sepharose bead suspension 20ul, on centrifugal segregation
Clearly, 1 ice-cold × PBS washes three times.The most each experimental group prepares holoprotein lysate 1000ug, adds 20ul the most cleaned
Pearl, 4 DEG C turn and shake 1-2h, and 4 DEG C of centrifugal 14000g × 10min, transfer supernatant is in new EP pipe.Then enter
Row co-immunoprecipitation: 1. adding 2ugDNA-PK antibody in supernatant, the mixed liquor of 4 DEG C of upper cleer and peaceful antibody of shaking is overnight.②
Mixed liquor always adds A-sepharose pearl cleaned for 20ul, 4 DEG C of shaking 2h.3. A-sepharose pearl is collected
Son, 12000rpm is centrifuged 5S, removes supernatant, and pearl IP lysis buffer cleans 5 times.The most resuspended pearl is at 20ul
In 5 × loading buffer, mixing.5. pearl boils 5min, and dissociate the albumen on pearl, and centrifugal 14000g ×
5min.6. transfer supernatant is in new EP pipe, loading, PAGE gel electrophoresis.After taking irradiation, 0.5h extracts simultaneously
Total protein of cell carries out the Western Blot detection of AMPK Thr172.
Result shows, in nasopharyngeal carcinoma cell, the DNA double chain fracture caused by x-ray can be with the physics of inducing DNA-PK Yu AMPK
In conjunction with, EBV-LMP1 significantly suppress this combination, decreases the phosphoric acid of the AMPK Thr172 of DNA damage induction simultaneously
Change.Result is shown in Fig. 3.
Embodiment 4 promotes EBV-LMP1 positive nasopharyngeal carcinoma's cellular radiosensitivity after activating AMPK
Using Clone forming Test, calculate the CNN surviving fraction of cell and draw survival curve, detection melbine activates
Impact on EBV-LMP1 positive nasopharyngeal carcinoma's cellular radiosensitivity after AMPK.
Experimental technique is as follows: take the cell of exponential phase of growth, and 0.25% trypsinization becomes single cell suspension.Gradient multiple dilutions
By 102～105Individual cell seeding in 6 orifice plates of diameter 3CM, the corresponding different exposure dose of different cell number.Each dose
Amount sets 3 parallel holes.1 hour training base of pre-irradiation adds melbine (5 μMs), uses Siemens primus.E
Linear accelerator, close rate 2Gy/min, cover 1～1.5cm thickness tissue glue during irradiation.Cell accepts irradiation
After, continuing to cultivate 10～14 days, naked eyes terminate cultivating when seeing Clone formation.Abandon culture medium, wash 2 times with PBS.Methyl alcohol
Fixing 15min, violet staining 10min, flowing water rinses dyeing liquor, and air is dried.Count the clone in each culture hole
Number, 50 cell number of ＞ are a clone, calculate every class mean.Calculate the CNN surviving fraction (surviving of every kind of cell
Fraction, SF) value.Utilizing Graphpad prism software, the survival using one-hit multitarget every kind of cell of models fitting is bent
Line.
Result shows: the activator melbine of AMPK can substantially reduce the CNN surviving fraction of cell, promotes EBV-LMP1 sun
The radiosensitivity of property nasopharyngeal carcinoma cell, result is shown in Fig. 4.
Embodiment 5 DNA-PK Thr2609 phosphorylation level reduces suppression EBV-LMP1 positive nasopharyngeal carcinoma's tissue DNA injury repair
Ability
The method using SABC detects in the tissue samples of 60 example nasopharyngeal carcinoma and 15 example normal nasopharyngeal epitheliums
EBV-LMP1, γ-H2AX, DNA-PK Thr2609 and the expression of AMPK Thr172, specify it in normal nasopharyngeal
Skin and the differential expression in tumour.
Experimental technique is as follows: toasted 30 minutes in 80 DEG C of insulating boxs by organization chip, is subsequently placed in turpentine oil immersion 5
Minute, soak 5 minutes again after changing turpentine oil；It is sequentially placed in absolute ethyl alcohol, 95% ethanol and 80% ethanol each immersion 5 again
Minute.Then chip is placed in 0.01M sodium citrate cushioning liquid (pH6.0) put into micro-wave oven high fire heating and put boiling,
Then in using instead, low fire continues heating 10～15 minutes.PBS washes 5 minutes × 3 times, drips 3%H2O2Middle room temperature stands 10 points
Clock, PBS washes 5 minutes × 3 times, and dropping one resists 50 μ L, and 4 DEG C of wet boxes are overnight.37 DEG C of rewarmings 45 minutes, PBS washes 5
Minute × 3 times, dropping two resists 40～50 μ L, and room temperature stands 1 hour, and PBS washes 5 minutes × 3 times, DAB colour developing 5～10
Minute (grasping dye levels under the microscope), running water rinses 10 minutes, and haematoxylin redyes 1～2 minute (micro-
Dye levels is grasped under mirror), running water rinses 10～15 minutes, is dried, diagosis after resinene mounting.SABC is commented
Divide the criterion with reference to Xu Liangzhong: according to the degree of strength scoring of coloring: non-coloring 0 point, faint yellow 1 point, brown color 2
Point, sepia 3 points；Percentage score as shared by positive cell simultaneously: positive cell rate≤10% is 1 point, positive cell
Rate > 10% and≤50% it is 2 points, positive cell rate > 50% and≤75% it is 3 points, positive cell rate > 75% it is 4 points.Utilize
SPSS 16.0 software analysis immunohistochemistry results, correlation analysis uses Spearman method, calculates with Log-rank
P is worth significant difference.
Result shows: the EBV-LMP1 in tissues of nasopharyngeal carcinoma expresses apparently higher than normal nasopharyngeal epithelium (p=0.001), and
DNA-PK Thr2609 and AMPK Thr172 phosphorylation level generally reduce (p ＜ 0.05), and with the expression of EBV-LMP1
There is negative correlation；γ H2AX horizontal compared with normal nasopharyngeal epithelium in tissues of nasopharyngeal carcinoma significantly raises (p=0.0005), with
There is negative correlation in DNA-PK Thr2609 phosphorylation level.The reduction suppression of DNA-PK Thr2609 phosphorylation level
The DNA damage repair ability of EBV-LMP1 positive nasopharyngeal carcinoma's cell.Result is shown in Fig. 5.
Embodiment 6 DNA-PK Thr2609 and the prediction Radiotherapy for Nasopharyngeal Carcinoma prognosis of AMPK Thr172 phosphorylation level
On the basis of above-mentioned SABC, according to the expression of DNA-PK Thr2609 and AMPK Thr172 by patient
Be divided into negative expression and positive expression group, analyze respectively its with Patients Treated by Radiotherapy after the relation of 5 years survival rates, clearly both and nose
The relation of pharynx cancer Patients Treated by Radiotherapy prognosis.
Experimental technique is as follows: ImmunohistochemistryMethods Methods and standards of grading ibid, utilize SPSS 16.0 software analysis immuning tissue
Chemical results, Overall survival uses Kaplan Meier method to be analyzed, and calculating p with Log-rank is worth statistics
Difference.
Result shows: there is positive correlation (p=0.000) between DNA-PK Thr2609 and AMPK Thr172；DNA-PK
5 years survival rates of Thr2609 feminine gender group and positive group are respectively 66.67% and 70.37% (p=0.757), and single factor test divides
5 years survival rates of analysis display DNA-PK Thr2609 and patient are without significant correlation；And AMPK Thr172 feminine gender group and sun
Property group 5 years survival rates be respectively 53.57% and 78.26% (p=0.037), single factor analysis display AMPK Thr172
Significant correlation is there is with 5 years survival rates of patient.Although prompting individually DNA-PK Thr2609 level can not evaluate nose
The prognosis of pharynx cancer patient, but by interacting therewith the detection of AMPK Thr172 level, the existence of patient can be predicted
Phase.Result is shown in Fig. 6.
Claims (4)
1. evaluate Epstein-Barr virus positive nasopharyngeal carcinoma's DNA damage repair ability and the biomarker of radiotherapy prognosis, it is characterised in that institute
State DNA-PK and AMPK that biomarker is phosphorylation.
2. biomarker as claimed in claim 1, it is characterised in that described biomarker is the DNA-PK of phosphorylation
Thr2609 and AMPK Thr172.
3. biomarker described in claim 1 or 2 is evaluated Epstein-Barr virus positive nasopharyngeal carcinoma's DNA damage repair ability in preparation and puts
Treat the application in prognosis detection medicine.
4. biomarker described in claim 1 or 2 is evaluated Epstein-Barr virus positive nasopharyngeal carcinoma's DNA damage repair ability in preparation and puts
Treat the application in prognosis detection kit.
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| CN101956014A (en) * | 2010-09-30 | 2011-01-26 | 中山大学 | Kit for detecting 7 genetic markers of peripheral blood in early diagnosis of nasopharyngeal darcinoma |
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