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EP2734642A1 - Snrna rnu2 - 1 en tant que marqueur tumoral - Google Patents

Snrna rnu2 - 1 en tant que marqueur tumoral

Info

Publication number
EP2734642A1
EP2734642A1 EP12747987.1A EP12747987A EP2734642A1 EP 2734642 A1 EP2734642 A1 EP 2734642A1 EP 12747987 A EP12747987 A EP 12747987A EP 2734642 A1 EP2734642 A1 EP 2734642A1
Authority
EP
European Patent Office
Prior art keywords
fragments
seq2
seq10
rnu2
diagnosis
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.)
Withdrawn
Application number
EP12747987.1A
Other languages
German (de)
English (en)
Inventor
Alexander Baraniskin
Stephan Hahn
Wolff Schmiegel
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.)
Ruhr Universitaet Bochum
Original Assignee
Ruhr Universitaet Bochum
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
Priority claimed from DE102011108254A external-priority patent/DE102011108254A1/de
Priority claimed from DE201210005153 external-priority patent/DE102012005153A1/de
Application filed by Ruhr Universitaet Bochum filed Critical Ruhr Universitaet Bochum
Publication of EP2734642A1 publication Critical patent/EP2734642A1/fr
Withdrawn legal-status Critical Current

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
    • 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
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • the invention relates to a method for the diagnosis of malignant diseases, which is based on the determination of the content of a particular snRNA, RNU2-1 f or partial sequences thereof in different body fluids and a kit for the diagnosis of such diseases and the use of this snRNA and its partial sequences and more specific Primer for the diagnosis of such
  • SnRNAs Small nuclear RNAs
  • SnRNAs are small RNA molecules that are involved in a number of important processes. SnRNAs are catalytically active as part of the spliceosome. They are responsible for the recognition and splicing of the intron of pre-mRNA contained in the nucleus. They also form complexes with several specific proteins, called snRNPs (small nuclear ribonucleoprotein particles) or snurps.
  • snRNPs small nuclear ribonucleoprotein particles
  • snRNAs In different body fluids such as blood or serum / plasma, ascites, pleural effusion, cerebrospinal fluid and urine are snRNAs and their
  • Subsequences detectable by RNA or DNA amplification techniques. In various types of tumors there are specific expression patterns of snRNAs.
  • Tumor markers used to assess the course of the disease are endogenous substances that can be found more frequently in some tumor diseases in the blood. The tumor cells form these substances themselves or stimulate their formation.
  • a tumor marker that is highly sensitive and specific for the presence of a malignant disease.
  • a tumor marker that provides meaningful values regardless of the type of tumor and in which
  • Differential diagnosis can be used.
  • the invention relates to a method for the diagnosis of malignant diseases, comprising the following steps:
  • the snRNA serving as marker according to the invention has the following nucleotide sequence:
  • RNU2-1f Meaningful fragments (RNU2-1f) of RNU2-1 are the following:
  • the snRNA RNU2-1 has in its nucleus a nucleotide sequence that provides (among other sequences?) For the tumor events a relevant statement.
  • This nucleotide sequence is shown in bold in SEQ1 and reproduced in the above table as SEQ4.
  • the nucleotide sequence of SEQ4 corresponds in all nucleotides of a microRNA miR-1246, which is part of the nucleotide sequence pre-miR-1246. However, this pre-miR-1246 has nothing to do with the tumor activity of interest here; the correspondence of the central nucleotide sequences is purely coincidental.
  • the diagnostics developed for miR-1246 can be used for purposes according to the invention for detecting a tumor activity of the patient.
  • the fragments SEQ2 to SEQ10 reproduced above are sequences derived from Qiagen PCR products which can be used for tumor diagnosis.
  • the sequences SEQ2 and SEQ3 are compared to the sequence SEQ4 shortened by one or two nucleotides at the 3 'end, the sequences SEQ5 to SEQ10 have SEQ4
  • sequences SEQ 2 to SEQ 10 listed above appear in the frequencies given in the cloning of PCR products with the Qiagen miR-1246 qRT-PCR assay. In 76% of the sequences is one
  • SnRNAs were quantitatively real-time
  • qRT-PCR Polymerase chain reaction
  • Cholangiocarcinoma hepatocellular carcinoma, renal cell and bladder carcinoma and GIST. This not only distinguished healthy patients from the sick, but also patients with malignancies of patients with inflammatory changes relevant for differential diagnosis.
  • biomarkers SEQ1 and SEQ2 to SEQ 10 are not limited to the first or early diagnosis of malignant diseases.
  • the data from the experiments show that they can also be used as progression markers and predicative markers. It has been found that the expression of the fragments SEQ2 to SEQ10 in colorectal carcinoma, bronchial and pancreatic carcinoma correlates with the course of the disease (course marker), the concentration of these fragments decreases with good clinical response to chemotherapy. Accordingly, the method according to the invention is also of value for the treatment of malignant diseases. It was also
  • the method according to the invention provides that the concentration of at least one fragment expressing the presence or absence of a malignant disease of the snRNA RNU2-1 is determined.
  • a qualitative determination can be made using a miR-1246 (SEQ4) specific probe. Since RNU2-1 and its fragments are detectable even in healthy patients in small amounts, the determined content is compared with a reference value.
  • a reference value can be a standard value, the value found being compared with the value of a healthy patient (standard value), but as a rule it is a relative value, since this is easier to determine.
  • a reference value can be a standard value, the value found being compared with the value of a healthy patient (standard value), but as a rule it is a relative value, since this is easier to determine.
  • Reference value can be the expression of a
  • unaffected micro-RNA ie a micro-RNA that is expressed unchanged in healthy and diseased patients, be, or even an added synthetic micro-RNA whose relative concentration in the sample is known.
  • a synthetic microRNA is syn-cel-54, which has been developed as a miRNA mimic for such reference purposes.
  • Relative quantifications can be carried out, for example, in the form of quantitative real-time PCR (qRT-PCR).
  • RNAs in a sample is determined, if appropriate after amplification by fluorescence measurement. Quantitative determinations are made using primers specific for the corresponding RNA fragments, ie by primers for miR-1246 (SEQ4) or one on at least one of
  • Quantitative determinations may also be made by specific primers to fragments other than SEQ2 to SEQ10 of RNU2-1. Also in this case, it is advantageous if such a primer detects a plurality of fragments which have a similarity in the sequence of a few nucleotides, as is also present in SEQ 1 to 10.
  • the invention further relates to a kit for the diagnosis of malignant diseases, comprising at least one probe and / or a primer for detecting the sn RNA RNU2-1, the SEQ1 and / or at least one of the fragments SEQ2 to
  • SEQ10 contains the snRNA RNU2-1.
  • the kit expediently also contains a reference RNA together with the associated probe and / or associated primer.
  • the invention relates to the use of RNU2-1 and / or at least one of its fragments for the diagnosis of malignant diseases and to the use of probes and / or primers specific for these fragments.
  • the probes are in particular also miR-1246 (SEQ4) specific primers, thus hybridizing oligonucleotides.
  • the invention enables a differential diagnosis between non-malignant diseases and neoplasias.
  • overexpression of RNU2-1 resulted from its fragments SEQ1 and SEQ2 to SEQ10 in the sample of a non-cancer patient or a healthy subject.
  • RNA content was extracted using a miRVana RNA isolation kit (Ambion, Austin, USA) according to the manufacturer's instructions. Frozen samples were thawed on ice and 0.17 ml of it was diluted with the same volume miRVana PARIS 2X denaturing Solution and then incubated on ice for 5 min. Thereafter, 5 ⁇ each of the synthesized miRNA mimic syn-cel-54 were added to the samples for the purpose of normalization
  • microRNA used 2 ⁇ of the total RNA solution were in the Reverse transcription reaction (37 ° C for 60 min and 95 ° C for 5 min, followed by 4 ° C) was used.
  • the real-time qPCR was tested on an Opticon 2 system (MJ Research, Waltham, MA) according to the protocol of the
  • the miR-1246 has the following nucleotide sequence: miR-1246 AAUGGAUUUUUGGAGCAGG This sequence is completely identical to the fragment SEQ4 of RNU2-1 and is present in the fragments SEQ1 and SEQ2, 3 and 5 to 10 almost completely or completely included.
  • Non-Hodgkin's lymphoma o Primary CNS lymphoma
  • Figure 1 shows the result of a serum test of a healthy
  • FIG. 2 shows a corresponding test with the miR-1246 assay from Qiagen.
  • the assay related to Figure 1 is specific for the fragments of RNU2-1 but less sensitive overall. Since the Qiagen assay on miR-1246 detects more fragments, the specificity is increased.
  • Figure 3 shows the result of serum tests on various malignant
  • the threshold (cut off) is -2.995 and is indicated by the dashed line.
  • PDAC pancreatic ductal adenocarcinoma
  • CRC colorectal carcinoma
  • R rectal carcinoma
  • CRC I to IV for the UICC stages I to IV of the CRC
  • HC healthy controls
  • DC diseased controls (differential diagnoses)
  • CRP for c -reactive protein.

Landscapes

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

Abstract

L'invention concerne un procédé de diagnostic de maladies malignes comportant les étapes consistant à préparer un échantillon obtenu à partir d'un liquide organique ou d'une sécrétion corporelle et à déterminer la concentration de snRNA RNU2-1 ou de ses fragments dans l'échantillon, ainsi qu'une trousse destinée à la réalisation de ce diagnostic et l'utilisation de sondes spécifiques au RNU2-1 et à ses fragments.
EP12747987.1A 2011-07-22 2012-07-20 Snrna rnu2 - 1 en tant que marqueur tumoral Withdrawn EP2734642A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE102011108254A DE102011108254A1 (de) 2011-07-22 2011-07-22 Pantumormarker
DE201210005153 DE102012005153A1 (de) 2012-03-16 2012-03-16 Pantumormarker
PCT/EP2012/064321 WO2013014091A1 (fr) 2011-07-22 2012-07-20 Snrna rnu2 - 1 en tant que marqueur tumoral

Publications (1)

Publication Number Publication Date
EP2734642A1 true EP2734642A1 (fr) 2014-05-28

Family

ID=46682802

Family Applications (1)

Application Number Title Priority Date Filing Date
EP12747987.1A Withdrawn EP2734642A1 (fr) 2011-07-22 2012-07-20 Snrna rnu2 - 1 en tant que marqueur tumoral

Country Status (3)

Country Link
US (1) US20140371084A1 (fr)
EP (1) EP2734642A1 (fr)
WO (1) WO2013014091A1 (fr)

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006034278A2 (fr) * 2004-09-21 2006-03-30 Matritech, Inc. Procedes et compositions de detection du cancer au moyen de composants de la particule spliceosomale u2
WO2010098862A2 (fr) * 2009-02-24 2010-09-02 Combimatrix Corporation Procédé d'utilisation d'une micropuce d'oligonucléotides pour détecter un cancer à partir d'un acide nucléique de sérum
CA2765870A1 (fr) * 2009-06-19 2010-12-23 Leroi V. Desouza Biomarqueurs d'hypernephrome
US20120231970A1 (en) * 2009-09-30 2012-09-13 Japan Health Sciences Foundation Colon cancer marker and method for testing for colon cancer
CA2825522A1 (fr) * 2011-01-26 2012-08-02 Cepheid Procedes de detection du cancer du poumon

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2013014091A1 *

Also Published As

Publication number Publication date
WO2013014091A1 (fr) 2013-01-31
US20140371084A1 (en) 2014-12-18

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