CN107164480A - Application of the ZEB2 gene expression variations in pancreatic cancer chemotherapy Prognosis - Google Patents

Application of the ZEB2 gene expression variations in pancreatic cancer chemotherapy Prognosis Download PDF

Info

Publication number
CN107164480A
CN107164480A CN201710403330.9A CN201710403330A CN107164480A CN 107164480 A CN107164480 A CN 107164480A CN 201710403330 A CN201710403330 A CN 201710403330A CN 107164480 A CN107164480 A CN 107164480A
Authority
CN
China
Prior art keywords
plko
zeb2
purposes
puro
rna
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN201710403330.9A
Other languages
Chinese (zh)
Other versions
CN107164480B (en
Inventor
邹叶青
黄波
付晓伟
王小中
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Second Affiliated Hospital to Nanchang University
Original Assignee
Second Affiliated Hospital to Nanchang University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Second Affiliated Hospital to Nanchang University filed Critical Second Affiliated Hospital to Nanchang University
Priority to CN201710403330.9A priority Critical patent/CN107164480B/en
Publication of CN107164480A publication Critical patent/CN107164480A/en
Application granted granted Critical
Publication of CN107164480B publication Critical patent/CN107164480B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1135—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against oncogenes or tumor suppressor genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Veterinary Medicine (AREA)
  • Pathology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Plant Pathology (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Oncology (AREA)
  • Medicinal Chemistry (AREA)
  • Hospice & Palliative Care (AREA)
  • Virology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Expression of the present invention by detecting ZEB2 genes, with preferable prediction effect, preliminary guidance is provided for later stage further treatment for treatment of pancreatic cancer patient progress prognosis.Simultaneously the invention provides a kind of siRNA of blocking ZEB2 gene expressions, with the preferable effect for suppressing ZEB2 gene expressions.The present invention has preferable inhibition and application prospect.

Description

Application of the ZEB2 gene expression variations in pancreatic cancer chemotherapy Prognosis
Technical field
Blocked the present invention relates to purposes of the ZEB2 genes in diagnosis of pancreatic cancer and/or prognosis kit is prepared and ZEB2 Purposes of the agent in treatment pancreatic cancer drug is prepared, belongs to technical field of pharmaceutical biotechnology.
Background technology
Cancer of pancreas is that a kind of grade malignancy is very high, diagnoses and treat all highly difficult malignant tumor of digestive tract, about 90% is Duct adenocarcinoma originating from glandular tube epithelium.Its morbidity and mortality substantially rises in recent years.5 years survival rates<1%, it is prognosis One of worst malignant tumour.The diagnosis rate of cancer of pancreas early stage is not high, and operative mortality rate is higher, and cure rate is very low.This disease is sent out Sick rate male is higher than women, and the ratio between men and women is 1.5~2:1, male patient is common far beyond premenopausal women, postmenopausal women's The incidence of disease is similar with male.Cancer of pancreas is high because of its grade malignancy, and early diagnosis is difficult, and what poor prognosis turned into neoplastic disease one chooses greatly War.Although the cancer of pancreas less than 2cm clinically is considered as into early carcinoma at present, the patient that now there are about 3 0%~4 0% has Lymphatic metastasis.Therefore, there is its limitation by stages to cancer of pancreas according only to tumor size.High risk factor and pancreas on cancer of pancreas Relation between gland cancer morbidity is still disputable, but to these crowds preferably periodically ultrasound diagnosis.There is the alarm features of cancer of pancreas, such as disappear Thin, diarrhoea, indigestion etc. should highly doubt and examine cancer of pancreas.
Conventional diagnosis of pancreatic cancer method includes imaging diagnosis, such as B ultrasound, CT etc. at present.Tumor markers is reflected Generation, evolution and the gene activation degree of cancer, can be detected in the tissue, body fluid and excreta of tumor host.Have a variety of Pancreatic tumour mark has been used for clinic, but its sensitiveness, specificity are still undesirable.Common mark carcinomebryonic antigen (CEA)：Have higher expression rate in Pancreas cancer patients serum, but its specificity is low, its increase also see liver, Colon and rectum, stomach, Gallbladder cancer and non-digestive tract cancer.Thus limit its application value.Another mark CA19-9：It is pancreas generally acknowledged at present Cancer related neoplasms mark, practical value of other tumor markerses to diagnosis of pancreatic cancer is judged frequently as standard sign thing, Current the sixth of the twelve Earthly Branches is widely used in clinic.The sensitiveness of CA19-9 diagnosis of pancreatic cancer is 69%~93%, and specificity is 81%~85%.It is right High-risk patient can be with CA19-9 as screening test, and its result is better than CEA.In addition to cholangiocarcinoma, with other malignants tumor of digestive tract There is a good significance of differential diagnosis, and also there are certain values to the diagnosis of Early pancreatic carcinoma, but diagnosis effect also has and changed The space entered.
ZEB2 genes in the prior art respectively together with other small molecules breast cancer, carcinoma of urinary bladder, carcinoma of endometrium, There is certain indicative function in cervical carcinoma and nasopharyngeal carcinoma, but also without relevant report in cancer of pancreas.Particularly in pancreas Application in cancer chemotherapy Prognosis also without reference to.
The content of the invention
ZEB2 gene expression doses predict the mark purposes of Pancreas cancer patients prognosis, can based on ZEB2 gene expression doses To predict the prognosis of Pancreas cancer patients, the low patient's good prognosis of ZEB2 expressions, the high patient's poor prognosis of expression.
Detection and the application process of Pancreas cancer patients prognosis are predicted based on ZEB2 gene expression doses, a. extracts tumor patient The cell total rna of primary carcinoma tissue specimen；CDNA is synthesized by template reverse transcription of RNA；Using cDNA as template, with ZEB2 genes and The specific primer and fluorescence labeling probe of house-keeping gene carry out real-time quantitative PCR amplification, calculate the relative of ZEB2 gene mRNAs Expression quantity；B. according to Pancreas cancer patients relapse and metastasis positive case and the ZEB2mRNA expression quantity of relapse and metastasis negative case, really Determine the critical value of Index for diagnosis.
ZEB2mRNA expression quantity is less than patient's good prognosis of critical value, and higher than patient's poor prognosis of critical value.
Described detection method, with ZEB2mRNA expression quantity in primary cancerous tissue to tumor patient Index for diagnosis, expression quantity Low patient is then judged as good prognosis, and expression quantity is low high to be then judged as poor prognosis.
The present invention confirms that ZEB2 can be applied to the index prediction prognosis of diagnosis of pancreatic cancer therapeutic effect by grinding to make internal disorder or usurp；ZEB2 can The novel targets treated as treatment of pancreatic cancer new drug and biological immune.Block ZEB2 expression to suppress pancreatic tumor growth, suppress Pancreatic tumor cell, the novel targets that can be treated as treatment of pancreatic cancer new drug and biological immune.
In addition, the present invention provides the siRNA, its sequence such as SEQ ID NO that a species specificity suppresses ZEB2 gene expressions：1 With shown in 2.
In addition, the present invention provides a kind of gene interference slow virus carrier, for coding specificity is suppressed into ZEB2 gene expressions Interference microRNA gene fragment clone enter after slow virus carrier obtain, can produce people's ZEB2 genes small molecule interference RNA。
The slow virus carrier can be selected from：pLKO.1-puro、pLKO.1-CMV-tGFP、pLKO.1-puro-CMV- tGFP、pLKO.1-CMV-Neo、pLKO.1-Neo、pLKO.1-Neo-CMV-tGFP、pLKO.1-puro-CMV-TagCFP、 pLKO.1-puro-CMV-TagYFP、pLKO.1-puro-CMV-TagRFP、pLKO.1-puro-CMV-TagFP635、 pLKO.1-puro-UbC-TurboGFP、pLKO.1-puro-UbC-TagFP635、pLKO-puro-IPTG-1xLacO、pLKO- puro-IPTG-3xLacO、pLP1、pLP2、pLP/VSV-G、pENTR/U6、pLenti6/BLOCK-iT-DEST、pLenti6- GW/U6-laminshrna, pcDNA1.2/V5-GW/lacZ, pLenti6.2/N-Lumio/V5-DEST, pGCSIL-GFP or Any in pLenti6.2/N-Lumio/V5-GW/lacZ.
The present invention carries out prognosis by detecting that the expression of ZEB2 genes is directed to treatment of pancreatic cancer patient, with preferable Prediction effect, preliminary guidance is provided for later stage further treatment.Simultaneously ZEB2 gene tables are blocked the invention provides one kind The siRNA reached, with the preferable effect for suppressing ZEB2 gene expressions.The present invention has before preferable inhibition and application Scape.
Brief description of the drawings
Fig. 1 is that qRT-PCR is contrasted in normal pancreatic tissue, pancreatitis, pancreatic tumor tissue and pancreatic neoplasm stem cell ZEB2 expression change comparison diagram.
Fig. 2 is the ZEB2 expressions and Prognostic significance figure of Patients with Pancreatic Cancer.
Fig. 3 is that siRNA disturbs ZEB2 gene expression design sketch.
Embodiment
Embodiment 1 detects ZEB2 expressions in pancreatic cancer cell, pancreas pulmonary metastases cell and normal pancreatic cells
Clinical samples inclusion criteria：Receive surgical resection case, postoperative pathological is diagnosed as ductal adenocarcinoma of pancreas；Puncture Biopsy obtains complete tissue sample, and pathological diagnosis is ductal adenocarcinoma of pancreas.Participation is originally ground before the corrective surgery made internal disorder or usurp or biopsy Receiving, radiotherapy and immunization therapy, patient endorsed informed consent form, and by Ethics Committee's examination ＆ verification.
Method：Obtain postoperative and biopsy normal pancreatic tissue, pancreatic tumor tissue.QRT-PCR is carried out after extracting RNA, QRT-PCR operating procedures are the conventional operating method in this area, and required primer is respectively：ZEB2 sense primers：5’- AGCCAAGGAATGCTACCAA-3 ', anti-sense primer：5’-GGCCCCAGAGCATCATAA TC-3’；β-actin sense primers： 5 '-CAGCTGAGAGGGAAATCGTG-3 ', anti-sense primer：5’-CGTTGCCAATAGTGATGACC-3’.
ZEB2 sense primers：5’-CAA GAG GCG CAA ACA AGC C-3’
Anti-sense primer：5’-GGT TGG CAA TAC CGT CAT CC-3’
β-actin sense primers：5’-CAT GTA CGT TGC TAT CCA GGC-3’、
Anti-sense primer：5’-CTC CTT AAT GTC ACG CAC GAT-3’.
ZEB2 expression water in qRT-PCR contrasts normal pancreatic tissue, pancreatic tumor tissue and pancreas pulmonary metastases cell Flat change.
As a result：As shown in figure 1, quantitative PCR data finds that ZEB2 expression is relatively low in normal pancreatic tissue, relatively Raised in normal structure, Pancreatic Adenocarcinoma close to 9 times, nearly 9.5 times are raised in cancer of pancreas Lung metastases tissue.Every group of quantitative PCR 8 samples are analyzed, β-actin indicate that detection is accurate as internal reference, met the requirements.
Immunohistochemical staining uses Dako companies of U.S. EnVisionTMTwo step method detecting system detects ZEB2 albumen tables Reach.The positive percentage statistics of sample is carried out according to IRS commonly used in the art.Staining power and sun can be taken into account using IRS Property cell percentage.
As shown in table 1, SABC inspection finds that normal pancreatic tissue only has 2.9%, Pancreatic Adenocarcinoma positive for ZEB2 For 86.9%, cancer of pancreas Lung metastases are organized as 90.5% (table 1).SABC checks that every group is 10 samples.
As can be seen from the above results, ZEB2 quantitative PCRs and SABC inspection can be used to diagnosis of pancreatic cancer.
Embodiment 2 detects ZEB2 in pancreatic tumor cell Positive Level and the relation of prognosis
There is complete follow-up to record 30 Patients with Pancreatic Cancer for selection, obtains pathological section and carries out ZEB2 SABC inspections, SABC operating procedure is as above.Selected patient postoperative carries out conventional gemcitabine chemotherapy, no other treatment record.30 The ZEB2 SABC inspections of Patients with Pancreatic Cancer are divided into three groups, and (strong positive is 3.5, and the positive is 2.5, and weakly positive is 1.5) every group 10, prognosis tracking is carried out, the life span of patient is tracked.
As a result：As shown in Fig. 2 prognosis tracking data shows pancreatic tumor cell Positive Level and prognosis life cycle is in inverse ratio Relation, strong positive patient's poor prognosis, life cycle is short, and weakly positive patient prognosis is preferably, and life cycle is longer.Conclusion, ZEB2 is immunized Groupization inspection AQL can be used to predict prognosis, and can as predicted treatment effect index.
The ZEB2 of embodiment 3 disturbs the influence for tumour
1st, the preparation of people ZEB2 gene RNAis slow virus
(1) effective siRNA target spot of the screenings for people's ZEB2 genes
People ZEB2 (BC127102) gene information is transferred from Genbank；Having for ZEB2 genes is designed using DNAMAN The siRNA target spots of effect.In coded sequence (CDS) region of ZEB2 genes, 2 are devised for the effective of ZEB2 genes SiRNA target sequences.Respectively：ZEB2-1：cccagaagcccctgaggagctg；ZEB2-2： tactgcaagcgggaggcggagg。
The double-stranded DNA Oligo sequences of two ends I containing Age and EcoR I restriction enzyme site cohesive ends are synthesized for siRNA target spots； PGCSIL-GFP carriers are acted on Age I and EcoR I restriction enzymes, it is linearized, agarose gel electrophoresis identification Endonuclease bamhi.And build the RNAi carrier containing ZEB2-1 or ZEB2-2, be named as pGCSIL-GFP-ZEB2-1-shRNA and PGCSIL-GFP-ZEB2-2-shRNA, while building pGCSIL-GFP-control according further to the conventional test method in this area Negative control plasmids.Simultaneously by the vector introduction into slow virus it is standby.
2nd, real-time fluorescence quantitative RT-PCR method detects the silence efficiency of ZEB2 genes
Human pancreas cancer Panc-1 cells and normal human oral epithelial cells in exponential phase carry out pancreatin digestion, system It is inoculated in into cell suspension (cell number is about 6.0 × 104/ml) in 6 orifice plates, culture to cell fusion degree reaches about 40%.Root According to plural number (Panc-1 MOI is 20) value is infected, culture medium is changed after adding the virus of Sq, culture 24h, when infecting Between reach after 5 days, collect cell.According to the Trizol operational manuals of Invitrogen companies, extracted total RNA.According to The M-MLV operational manuals of Promega companies, RNA reverse transcriptions are obtained into cDNA, and (reverse transcription reaction system is shown in Table 7,42 DEG C of reactions 1h, then water-bath 10min inactivates reverse transcriptase in 70 DEG C of water-baths).
Real_time quantitative detection is carried out using ABI7500 type Real time PCR instruments (Life).The primer of gene is real as described above Apply described in example.Experimental result (Fig. 3) shows, in experimental group the ZEB2mRNA expressions of human pancreas cancer Panc-1 cells respectively under 99.3% and 99.5% have been dropped, expression effect is suppressed with preferable.
3rd, the multiplication capacity of the tumour cell of ZEB2-shRNA slow virus is infected in detection
Cell prepared by above-mentioned experiment 2, after time of infection reaches 5 days, collects each experiment in exponential phase Group cell.Cell suspension (2 × 104/ml) is resuspended into complete medium, is about 2000/hole with cell density, is inoculated with 96 holes Plate.Every group of 5 multiple holes, per the μ l of hole 100.Complete after plate, put 37 DEG C, 5%CO2 incubator cultures.Since after bed board second day, Daily with Cellomics instruments (Thermo Fisher) detection read plate once, continuous detection read plate 5 days.Pass through adjustment Cellomics arrayscan input parameter, calculates the cell with green fluorescence in scanning orifice plate every time exactly Data are carried out statistics drawing, draw cell growth curve by quantity.As a result show, the people that two ZEB2shRNA slow virus are infected Cancer of pancreas Panc-1 cells were cultivated after 5 days in vitro, and vigor cell number have dropped 88.9% and 86.4% respectively, show ZEB2 Gene silencing causes tumor cell proliferation ability to be suppressed.
The present invention is described in detail above with specific embodiment.It will be apparent to one skilled in the art that the present invention is not limited to Listed embodiment herein, its protection domain is defined by the appended claims, and the following examples are only with example Mode illustrates the present invention, so that the present invention is more readily understood.
Sequence table
The ＞ applicants of ＜ 110
Application of the ＞ ZEB2 gene expression variations of ＜ 120 in pancreatic cancer chemotherapy Prognosis
〈210〉1
〈211〉22
〈212〉DNA
The ＞ artificial sequences of ＜ 213
〈400〉ZEB2-1 siRNA
cccagaagcccctgaggagctg
〈210〉2
〈211〉22
〈212〉DNA
The ＞ artificial sequences of ＜ 213
〈400〉ZEB2-2 siRNA
tactgcaagcgggaggcggagg
〈210〉3
〈211〉19
〈212〉DNA
The ＞ artificial sequences of ＜ 213
The ＞ ZEB2 sense primers of ＜ 400
5’-AGCCAAGGAATGCTACCAA-3’
〈210〉4
〈211〉20
〈212〉DNA
The ＞ artificial sequences of ＜ 213
The ＞ ZEB2 anti-sense primers of ＜ 400
5’-GGCCCCAGAGCATCATAA TC-3’
〈210〉5
〈211〉20
〈212〉DNA
The ＞ artificial sequences of ＜ 213
＞ β-actin the sense primers of ＜ 400
5’-CAGCTGAGAGGGAAATCGTG-3’
〈210〉6
〈211〉20
〈212〉DNA
The ＞ artificial sequences of ＜ 213
＞ β-actin the anti-sense primers of ＜ 400
5’-CGTTGCCAATAGTGATGACC-3’。

Claims (14)

  1. Purposes of the 1.ZEB2 genes in diagnosis of pancreatic cancer and/or prognosis kit is prepared.
  2. 2. purposes as claimed in claim 1, it is characterised in that included in the kit for qRT-PCR primer pair and Corresponding reagent.
  3. 3. purposes as claimed in claim 2, it is characterised in that contain following primer sequence in the kit：ZEB2 is detected Primer pair：Sense primer：5’-AGCCAAGGAATGCTACCAA-3’；Anti-sense primer：5’-GGCCCCAGAGCATCATAA TC- 3’；Internal control primer pair：β-actin sense primers：5 '-CAGCTGAGAGGGAAATCGTG-3 ', anti-sense primer：5’- CGTTGCCAATAGTGATGACC-3’。
  4. 4. purposes as claimed in claim 1 or 2, it is characterised in that：Also including the use of specification in the kit.
  5. Purposes of the 5.ZEB2 gene expressions blocking agent in treatment pancreatic cancer drug is prepared.
  6. 6. purposes as claimed in claim 2, it is characterised in that：ZEB2 gene expressions blocking agent is ZEB2 gene RNA interferings.
  7. 7. purposes as claimed in claim 6, it is characterised in that：RNA interfering is siRNA, shRNA, RNAi or chemistry disruption Inhibitor.
  8. 8. purposes as claimed in claim 7, it is characterised in that：The small molecules interference RNA includes justice RNA fragments and antisense RNA fragments, the just RNA fragments and the antisense RNA fragment are complementary.
  9. 9. the purposes as described in claim 6-8 is any, it is characterised in that：The length of the just RNA fragments and antisense RNA fragment Degree is 15-27 nucleotides；Preferably, length is 19-23 nucleotides；Optimal, length is 19,20 or 21 Nucleotides.
  10. 10. the purposes as described in claim 6-8 is any, it is characterised in that：The small molecules interference RNA is that hair clip type is single-stranded By stem ring in the middle of RNA, including just RNA fragments, stem ring fragment and antisense RNA fragment, just RNA fragments and antisense RNA fragment Fragment separates；Wherein, just RNA fragments and antisense RNA fragment are complementary.
  11. 11. purposes as claimed in claim 10, it is characterised in that：The sequence of small molecules interference RNA is SEQ ID NO：1-2 appoints Shown in one.
  12. 12. purposes as claimed in claim 11, it is characterised in that：Further, the siRNA sequence is built Slow virus carrier is disturbed as gene, the carrier is that will encode the specific interference microRNA for suppressing ZEB2 gene expressions Gene fragment clone enter after slow virus carrier obtain, people's ZEB2 gene small molecules interference RNAs can be produced.
  13. 13. purposes as claimed in claim 12, it is characterised in that：The slow virus carrier can be selected from：
    pLKO.1-puro、pLKO.1-CMV-tGFP、pLKO.1-puro-CMV-tGFP、
    pLKO.1-CMV-Neo、pLKO.1-Neo、pLKO.1-Neo-CMV-tGFP、
    pLKO.1-puro-CMV-TagCFP、pLKO.1-puro-CMV-TagYFP、
    pLKO.1-puro-CMV-TagRFP、pLKO.1-puro-CMV-TagFP635、
    pLKO.1-puro-UbC-TurboGFP、pLKO.1-puro-UbC-TagFP635、
    pLKO-puro-IPTG-1xLacO、pLKO-puro-IPTG-3xLacO、pLP1、pLP2、
    pLP/VSV-G、pENTR/U6、pLenti6/BLOCK-iT-DEST、
    pLenti6-GW/U6-laminshrna、pcDNA1.2/V5-GW/lacZ、
    Appointing in pLenti6.2/N-Lumio/V5-DEST, pGCSIL-GFP or pLenti6.2/N-Lumio/V5-GW/lacZ One.
  14. 14. purposes as claimed in claim 1, it is characterised in that：The cancer of pancreas is ductal adenocarcinoma of pancreas.
CN201710403330.9A 2017-06-01 2017-06-01 Application of the ZEB2 gene expression variations in pancreatic cancer chemotherapy Prognosis Active CN107164480B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201710403330.9A CN107164480B (en) 2017-06-01 2017-06-01 Application of the ZEB2 gene expression variations in pancreatic cancer chemotherapy Prognosis

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201710403330.9A CN107164480B (en) 2017-06-01 2017-06-01 Application of the ZEB2 gene expression variations in pancreatic cancer chemotherapy Prognosis

Publications (2)

Publication Number Publication Date
CN107164480A true CN107164480A (en) 2017-09-15
CN107164480B CN107164480B (en) 2018-11-06

Family

ID=59822282

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201710403330.9A Active CN107164480B (en) 2017-06-01 2017-06-01 Application of the ZEB2 gene expression variations in pancreatic cancer chemotherapy Prognosis

Country Status (1)

Country Link
CN (1) CN107164480B (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109459569A (en) * 2018-07-28 2019-03-12 南昌大学 A method of exempting from blood sample tumour early monitoring

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103800918A (en) * 2012-11-08 2014-05-21 上海交通大学医学院附属第九人民医院 Application of micro RNA in preparation of antitumor drugs
CN104911183A (en) * 2015-06-25 2015-09-16 中山大学孙逸仙纪念医院 Transcription template of shRNA for inhibiting expression of pancreatic cancer TAK1 gene
CN105899677A (en) * 2013-12-16 2016-08-24 博爱米拉索勒股份公司 Different levels in blood cell samples of EMT-markers for the diagnosis of cancer, in particular of colorectal (CRC) and pancreatic (PC) cancer
CN106442956A (en) * 2015-08-11 2017-02-22 钱朝南 Method for screening anti-tumor metastasis compounds

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103800918A (en) * 2012-11-08 2014-05-21 上海交通大学医学院附属第九人民医院 Application of micro RNA in preparation of antitumor drugs
CN105899677A (en) * 2013-12-16 2016-08-24 博爱米拉索勒股份公司 Different levels in blood cell samples of EMT-markers for the diagnosis of cancer, in particular of colorectal (CRC) and pancreatic (PC) cancer
CN104911183A (en) * 2015-06-25 2015-09-16 中山大学孙逸仙纪念医院 Transcription template of shRNA for inhibiting expression of pancreatic cancer TAK1 gene
CN106442956A (en) * 2015-08-11 2017-02-22 钱朝南 Method for screening anti-tumor metastasis compounds

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109459569A (en) * 2018-07-28 2019-03-12 南昌大学 A method of exempting from blood sample tumour early monitoring

Also Published As

Publication number Publication date
CN107164480B (en) 2018-11-06

Similar Documents

Publication Publication Date Title
Jiao et al. hsa_circ_0000745 promotes cervical cancer by increasing cell proliferation, migration, and invasion
Li et al. LncRNA‐MEG3 inhibits cell proliferation and invasion by modulating Bmi1/RNF2 in cholangiocarcinoma
Zou et al. The downregulation of microRNA-10b and its role in cervical cancer
CN108624688B (en) Application of hsa _ circ _0012755 as prostate cancer molecular target in preparation of medicines and kits
CN108728535A (en) Applications of the hsa_circ_0049154 as prostate cancer molecular target in preparing drug and kit
Xu et al. LncRNA TUG1 aggravates the progression of prostate cancer and predicts the poor prognosis.
CN109468382B (en) Application of lncRNA in diagnosis and treatment of lung adenocarcinoma
CN112501299A (en) Method for predicting recurrence and metastasis of liver cancer and application
Sassenberg et al. Upregulation of the long non-coding RNA CASC9 as a biomarker for squamous cell carcinoma
CN107630092A (en) The 3p of miR 505 are applied to diagnosis, prognosis and the treatment of prostate cancer with osseous metastasis
Wu et al. MiR-660-5p promotes the progression of hepatocellular carcinoma by interaction with YWHAH via PI3K/Akt signaling pathway
CN105154447B (en) Applications of the AC016745.3 as prostate cancer molecular target and its in diagnostic kit
Zhang et al. LncRNA SNHG7 participates in osteosarcoma progression by down-regulating p53 via binding to DNMT1.
Zhang et al. MiR-99a suppressed cell proliferation and invasion by directly targeting HOXA1 through regulation of the AKT/mTOR signaling pathway and EMT in ovarian cancer.
Yin et al. Peripheral blood circulating microRNA‐4636/− 143 for the prognosis of cervical cancer
Cui et al. HOXA10 promotes the development of bladder cancer through regulating FOSL1.
CN109266743B (en) A kind of cancer markers and application thereof
Tian et al. CircularRNA-9119 promotes the proliferation of cervical cancer cells by sponging miR-126/MDM4
Tian et al. MicroRNA-497-5p is downregulated in hepatocellular carcinoma and associated with tumorigenesis and poor prognosis in patients
Li et al. Long noncoding RNA SNHG16 regulates the growth of human lung cancer cells by modulating the expression of aldehyde dehydrogenase 2 (ALDH2)
Zhang et al. MicroRNA-378a-3p downregulation as a novel biomarker with poor clinical outcomes in cervical cancer
CN107164480B (en) Application of the ZEB2 gene expression variations in pancreatic cancer chemotherapy Prognosis
US20190316207A1 (en) Mir-320e and colorectal cancer
Bae et al. RNA-based biomarkers for the diagnosis, prognosis, and therapeutic response monitoring of prostate cancer
Ye et al. Metabolism-Related Bioinformatics Analysis Reveals That HPRT1 Facilitates the Progression of Oral Squamous Cell Carcinoma In Vitro

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant