CN108931633A - Diagnostic of Carcinoma of Gallbladder and Index for diagnosis marker PIM1 - Google Patents
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Abstract
The invention discloses Diagnostic of Carcinoma of Gallbladder and Index for diagnosis marker PIM1, also disclose the kit of Diagnostic of Carcinoma of Gallbladder and Index for diagnosis, can be used for diagnosing, predict, detecting or screening Human gallbladder carcinoma cellular invasion, gallbladder cancer clinical stages or Gallbladder Carcinoma Patients prognosis.The invention also discloses PIM1 protein inhibitors to prepare the purposes in anti-gall-bladder cancer drug.Present invention demonstrates that PIM1 has very important meaning in clinical practice, it is likely to become the knubble biological diagnosis marker of a new generation and the molecular target for oncotherapy, the research of the completely new target of gallbladder cancer disease based on PIM1 is opened, also opens up new approach to develop anti-cancer agent.
Description
Technical field
The present invention relates to biomedicine technical fields, and in particular to gall-bladder carcinoma marker.
Background technique
Gallbladder cancer (gallbladder cancer, abbreviation GBC) is the most common form of biliary tract malignancy, accounts for biliary tract evil
The 80%-95% of property tumour.In the past few decades, Gallbladder Cancer strategy does not make substantial progress, and due to a lack of allusion quotation
Type symptom, early diagnostic rate only have 19.1%, and the middle and advanced stage stage has been in when most of patients is clarified a diagnosis.So far, it performs the operation
Excision is still the primary treatments of Gallbladder Carcinoma Patients, however excision of performing the operation is not appropriate for patients with terminal.Gallbladder cancer to radiotherapy,
The remedy measures such as chemotherapy are insensitive, therefore whole poor prognosis, and Foreign Epidemic disease is research shows that the postoperative 5 years survival rates of gallbladder cancer
Only 5%.In addition to this, postoperative easy to recur or transfer has been largely fixed the poor prognosis of Advanced Gallbladder Cancer.It is reported that
Gallbladder cancer is as caused by complicated inherent cause and environmental factor, however, the molecular mechanism of gallbladder cancer progress is still not
It is clear.Therefore, the biomarker and effective therapeutic strategy for probing into the early detection for making a definite diagnosis gallbladder cancer are vital.
PIM family, including PIM1, PIM2 and PIM3, they all include the activation site for being referred to as ATP anchor, are belonged to
Calcium/calmodulin adjusts kinases, highly conserved in the evolutionary process of multicellular tissue.PIM1 gene is as Moloney earliest
The provirus of mouse leukemia virus inserts people's point and is found.However, expression and function of the PIM1 in gallbladder cancer are made
With unclear, the more researchs of this needs.
Summary of the invention
Present invention seek to address that the technical problems existing in the prior art.
In order to probe into the expression of PIM1 in gallbladder cancer, we use tissue array technology and immunohistochemistry skill
Art probes into PIM1 protein expression level in Proteins in Carcinoma of Gallbladder and neighbouring cancer beside organism's sample, and is existed by western blotting technique
Result is further verified in Proteins in Carcinoma of Gallbladder and gallbladder carcinoma cells.It is aobvious in the expression of results that protein level probes into PIM1
Show, PIM1 expression is significantly higher than cancer beside organism in gallbladder cancer.
For the relationship further probed between the expression of PIM1 and gallbladder cancer Clinical symptoms and prognosis, present inventors have shown that
PIM1 expression in the sample that TNM stage is III-IV phase is significantly higher than I-II phase sample；Present inventors also established that
PIM1 expression in the sample that DISTANT METASTASES IN occurs for tumour is significantly higher than the sample that DISTANT METASTASES IN does not occur for tumour, the present invention
Confirmation PIM1 molecule can be used for preparing, predict, screening Human gallbladder carcinoma is spread, the kit of clinical stage.
The present inventor also carries out Kaplan-Meier survival analysis, as the result is shown when PIM1 gene high expression group survival of patients
Between be considerably shorter than PIM1 gene low expression patient, there is close for the increase of PIM1 expression and the clinical prognosis of gallbladder cancer during one's sickness
The correlation cut.PIM1 expression can be used as the reference judge index and the prediction of prognosis survival risk of gallbladder cancer prognosis life span
With reference to.
In order to further probe into effect of the PIM1 gene in gallbladder cancer occurrence and development, the present inventor is interfered using PIM1
RNA and PIM1 specific inhibitor SGI-1776 inhibits expression of the PIM1 in gallbladder carcinoma cells respectively, analyzes thin to gallbladder cancer
Born of the same parents' proliferation, migration, invasion and the influence of Apoptosis, present inventors have shown that inhibiting PIM1 expression, gallbladder carcinoma cells proliferation is moved
It moves, invasive ability significant decrease, Apoptosis dramatically increases, and gall-bladder carcinoma marker PIM1 is alternatively arranged as screening preparation and diagnoses, is slow
The molecular target of the medicament of solution or treatment gallbladder cancer.
The present invention also applies the GBC-SD cell inoculation of the PIM1 of slow virus knockout to nude mice by subcutaneous, it was confirmed that inhibits PIM1
Express influence to one-tenth knurl ability in gallbladder carcinoma cells body, the inventors discovered that the experimental group that knocks out of PIM1 with without knockout
Control group is compared, and gross tumor volume, luciferase expression significantly reduce, and the present invention demonstrates again that gall-bladder carcinoma marker PIM1 is alternatively arranged as sieving
The molecular target of the medicament of choosing preparation diagnosis, alleviation or treatment gallbladder cancer.In a first aspect, the invention discloses people's PIM1 albumen,
Purposes as Diagnostic of Carcinoma of Gallbladder and/or Index for diagnosis marker.
Second aspect, the invention discloses the purposes that people's PIM1 albumen is used to prepare kit, the kit is used for gallbladder
The diagnosis of capsule cancer and/or Index for diagnosis；Accordingly, the present invention provides kit, which includes the anti-of anti-human PIM1 albumen
Body, and it is used for Diagnostic of Carcinoma of Gallbladder and/or Index for diagnosis；Antibody can be monoclonal or polyclonal antibody.Kit can be used for diagnosing,
Prediction, detection or screening Human gallbladder carcinoma cellular invasion, gallbladder cancer clinical stages or Gallbladder Carcinoma Patients prognosis.
The third aspect, the present invention provides PIM1 protein inhibitors to prepare the purposes in anti-gall-bladder cancer drug, the suppression
Preparation is SGI-1776, its pharmaceutically acceptable salt or PIM1 specificity RNA interfering.Further, the drug is for delaying
Solution or treatment gallbladder cancer, or the prognosis for improving Gallbladder Carcinoma Patients.
It is noted that gallbladder cancer TNM stage described in the 1-3 either side of front can for III-IV phase (preferably) or
I-II phase.
Present invention demonstrates that PIM1 has very important meaning in clinical practice, it is possible to the knubble biological as a new generation
Diagnosis marker and molecular target for oncotherapy；Especially open the completely new target of gallbladder cancer disease based on PIM1
Research, also for develop anti-cancer agent open up new approach.
Detailed description of the invention
Fig. 1 .1 is western blot figure, wherein the corresponding expression for indicating PIM1 albumen in Proteins in Carcinoma of Gallbladder of PIM1；
Fig. 1 .2 is western blot figure, wherein the corresponding expression for indicating four kinds of gallbladder carcinoma cells system PIM1 albumen of PIM1.
Fig. 2 .1 is indicated through tissue array technology and immunohistochemistry technology to PIM1 egg in gallbladder cancer and cancer beside organism
White differential expression is for statistical analysis, it has been found that：PIM1 expression in gallbladder cancer sample is significantly higher than sample by cancer；
Fig. 2 .2A is the expression of PIM1 and the correlation analysis of TNM stage, it has been found that：PIM1 is in TNM stage
Expression is significantly higher than the gallbladder cancer sample of I-II in the gallbladder cancer sample of III-IV；
Fig. 2 .2B indicates to pass through tissue array technology and immunohistochemistry technology to the expression and gallbladder cancer of PIM1
The correlation of DISTANT METASTASES IN situation is for statistical analysis, it has been found that：PIM1 expresses water in the gallbladder cancer sample for merging transfer
It is flat to be significantly higher than the gallbladder cancer sample for not merging transfer；
Correlation analysis of Fig. 2 .3 between PIM1 expression and patient's overall survival, Kaplan-Meier existence point
The total life span for analysing PIM1 high expression group patient as the result is shown is considerably shorter than the patient of PIM1 low expression group, i.e. PIM1 high expression
Prompt worse clinical prognosis.
Fig. 3 .1A is that experiment 3.1GBC-SD gallbladder carcinoma cells are proliferated comparison diagram, and Fig. 3 .1B is that experiment 3.1NOZ gallbladder cancer is thin
Born of the same parents are proliferated comparison diagram；Fig. 3 .2 is 3.2 gallbladder carcinoma cells migration situation comparison diagrams of experiment；Fig. 3 .3 is 3.3 gallbladder carcinoma cells of experiment
Invade situation comparison diagram；Experimental group is to inhibit the expression of PIM1 using RNA interfering in each figure, and control group is not apply RNA interfering
Gallbladder carcinoma cells, as the result is shown：After PIM1 expression is lowered, cell Proliferation, migration, invasive ability are obviously reduced.
Fig. 4 is PIM1 experimental group and nude mice of control group standardizes photon flux change curve, and experimental study PIM1 is lowered
Afterwards to the influence of nude mice one-tenth knurl ability, as the result is shown：The experimental group that PIM1 is knocked out, gross tumor volume are substantially less than control group.
Specific embodiment
The present invention is explained below by several model experiments, and the present invention is not limited thereto.
One, Western blot detects the expression of PIM1 albumen in Proteins in Carcinoma of Gallbladder and cell
Experiment 1.1：
Histone lysate is added in 9 Proteins in Carcinoma of Gallbladder of Liquid nitrogen storage, is filled on ice with electric homogenizer
Divide grinding, is placed in the 1h of shaking table reaction on ice, takes supernatant after 4 DEG C of 10000r/min centrifugation 15min.It is examined using BCA method microplate reader
Protein concentration is surveyed, and albumen is adjusted into consistent 100 DEG C of buffer of addition and boils 5min.Protein sample is added to respectively prefabricated
12% polyacrylamide gel swimming lane, the electrophoresis 90min under 110V voltage.Electrotransfer is to nitrocellulose after the completion of protein electrophoresis
Film, film is placed in the PBST liquid containing 5%BSA and closes 1h processing, and primary antibody is added in 4 DEG C of environment and is incubated overnight, glimmering after rewarming
Light secondary antibody is incubated for the optical density after 2h using luminescence image analysis instrument analysis antibody specificity band, and with PIM1 albumen/β-
The optical density of actin compares opposing proteins expression and analysis is normalized.
As a result, it has been found that：There are 6 Proteins in Carcinoma of Gallbladder to detect the expression of apparent PIM1, as shown in attached drawing 1.1.Experiment
1.2：
Referring to experiment 1.1, PIM1 protein expression level in gallbladder carcinoma cells is analyzed using Westernblot, gallbladder
Capsule cancer cell comes from cell line GBC-SD, NOZ, OCGU1 and SGC-996, as a result prompts PIM1 equal in aforementioned four cell line
It is expressed in height, and the expression in Gallbladder carcinoma cell line GBC-SD, NOZ, it is higher than in SGC-996, OCGU1 cell, such as attached drawing 1.2
It is shown.
Two, array experiment
With expression of the ImmunohistochemistryMethods Methods analysis PIM1 albumen in Proteins in Carcinoma of Gallbladder and cancer beside organism
Organization chip:1. constructing gallbladder tumors organization chip, tissue samples source is the first affiliated hospital of Zhengzhou University,
It include wherein Proteins in Carcinoma of Gallbladder 52, neighbouring cancer beside organism 27, clinical information includes gender, age, TNM stage, tumour
Size, classification, follow-up information etc.；The diameter of chip sample point is all 1.5mm, and fixed form is fixed for formalin.2. simultaneously
Use commercialization organization chip (Shanghai Xin Chao biotech firm), including 79 Proteins in Carcinoma of Gallbladder, 20 cancer beside organisms, clinical information
Including gender, age, TNM stage, tumor size, classification, follow-up information etc.；The diameter of chip sample point is all 1.5mm, fixed
Mode is fixed for formalin.
Immunohistochemistry main operational steps：
After organization chip to be carried out to roasting piece, dewaxing, aquation, reparation antigen, closing respectively, it is polyclonal that rabbit-anti people PIM1 is added
Antibody (1:100 dilutions), 4 DEG C of overnight incubations；The goat anti-rabbit igg working solution of HRP label is added dropwise, in 37 DEG C of incubations 60min, SABC
37 DEG C of reagent are incubated for 30 minutes, are then developed the color with DAB；Haematoxylin is redyed；Dimethylbenzene is transparent, neutral gum mounting.Utilize tissue
Chip scanner carries out panoramic scanning, is independently scored by 2 senior pathologists, then unified score result through discussion.
Five grades are divided according to dye levels, 1 point indicates almost dye-free；2 points indicate weak dyeing；3 points of expression moderate dyeing；4 points
Indicate that strong dyeing and 5 split poles are dyed by force.Meanwhile by 1 point, 2 points and 3 points of definition be low expression, 4 points and 5 points are considered as high expression
For statisticalling analyze.
Statistical procedures：
Mapping software uses Graph pad Prism5, and photo finishing uses Adobe Illustrator CS6, statistical
Analysis uses SPSS23.0.Clinical symptoms and the difference of PIM1 expression use Chi-square Test；Probe into gallbladder cancer increased risk because
Element uses single-factor regressioning analysis and multinomial logistic regression；The phase of PIM1 expression and Gallbladder Carcinoma Patients Overall survival
Closing property uses Kaplan-Meier survival analysis.Results of statistical analysis P<0.05, it is believed that difference has statistical significance.
2.1：Coloration result shows that PIM1 subcellular localization is mainly distributed on cytoplasm and cell membrane, and positive cell presents bright
Aobvious brown color or brown granular, negative cells are rendered as lighter colored particles or colourless；Table of the PIM1 albumen in Proteins in Carcinoma of Gallbladder
Up to horizontal high (positive rate 75.8%), and low expression level (positive rate 53.2%) is shown as in cancer beside organism's major part,
Two comparison among groups, difference have statistical significance (P<0.05), as shown in Fig. 2 .1.
2.2 the expression of PIM1 and the relationship of Clinical symptoms
The expression of the Clinical symptoms of follow-up patient and PIM1 are subjected to Chi-square Test analysis, the results show that patient
The differential expression of age, gender and tumor size and PIM1 are not statistically significant, the TNM stage and DISTANT METASTASES IN and PIM1 of tumour
Differential expression is statistically significant.
2.2.1 the correlation of the expression of PIM1 and TNM stage
Patient's PIM1 expression of III~IV phase is apparently higher than the patient of I~II phase in TNM stage, and difference has statistics
Learn meaning (P<0.05) it, is mapped using Graph pad Prism5, sees Fig. 2 .2A.
Fig. 2 .2A is the expression of PIM1 and the correlation analysis of TNM stage.
2.2.2 the expression of PIM1 and the correlation that DISTANT METASTASES IN occurs
Patient's PIM1 expression that DISTANT METASTASES IN occurs is apparently higher than the patient that DISTANT METASTASES IN does not occur, and difference has system
Meter learns meaning (P<0.05), see Fig. 2 .2B.
Correlation between 2.3 Clinical symptoms and patient's overall survival
66 patients complete to Follow-up Data carry out single-factor regressioning analysis, discovery age, gender, tumor size and trouble
The overall survival of person is without significant correlation (P>0.05), the TNM stage of tumour, DISTANT METASTASES IN and PIM1 express total with patient
There are significant correlation (P for body survival rate<It 0.05), is latent dangerous factor；Further multinomial logistic regression confirms, high-level
There are significant correlations for the overall survival of the PIM1 of expression and patient, are the independent hazard factors of gallbladder cancer overall survival.
Correlation between PIM1 expression and patient's overall survival is as shown in Fig. 2 .3：The trouble of PIM1 high level expression
Person's overall survival is substantially less than the patient of PIM1 low expression level, and difference has statistical significance (P<0.05).
Three, In vitro cell experiment
By taking the cancer cell from gallbladder cancer two kinds of cell line of GBC-SD and NOZ as an example, experimental group is interfered with PIM1 specificity
RNA inhibits expression of the PIM1 in gallbladder carcinoma cells, and the sense strand sequence from 5 ' to 3 ' of RNA interfering used is
GAUAUGGUGUGUGGAGAUAtt, antisense strand are UAUCUCCACACACCAUAUCtt (after removal tt as shown in sequence 1), control
Group then corresponds to the normal cancer cell without RNA interfering processing, analyzes to gallbladder carcinoma cells proliferation, migration and the influence of invasion；
As a result, it has been found that cell Proliferation, migration, invasive ability are obviously reduced, and are described in detail below after PIM1 expression is lowered：
Experiment 3.1：CCK-8 detects cell proliferative conditions.
By cell dissociation after the transfection screening in logarithmic growth phase, cell density is adjusted after cell count to appropriate dense
Degree.With the volume in the hole 200ul/ by cell suspension inoculation into 96 orifice plates, 37 DEG C, cultivate in 5%CO2 incubator；It is being protected from light item
Under part, above-mentioned 96 orifice plate is added with the volume in the hole 10ul/ in CCK8 solution, after being incubated for 2h in incubator, by enzyme linked immunosorbent detection
It is set as 450nm wavelength on instrument, detects each hole absorbance (A450) value.While zeroing hole and control wells are set, every group setting 3
Multiple holes.Two groups of mean value and standard deviation are analyzed respectively, and difference has statistical significance (P<0.05).
Absorbance experimental result is as shown in Fig. 3 .1A/3.1B.
Experiment 3.2：Cell scratch detection cell migration situation.
Cellular processes add in the hole cell suspension 2ml/ in 6 orifice plates referring to above-mentioned experiment, to every hole cell confluency degree
When reaching 80-90%, using Tip pipette tips in hole standardized straight line, formed cell monolayer between scratch；Then it is rushed with sterile PBS
The cell to fall off in scratching process is washed, up to no cast-off cells exist.According to pre-designed time point, 6 orifice plates are set
It in microscopically observation and takes pictures, measures the migration distance between cell scratch, experiment is in triplicate.Two groups of mean value is analyzed respectively
And standard deviation, difference have statistical significance (P<0.05).As a result as shown in Figure 3 .2.
Experiment 3.3：Transwell experiment detection cell invasion situation.
The Matrigel glue that -20 DEG C save first is placed in 4 DEG C of refrigerator overnights, then Matrigel glue is diluted in not in proportion
It in culture solution containing serum, is then equably paved on the cell Transwell film, places solidification；
Removing the residual media in culture plate, every hole is added the serum-free medium that 50ul contains 10g/L BSA, and 37 DEG C
It is incubated for 30min；After experimental group and cellular control unit are carried out conventional digestion, centrifugation, with the BSA free serum culture containing 10g/L
Cell is resuspended in liquid, adjusts cell concentration；It takes the cell suspension inoculation of 200ul into the upper chamber of the cell Transwell, then will
The cell Transwell is placed in 24 orifice bores, and room adds culture solution of the 500ul containing 10%FBS under 24 orifice plates；It is placed in 37 DEG C, 5%
CO2In cell incubator, routine culture 36h；Cell is taken out, is rinsed with PBS, carefully wipes micropore film inner layer with wet cotton swab
Cell, fixed with 95% ethyl alcohol, dye；5 visuals field are randomly selected under microscope, count the cell across microporous barrier lower layer
Number, takes its average value, every group of 3 cells, experiment is repeated 3 times.Two groups of mean value and standard deviation are analyzed respectively, and difference has statistics
Learn meaning (P<0.05).As a result as shown in Fig. 3 .3.
Experimental group uses PIM1 protein inhibitor SGI-1776 instead and inhibits PIM1 expression, repeats above-mentioned 3 experiments, as a result also sends out
It is existing：After PIM1 expression is lowered, cell Proliferation, migration, invasive ability are obviously reduced.
Four, tumor formation in nude mice：
To the cancer cell from gallbladder carcinoma cells system GBC-SD, experimental group knocks out PIM1 with slow virus, expands training after screening
It supports, control group is then knocked out without slow virus, takes each group logarithmic growth phase cell that cell suspension is made, and is inoculated in armpit on the right side of nude mice
Nest is subcutaneous, establishes Xenografts in nude mice model.It is imaged using small animal living body within periodically (1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks)
Instrument measures and records the standardized photon flux of each group nude mice, for reflecting nude mouse tumor size indirectly, draws curve.
The results show that PIM1 knockout group, tumour standardization photon flux is substantially less than control group, the result of experiment such as Fig. 4
It is shown.
Sequence table
<110>The first affiliated hospital of Zhengzhou University
<120>Diagnostic of Carcinoma of Gallbladder and Index for diagnosis marker PIM1
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> RNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
gauauggugu guggagaua 19
Claims (10)
1. kit, which includes the antibody of anti-human PIM1 albumen, and is used for Diagnostic of Carcinoma of Gallbladder and/or Index for diagnosis.
2. kit as described in claim 1, characterized in that the kit is used to diagnose, predict, detect or screening people's gallbladder
The diffusion of capsule cancer cell, gallbladder cancer clinical stages or Gallbladder Carcinoma Patients prognosis.
3.PIM1 protein inhibitor is preparing the purposes in anti-gall-bladder cancer drug.
4. purposes as claimed in claim 3, characterized in that the inhibitor be SGI-1776, its pharmaceutically acceptable salt,
Or PIM1 specificity RNA interfering.
5. purposes as claimed in claim 4, characterized in that the sense strand sequence from 5 ' to 3 ' of the RNA is
The antisense strand sequence from 5 ' to 3 ' of GAUAUGGUGUGUGGAGAUAtt, the RNA are UAUCUCCACACACCAUAUCtt.
6. the purposes as described in any first claim, characterized in that the anti-gall-bladder cancer drug is for alleviating or treating gallbladder
Capsule cancer, or the prognosis for improving Gallbladder Carcinoma Patients.
7. anti-PIM1 antibody is used to prepare the purposes of kit, the kit is used for Diagnostic of Carcinoma of Gallbladder and/or Index for diagnosis.
8. purposes as described in claim 1, characterized in that the kit is used to diagnose, predict, detect or screening people's gall-bladder
Cancer cell diffusion, gallbladder cancer clinical stages or Gallbladder Carcinoma Patients prognosis.
9. people's PIM1 albumen, the purposes as Diagnostic of Carcinoma of Gallbladder and/or Index for diagnosis marker.
10. kit or purposes as described in any first claim, characterized in that the TNM stage of the gallbladder cancer is
III-IV phase or I-II phase.
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| CN115088678A (en) * | 2022-07-21 | 2022-09-23 | 吉林大学 | Method for establishing and analyzing subcutaneous tumor formation animal model of gallbladder cancer |
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| WO2017117344A1 (en) * | 2015-12-30 | 2017-07-06 | Icahn School Of Medicine At Mount Sinai | Fly avatars for cancer and uses thereof |
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| CN103298816A (en) * | 2010-12-17 | 2013-09-11 | 内尔维阿诺医学科学有限公司 | Substituted pyrazolo-quinazoline derivatives as kinase inhibitors |
| CN103874770A (en) * | 2011-08-08 | 2014-06-18 | 卡里斯生命科学卢森堡控股有限责任公司 | Biomarker compositions and methods |
| CN103212064A (en) * | 2012-01-19 | 2013-07-24 | 中国科学院上海巴斯德研究所 | Application of phosphorylation-pathway-related factor in regulating regulatory T-cell function |
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| CN115088678A (en) * | 2022-07-21 | 2022-09-23 | 吉林大学 | Method for establishing and analyzing subcutaneous tumor formation animal model of gallbladder cancer |
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| CN113855675A (en) | 2021-12-31 |
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