CN105861705B - Evaluate biomarker and the application of Epstein-Barr virus positive nasopharyngeal carcinoma DNA damage repair ability and radiotherapy prognosis - Google Patents
Evaluate biomarker and the application of Epstein-Barr virus positive nasopharyngeal carcinoma DNA damage repair ability and radiotherapy prognosis Download PDFInfo
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Abstract
The invention discloses biomarker and the applications of evaluation Epstein-Barr virus positive nasopharyngeal carcinoma DNA damage repair ability and radiotherapy prognosis.The biomarker is the DNA-PK and AMPK of phosphorylation, is preferably DNA-PK Thr2609 and the AMPK Thr172 of phosphorylation.The biomarker can be used for preparing evaluation Epstein-Barr virus positive nasopharyngeal carcinoma DNA damage repair ability and radiotherapy prognosis detection drug and kit.
Description
Technical field
The present invention relates to biomarkers, will specifically participate in the DNA protein kinase (DNA- of DNA damage reaction
Activated protein kinase, DNA-PK) and AMP activation protein kinase (AMP-activated protein
Kinase, AMPK) phosphorylation level conduct evaluation Epstein-Barr virus (Epstein-Barr virus, EBV) positive nasopharyngeal carcinoma's DNA damage
The biomarker of repair ability and radiotherapy prognosis.
Background technique
DNA damage reaction (DNA damage response, DDR) is detected and is transmitted by a plurality of signal transduction pathway
DNA damage signal, active cell cycle check point carry out DNA damage reparation or active cell apoptosis, maintain the stabilization of genome
Property and cell homeostasis.DNA Damage repair ability defect is the important mechanisms of tumor invasion.DNA damage be not repaired or
The unstability that may cause genome is mistakenly repaired, oncogenic generation is led；The exception of DNA damage reparation simultaneously is related to
Sensibility of the tumour to chemicotherapy.The novel targets of oncotherapy are being converted into the achievement of DNA damage repair mechanism research.
A kind of DNA damage that DNA double chain fracture is eukaryocyte most serious and ionising radiation are to caused by chromosome
The fatal injury of most critical.It is human somatic cell that nonhomologous end, which recombinates (nonhomologous end-joining, NHEJ),
The main path of DNA plerosis double-strand break, DNA-PK are the central elements of NHEJ.
AMPK is a serine threonine kinases, and major function is the variation of permissive cell energy state, remains thin
Born of the same parents' metabolic homeostasis.Newest research discovery AMPK plays a significant role in DDR, and there are DNA-PK's and AMPK in kinds of tumors
The interaction of unconventionality expression and the two.
Nasopharyngeal carcinoma (nasopharyngeal carcinoma, NPC) is the common malignant tumour in China, and annual morbidity is
(10-25)/100,000, the death rate account for the 2.81% of the national malignant tumor death rate, wherein Epstein-Barr virus (Epstein-Barr
Virus, EBV) positive nasopharyngeal carcinoma accounts for 80% or more.
EBV belongs to mankind's gamma herpes viruses, is first DNA tumorigenesis disease for being proved to fall ill directly related with human tumor
Poison.Ebv infection related neoplasms account for about the 1.5% of global malignant tumour, account for the 5.6% of all infection associated tumors, at present
It 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 mediates host cell
Proliferation, invasion transfer and to anti-apoptotic, remarkably promote the Radioresistance of tumour cell.EBV-LMP1 is illustrated to DNA damage
The Regulation Mechanism of reaction facilitates the novel targets for finding treatment of nasopharyngeal carcinoma and prognosis.
Summary of the invention
The present invention is directed to overcome the deficiencies of the prior art and provide evaluation Epstein-Barr virus positive nasopharyngeal carcinoma DNA damage repair ability
Biomarker and application with radiotherapy prognosis.The biomarker is the DNA-PK and AMPK of phosphorylation, preferably phosphoric acid
DNA-PK Thr2609 and the AMPK Thr172 of change.The biomarker can be used for preparing evaluation Epstein-Barr virus positive nasopharyngeal carcinoma
DNA damage repair ability and radiotherapy prognosis detection drug and kit.
The invention will be further described below:
Present invention determine that EBV-LMP1 regulation nasopharyngeal carcinoma DNA damage reacts new molecular mechanism, specify DNA-PK and
The reduction of AMPK phosphorylation level is related to EBV positive nasopharyngeal carcinoma's DNA damage repair ability and Radiotherapy for Nasopharyngeal Carcinoma prognosis
Property, feasibility of both evaluations as new biomarker.
The present invention detects nasopharyngeal carcinoma cell DNA double chain fracture restoration ability using γ-H2AX analytical technology, finds EBV-
The DNA double chain fracture restoration ability of LMP1 positive cell is obviously inhibited.
By the central element DNA-PK protein phosphorylation level detection to DNA double chain fracture restoration, EBV-LMP1 is found
The DNA Damage repair ability of DNA-PK Thr2609 phosphorylation level and its mediation can be significantly reduced；EBV- simultaneously
LMP1 reduces the combination of DNA-PK and AMPK, makes the reduction of nasopharyngeal carcinoma cell AMPK Thr172 phosphorylation level, can after activation AMPK
Significantly to reverse the Radioresistance of EBV-LMP1 positive nasopharyngeal carcinoma's cell.
It is significantly increased using the more normal nasopharyngeal epithelium of γ H2AX level in ImmunohistochemistryMethods Methods discovery tissues of nasopharyngeal carcinoma, and
DNA-PK Thr2609 phosphorylation level is substantially reduced, and there are negative correlations for the two, prompts the drop of DNA-PK Thr2609 level
The low DNA damage repair ability for inhibiting EBV positive nasopharyngeal carcinoma's cell.
It is also significantly reduced with the AMPK Thr172 phosphorylation level in ImmunohistochemistryMethods Methods detection tissues of nasopharyngeal carcinoma, DNA-
There are negative correlations with the expression of EBV-LMP1 with AMPK Thr172 phosphorylation level by PK Thr2609；To DNA-PK
Thr2609 and AMPK Thr172 carries out correlation analysis discovery, AMPK Thr172 with 5 years survival rates after Radiotherapy for Nasopharyngeal Carcinoma
5 years survival rates of negative patient significantly reduce, and prompting AMPK Thr172 phosphorylation level is evaluation Radiotherapy for Nasopharyngeal Carcinoma prognosis
A new biomarker.
In short, DNA double chain fracture restoration ability is substantially less than normal nasopharyngeal in research discovery nasopharyngeal carcinoma cell of the invention
Epithelium, while the expression of DNA-PK Thr2609 and AMPK Thr172 reduce, and the Latent membrane protein1 with EBV coding
(latent membrane protein 1, LMP1) is horizontal negatively correlated.EBV-LMP1 is to DNA-PK Thr2609 phosphorylation
Inhibition is the important molecule basis that cell DNA double-strand break repair ability reduces, and is to inhibition of the DNA-PK in conjunction with AMPK
One of the reason of AMPK Thr172 low expression, the nasopharyngeal carcinoma cell Radioresistance that activation AMPK can reverse EBV-LMP1 to mediate,
There are significant positive correlations for 5 years survival rates after AMPK Thr172 level and Patients Treated by Radiotherapy.Therefore, in tissues of nasopharyngeal carcinoma
The low expression of DNA-PK Thr2609 can be used for assessing DNA double chain fracture restoration ability, and AMPK Thr172 phosphorylation level can
For predicting the radiotherapy prognosis of Nasopharyngeal Carcinoma Patients.
Detailed description of the invention
Fig. 1 is using Western Blot detection γ-H2AX evaluation EBV-LMP1 to nasopharyngeal carcinoma cell DNA double chain fracture restoration
The influence of ability, A show that the γ-H2AX level of CNE1-LMP1 cell is increased compared with CNE1 cell；B shows HNE2-LMP1 cell
γ-H2AX level is increased compared with HNE2 cell；
Fig. 2 stablizes expression LMP1 using Western Blot detection and knocks out the nasopharyngeal carcinoma cell phosphorylated cdna-PK of LMP1
The level of Thr2609, A shows that the DNA-PK Thr2609 level of CNE1-LMP1 cell is reduced compared with CNE1 cell, after knocking out LMP1
It increases；B shows that the DNA-PK Thr2609 level of HNE2-LMP1 cell is reduced compared with HNE2 cell, increases after knocking out LMP1；
Combination of the Fig. 3 using the nasopharyngeal carcinoma cell DNA-PK and AMPK of the detection DNA damage induction of co-immunoprecipitation method, A
Show the combination of DNA-PK and AMPK in CNE1-LMP1 cell compared with CNE1 Leukopenia；B shows DNA- in HNE2-LMP1 cell
The combination of PK and AMPK is compared with HNE2 Leukopenia；C shows that EBV-LMP1 inhibits the AMPK of nasopharyngeal carcinoma cell DNA damage induction
Thr172 phosphorylation；
Fig. 4 use Clone forming Test, draw cell survival curve detection activation AMPK after to EBV-LMP1 positive nose
The influence of pharynx cancer cellular radiosensitivity, A show that melbine remarkably promotes the radiosensitivity of CNE1-LMP1 cell；B is shown
Melbine remarkably promotes the radiosensitivity of HNE2-LMP1 cell；
Fig. 5 is using 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, and case2 and case3 are low table respectively
Up to the tissues of nasopharyngeal carcinoma of EBV-LMP1 and high expression EBV-LMP1；
The ImmunohistochemistryResults Results that Fig. 6 is expressed according to tissues of nasopharyngeal carcinoma DNA-PK Thr2609 and AMPK Thr172, by patient
It is divided into negative and positive expression group, analyzes the relationship of 5 years survival rates of itself and patient respectively using Kaplan Meier method.
Specific embodiment
It is CNE1- that Nasopharyngeal Carcinoma Cell Line CNE1 cell used in the present invention is built for Tumour Inst., Chinese Medical Academy
LMP1, HNE2, HNE2-LMP1 cell are built by Central South University's tumour.
1 EBV-LMP1 of embodiment inhibits nasopharyngeal carcinoma cell DNA double chain fracture restoration
H2AX albumen is the member in eukaryocyte in H2A histone family, and it is silk that carboxyl terminal, which includes one 139,
One glutamine of serine, one glutamic acid (Ser-Gin-Glu, SQE) structural domain of histidine residue, can be by PI3K kinases man
Race includes ATM, ATR, DNA-PK phosphorylation, i.e. γ-H2AX.Ser139 phosphorylations of H2AX are ionising radiation or chemicals
An earliest events for causing DNA double chain to be broken form γ-H2AX foci in breaking part, recruit DNA damage identification and repair
Albumen.It is now recognized that γ-H2AX is one of detection DNA double chain fracture-sensitive and the highest index of specificity.By to γ-
The Western Blot of H2AX is detected, and specifies influence of the EBV-LMP1 to nasopharyngeal carcinoma cell DNA double chain fracture restoration.
Experimental method is as follows: nasopharyngeal carcinoma cell is cultivated in 6 orifice plates, until being shone when 70~80% fusion using the x-ray of 6-MV
4Gy is penetrated to induce DNA double chain to be broken, after irradiation 0.5h, 1h, 4h and extract total protein of cell for 24 hours, is placed in -20 DEG C and freeze.To
The Western Blot analysis of γ-H2AX is carried out after the completion of each time point collecting protein.
The results show that the 0.5h after roentgen radiation x, 1h, 4h and for 24 hours, the γ H2AX in 2 plants of EBV-LMP1 positive cells is horizontal
It is increased compared with EBV-LMP1 negative cells, prompts EBV-LMP1 that can inhibit the DNA double chain fracture restoration of nasopharyngeal carcinoma cell.As a result see
Fig. 1.
2 EBV-LMP1 of embodiment inhibits nasopharyngeal carcinoma cell DNA-PK phosphorylation
The phosphorylation of DNA-PK Thr2609 is the key that it plays a role in DNA double chain fracture restoration, by steady
Surely expression LMP1 and the Western Blot detection for knocking out DNA-PK Thr2609 in LMP1 nasopharyngeal carcinoma cell, specify EBV-LMP1
Regulation to nasopharyngeal carcinoma cell DNA-PK phosphorylation.
Experimental method is as follows: nasopharyngeal carcinoma cell is cultivated in 6 orifice plates, and EBV-LMP1 positive cell transfects the siRNA of LMP1
After continue culture 48 hours, then use 6-MV roentgen radiation x 4Gy, after irradiation 0.5h extract total protein of cell, carry out DNA-
The Western Blot of PK Thr2609 is analyzed.
The results show that EBV-LMP1 can inhibit the phosphoric acid of DNA-PK Thr2609 in 2 plants of EBV-LMP1 positive cells
Change, the phosphorylation for promoting DNA-PK Thr2609 after it is expressed is reduced using the siRNA of selectively targeted LMP1.As a result see figure
2。
3 EBV-LMP1 of embodiment inhibits the combination of nasopharyngeal carcinoma cell DNA-PK and AMPK, reduces the phosphorylation of AMPK
Using the physical bond of the DNA-PK and AMPK of the detection DNA damage induction of co-immunoprecipitation method and EBV-LMP1 pairs
The regulation of this combination detects the AMPK Thr172 phosphorylation of DNA damage induction by Western Blot.
Experimental method is as follows: nasopharyngeal carcinoma cell is cultivated in 10cm culture dish, when cell reaches 90% fusion, gives 6-
The roentgen radiation x 4Gy of MV, 0.5h extracts total protein of cell after irradiation, and progress pre cleaning first is to remove in holoprotein lysate
The nonspecific proteins that can be combined with pearl: 1. taking A-sepharose bead suspension 20ul, is centrifuged and removes supernatant, and ice-cold 1
× PBS is washed three times.2. each experimental group prepares holoprotein lysate 1000ug, the pearl that 20ul had been cleaned is added, 4 DEG C turn to shake
1-2h, 4 DEG C of centrifugation 14000g × 10min shift supernatant into new EP pipe.Then it carries out co-immunoprecipitation: being 1. added
2ugDNA-PK antibody into supernatant, stay overnight by the mixed liquor of 4 DEG C of shaking supernatant antibody.2. mixed liquor is always added 20ul and cleaned
A-sepharose pearl, 4 DEG C of shaking 2h.3. collecting A-sepharose pearl, 12000rpm is centrifuged 5S, removes supernatant, pearl
It is cleaned 5 times with IP lysis buffer.4. pearl is resuspended in 20ul5 × loading buffer, mix.5. pearl boils
5min dissociates the albumen on pearl, is centrifuged 14000g × 5min.6. shifting supernatant into new EP pipe, loading, SDS-PAGE coagulates
Gel electrophoresis.The total protein of cell that 0.5h is extracted after irradiating is taken to carry out the Western Blot detection of AMPK Thr172 simultaneously.
The results show that the fracture of DNA double chain caused by x-ray can induce the physics of DNA-PK and AMPK in nasopharyngeal carcinoma cell
In conjunction with EBV-LMP1 significantly suppresses this combination, while reducing the phosphorylation of the AMPK Thr172 of DNA damage induction.Knot
Fruit sees Fig. 3.
Embodiment 4 promotes EBV-LMP1 positive nasopharyngeal carcinoma cellular radiosensitivity after activating AMPK
Using Clone forming Test, calculates the CNN surviving fraction of cell and draw survival curve, detection is activated with melbine
To the influence of EBV-LMP1 positive nasopharyngeal carcinoma's cellular radiosensitivity after AMPK.
Experimental method is as follows: taking the cell of exponential phase of growth, 0.25% pancreatin is digested to single cell suspension.Gradient multiple is dilute
It releases 102~105For a cell seeding into the 6 orifice plates of diameter 3CM, different cell numbers correspond to different exposure doses.Each dosage
If 3 parallel holes.It irradiates in preceding 1 hour training base and melbine (5 μM) is added, added using Siemens primus.E electronic line of sight
The irradiation of fast device, dosage rate 2Gy/min, when irradiation, cover 1~1.5cm thick tissue glue.Cell receive irradiation after, continue culture 10~
14 days, culture was terminated when visually seeing Clone formation.Culture medium is abandoned, is washed 2 times with PBS.Methanol fixes 15min, violet staining
10min, flowing water rinse dyeing liquor, are air-dried.Clone's number in each culture hole is counted, 50 cell numbers of > are one gram
It is grand, calculate every class mean.Calculate CNN surviving fraction (surviving fraction, SF) value of every kind of cell.Utilize Graphpad
Prism software, using the survival curve of every kind of cell of one-hit multitarget models fitting.
As the result is shown: the agonist melbine of AMPK can substantially reduce the CNN surviving fraction of cell, promote EBV-LMP1
The radiosensitivity of positive nasopharyngeal carcinoma's cell, is as a result shown in Fig. 4.
5 DNA-PK Thr2609 phosphorylation level of embodiment, which reduces, inhibits the damage of EBV-LMP1 positive nasopharyngeal carcinoma tissue DNA
Repair ability
EBV- is detected in the tissue samples of 60 nasopharyngeal carcinoma and 15 normal nasopharyngeal epitheliums using the method for immunohistochemistry
The expression of LMP1, γ-H2AX, DNA-PK Thr2609 and AMPK Thr172 specify it in normal nasopharyngeal epithelium and tumour
In differential expression.
Experimental method is as follows: organization chip being toasted 30 minutes in 80 DEG C of insulating boxs, is subsequently placed in turpentine oil and impregnates 5
Minute, it is impregnated again 5 minutes after replacing turpentine oil；It is sequentially placed into dehydrated alcohol, 95% ethyl alcohol and 80% ethyl alcohol again and respectively impregnates 5 points
Clock.Then chip is placed in 0.01M sodium citrate buffer solution (pH6.0) and is put into micro-wave oven high fire heating and set boiling, then
Low fire continues heating 10~15 minutes in using instead.PBS is washed 5 minutes × 3 times, and 3%H is added dropwise2O2In be stored at room temperature 10 minutes, PBS is washed
5 minutes × 3 times, 50 μ L of primary antibody is added dropwise, 4 DEG C of wet box are stayed overnight.37 DEG C rewarming 45 minutes, PBS is washed 5 minutes × 3 times, be added dropwise secondary antibody 40
~50 μ L are stored at room temperature 1 hour, and PBS is washed 5 minutes × 3 times, and DAB develops the color 5~10 minutes (grasps dyeing journey under the microscope
Degree), tap water rinses 10 minutes, and haematoxylin is redyed 1~2 minute (grasping dye levels under the microscope), and tap water rinses 10
It is~15 minutes, dry, diagosis after resinene mounting.Immunohistochemistry scoring refers to the judgment criteria of Xu Liangzhong: according to coloring
Degree of strength scoring: non-coloring 0 divides, and faint yellow 1 point, brown color 2 is divided, and sepia 3 is divided；Simultaneously by percentage shared by positive cell
Than score: positive cell rate≤10% be 1 point, positive cell rate > 10% and≤50% be 2 points, positive cell rate > 50% and≤
75% is 3 points, and positive cell rate > 75% is 4 points.Immunohistochemistry results, correlation point are analyzed using 16.0 software of SPSS
Analysis uses Spearman method, calculates p value with Log-rank and obtains statistical difference.
As the result is shown: the EBV-LMP1 expression in tissues of nasopharyngeal carcinoma is apparently higher than normal nasopharyngeal epithelium (p=0.001), and
DNA-PK Thr2609 and AMPK Thr172 phosphorylation level generally reduces (p < 0.05), and exists with the expression of EBV-LMP1
Negative correlation；The more normal nasopharyngeal epithelium of γ H2AX level in tissues of nasopharyngeal carcinoma significantly increases (p=0.0005), with DNA-PK
There are negative correlations for Thr2609 phosphorylation level.The reduction of DNA-PK Thr2609 phosphorylation level inhibits EBV-LMP1 positive
The DNA damage repair ability of nasopharyngeal carcinoma cell.As a result see Fig. 5.
6 DNA-PK Thr2609 and AMPK Thr172 phosphorylation level of embodiment predicts Radiotherapy for Nasopharyngeal Carcinoma prognosis
On the basis of above-mentioned immunohistochemistry, it will be suffered from according to the expression of DNA-PK Thr2609 and AMPK Thr172
Person is divided into negative expression and positive expression group, analyzes the relationship of itself and 5 years survival rates after Patients Treated by Radiotherapy respectively, both clear and nose
The relationship of pharynx cancer Patients Treated by Radiotherapy prognosis.
Experimental method is as follows: ImmunohistochemistryMethods Methods and standards of grading are same as above, and analyze immuning tissue using 16.0 software of SPSS
Chemical results, Overall survival are analyzed using Kaplan Meier method, are calculated p value with Log-rank and are obtained statistical difference.
As the result is shown: there are 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 analysis is aobvious
Show 5 years survival rates of DNA-PK Thr2609 and patient without significant correlation；And the 5 of AMPK Thr172 feminine gender group and positive group
Year, survival rate was respectively 53.57% and 78.26% (p=0.037), and single factor analysis shows 5 years of AMPK Thr172 and patient
There are significant correlations for survival rate.Although prompting individual DNA-PK Thr2609 level that cannot evaluate the pre- of Nasopharyngeal Carcinoma Patients
Afterwards, but the detection of AMPK Thr172 level by interacting therewith, the life cycle of patient can be predicted.As a result see Fig. 6.
Claims (2)
1. the DNA-PK Thr2609 of phosphorylation and the AMPK Thr172 of phosphorylation evaluate EB virus-positive nasopharyngeal carcinoma in preparation
Application in DNA injury repair ability and radiotherapy prognosis detection drug.
2. the DNA-PK Thr2609 of phosphorylation and the AMPK Thr172 of phosphorylation evaluate EB virus-positive nasopharyngeal carcinoma in preparation
Application in DNA injury repair ability and radiotherapy prognosis detection kit.
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