CN111304250A - Screening method of pancreatic cancer tumorigenic gene and application of screened gene - Google Patents
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Abstract
A screening method of pancreatic cancer tumorigenic genes and application of screened genes, belonging to the technical field of cancer diagnosis. Aiming at the technical problem of how to detect pancreatic cancer occurrence in a patient, the invention screens and obtains a key gene BRE gene related to pancreatic cancer tumorigenesis by utilizing a random gene mutation regulation technology, and proves that the increased expression of the BRE gene can be used as an index for pancreatic cancer occurrence detection, screening or pancreatic cancer patient prognosis judgment. The BRE gene is used for detecting in the high risk group of pancreatic cancer, the early diagnosis of pancreatic cancer can be quickly and effectively realized, a treatment target point and an important basis are provided for the prediction and diagnosis of the occurrence and development of pancreatic cancer, and the BRE gene can be applied to the diagnosis, treatment, monitoring and prognosis of pancreatic cancer.
Description
Technical Field
The invention belongs to the technical field of cancer diagnosis, and particularly relates to a screening method of pancreatic cancer tumorigenic genes and application of screened genes.
Background
Pancreatic cancer is highly malignant, difficult to diagnose and treat, and its morbidity and mortality rate have increased significantly in recent years. The survival rate of 5 years is less than 1 percent, is one of the worst malignant tumors in prognosis, and is called as 'king of cancer'. Pancreatic cancer is difficult to diagnose in early stage, is found to be in late stage, loses the chance of operation, has no specific chemotherapeutic drug, and has low cure rate. Pancreatic cancer onset is on a rapid rising trend. Data published by the american cancer society in 2017 show that new cases of american pancreatic cancer are listed 11 th in men, 8 th in women, and 4 th in mortality of malignant tumors. The pancreatic cancer is 8 th of the incidence rate of male malignant tumors in China cities, and 5 th of the mortality rate of malignant tumors in people in large cities (Beijing, Shanghai). The three major problems facing cancer in humans are carcinogenesis, cancer metastasis and chemotherapy resistance in therapy, which are also the leading causes of death in cancer patients. Most patients have metastases at the time of diagnosis, and since surgical treatment is not suitable, chemotherapy is the most important means for treating cancer patients. However, chemotherapy resistance of cancer cells during chemotherapy results in poor or even ineffective chemotherapeutic drug treatment. Therefore, the study of the mechanism of carcinogenesis, especially the molecular mechanism, is an important breakthrough to solve this problem.
The pathogenesis of the tumor is complex, single physical factors, chemical factors and virus infection are considered to cause mutation carcinogenesis in the past, and the present theory is that the occurrence of the cancer is a multi-step and multi-factor comprehensive effect. Taking the study of colon cancer as an example by professor Vogelstein, university of hopkins, usa, cancer undergoes multiple steps during development, such as hyperplasia, benign adenoma, carcinoma in situ, and invasive carcinoma. The normal epidermis is proliferated due to the deletion of an APC gene or the action of a Wnt signal pathway, then KRAS gene mutation causes gradually-increased benign adenoma, and the subsequent series of gene mutations such as SMAD4, CDC4, TP53 cause malignant adenoma. There are multiple genes that function differently in the progression from adenoma to carcinoma, and therefore, cancer is a multistep process with multiple genes involved. Screening and identifying key genes controlling carcinogenesis and further researching the molecular mechanism of carcinogenesis are important breakthrough in researching carcinogenesis and are key steps in developing novel products for preventing, diagnosing and treating cancers.
With the development of molecular biology, genetics, genomics, transcriptomics and proteomics, many methods have been used to screen for the genes involved in carcinogenesis, including gene chip, microRNA chip, small RNA interference (siRNAs), chemical mutagenesis DNA, high throughput sequencing, and retroviral insertion mutation. These technologies can be broadly classified into forward genetics and reverse genetics, the former starting from malignant tumor cells and studying gene changes, including gene mutation analysis based on DNA sequencing, gene expression profiling, whole genome association studies, and the like; the latter studies carcinogenesis reversely from gene change, including cDNA library technology, RNAi library technology, antisense RNA technology, transgenic technology, insertion mutation technology and the like. The high-throughput genome sequencing and expression profiling analysis is to obtain a large number of differential molecules by comparing the difference of genes, proteins or MicroRNA of tumor cells and normal cells, and then the method has the biggest defect of the difference of the spatial-temporal expression of the genes, the proteins or the MicroRNA molecules, the occurrence of cancer is a dynamic process, two or more static states are compared, the biological process cannot be reproduced, the screened characteristic related molecules have no causal relationship with phenotypes, and a large amount of verification work is needed subsequently. In addition, the amount of data generated by these techniques is huge, and how to mine meaningful information from the data is also a core problem in subsequent analysis. The technologies of cDNA library, RNAi library, antisense RNA, etc. can start from normal cells to study cancer occurrence and even reproduce the cancer occurrence process, however, the technology needs to know the sequence of the target gene in advance, which reduces the range of gene screening and often misses some unknown genes. Therefore, in order to identify genes responsible for pancreatic cancer development, a need exists for a method of identifying key genes that are causally related to cancer development.
The Random Gene mutation regulation Technology (CRGP) is a functional genomics Technology based on the integration transposon insertion mutation Technology, the antisense RNA Technology and the eukaryotic Gene expression regulation Technology. CRGP can generate a whole genome homozygous gene mutation; providing whole genome gene screening and gene function analysis; simultaneously, the relationship between gene mutation and functional expression is discovered and confirmed; systemic gene function localization and analysis of its functional characteristics in genetic and biochemical pathways; quickly separating disease-related genes and rheostat gene expression regulation. The method utilizes a random gene mutation regulation and control method to screen key genes for controlling pancreatic cancer neoplasia, and lays a foundation for developing novel pancreatic cancer prevention, diagnosis and treatment products.
Disclosure of Invention
Aiming at the technical problem of how to detect pancreatic cancer occurrence in a patient, the invention provides a pancreatic cancer tumorigenic gene and application thereof, and the specific technical scheme is as follows:
the invention aims to provide a method for screening a pancreatic cancer tumorigenesis related gene, which comprises the following steps:
1) transfecting a human tumor cell strain by CAT-tTA plasmid to obtain a Tet-off cell strain for stably expressing a transcription activating factor;
2) the gene search vector and the plasmid for effectively expressing transposase MPB were each 50ng/105Transfecting the Tet-off cell strain obtained in the step 1) for 24 hours, adding G418 with the final concentration of 600 mu G/ml for screening, and establishing a whole genome random gene mutation library;
3) screening pancreatic cancer cell strains with high tumor forming capability: the lower agar was used at a final mass concentration of 0.6% and the upper agar containing cells at a final mass concentration of 0.3%, with the number of cells being 6X 104Adding 2ml of culture solution into upper agar in a 10cm cell culture plate, picking out single clone after 14-21 days, and performing expanded culture, passage and cryopreservation to obtain a single clone cell strain;
4) and (3) detecting the nodulation capacity of the candidate clone cell strain: the lower agar was used at a final mass concentration of 0.6% and the upper agar containing cells at a final mass concentration of 0.3%, with the number of cells being 6X 104A 10cm cell culture plate, adding 500 μ l of culture solution to the upper agar, and culturing at 37 deg.C in 5% CO2 incubator; setting a doxycycline experimental group and a non-doxycycline control group in the experiment, respectively marking as + DOX and-DOX groups, setting three multiple holes in each group, carrying out statistical analysis after 14-21 days, and screening candidate mutant clones which are regulated and controlled by doxycycline and have high tumorigenic capacity;
5) cloning of pancreatic cancer tumorigenesis-associated candidate genes: extracting candidate mutant clones obtained by screening in the step 4), extracting genome DNA, connecting joint DNA after Sau3A1 restriction endonuclease, and obtaining DNA sequences at two ends of GSV insertion by Splinkette PCR and sequencing to obtain pancreatic cancer tumorigenesis related candidate genes;
the gene searching vector comprises a tetracycline reaction element, a piggybac transposon, a neomycin resistance gene and a plasmid replication origin, wherein the tetracycline reaction element is obtained by splicing two sections of tetracycline reaction elements; the plasmid for effectively expressing the transposase MPB is MPB; the human tumor cell strain is a human pancreatic cancer Aspc-1 cell strain.
Further limited, the related gene of pancreatic cancer tumorigenesis is human BRE gene.
The invention also provides application of the human BRE gene in preparation of a pancreatic cancer diagnosis or screening kit.
The invention also provides application of the human BRE gene in preparation of a kit for determining existence of pancreatic cancer tumors.
The invention also provides application of the human BRE gene in preparation of a kit for prognosis judgment of a pancreatic cancer patient.
The invention also provides application of the human BRE gene in preparation of a kit for determining whether pancreatic cancer patients are effective after operation, radiotherapy or chemotherapy treatment.
The invention also provides a kit for detecting or assisting in diagnosing pancreatic cancer, which contains a BRE gene reagent, wherein the BRE gene is used as a marker for detecting pancreatic cancer neoplasia.
Advantageous effects
The invention screens a gene BRE related to pancreatic cancer by random gene mutation regulation and control technology, and proves that the expression increase of the BRE gene can be used as an index for pancreatic cancer occurrence detection, screening or pancreatic cancer patient prognosis judgment. The BRE gene is used for detecting in the high risk group of pancreatic cancer, the early diagnosis of pancreatic cancer can be quickly and effectively realized, a treatment target point and an important basis are provided for the prediction and diagnosis of the occurrence and development of pancreatic cancer, and the BRE gene can be applied to the diagnosis, treatment, monitoring and prognosis of pancreatic cancer.
Drawings
FIG. 1 candidate mutant clone A2 screened for high tumorigenicity;
FIG. 2 regulatory response of candidate mutant clone A2 to DOX, wherein 2D2 represents Tet-off- #2D2 cell line, and the ordinate is the number of clones formed;
FIG. 3 expression of BRE gene in candidate clone A2.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the reagents used are commercially available. Wherein:
restriction enzyme, T4DNA ligase was purchased from NEB, primers, sequencing, Taq polymerase from Invitrogen, plasmid extraction kit and gel recovery kit from OMEGA, Aspc-1 cell line (from the cell center of the institute of basic medicine, national academy of medicine), fetal bovine serum, DMEM medium, PBS from Hyclone, FugenHD from Roche, various cell culture consumables from Corning, puromycin (puromycin) and Luciferin substrates from Invivo, and neomycin (neomycin, G418) from Merck.
The experimental procedures, for which specific conditions are not indicated in the examples, are generally conventional in the art, according to conventional conditions such as those described in Sambrook et al, molecular cloning, A laboratory Manual (third edition) (scientific Press, 2002), or according to conditions recommended by the reagent manufacturers.
The BRE gene is completely called as: the brain regeneration expression gene (The brain regenerative expression), also known as BABAM2 gene, is a known gene prior to The present invention, Genbank accession number: NM — 004899, derived from the human genome.
The CAG-tTA plasmid, the Luciferase reporter gene pUHC13-3 plasmid, the gene search vector GSV based on PiggyBac and the plasmid of transposase (MPB) are described in the published patent CN102747096A "Gene search vector, random gene mutation regulation method and application". The full-length nucleotide sequence of the BRE gene of the invention can be obtained by a PCR amplification method or an artificial synthesis method. According to the invention, research and analysis through a random gene mutation regulation technology confirm that the BRE gene has the effect of promoting pancreatic cancer.
Example 1 pancreatic cancer tumorigenic Gene BRE screening and functional Studies thereof.
Before screening a target gene, screening conditions of pancreatic cancer cell strains with high tumor forming capability are searched, and the method comprises the following steps:
spreading agar with final concentration of 0.6% into 6-well plate (2 ml/well) containing 1 × DMEM and 10% FBS, cooling at room temperature to solidify, and placing in CO2 incubator; culturing Asp-1 cells to logarithmic growth phase, digesting, centrifuging, collecting cells, resuspending and counting the cells; six-well plates are used to be mixed in culture media (containing 1 × DMEM and 10% FBS) with different agar concentrations according to the cell concentration of 104/well, the final agar concentrations are set to be 0.6%, 0.5%, 0.4%, 0.3%, 0.2% and 0.1% in sequence, 500 μ l of culture solution is slowly and lightly added to the upper agar, the culture solution is changed every 3-4 days, the result is observed after 14-21 days, and the data is photographed, recorded and counted. The results suggest that most cells were dead and unable to form tumors at the concentration of 0.3%, and therefore, the upper agar concentration of 0.3% was set as the screening concentration.
Screening of BRE gene related to pancreatic cancer tumorigenesis:
establishing a random gene mutation library: transfecting a human tumor cell strain by using CAG-tTA plasmid to obtain a Tet-off cell strain for stably expressing a transcription activator; the specific method comprises the following steps:
1) the linear CAG-tTA plasmid is transfected into an Aspc-1 cell, and a luciferase reporter gene technology is utilized to screen a Tet-off cell strain with high expression efficiency. The day before transfection was inoculated with 5X 10 according to the Roche FugeneHD protocol5Diluting the CAG-tTA plasmid to 0.02 mu g/mu l by using a serum-free and antibiotic-free DMEM (DMEM) culture medium when the cells grow to about 80 percent on an Aspc-1 cell to 60mm cell culture dish, taking 250 mu l, adding 15 mu l FugeneHD into the diluted CAG-tTA plasmid, gently mixing the diluted CAG-tTA plasmid and the FugeneHD, incubating the mixture at normal temperature for 15min, slowly dropping the diluted CAG-tTA plasmid into the prepared cells, gently mixing the mixed cells, and placing the mixed cells at 37 ℃ in a CO (carbon monoxide) culture2Continuously culturing in an incubator; after 24h, the cells were digested, divided into 3 10cm dishes on average, and the mixture was added to a puromycin (puromycin, 2. mu.g/ml) medium to continue the cultureCulturing, changing the liquid every 2 days and continuously screening; after 2 weeks, colonies that could be observed with the naked eye were formed.
2) Luciferase reporter gene is screened for stably expressed Tet-off Aspc-1 cell strain, puromycin screened clone is picked and cultured in an amplification way, digested and inoculated with 104Cells were plated in 96-well plates, 6 parallel wells were seeded per clone, and placed in 5% CO2Culturing overnight in an incubator at 37 ℃ with saturated humidity, when the cell density reaches 80%, transfecting Luciferase reporter gene-pUHC 13-3 plasmid with plasmid DNA0.05 μ g per well and FuGeneHD: DNA 3: 1 according to FuGeneHD instruction by the same method, and adding Doxycycline (DOX) solution (0.2 μ g per well) to one half of each clone; culturing in an incubator, washing cells with ice-cold PBS after 48 hours, discarding supernatant, lysing the cells by using a Harvest Buffer (4 ℃, 30min), uniformly mixing lysis solutions, taking 50 mu l of each well to a 96-well enzyme label plate, preparing a proper reaction solution according to the proportion of ATP to luciferase Buffer which is 1: 3.6, reading absorbance values on a bioluminescence detector, and calculating the down-regulation multiple of luciferase expression intensity of each DOX-added well and each DOX-not-added well according to the result; among the selected clones, 3 clones have relatively high fluorescence intensity (clone numbers are 2B6, 2D2 and 5A3), and after DOX is added, the relative light unit energy of the 3 clones is greatly reduced, and the difference between the two clones before and after the DOX is added can reach hundreds to thousands of times.
(II) the gene search vector GSV based on PiggyBac is transfected into ASPC-1Tet-off- #2D2 cell strain according to FugeneHD instruction, and the gene search vector GSV is transfected into 10 th cell strain5Co-transfecting 50ng GSV and 50ng transposase (MPB), transferring the cells into a 10cm culture dish after 24h of transfection, adding G418 with the final concentration of 600 mug/ml for screening, replacing the culture solution once every 2-3 days, establishing a whole genome random gene mutation library, estimating the size of the mutation library according to G418 resistant clone, and obtaining more than 10 ten thousand mutant cell clones in total.
(III) screening pancreatic cancer cell strains with high tumor forming capability: the lower layer was continued to use (final mass concentration) 0.6% agar, and the upper layer (containing cells) agar as a final materialThe quantitative concentration is 0.3%, and the number of cells is 6X 104A cell culture plate of/10 cm, slowly and gently adding 2ml of culture solution to upper agar, changing the culture solution once every 3-4 days, leading most cells to die after 14-21 days, leading a small amount of clone to be formed, picking single clone, carrying out expanded culture, passaging and freezing to obtain 5 clones in total, and sequentially naming the 5 clones as A1, A2, A3, A4 and A5, wherein the candidate A2 is shown in figure 1.
And (IV) detecting the nodulation capacity of the candidate clone cell strain: according to the method for detecting agar clone forming ability, 0.6% agar is continuously used in the lower layer (final mass concentration), the agar in the upper layer (containing cells) has a final mass concentration of 0.3%, and the number of cells in a six-well plate is 104Perwell, plate, Add gently 500. mu.l of culture medium slowly onto the upper agar, prepare in advance the DMEM medium containing DOX (DOX concentration in the medium is 1ug/ml) to add DOX group, stand at 37 deg.C with 5% CO2Culturing in an incubator; changing the culture solution every 3-4 days, setting + DOX and-DOX groups in the experiment, setting three multiple holes in each group, observing the result after 14-21 days, photographing, recording and counting the data, wherein the result is shown in figure 2<0.01, ns represents no statistical significance, and the statistical difference exists between the candidate clone A2+ DOX and A2-DOX groups, which indicates that DOX has regulation and control performance on the mutant clone A2, and the gene mutated by GSV in the A2 clone has a clear causal relationship with the tumor forming capability of a tumor cell line.
(V) cloning of pancreatic cancer tumorigenesis related candidate genes: extracting A2 cell line genome with Tiangen genome extraction kit, digesting 1ug of genome DNA according to the specification of Sau3A1 restriction enzyme of NEB, and then performing T4DNA ligase instruction and Adaptor (5 μ l 100 μm SPL and 5 μ l 100 μm SPR, adding 90 μ l water, heating to 100 deg.C, reacting for 10min, slowly cooling to room temperature) are connected at room temperature overnight, and the specific reaction system comprises 5 μ l Sau3AI single-cut genomic DNA, 1 μ l Adaptor, 10 XT4Ligation Buffer 1. mu.l, ATP (0.01M) 0.1. mu.l, T4Ligase 0.5. mu.l and deionized water was added to a total volume of 10. mu.l. Obtaining DNA sequences at two ends of GSV insertion by Splinkette PCR and sequencing, and selecting insertion sites at the common integration sites of piggyBac transposonsAt point TTAA, see FIG. 3 (the sequence of the adaptor and the primer used in Splinkette PCR are described in published patent CN102747096A, "Gene search vector, random Gene mutation control method and use").
The sequence obtained after sequencing is aligned and positioned at an insertion site through Blat of a UCSC genome, the GSV insertion site is positioned at the third intron of a gene BRE, and the specific position is 27931921 site of chromosome 2 (Chr 2: 27931921), so that antisense RNA is generated, the transcription and translation of mRNA of the gene are interfered, and the expression level of the gene is regulated.
Experiment shows that the BRE gene is expressed in candidate clone A2 in four groups, namely candidate clone A2(+ DOX), A2(-DOX), control ASPC-1-2D2(+ DOX) and ASPC-1-2D2(-DOX), wherein the ASPC-1-2D2 is the Tet-off- #2D2 cell strain, the Actin is control β -Actin (β -Actin is cytoskeletal protein, the expression is fixed, and when Western Blot detection is carried out, the cells are usually used as internal reference.) to be cultured to logarithmic growth phase, digested, centrifuged, collected and counted, and inoculated with 5 × 105Placing the cells into a 60mm culture dish, placing the culture dish at 37 ℃ and 5% CO2Culturing in an incubator. After the cells are fully paved, adding 200 mul of lysate to lyse the cells on ice for 15min, wherein the culture dish is continuously flapped every 3-5min to ensure that the lysate is uniformly distributed; transferring the cracked product to a 1.5ml EP tube, and centrifuging for 10min to 12000g at 4 ℃; the supernatant was transferred to a new centrifuge tube and placed on ice. Measuring the concentration of a protein sample by using a BCA kit of Thermofish company, taking 50 mu g of total protein, diluting the sample by using 5 Xprotein loading Buffer, heating at 100 ℃ for 3min to denature the protein sample, and preparing the protein sample for loading; after electrophoresis and membrane conversion, 5% skimmed milk powder (5g skimmed milk powder, 100ml TBS-T) was used for sealing at 37 deg.C for 1h, and TBS-T was washed 3 times for 3min each time; hybridizing the BRE antibody diluted by 5% skimmed milk powder at 4 ℃ overnight, and washing with TBS-T for 3 times (3 min each time) the next day; after hybridizing for 1h by using a secondary antibody, washing for 3 times for 3min by TBS-T; performing tabletting with ThermoWB color development kit (several drops of solution A and B are mixed uniformly), and performing tabletting with X-ray film automatic developing machine to obtain BRE gene expression in candidate clone A2 with DOX addedThe regulation and control are reduced, which shows that the up-regulation of BRE gene leads to the increase of the tumor forming capability of tumor cell strains, namely, the generation and growth of tumor cells can be promoted.
Claims (7)
1. A method for screening a pancreatic cancer tumorigenic gene is characterized by comprising the following steps:
1) transfecting a human tumor cell strain by CAT-tTA plasmid to obtain a Tet-off cell strain for stably expressing a transcription activating factor;
2) the gene search vector and the plasmid for effectively expressing transposase MPB were each 50ng/105Transfecting the Tet-off cell strain obtained in the step 1) for 24 hours, adding G418 with the final concentration of 600 mu G/ml for screening, and establishing a whole genome random gene mutation library;
3) screening pancreatic cancer cell strains with high tumor forming capability: the lower agar was used at a final mass concentration of 0.6% and the upper agar containing cells at a final mass concentration of 0.3%, with the number of cells being 6X 104Adding 2ml of culture solution into upper agar in a 10cm cell culture plate, picking out single clone after 14-21 days, and performing expanded culture, passage and cryopreservation to obtain a single clone cell strain;
4) and (3) detecting the nodulation capacity of the candidate clone cell strain: the lower agar was used at a final mass concentration of 0.6% and the upper agar containing cells at a final mass concentration of 0.3%, with the number of cells being 6X 104A 10cm cell culture plate, adding 500 μ l of culture solution to the upper agar, and culturing at 37 deg.C in 5% CO2 incubator; setting a doxycycline experimental group and a non-doxycycline control group in the experiment, respectively marking as + DOX and-DOX groups, setting three multiple holes in each group, carrying out statistical analysis after 14-21 days, and screening candidate mutant clones which are regulated and controlled by doxycycline and have high tumorigenic capacity;
5) cloning of pancreatic cancer tumorigenesis-associated candidate genes: extracting candidate mutant clones obtained by screening in the step 4), extracting genome DNA, connecting joint DNA after Sau3A1 restriction endonuclease, and obtaining DNA sequences at two ends of GSV insertion by Splinkette PCR and sequencing to obtain pancreatic cancer tumorigenesis related candidate genes;
the gene searching vector comprises a tetracycline reaction element, a piggybac transposon, a neomycin resistance gene and a plasmid replication origin, wherein the tetracycline reaction element is obtained by splicing two sections of tetracycline reaction elements; the plasmid for effectively expressing the transposase MPB is MPB; the human tumor cell strain is a human pancreatic cancer Aspc-1 cell strain.
2. The screening method according to claim 1, wherein the pancreatic cancer tumorigenic gene is a human BRE gene.
3. Application of the human BRE gene in preparation of a kit for diagnosing or screening pancreatic cancer.
4. Application of human BRE gene in preparation of kit for determining pancreatic cancer tumor.
5. Application of the human BRE gene in preparation of a kit for prognosis of pancreatic cancer patients.
6. The application of the human BRE gene in preparing a kit for determining whether pancreatic cancer patients are effective after operation, radiotherapy or chemotherapy treatment.
7. A kit for detecting or assisting in diagnosing pancreatic cancer is characterized in that the kit contains a BRE gene reagent, and the BRE gene is used as a marker for detecting pancreatic cancer neoplasia.
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CN102747096A (en) * | 2011-04-18 | 2012-10-24 | 中国医学科学院基础医学研究所 | Gene search vector, random gene mutation control method and application thereof |
CN105838735A (en) * | 2016-02-19 | 2016-08-10 | 浙江中医药大学 | Human pancreatic cancer nude mouse model construction method and use thereof |
CN108841955A (en) * | 2018-07-02 | 2018-11-20 | 中国医学科学院北京协和医院 | Application of the C22orf41 as Pancreatic Cancer Tumor Markers object |
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